WO2024258911A1 - Protéines de liaison à l'adn spécifiques d'une séquence - Google Patents

Protéines de liaison à l'adn spécifiques d'une séquence Download PDF

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WO2024258911A1
WO2024258911A1 PCT/US2024/033519 US2024033519W WO2024258911A1 WO 2024258911 A1 WO2024258911 A1 WO 2024258911A1 US 2024033519 W US2024033519 W US 2024033519W WO 2024258911 A1 WO2024258911 A1 WO 2024258911A1
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seq
polypeptide
engineered
tag
mutation
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Ruijie ZHANG
Derek Hunter VALLEJO
Katherine NAKAMA
Lorita BOGHOSPOR
Lauren GUTGESELL
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10X Genomics Inc
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10X Genomics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the specific position of a cell within a tissue can also affect the cell’s morphology, differentiation, fate, viability, proliferation, behavior, signaling, and cross-talk with other cells in the tissue.
  • This spatial heterogeneity has been previously studied using techniques that only provide data for a small handful of analytes in the context of an intact tissue or a portion of a tissue. Specifically, these techniques can provide substantial analyte data for dissociated tissues (i.e., single cells). However, they fail to provide information regarding the position of a single cell in a biological sample (e.g., tissue sample) caused in part by inefficient transcript capture.
  • RT polypeptide comprising: (a) an RT polypeptide sequence; (b) a DNA binding domain, where the DNA binding domain is from a molecule capable of binding a minor groove of a nucleic acid; and (c) a linker connecting the RT polypeptide sequence and the DNA binding domain.
  • the DNA binding domain is located at the N-terminus of the RT polypeptide sequence. In some embodiments, the DNA binding domain is located at the C-terminus of the RT polypeptide sequence.
  • the linker is a glycine-serine (GS) linker selected from the group consisting of (GS)n, (GSGGS)n, (SGGSG)n, (GGGS)n, (GGSG)n, (GGSGG)n, (GSGSG)n, (GSGGG)n, GGGSG)n, and (GSSSG)n, where n represents an integer of at least 1.
  • the linker is GGGS.
  • the linker is SGGSG.
  • the DNA binding domain specifically recognizes adenine-thymine-rich region on a nucleic acid molecule.
  • the DNA binding domain specifically recognizes oligo(dA) or oligo(dT) tracts on a nucleic acid molecule.
  • the DNA binding domain comprises at least one AT-rich interaction domain.
  • the DNA binding domain comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 AT-rich interaction domains.
  • the AT-rich interaction domain comprises a core sequence, wherein the core sequence is a two base core sequence, a three base core sequence, a four base core sequence, or a five base core sequence.
  • At least one of the bases of the core sequence comprises an arginine; a glycine and an arginine; a proline and an arginine; a lysine and an arginine; or any combination thereof.
  • the AT-rich interaction domain comprises a GRKPG (Gly-Arg-Lys-Pro-Gly) repeat, a RKRGRPKK repeat, a KKRGRPKK repeat, a RKRGR repeat, a GR*R/PPK repeat, a GR*RPK repeat, a GR*PPK repeat, a KRPR* repeat, or a K/RKRGRPKK repeat.
  • the AT-rich interaction domain comprises a core sequence comprising an amino acid selected from the group consisting of SEQ ID NO: 11- 24.
  • the DNA binding domain is a DNA binding domain of any one of Saccharomyces cerevisiae datin (DAT1), high mobility group AT hook 1 (HMGA1), lysine-specific methyltransferase 2a ( KMT2A), Myocyte Enhancer Factor 2C (MEF2C), Heterogeneous Nuclear Ribonucleoprotein D (HNRNPD), Structural Maintenance of Chromosomes 1A (SMC1), Structural Maintenance Of Chromosomes 2 (SMC2), Caenorhabditis elegans tbp-1, Drosophila melanogaster D1 protein, Salmonella typhimurium Hin recombinase, S.
  • DAT1 Saccharomyces cerevisiae datin
  • HMGA1 high mobility group AT hook 1
  • KMT2A lysine-specific methyltransferase 2a
  • MEF2C Myocyte Enhancer Factor 2C
  • HNRNPD Heterogeneous Nuclear
  • the DNA binding domain is from a S. cerevisiae DAT1.
  • the amino acid sequence of the DNA binding domain comprises a DNA binding domain consensus motif set forth in SEQ ID NO: 13, 14, 16, or 22.
  • the DNA binding domain comprises: (a) a full-length DAT1 sequence or SEQ ID NO: 2; (b) an N-terminal truncated variant of DAT1 (D90) comprising the first 90 amino acids of the full length DAT1, or SEQ ID NO: 3; (c) a truncated variant of DAT1 (D60) comprising the first 60 amino acids of the full length DAT1 or SEQ ID NO: 5; (d) a truncated variant of DAT1 (D48) comprising the first 48 amino acids of the full length DAT1 or SEQ ID NO: 6; (e) a truncated variant of DAT1(D36) comprising the first 36 amino acids of the full length DAT1 or SEQ ID NO: 8; (f) a truncated variant of DAT1 (D35) comprising the first 35 amino acids of full length DAT1 or SEQ ID NO: 9; (g) an amino acid sequence having at least about 90%,
  • the DNA binding domain comprises the amino acid sequence of SEQ ID NO: 11 (GRKPG). In some embodiments, the DNA binding domain optionally comprise at least 2 domains or at least 3 domains comprising SEQ ID NO: 11. [00015] In some embodiments, the DNA binding domain comprises a mutation in any of one of SEQ ID NO: 2, 3, 5, 6, 8, 9, or 11. In some embodiments, the mutation is selected from a substitution, an insertion, a deletion, or any combination thereof. [00016] In some embodiments of the engineered RT polypeptide described herein, the DNA binding domain comprises SEQ ID NO: 2. In some embodiments of the engineered RT polypeptide described herein, the DNA binding domain comprises the amino acid sequence of SEQ ID NO: 3, 8, or 9.
  • the RT polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 7, and further comprises a combination of mutations selected from the group consisting of: (i) E69K, L139P, E302R, T306K, W313F, T330P, and N454K; and additionally one or more of M39V, P47L, Q91R, M66L, F155Y, D200N, D200E, H204R, G429S, L435G, L435K, P448A, D449G, H503V, D524N, T542D, E545G, D583N, H594Q, L603W, L603F, E607K, E607G, P627S, H634Y, H638G, A644V, D653H, K658R and L671P; and (ii) E69K, L139P, D
  • the amino acid sequence of the RT polypeptide sequence is: (a) at least 90% identical to SEQ ID NO: 1 or 143; (b) about 90% to about 99.99% identical to SEQ ID NO: 1 or 143, about 92% to about 99.99% identical to SEQ ID NO: 1 or 143, about 93% to about 99.99% identical to SEQ ID NO: 1 or 143, about 94% to about 99.99% identical to SEQ ID NO: 1 or 143, about 95% to about 99.99% identical to SEQ ID NO: 1 or 143, about 96% to about 99.99% identical to SEQ ID NO: 1 or 143, about 97% to about 99.99% identical to SEQ ID NO: 1 or 143, or about 98% to about 4 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC 99.99% identical to SEQ ID NO: 1 or 143; or (c) about
  • the RT polypeptide sequence comprises: (a) an amino acid sequence that is at least 95% identical to SEQ ID NO:1, 7, or 179, and; (b) a combination of mutations indexed to SEQ ID NO:7 or 178 selected from the group consisting of: (i) a combination of variants consisting of a T542D mutation, a D583N mutation, an E607G mutation, an A644V mutation, a D653H mutation, and a K658R mutation; and (ii) a combination of variants consisting of an E545G mutation, a D583N mutation, an H594Q mutation, an L603F mutation, and a S679P mutation.
  • the amino acid sequence of the RT polypeptide sequence comprises E69K, L139P, D200N, E302R, T306K, W313F, T330P, N454K, H503V, D524N, L603W, E607K, and H634Y.
  • the amino acid variations are at any one position or combination thereof as identified in an alignment of SEQ ID NO: 1 or 143 to any one of the RT polypeptide sequences in Table 1 or Table 2.
  • the RT polypeptide sequence comprises: (a) M39V, M66I, Q91R, I347V, and H594Q substitution in SEQ ID NO: 143; or (b) SEQ ID NO: 129 (SOLD 034).
  • the RT polypeptide sequence comprises M39V, T542D, D583N, E607G, A644V, D653H, K658R, and L671P in SEQ ID NO: 143.
  • the RT polypeptide sequence comprises: (a) M39V, T542D, D583N, E607G, A644V, D653H, K658R, L671P in SEQ ID NO: 143; or (b) SEQ ID NO: 111 (SOLD 025).
  • the RT polypeptide sequence comprises T542D, D583N, E607G, A644V, D653H, K658R, E545G, D583N, H594Q, and a L603F in SEQ ID NO: 143.
  • an engineered RT polypeptide comprising: (a) an amino acid sequence that is at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identical to: (i) an amino acid sequence of an RT disclosed in Table 1, or Table 2; or (ii) SEQ ID NOs: 27-61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 141, 143, 145, 147, 149, 151, 157, 159, 172, or 173; and (b) a DNA binding domain comprising an amino acid selected from the group consisting of SEQ ID NO
  • Another aspect of the present disclosure provides an engineered RT polypeptide comprising: (a) an amino acid sequence of an RT disclosed in Table 1 or Table 2; and (b) an amino acid sequence of DNA binding domain disclosed in Table 1.
  • the engineered RT polypeptide comprises: (a) the amino acid sequence of any one of SEQ ID NO: 174-188; (b) an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: SEQ ID NO: 174-188; or (c) an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO: SEQ ID NO: 174-188.
  • the engineered RT comprises an amino acid sequence that is at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 27-61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 141, 143, 145, 147, 149, 151, 157, 159, 172, and 173.
  • the RT polypeptide is 42B L (SEQ ID NO: 145), 50A+G (SEQ ID NO: 147), SOLD 022 (SEQ ID NO: 105), SOLD 023 (SEQ ID NO: 107), SOLD 025 (SEQ ID NO: 111), SOLD 031 (SEQ ID NO: 123), SOLD 033 (SEQ ID NO: 127), SOLD 034 (SEQ ID NO: 129), SOLD 035 (SEQ ID NO: 131), SOLD 001 (SEQ ID NO: 65), and SOLD 33 VDG (SEQ ID NO: 173), or an RT polypeptide set forth in SEQ ID NO: 143, or SEQ ID NO: 172.
  • the engineered RT comprises at least two DNA binding domains.
  • at least one DNA binding domain is located at the N-terminus of the engineered RT and at least one DNA binding domain is located at the C-terminus of the engineered RT.
  • the at least two DNA binding domains are both located at the C-terminus or N-terminus of the engineered RT.
  • RT reverse transcriptase
  • RT reverse transcriptase
  • RT polypeptide is any one of the RT polypeptides listed in Table 1 or Table 2.
  • the DNA binding domain is a DNA binding protein selected from the group consisting of S. cerevisiae datin (DAT1); high mobility group AT hook 1 (HMGA1), lysine-specific methyltransferase 2a ( KMT2A), Myocyte Enhancer Factor 2C (MEF2C), Heterogeneous Nuclear Ribonucleoprotein D (HNRNPD), Structural Maintenance of Chromosomes 1A (SMC1), Structural Maintenance Of Chromosomes 2 (SMC2), C. elegans tbp-1, D.
  • DAT1 S. cerevisiae datin
  • HMGA1 high mobility group AT hook 1
  • KMT2A lysine-specific methyltransferase 2a
  • MEF2C Myocyte Enhancer Factor 2C
  • HNRNPD Heterogeneous Nuclear Ribonucleoprotein D
  • SMC1 Structural Maintenance of Chromosomes 1A
  • SMC2 Structural Maintenance Of
  • the linker is a glycine-serine (GS) linker selected from the group consisting of (GS)n, (GSGGS)n, (SGGSG)n, (GGGS)n, (GGSG)n, (GGSGG)n, (GSGSG)n, (GSGGG)n, GGGSG)n, and (GSSSG)n, where n represents an integer of at least 1.
  • the linker is GGGS.
  • the linker is SGGSG.
  • the DNA binding domain is a S. cerevisiae datin (DAT1) DNA binding domain or fragment thereof.
  • the DNA binding domain comprises: (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 3, 5, 6, 8, 9, and 11-24; or (b) a nucleic acid sequence of SEQ ID NO: 25.
  • the RT polypeptide is selected from the group consisting of 42B L (SEQ ID NO: 145), 50A+G (SEQ ID NO: 147), SOLD 022 (SEQ ID NO: 105), SOLD 023 (SEQ ID NO: 107), SOLD 025 (SEQ ID NO: 111), SOLD 031 (SEQ ID NO: 123), SOLD 033 7 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC (SEQ ID NO: 127), SOLD 034 (SEQ ID NO: 129), SOLD 035 (SEQ ID NO: 131), SOLD 001 (SEQ ID NO: 65), SOLD 33 VDG (SEQ ID NO:
  • Another aspect of the present disclosure provides a recombinant RT protein as described herein comprising, consisting essentially of, or consisting of SEQ ID NO: 174-188.
  • the engineered RT polypeptide described herein, or the recombinant RT protein described herein further comprises a tag protein selected from the group consisting of an affinity tag, a fluorescent tag, or an expression and/or solubility enhancement tag.
  • the tag is selected from hexahistidine tag (his-tag), small ubiquitin-like modifier tag (SUMO), a VariFlex C-Terminal solubility enhancement tag, a short peptide C- terminal tag, Thioredoxin (Trx) tag, Solubility-enhancer peptide sequences (SET) tag, IgG domain B1 of Protein G (GB1) tag, IgG repeat domain ZZ of Protein A (ZZ) tag, Solubility enhancing Ubiquitous Tag (SNUT tag), Seventeen kilodalton protein (Skp tag), Phage T7 protein kinase (T7PK) tag, E.
  • EspA Monomeric bacteriophage T70.3 protein (Orc protein) (Mocr) tag, E. coli trypsin inhibitor (Ecotin) tag, Calcium-binding protein (CaBP) tag, Stress-responsive arsenate reductase (ArsC) tag, N-terminal fragment of translation initiation factor IF2 (IF2-domain I) tag, N-terminal fragment of translation initiation factor IF2 (Expressivity) tag, Fasciola hepatica 8-kDa antigen tag (Fh8), Glutathione-S-transferase (GST) tag, maltose-binding protein tag (MBP), Flag tag peptide (FLAG), streptavidin binding peptide tag (Strep-II; strep), calmodulin-binding protein tag (CBP), mutated dehalogenase tag (HaloTag), staphylococcal Protein A (Protein A), inte
  • the tag is an affinity tag selected from hexahistidine tag (his-tag), Fasciola hepatica 8-kDa antigen tag (Fh8), Glutathione-S-transferase (GST) tag, maltose-binding protein tag (MBP), Flag tag peptide (FLAG), streptavidin binding peptide tag (Strep-II), calmodulin- binding protein tag (CBP), mutated dehalogenase tag (HaloTag), staphylococcal Protein A 8 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC (Protein A), intein mediated purification with the chitin-binding domain (IMPACT (CBD)), cellulose-binding module (CBM), dockerin domain of Clostridium josui tag (Dock
  • the engineered RT polypeptide or the recombinant RT protein comprises: (a) an hexahistidine tag (his-tag); or (b) an amino acid sequence of SEQ ID NO: 62; or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 62.
  • the engineered RT polypeptide or the recombinant RT protein comprises a solubility enhancer tag selected from the group consisting of a SUMO tag, a GST tag, a Trx tag, a VariFlex C-Terminal solubility enhancement tag, a short peptide C-terminal tag, an Fh8 tag, MBP tag, SET tag, GB1 tag, ZZ tag, HaloTag, SNUT tag, Skp tag, T7PK tag, EspA tag, Mocr tag, Ecotin tag, CaBO tag, ArsC tag, IF2-domain I tag, Expressivity tag, RpoA, tag, SlyD, tag, Tsf tag, RpoS tag, PotD tag, Crr tag, msyB tag, yigD tag, and rpoD tag.
  • a solubility enhancer tag selected from the group consisting of a SUMO tag, a GST tag, a Trx tag, a VariFlex C-Term
  • the engineered RT polypeptide or the recombinant RT protein comprises: (a) a short peptide C-terminal tag; (b) an amino acid sequence of SEQ ID NO: 193; or (c)an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 193.
  • the tag further comprises: (a) an endoprotein cleavage sequence; (b) a cleavage sequence recognized by an endoprotein selected from the group consisting of alanine carboxypeptidase, Armillaria mellea astacin, bacterial leucyl aminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosol alanyl aminopeptidase, elastase, endoproteinase Arg-C, enterokinase (EnTK), gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase, human rhinovirus 3C protease, hypodermin C, Iga-specific serine endopeptidase, leucyl aminopeptidase, leucyl endopeptidase, ly
  • the engineered RT polypeptide or the recombinant RT protein described herein exhibits increased template switching (TS) efficiency, increased processivity efficiency, increased binding affinity, increased transcription efficiency, increased chemical tolerance, improved ability to yield mitochondrial unique molecular identity (UMI) counts, improved ability to yield ribosomal unique molecular identity (UMI) counts, longer shelf life, higher strand displacement, higher end-to-end template jumping, or any combination thereof, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • TS template switching
  • UMI mitochondrial unique molecular identity
  • UMI ribosomal unique molecular identity
  • the engineered RT polypeptide or the recombinant RT protein comprises at least two or more of increased template switching (TS) efficiency, increased processivity efficiency, increased binding affinity, increased transcription efficiency, increased chemical tolerance, improved ability to yield mitochondrial unique molecular identity (UMI) counts, longer shelf life, higher strand displacement, higher end-to-end template jumping, or improved ability to yield ribosomal unique molecular identity (UMI) counts, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • TS template switching
  • UMI mitochondrial unique molecular identity
  • the recombinant RT protein or the engineered RT exhibits increased transcript capture during amplification, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • the DNA binding domain enhances the hybridization of a transcript and a primer during a nucleic acid amplification process, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • the primer comprises a poly-dT or a poly(dT)VN sequence and a non-poly(dT) sequence; and the transcript comprises a poly-dA sequence.
  • the DNA binding domain stabilizes the oligo(A)-oligo(T) based transcript- primer complex during a nucleic acid amplification process.
  • the primer is a barcoded molecule.
  • the transcript is a nucleic acid molecule selected from an RNA, a mRNA, or a DNA.
  • One aspect of the present disclosure provides an isolated nucleic acid molecule encoding: (a) an engineered RT polypeptide described herein; or (b) a recombinant RT protein described herein.
  • the nucleic acid molecule comprises a sequence selected from SEQ ID NO: 25, SEQ ID NO: 136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:167, SEQ ID NO:169, or SEQ ID NO: 171; or a nucleic acid sequence of Table 2.
  • One aspect of the present disclosure provides an expression vector comprising any isolated nucleic acid described herein.
  • One aspect of the present disclosure provides a host cell transfected with any expression vector described herein or any isolated nucleic acid described herein.
  • One aspect of the present disclosure provides a composition comprising: (a) any recombinant RT protein described herein; or (b) any engineered RT polypeptide described herein; or (d) any expression vector described herein; or (e) any host cell described herein; and (f) a buffer.
  • One aspect of the present disclosure provides a method for performing a reverse transcription reaction for generating a nucleic acid product from an RNA template comprising contacting under suitable conditions a biological sample or extract thereof with an engineered RT polypeptide described herein, or a recombinant RT protein describe herein.
  • the biological sample or extract thereof comprises a cell, optionally the cell is permeabilized and/or optionally the cell is fixed.
  • the biological sample or extract thereof comprises a cell bead, optionally the cell bead is fixed.
  • the biological sample or extract thereof comprises a nucleus, optionally the nucleus is permeabilized and optionally the nucleus is fixed. In some embodiments, the biological sample or extract thereof comprises (a) a suitable cellular preparation selected from cell populations and/or single cells, or (b) a tissue. [00058] In some embodiments, the biological sample or extract thereof comprises: (a) a cell, a cell bead, a permeabilized cell, a nucleus, where the nucleus is optionally permeabilized, and/or optionally the cell, the cell bead, the permeabilized cell and/or nucleus are fixed; (b) a suitable cellular preparation selected from cell populations and/or single cells; or (c) a tissue.
  • the biological sample or extract thereof comprises cells in suspension, fresh cells, fixed cells, or cells and tissues immobilized on various solid surfaces.
  • the reverse transcription reaction is part of a single cell RNA sequencing assay.
  • the single cell RNA sequencing assay further comprises, prior to the reverse transcription, partitioning the cell, the cell bead, or the nucleus into a partition.
  • the single cell RNA sequencing assay further comprises, after the reverse transcription reaction, hybridizing the nucleic acid product to an oligonucleotide molecule comprising a partition-specific barcode.
  • the reverse transcription reaction is part of a spatial RNA sequencing assay.
  • the engineered RT polypeptide or the recombinant RT protein enhances template switching (TS) efficiency, processivity 12 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC efficiency, binding affinity, transcription efficiency, chemical tolerance, ability to yield mitochondrial unique molecular identity (UMI) counts, ability to yield ribosomal unique molecular identity (UMI) counts, strand displacement, end-to-end template jumping, or any combination thereof, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • TS template switching
  • UMI mitochondrial unique molecular identity
  • UMI ribosomal unique molecular identity
  • the engineered RT polypeptide or the recombinant RT protein enhances at least two or more of template switching (TS) efficiency, processivity efficiency, binding affinity, transcription efficiency, chemical tolerance, ability to yield mitochondrial unique molecular identity (UMI) counts, strand displacement, end-to-end template jumping, or ability to yield ribosomal unique molecular identity (UMI) counts, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • TS template switching
  • UMI mitochondrial unique molecular identity
  • UMI ribosomal unique molecular identity
  • the recombinant RT protein or the engineered RT enhances transcript capture during amplification, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • the DNA binding domain of the recombinant RT protein or the engineered RT enhances the hybridization of a transcript and a primer during the amplification process, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • the engineered RT polypeptide or the recombinant RT protein comprises: (a) a DNA binding domain comprising an amino acid sequence selected from SEQ ID NO: 2, 3, 5, 6, 8, 9, or 11-24; and (b) an amino acid sequence selected from SEQ ID NOs: 27- 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 141, 143, 145, 147, 149, 151, 157, 159, 172, or 173.
  • the amino acid sequence of the engineered RT polypeptide or the recombinant RT protein comprises an amino acid sequence having at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 174-188.
  • the engineered RT polypeptide or the recombinant RT protein comprises: (a) M39V, M66I, Q91R, I347V, and H594Q substitution in SEQ ID NO: 143; (b) SEQ ID NO: 129 (SOLD 034); (c) SOLD 001 (SEQ ID NO: 65); or (d) SOLD 33 VDG (SEQ ID NO: 173).
  • the engineered RT polypeptide or the recombinant RT protein comprises M39V, T542D, D583N, E607G, A644V, D653H, K658R, and L671P in SEQ ID NO: 1 or 143.
  • the engineered RT polypeptide or the recombinant RT protein comprises: (a) M39V, T542D, D583N, E607G, A644V, D653H, K658R, L671P in SEQ ID NO: 143; or (b) SEQ ID NO: 111 (SOLD 025).
  • the engineered RT or the recombinant RT protein comprises a M39V, M66I, Q91R, I347V, H594Q in SEQ ID NO: 143.
  • the engineered RT polypeptide or the recombinant RT protein comprises an amino acid sequence that is at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical to an amino acid sequence disclosed in Table 1 or Table 2.
  • One aspect of the present disclosure provides a method of using an engineered RT polypeptide described herein, or a recombinant RT protein described herein, the method comprising contacting the engineered RT polypeptide or the recombinant RT protein with a nucleic acid template under suitable conditions to produce a polymerized nucleic acid product, where the nucleic acid template comprises an RNA, a DNA, or a nucleic acid comprising an unnatural nucleotide.
  • the nucleic acid template comprises an RNA.
  • One aspect of the present disclosure provides a nucleic acid extension method comprising: (a) contacting a target nucleic acid molecule with an engineered reverse transcriptase polypeptide or a recombinant RT protein and a plurality of nucleic acid barcoded molecules comprising a barcode sequence, and (b) incubating the target nucleic acid, the engineered RT polypeptide or the recombinant RT protein and barcoded molecules under suitable conditions in which the barcoded molecules are extended by the engineered RT polypeptide or the recombinant RT protein.
  • the engineered RT polypeptide comprises the amino acid sequence of an engineered RT polypeptide described herein, or a recombinant RT protein described herein. 14 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC [00073]
  • the recombinant RT protein or the engineered RT polypeptide exhibits increased transcript capture during amplification.
  • the DNA binding domain enhances the hybridization of a transcript and a primer during a nucleic acid amplification process.
  • the primer comprises a poly-dT or a poly(dT)VN sequence and a non-poly(dT) sequence; and the transcript comprises a poly-dA sequence.
  • the DNA binding domain stabilizes the oligo(A)-oligo(T) based transcript- primer complex during a nucleic acid amplification process.
  • the primer is a barcoded molecule.
  • the recombinant RT protein or the engineered RT polypeptide described herein performs the first strand complementary DNA (cDNA) reaction.
  • the first strand cDNA is amplified using a DNA polymerase to generate a second strand cDNA.
  • One aspect of the present disclosure provides a method of producing an engineered RT polypeptide or recombinant RT protein of the present disclosure, the method comprising providing a composition comprising a cell lysate and/or cellular fraction comprising the engineered RT polypeptide or the recombinant RT protein and subjecting the composition to protein purification steps so as to produce a substantially purified engineered RT enzyme.
  • kits comprising: (a) a recombinant RT protein described herein; or (b) an engineered reverse transcriptase polypeptide described herein; or (c) the isolated nucleic acid described herein; or (d) an expression vector described herein; or (e) a host cell described herein; or (f) a composition described herein; and (g) instructions.
  • FIG.1A shows an exemplary sandwiching process where a first substrate (e.g., a slide), including a biological sample, and a second substrate (e.g., array slide) are brought into proximity with one another.
  • FIG.1B shows a fully formed sandwich configuration creating a chamber formed from the one or more spacers, the first substrate, and the second substrate.
  • FIG.2A shows a perspective view of an exemplary sample handling apparatus in a closed position.
  • FIG.2B shows a perspective view of an exemplary sample handling apparatus in an open position.
  • FIG.3A shows the first substrate angled over (superior to) the second substrate.
  • FIG.3B shows that as the first substrate lowers, and/or as the second substrate rises, the dropped side of the first substrate may contact a drop of reagent medium.
  • FIG.3C shows a full closure of the sandwich between the first substrate and the second substrate with one or more spacers contacting both the first substrate and the second substrate.
  • FIG.4A shows a side view of the angled closure workflow.
  • FIG.4B shows a top view of the angled closure workflow. 16 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC
  • FIG.5 is a schematic diagram showing an example of a barcoded capture probe, as described herein.
  • FIG.6 shows a schematic illustrating a cleavable capture probe.
  • FIG.7 shows exemplary capture domains on capture probes.
  • FIG.8 shows an exemplary arrangement of barcoded features within an array.
  • FIG.9A shows and exemplary workflow for performing a templated capture and producing a ligation product
  • FIG.9B shows an exemplary workflow for capturing a ligation product from FIG.9A on a substrate.
  • FIG.10 is a schematic diagram of an exemplary analyte capture agent.
  • FIG.11 is a schematic diagram depicting an exemplary interaction between a feature- immobilized capture probe 1124 and an analyte capture agent 1126.
  • FIG.12 shows a schematic diagram of a non-limiting embodiment of a generalized capture probe used in spatial transcriptomics and/or single cell transcriptomic analyses, exemplary applications in addition to general reverse transcription reactions where the engineered reverse transcriptase of the disclosure could be used to extend a capture probe using a captured target nucleic acid as a template, thereby generating a cDNA product.
  • FIG.13 provides a schematic of an exemplary capillary electrophoresis (CE) validation assay process used to test the activity of candidate enzymes.
  • CE capillary electrophoresis
  • step 1 5’-end labeled DNA primers were hybridized to RNA templates at room temperature (approx.25°C); and poly rG- labeled template switching oligonucleotides (rG-TSO) were added to the reaction mixture.
  • step 2 the temperature was raised to about 53°C and first strand cDNA was synthesized with the addition of a poly-C tail (tailing).
  • step 3 template switching and TSO extension were performed.
  • step 4 the amplification product was transferred to a Genetic Analyzer for analysis.
  • FIGs.14A-B provide schematics of an exemplary single cell and spatial assay for transcript capture.
  • FIG.14A illustrates a schematic process of the 5’ single cell assay and FIG.
  • FIG. 14B illustrates a schematic process of the 3’ single cell assay and step 1 of Visium.
  • the first step of both assays is the hybridization of an oligo(A) from an mRNA to an oligo(T)) of a primer and 17 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC binding of reverse transcriptase to the annealed primer-template (which contains an oligo(A)- oligo(T) tract).
  • FIGs.15A-B provide schematics of exemplary Visium/ spatial 3’/5’ workflows.
  • FIG. 15A illustrates a schematic of a Visium 3’ workflow demonstrating a polyA capture (box).
  • FIG. 15A illustrates a schematic of a Visium 3’ workflow demonstrating a polyA capture (box).
  • FIGs.16A-B provide a non-limiting embodiment of a nucleic acid sequence (FIG. 16A) and an amino acid sequence (FIG.16B) of an exemplary engineered reverse transcriptase (RT) polypeptide described in the present disclosure (N-Dat_42BL) comprising a DNA binding domain (e.g., DAT; bold and underline) operably linked to a reverse transcriptase (42BL) via a linker (bold).
  • FIGs.17A-B provide a non-limiting embodiment of a nucleic acid sequence (FIG.
  • FIG.17A an amino acid sequence (FIG.17B) of an exemplary engineered reverse transcriptase (RT) polypeptide described in the present disclosure (C-Dat_42BL) comprising a reverse transcriptase (42BL) operably linked to a DNA binding domain (e.g., DAT; bold and underline) via a linker (bold).
  • RT reverse transcriptase
  • C-Dat_42BL exemplary engineered reverse transcriptase polypeptide described in the present disclosure
  • 42BL a reverse transcriptase operably linked to a DNA binding domain (e.g., DAT; bold and underline) via a linker (bold).
  • FIGs.18A-B provide a non-limiting embodiment of a nucleic acid sequence (FIG.
  • FIG.18B an amino acid sequence (FIG.18B) of an exemplary engineered reverse transcriptase (RT) polypeptide described in the present disclosure (N-DAT1full_42BL) comprising a DNA binding domain (e.g., full length DAT1 protein; bold and underline) operably linked to a reverse transcriptase (42BL) via a linker (bold).
  • RT reverse transcriptase
  • N-DAT1full_42BL exemplary engineered reverse transcriptase polypeptide described in the present disclosure
  • FIGs.19A-B provide a non-limiting embodiment of a nucleic acid sequence (FIG.
  • FIG.19B an amino acid sequence of an exemplary engineered reverse transcriptase (RT) polypeptide described in the present disclosure (C-DAT1full_42BL) comprising a reverse transcriptase (42BL) operably linked to a DNA binding domain (e.g., full length DAT1 protein; bold and underline) via a linker (bold).
  • RT reverse transcriptase
  • FIG.20 shows the performance of two engineered RTs described herein (N-DAT 42BL; SEQ ID NO: 175) and C-DAT 42BL (SEQ ID NO: 174)) in a Single Cell 5’ (SC-5’) gene expression assay when compared to two control MMLV variants (SOLD 001 (SEQ ID NO: 65) and SOLD 33 VDG (SEQ ID NO: 175)); and illustrates the superiority of the DAT engineered 18 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC RTs single cell assays over control non-DAT engineered RTs based on the median genes and UMIs per cell at 20k and 50k raw-reads per cell (rrpc).
  • FIG.21 shows the performance of two engineered RTs described herein (N-DAT 42BL; SEQ ID NO: 175) and C-DAT 42BL (SEQ ID NO: 174)) in a Single Cell 5’ (SC-5’) gene expression assay when compared to two control MMLV variants (SOLD 001 (SEQ ID NO: 65) and SOLD 33 VDG (SEQ ID NO: 175)); and illustrates the superiority of the DAT engineered RTs for single cell assays over control non-DAT engineered RTs based on spatial transcriptomics and single cell transcriptomic analyses.
  • FIGs.22A-B provide a bar graph (FIG.22A) and a quantification (FIG.22B) of the relative differences in performance between three reverse transcriptases when compared to a control RT and illustrating that a clear performance gains can be seen for a DAT fusion on either the N-terminal or C-terminal domain.
  • FIGs.22A-B show median genes and UMIs/cell at 50k raw-reads per cell of three reverse transcriptases with and without a DAT fusion domain.
  • FIGs.23A-D provide graphs illustrating the performance of various engineered RTs disclosed herein at maximum normalization depth; and showing median genes (FIG.23A) and UMIs/cell (FIG.23B) at maximum normalized read depth comparing library complexity of three reverse transcriptases with and without the DAT fusion domain.
  • FIGs.23C-D show saturation curves of the median genes (FIG.23C) and counts/cell (FIG.23C) as a function of read depth. At maximum normalized read depth, the benefit of the DAT fusion on each RT backbone can clearly be seen.
  • FIGs.24A-F provide graphs illustrating the differential gene expression of some engineered RTs comprising DAT1 at the N-terminus.
  • FIGs.24A, C, and E feature scatter plots showing gene expression correlation of three reverse transcriptases with and without the DAT fusion domain.
  • FIGs.24B, D, and F feature volcano plots showing the number of differentially expressed genes between three reverse transcriptases with and without the DAT fusion domain.
  • FIGs.25A-F provide graphs illustrating the differential gene expression of some engineered RTs comprising DAT1 at the C-terminus.
  • FIGs.25A, C, and E feature scatter plots showing gene expression correlation comparison of engineered RTs comprising N-Terminal 19 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC DAT Domain and C-Terminal DAT Domain.
  • FIGs.25B 42BL versus 42BL NDAT
  • D and F feature volcano plots comparing the number of differentially expressed genes between various RTs. Gains in performance were still present but were not as significant as a C-terminal DAT fusion.
  • the C-terminal DAT fusion did not perform as well as the N-terminal DAT fusion, but both N-terminal DAT fusion and C-terminal DAT fusion showed performance gains when compared to a control RT variant.
  • the control RT variant was a non-DAT fusion RT comprising the same RT backbone (e.g., RT backbone alone; SEQ ID NO: 145).
  • FIGs.26A-D provide performance comparison of the impact of the DAT domain across three reverse transcriptase backbones.
  • FIGs.26A-B summarize median genes (FIG.26A) and UMIs/cell (FIG.26B) at maximum normalization depth comparing the performance among three MMLV RT variants 42B, 42BL, and 50A+ G backbones with and without a N-terminal DAT fusion domain; and illustrate a clear performance benefit from the DAT domain.
  • FIG.26C shows gene expression correlation.
  • FIG.26D shows differential gene expression. MMLV RT variants without a DAT fusion domain were used as controls.
  • FIG.27 provides a schematic of exemplary Visium/ spatial 3’ workflows highlighting the improvements associated with the engineered RT variant comprising DAT1 at the N- terminus (NDAT1) disclosed herein.
  • NDAT1 RT variant reduced transcript mislocalization and increased target capture. Cleaving oligos after annealing and reducing steric hindrance also enhanced optimization.
  • reverse transcription there was an improved template switching and increased RT efficiency.
  • second strand synthesis there was an improved synthesis efficiency and increased product.
  • FIGs.28A-F provide UMI heat maps showing globally detected gene expression in two replicates of spatial assay performed using a first slide configuration, on a human tonsil tissue, using an NDAT1 MMLV RT variant (FIGs.28C-F) or a control MMLV RT variant (FIGs.28A-B) and three different buffer formulations – a commercially available RT reagent (FIG.28A and FIG.28C), Buffer X.4 (FIG.28D), and Buffer X.5 (FIG.28B, FIG.28E, and 20 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC FIG.28F).
  • FIGs.28A-F show that NDAT1 RT variant improved the sensitivity of the assay while maintaining good spatial resolution when compared to the control RT variant, with all three buffer conditions.
  • FIGs.29A-E provide graphs quantifying the quality, sensitivity, and detection of gene expression under the same six conditions shown in FIGs.28A-F.
  • fraction reads in spots under tissue (FIG.29A), reads mapped confidently to transcriptome (FIG.29B), total genes detected (FIG.29C), GRch38 median UMI counts per spot (30k mapped spot-reads per spot) (FIG.29D), and GRch38 median genes per spot (30k mapped spot-reads per spot) (FIG.29E).
  • the quantification confirmed that NDAT1 RT variant improved the sensitivity of the assay while maintaining good spatial resolution when compared to the control RT variant (FIGs.29D-E).
  • a MMLV RT variant without a DAT fusion domain was used as a control.
  • FIGs.30A-F provide UMI heat maps showing gene expression of RGS3 in a human tonsil tissue analyzed using NDAT1 RT variant (FIGs.30C-F) or a control RT variant (FIGs. 30A-B) and three different buffer formulations - RT reagent (FIG.30A and FIG.30C), Buffer X.4 (FIG.30D), and Buffer X.5 (FIG.30B, FIG.30E, and FIG.30F).
  • a MMLV RT variant without a DAT fusion domain was used as a control.
  • UMI heat maps are shown as a log10(UMI) ranging from cold to hot (0, 0.5, 1.0, 1.5, and 2.0).
  • FIGs.31A-C provide UMI heat maps showing gene expression of KRT5 in two replicates of a spatial assay performed using a first slide configuration on human tonsil tissues analyzed using NDAT1 RT variant (FIGs.31C-F) or a control RT variant (FIGs.31A-B; SEQ ID NO: 1, 142, 143, or 172) and three different buffer formulations - RT reagent (FIG.31A and FIG.31C), Buffer X.4 (FIG.31D), and Buffer X.5 (FIG.31B, FIG.31E, and FIG.31F).
  • a MMLV RT variant without a DAT fusion domain was used as a control.
  • FIGs.32A-E provide UMI heat maps showing globally detected gene expression (FIGs.32A-B) or gene expression of Crgm1 (FIG.32C), KRT5 (FIG.32D), and Rho (FIG.
  • FIGs.33A-D provide UMI heat maps showing globally detected gene expression (FIG.33A) or gene expression of CRGM1 (FIG.33B), KRT5 (FIG.33C), and RHO (FIG. 33D) in a spatial assay performed using a second slide configuration (e.g., Visium high definition (HD) spatial gene expression or next generation spatial gene expression) on Zebrafish (non-human or mouse tissue) analyzed using NDAT1 RT variant (42BL-NDAT1) and RT reagent buffer under sandwich configuration conditions.
  • UMI heat maps are shown as a log normalized per experiment for CRYGM1 (0.00-4.0), KRT5 (0.0-9.0) or RHO (0.0-7.0).
  • the present disclosure leverages 22 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC functional characteristics of DNA binding proteins to engineer RT variants that increase the sensitivity of the RT in single cell applications.
  • the present disclosure provides engineered RT variants (e.g., engineered reverse transcriptase (RT) polypeptide or recombinant RT proteins) to increase transcript capture during single application using an accessory protein that is sequence specific for oligo(A)-oligo(T) tract.
  • engineered RT variants e.g., engineered reverse transcriptase (RT) polypeptide or recombinant RT proteins
  • the accessory protein contemplated by the present disclosure is a DNA binding protein or amino acid motif which is sequence specific to oligo(A)-oligo(T) tract.
  • the accessory protein can function as a “fusion” partner with the reverse transcriptase (e.g., MMLV RT or variant thereof) in single cell 5', single cell 3', and/or spatial assays (e.g., 10X Genomics single cell 5', single cell 3', and/or spatial assays).
  • This strategy enabled improvement of transcript capture, and therefore sensitivity of the assays.
  • DAT1 [000122] Dating (DAT1) or a truncation thereof was identified as a candidate sequence specific DNA binding motif.
  • DAT1 is a yeast protein (e.g., Saccharomyces cerevisiae) that specifically recognizes the minor groove of non-alternating oligo(A)-oligo(T) tracts (e.g., >10 bp oligo(A)- oligo(T) tract).
  • yeast protein e.g., Saccharomyces cerevisiae
  • the sequence specific recognition may be determined by three repeated pentads of G-R-K-P-G (SEQ ID NO: 11).
  • the N-terminal 90 amino acids (D90) and/or the N-terminal 36 amino acids (D36) can bind in a sequence specific manner to oligo(A)-oligo(T) tract.
  • DAT1(D-90) can specifically bind to A-T tracts with Kd of about 3 x 10 -10 M (or 3 x 10 -9 M); and DAT1(D-36) protein can bind to A- T tracts with Kd of 4 x 10 -10 M.
  • DAT1(D-90) can also be more resistant to degradation by bacterial proteases than longer and shorter DAT1 derivatives.
  • DAT1(D-90) The DNA binding activity of DAT1(D-90) can also be resistant to heat (boiling in water bath for 10 min) and chemical treatment (6 M guanidine HCl). DAT1(D-90) can also be highly soluble in physiologic salt and pH conditions. [000123] While DNA binding proteins have been used in combination with a reverse transcriptase as fusion proteins to improve processivity of the reverse transcriptase, sequence specific DNA binding proteins have not been explored. See e.g., Oscorbin et al., FEBS Lett., 594, 4338 (2020).
  • sequence specific DNA binding proteins have not been applied 23 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC to sequencing applications of any type. Fusion proteins with DAT1 have also not been constructed. [000124] As such, engineered RT molecules comprising DAT1 or fragments thereof were investigated as methods to improve the sensitivity of single cell assays. The binding properties of DAT1 was found to be highly tunable. DAT1 was combined with reverse transcriptase (42B or other MMLV variants). Both N-terminal and C-terminal fusions were explored.
  • DAT1(90), first 90 N-terminal amino acids; or DAT1(36), a 36 residue minimal DAT1 binding domain i.e., DAT1(90), first 90 N-terminal amino acids; or DAT1(36), a 36 residue minimal DAT1 binding domain.
  • DAT1 binding domain can assist a reverse transcriptase in binding to primed transcripts (FIGs. 14A-B and 15A-B; box), thereby increasing the assay sensitivity.
  • the DAT1 constructs were optimized for: (1) truncation of DAT1 binding domain; (2) tuning the binding strength of DAT1 to oligo(A)-oligo(T) tract through sequence modification (e.g., removal or alteration of G-R-K- P-G binding pentad); and (3) identification and testing of other sequence-specific DNA binding proteins.
  • the DNA binding domain is from a molecule capable of binding a minor groove of a nucleic acid (e.g., DAT 1).
  • the present disclosure provides recombinant reverse transcriptase (RT) proteins comprising a RT polypeptide, fused to a DNA binding domain, where the RT polypeptide and the DNA binding domain are separated by an amino acid linker, and the DNA binding domain is fused to the N-terminus of the RT polypeptide.
  • RT reverse transcriptase
  • the present disclosure provides recombinant reverse transcriptase (RT) proteins comprising a RT polypeptide, fused to a DNA binding domain, where the RT polypeptide and the DNA binding domain are separated by an amino acid linker, and the DNA binding domain is fused to the C-terminus of the RT polypeptide.
  • compositions comprising the engineered RT or the recombinant RT protein and methods of using the engineered RT or the recombinant RT protein for performing reverse transcription reactions in a variety of applications.
  • An exemplary DNA binding protein is DAT1.
  • a full-length DNA binding protein, truncations or fragments thereof, 24 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC and/or peptide motifs with sequence specific binding can be used to engineer the RT contemplated by the present disclosure.
  • FIG.20 and FIG.21 show that DAT1 in combination with reverse transcriptase MMLV, or MMLV variants thereof (e.g., SOLD 001 or SOLD 033 VDG), either fused at the N- terminus or the C-terminus of the RT improved the assay (GEX) sensitivity even at low sequencing depth.
  • the engineered RT molecules disclosed herein exhibited large change in differential gene expression in single cell assays.
  • the engineered RT molecules comprising DAT1 or variants thereof disclosed herein picked-up up to about 5000 additional genes when compared to a non-DAT1 RT control (e.g., MMLV variant alone).
  • FIGs.20-26 The engineered RT molecules disclosed herein also exhibited increase in median UMI counts per spot and median gene counts per spot in spatial assay.
  • the engineered RT molecules disclosed herein gave decrease in fraction of reads mapped to exons with gain in fraction mapped to introns. See e.g., FIG.21. A performance difference between FPLC and plate purified proteins was also demonstrated. [000128]
  • the engineered RT polypeptides and/or the recombinant RT proteins disclosed herein were tested in spatial platforms using the 3’ workflow shown in FIG.27 under sandwich configuration conditions and using three different buffer formulations described herein.
  • the engineered N-DAT1 RT variants disclosed herein enhanced the quality and the sensitivity of the spatial assay metrics while maintaining good spatial resolution when compared to a control RT lacking the DAT1 domain (also referred to as “control RT”).
  • a control RT in the context of DAT1-RT fusion, refers to a non-DAT1 fusion RT comprising the same RT backbone as the N-DAT1 RT or the C-DAT1 RT.
  • the control RT is a MMLV variant (SEQ ID NOs: 1, 143, or 172) or MMLV variant L (SEQ ID NO: 145).
  • FIGs.28A-F show UMI heat maps showing globally detected gene expression in a spatial assay performed using a first slide configuration, on human tonsil tissue analyzed using NDAT1 RT variant (FIGs.28C-F) compared to a control RT variant (FIGs.28A-B) using three different buffer formulations.
  • FIGs.29A-F show that the engineered RT polypeptides and/or the recombinant RT proteins disclosed herein (e.g., an engineered RT variant comprising DAT-1 at the N-terminus (N-DAT)) also significantly enhanced the quality and the sensitivity metrics of the spatial assays.
  • FIGs.29A-F quantify the quality, sensitivity, and detection of gene expression metrics under the same six conditions shown in FIGs.28A-F. In particular, fraction reads in spots under tissue (FIG.29A) was substantially the same among the 6 conditions tested. However, the standard deviation of the sample comprising the N-DAT RT and RT reagent buffer (N-DAT_RTR) was smaller.
  • the “fraction reads under tissue” refers to the ratio between reads in the area of direct interaction between the array and the tissue over total reads.
  • the fraction reads under tissue showed the diffusion or transcript mislocalization that may have occurred during transcript release from the tissue or transcript capture onto the array.
  • Reads mapped confidently to transcriptome (FIG.29B) were significantly increased in the samples containing the N-DAT_RT when compared to the five other conditions.
  • the total genes detected appeared the same in all conditions tested, except in samples including the control RT variant and using RT reagent buffer (42B_RTR), which showed significantly reduced total genes detected.
  • FIG.29D Analysis of GRch38 median UMI counts per spot (30k mapped spot-reads per spot) (FIG.29D) showed that median UMI counts per spot were significantly enhanced in all samples comprising an N-DAT RT variant when compared to the control RT samples regardless of the buffer used. Unexpectedly, samples comprising the N- DAT_RT variant and Buffer X.4 showed less variability.
  • the GRch38 median genes per spot (30k mapped spot-reads per spot) (FIG.29E) analysis showed that median genes per spot were significantly enhanced in all samples comprising an N-DAT1 RT variant when compared to the control RT samples regardless of the buffer used. Assessment of individual gene expression also showed substantially similar results as globally detected gene expression.
  • FIGs.29E Analysis of GRch38 median UMI counts per spot (30k mapped spot-reads per spot) (FIG.29D) showed that median UMI counts per spot were significantly enhanced in all samples comprising an N-DAT RT variant when compared to the control RT samples regardless
  • FIGs.31A-C show UMI heat maps illustrating the gene expression of KRT5 in a human tonsil 26 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC tissue.
  • spatial assays conducted with an N-DAT RT variant were successful using two different slide configurations: the first slide configuration (e.g., Visium standard definition (SD) spatial gene expression assay) or the second slide configuration (e.g., Visium high definition (HD) or next generation spatial gene expression assay), with a sample from Zebrafish (FIGs.32A-E and FIGs.33A-D).
  • SD Visium standard definition
  • HD Visium high definition
  • FIGs.32A-E and FIGs.33A-D next generation spatial gene expression assay
  • the engineered RT polypeptides and/or the recombinant RT proteins disclosed herein improved the sensitivity of the spatial assay while maintaining good spatial resolution when compared to the control RT variant (FIGs.29D-E). Furthermore, in some metrics, such as reads mapping confidently to the genome, use of the N-DAT RT variant with RT Reagent was particularly superior.
  • the present disclosure demonstrates for the first time that incorporation of DAT1 or any molecule having substantially similar molecular function, in single and spatially assay increased transcript capture and single cell assay sensitivity.
  • the DNA binding domain enhances the enzymatic activity of the engineered reverse transcriptase.
  • the addition of the DNA binding domain can enhance the template switching (TS) efficiency, higher end-to-end template jumping/switching, processivity efficiency, binding affinity, transcription efficiency, chemical tolerance, ability to yield mitochondrial unique molecular identifier (UMI) counts, ability to yield ribosomal unique molecular identifier (UMI) counts, shelf life, higher strand displacement, increased thermostability, improved thermoreactivity, and any combination thereof, for the engineered (i.e., recombinant) reverse transcriptase when compared to a control RT (SEQ ID NO: 1, 143, 145, or 172), WT MMLV, or known MMLV variants.
  • TS template switching
  • UMI mitochondrial unique molecular identifier
  • UMI ribosomal unique molecular identifier
  • TS efficiency Small RNAs ( ⁇ 200 nucleotides) are for the most part non-coding regulatory elements and play a key role in gene expression. Small RNAs regulate gene expression in plants, animals, and many fungi—including several roles in development, proliferation, differentiation, immune reaction, apoptosis, tumorigenesis and adaptation to stress. Given their importance in regulation, miRNAs are candidates as biomarkers for several human diseases. Thus, developing accurate and reproducible ways to study these and other small RNAs is necessary to further decipher their biological consequences. [000135] The main sources of bias in a typical library preparation workflow are the enzymatic ligations that introduce 5′ and 3′ sequencing adaptors to single-stranded templates.
  • Template 27 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC switching permits ligation-free incorporation of the 5′ adapter during reverse transcription.
  • Template switching-based methods depend upon the natural tendency of MMLV-type reverse transcriptases to add nontemplated nucleotides at the 3′ end of the emerging cDNA strand. These nontemplated additions serve as an anchoring unit for annealing complementary nucleotides in a provided template switching oligonucleotide (TSO); upon reaching the cDNA-TSO cross- junction, the reverse transcriptase effectively switches templates, continuing cDNA synthesis out of the TSO sequence.
  • TSO template switching oligonucleotide
  • End-to-end template jumping or switching refers to the ability of a reverse transcriptase to template-switch from the 5’ end of one template to the 3’ end of another. Improved end-to-end template jumping or switching can result in an improved process efficiency.
  • the engineered reverse transcriptase described herein exhibiting improved or higher end-to-end template jumping or switching, is highly desirable.
  • DNA binding affinity To initiate reverse transcription, reverse transcriptases require a short DNA oligonucleotide called a primer to bind to its complementary sequences on the RNA template and serve as a starting point for synthesis of a new strand. Improved binding affinity results in a more efficient process, particularly when limited amounts of RNA are available. Thus, the engineered reverse transcriptase described herein, exhibiting improved DNA binding affinity, is highly desirable.
  • Transcription efficiency The RNA-to-cDNA conversion step in transcriptomics experiments is widely recognized as inefficient and variable.
  • Transcriptomics measurements almost invariably include a reverse transcription (RT) step, where RNA transcripts are used as templates to generate cDNA transcripts for quantification.
  • RT reverse transcription
  • RNA transcripts are used as templates to generate cDNA transcripts for quantification.
  • the engineered reverse transcriptase described herein exhibiting improved transcription efficiency, is highly desirable.
  • Reverse transcriptases function in an environment that may include processing chemicals, such as cell fixation chemicals or processing reagents, which can negatively impact the function and activity of the enzyme. Thus, the engineered reverse transcriptase described herein, exhibiting improved chemical tolerance, is highly desirable.
  • Ability to yield mitochondrial and/or ribosomal unique molecular identifier (UMI) counts Unique molecular identifier (UMI) counting is a gene expression quantification scheme used in single-cell RNA-sequencing (scRNA-seq) analysis. Single-cell RNA-sequencing (scRNA-seq) technology provides transcriptome profiles of individual cells, enabling the dissection of the heterogeneity of different cell populations and tissues.
  • scRNA-seq protocols The paucity of starting material for reverse transcription remains an inherent limitation of scRNA-seq protocols and contributes to the relatively low rate at which messenger RNA (mRNA) molecules in individual cells are converted to cDNA molecules that can be captured and sequenced.
  • mRNA messenger RNA
  • mRNA-seq protocols employ an additional step in which individual transcripts are barcoded with unique molecular identifiers (UMIs) before amplification, resulting in a more accurate quantification of the transcript count.
  • UMIs incorporate a unique barcode onto each molecule within a given sample library.
  • variant alleles present in the original sample can be distinguished from errors introduced during library preparation, target enrichment, or sequencing.
  • the engineered reverse transcriptase described herein exhibiting an improved ability to yield mitochondrial and/or ribosomal UMI counts, is highly desirable.
  • Shelf life and/or stability In another aspect of the disclosure, the engineered reverse transcriptase described herein, exhibit improved stability and/or shelf life. A longer period of stability, and/or shelf life, is desirable as it can result in more efficient processes.
  • Strand displacement is the process through which two strands with partial or full complementarity hybridize to each other, displacing one or more pre- hybridized strands in the process.
  • Reverse transcriptase first transcribes a complementary strand 29 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC of DNA to make an RNA:DNA hybrid.
  • reverse transcriptase or RNase H degrades the RNA strand of the hybrid.
  • the single-stranded DNA is then used as a template for synthesizing double-stranded DNA (cDNA).
  • RT reverse transcriptase catalyzes the conversion of RNA into an integration-competent double-stranded DNA, with a variety of enzymatic activities that include the ability to displace a non-template strand concomitantly with polymerization.
  • RT are capable of efficiently unwinding duplexes in the template during polymerization.
  • This strand displacement synthesis activity by RT is required for the polymerization on the highly structured RNA and the removal of RNA fragments which cannot be cleaved by the enzymes’ RNase H activity.
  • strand displacement synthesis on a DNA duplex is particularly important to complete the plus- and minus-strands by polymerizing on the long terminal repeats.
  • any of the engineered RT enzymes of the present disclosure including without limitation any of the enzymes comprising the amino acid sequence and/or nucleic acid sequences shown in Table 1 or Table 2 could be analyzed in any suitable assay, including without limitation the assays described herein. Assays include without limitation 5’ gene expression analyses, with or without VDJ analysis, 3’ gene expression analysis, epigenetic analysis, or multiomic analyses.
  • experiments are carried out as found in the manufacturer’s instructions for the Chromium Single Cell 5’ Gene Expression Assay kit (10X Genomics); Chromium Single Cell 3’ Gene Expression Assay kit (10X Genomics), including any of multiomic extensions or applications.
  • SPATIAL ANALYSIS METHODS [000144] Spatial analysis methodologies described herein can provide a vast amount of analyte and/or expression data for a variety of analytes within a biological sample at high spatial resolution, while retaining native spatial context.
  • Spatial analysis methods can include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte (e.g., a protein and/or a nucleic acid) produced by and/or present in a cell.
  • a spatial barcode e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample
  • a capture domain that is capable of binding to an analyte (e.g., a protein and/or a nucleic acid) produced by and/or present in a cell.
  • Spatial 30 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC analysis methods and compositions can also include the use of a capture probe having a capture domain that captures an intermediate agent for indirect detection of an analyte.
  • the intermediate agent can include a nucleic acid sequence (e.g., a barcode) associated with the intermediate agent. Detection of the intermediate agent is therefore indicative of the analyte in the cell or tissue sample.
  • a nucleic acid sequence e.g., a barcode
  • a “barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and/or a capture probe).
  • a barcode can be part of an analyte, or independent of an analyte.
  • a barcode can be attached to an analyte.
  • a particular barcode can be unique relative to other barcodes.
  • an “analyte” can include any biological substance, structure, moiety, or component to be analyzed.
  • the term “target” can similarly refer to an analyte of interest. 31 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC [000147]
  • Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes.
  • non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments.
  • viral proteins e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.
  • the analyte(s) can be localized to subcellular location(s), including, for example, organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc.
  • organelles e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc.
  • analyte(s) can be peptides or proteins, including without limitation antibodies and enzymes. Additional examples of analytes can be found in Section (I)(c) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No.2020/0277663.
  • an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.
  • an intermediate agent for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.
  • an intermediate agent for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.
  • an intermediate agent for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.
  • an intermediate agent for example, a ligation product or an analyte capture agent (e.g., an
  • a tissue microarray contains multiple representative tissue samples – which can be from different tissues or organisms – assembled on a single histologic slide.
  • the TMA can therefore allow for high throughput analysis of multiple specimens at the same time.
  • Tissue microarrays are paraffin blocks produced by extracting cylindrical tissue cores from different paraffin donor blocks and re-embedding these into a single recipient (microarray) block at defined array coordinates.
  • the biological sample as used herein can be any suitable biological sample described herein or known in the art.
  • the biological sample is a tissue.
  • the tissue sample is a solid tissue sample.
  • the biological sample is a tissue section.
  • the tissue is flash-frozen and sectioned.
  • any 32 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC suitable method described herein or known in the art can be used to flash-freeze and section the tissue sample.
  • the biological sample e.g., the tissue
  • the biological sample is flash-frozen using liquid nitrogen before sectioning.
  • the biological sample e.g., a tissue sample
  • nitrogen e.g., liquid nitrogen
  • isopentane e.g., or hexane.
  • the biological sample, e.g., the tissue is embedded in a matrix e.g., optimal cutting temperature (OCT) compound to facilitate sectioning.
  • OCT optimal cutting temperature
  • OCT compound is a formulation of clear, water-soluble glycols and resins, providing a solid matrix to encapsulate biological (e.g., tissue) specimens.
  • the sectioning is performed using cryosectioning.
  • the methods further comprise a thawing step, after the cryosectioning.
  • the biological sample can be from a mammal. In some instances, the biological sample is from a human, mouse, or rat.
  • the biological sample can be obtained from non-mammalian organisms (e.g., a plants, an insect, an arachnid, a nematode (e.g., Caenorhabditis elegans), a fungi, an amphibian, or a fish (e.g., zebrafish)).
  • a biological sample can be obtained from a prokaryote such as a bacterium, e.g., Escherichia coli, Staphylococci or Mycoplasma pneumoniae; an archaea; a virus such as Hepatitis C virus or human immunodeficiency virus; or a viroid.
  • a biological sample can be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX).
  • the biological sample can include organoids, a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy.
  • Organoids can be generated from one or more cells from a tissue, embryonic stem cells, and/or induced pluripotent stem cells, which can self-organize in three-dimensional culture owing to their self-renewal and differentiation capacities.
  • an organoid is a cerebral organoid, an intestinal organoid, a stomach organoid, a lingual organoid, a thyroid organoid, a thymic organoid, a testicular organoid, a hepatic organoid, a pancreatic organoid, an epithelial organoid, a lung organoid, a kidney organoid, a gastruloid, a cardiac organoid, or a retinal organoid.
  • Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre-disposition to a disease, and/or individuals that are in need of therapy or suspected of needing therapy.
  • a disease e.g., cancer
  • a pre-disposition to a disease e.g., cancer
  • 10X Genomics Ref.: 100-165501PC Biological samples can be derived from a homogeneous culture or population of the subjects or organisms mentioned herein or alternatively from a collection of several different organisms, for example, in a community or ecosystem.
  • Biological samples can include one or more diseased cells.
  • a diseased cell can have altered metabolic properties, gene expression, protein expression, and/or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells.
  • the biological sample e.g., the tissue sample
  • the biological sample is fixed in a fixative including alcohol, for example methanol. In some embodiments, instead of methanol, acetone, or an acetone-methanol mixture can be used. In some embodiments, the fixation is performed after sectioning. In some instances, the biological sample is not fixed with paraformaldehyde (PFA).
  • PFA paraformaldehyde
  • the biological sample when the biological sample is fixed with a fixative including an alcohol (e.g., methanol or acetone-methanol mixture), it is not decrosslinked afterward.
  • the biological sample is fixed with a fixative including an alcohol (e.g., methanol or an acetone-methanol mixture) after freezing and/or sectioning.
  • the biological sample is flash-frozen, and then the biological sample is sectioned and fixed (e.g., using methanol, acetone, or an acetone-methanol mixture).
  • methanol, acetone, or an acetone-methanol mixture when methanol, acetone, or an acetone-methanol mixture is used to fix the biological sample, the sample is not decrosslinked at a later step.
  • the biological sample is frozen (e.g., flash frozen using liquid nitrogen and embedded in OCT) followed by sectioning and alcohol (e.g., methanol, acetone-methanol) fixation or acetone fixation
  • fresh frozen e.g., acetone and/or alcohol (e.g., methanol, acetone-methanol)
  • fixation of the biological sample e.g., using acetone and/or alcohol (e.g., methanol, acetone-methanol) is performed while the sample is mounted on a substrate (e.g., glass slide, such as a positively charged glass slide).
  • the biological sample e.g., the tissue sample
  • the biological sample is fixed e.g., immediately after being harvested from a subject.
  • the fixative is preferably an aldehyde fixative, such as paraformaldehyde (PFA) or formalin.
  • the fixative induces crosslinks within the biological sample.
  • the biological sample is dehydrated via sucrose gradient.
  • the fixed biological sample is treated with a sucrose gradient and then embedded in a matrix e.g., OCT compound.
  • the fixed biological sample is not treated with a sucrose gradient, but rather is embedded in a matrix e.g., OCT compound after fixation.
  • a fixed frozen tissue sample when a fixed frozen tissue sample is treated with a sucrose gradient, it can be rehydrated with an ethanol gradient.
  • the PFA or formalin fixed biological sample which can be optionally dehydrated via sucrose gradient and/or embedded in OCT compound, is then frozen e.g., for storage or shipment.
  • the biological sample is referred to as “fixed frozen”.
  • a fixed frozen biological sample is not treated with methanol.
  • a fixed frozen biological sample is not paraffin embedded.
  • a fixed frozen biological sample is not deparaffinized.
  • a fixed frozen biological sample is rehydrated in an ethanol gradient.
  • the biological sample e.g., a fixed frozen tissue sample
  • Citrate buffer can be used for antigen retrieval to decrosslink antigens and fixation medium in the biological sample.
  • any suitable decrosslinking agent can be used in addition to or alternatively to citrate buffer.
  • the biological sample e.g., a fixed frozen tissue sample
  • the biological sample can further be stained, imaged, and/or destained.
  • a fresh frozen tissue sample or fixed frozen tissue sample is stained (e.g., via eosin and/or hematoxylin), imaged, destained (e.g., via HCl), or a combination thereof.
  • a fresh frozen tissue sample is fixed in methanol, it is treated with isopropanol prior to being stained (e.g., via eosin and/or hematoxylin), imaged, destained (e.g., via HCl), or a combination thereof.
  • a fixed frozen tissue sample when a fixed frozen tissue sample is treated with a sucrose gradient, it can be rehydrated with an ethanol gradient before being stained, (e.g., via eosin and/or hematoxylin), imaged, destained (e.g., via HCl), decrosslinked (e.g., via TE buffer or citrate buffer), or a combination thereof.
  • the biological sample can undergo further fixation (e.g., while mounted on a substrate), stained, imaged, and/or destained.
  • a fixed frozen biological sample may 35 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC be subject to an additional fixing step (e.g., using PFA) before optional ethanol rehydration, staining, imaging, and/or destaining.
  • the biological sample can be fixed using PAXgene ® .
  • the biological sample can be fixed using PAXgene ® in addition, or alternatively to, a fixative disclosed herein or known in the art (e.g., alcohol, acetone, acetone-alcohol, formalin, paraformaldehyde).
  • PAXgene ® is a non-cross-linking mixture of different alcohols, acid and a soluble organic compound that preserves morphology and bio-molecules. It is a two-reagent fixative system in which tissue is firstly fixed in a solution containing methanol and acetic acid then stabilized in a solution containing ethanol. See, Ergin B. et al., J Proteome Res.2010 Oct 1;9(10):5188-96; Kap M. et al., PLoS One.; 6(11):e27704 (2011); and Mathieson W.
  • the fixative is PAXgene ® .
  • a fresh frozen tissue sample is fixed with PAXgene ® .
  • a fixed frozen tissue sample is fixed with PAXgene ® .
  • the biological sample e.g., the tissue sample is fixed, for example in methanol, acetone, acetone-methanol, PFA, PAXgene ® or is formalin-fixed and paraffin-embedded (FFPE).
  • the biological sample comprises intact cells.
  • the biological sample is a cell pellet, e.g., a fixed cell pellet, e.g., an FFPE cell pellet. FFPE samples are used in some instances in the RTL methods disclosed herein.
  • RNA integrity of fixed (e.g., FFPE) samples can be lower than a fresh sample, thereby making it more difficult to capture RNA directly, e.g., by capture of a common sequence such as a poly(A) tail of an mRNA molecule.
  • RTL probes can be utilized to beneficially improve capture and spatial analysis of fixed samples.
  • the biological sample e.g., tissue sample, can be stained, and imaged prior, during, and/or after each step of the methods described herein.
  • tissue sample can be obtained from any suitable location in a tissue or organ of a subject, e.g., a human subject.
  • the sample is a mouse sample.
  • the sample is a human sample.
  • the sample can be derived from skin, brain, breast, lung, liver, kidney, prostate, tonsil, thymus, testes, bone, lymph node, ovary, eye, heart, or spleen.
  • the sample is a human or mouse breast tissue sample.
  • the sample is a human or mouse brain tissue sample.
  • the sample is a human or mouse lung tissue sample.
  • the sample is a human or mouse tonsil tissue sample.
  • the sample is a human or mouse liver tissue sample.
  • the sample is a human or mouse bone, skin, kidney, thymus, testes, or prostate tissue sample.
  • the tissue sample is derived from normal or diseased tissue.
  • the sample is an embryo sample.
  • the embryo sample can be a non-human embryo sample.
  • the sample is a mouse embryo sample.
  • stains include histological stains (e.g., hematoxylin and/or eosin) and immunological stains (e.g., fluorescent stains).
  • the biological sample is imaged.
  • the biological sample is visualized or imaged using bright field microscopy.
  • the biological sample is visualized or imaged using fluorescence microscopy. Additional methods of visualization and imaging are known in the art. Non-limiting examples of visualization and imaging include expansion microscopy, bright field microscopy, dark field microscopy, phase 37 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy and confocal microscopy.
  • the sample is stained and imaged prior to adding the primer to the biological sample.
  • the method includes staining the biological sample. In some embodiments, the staining includes the use of hematoxylin and eosin.
  • a biological sample can be stained using any number of biological stains, including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin.
  • biological stains including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osm
  • the biological sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner’s, Leishman, Masson’s trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright’s, and/or Periodic Acid Schiff (PAS) staining techniques.
  • PAS staining is typically performed after formalin or acetone fixation.
  • the staining includes the use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.
  • a biological sample is permeabilized with one or more permeabilization reagents.
  • permeabilization of a biological sample can facilitate analyte capture.
  • Exemplary permeabilization agents and conditions are described in Section (I)(d)(ii)(13) or the Exemplary Embodiments Section of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No.2020/0277663.
  • the method includes a step of permeabilizing the biological sample.
  • the biological sample can be permeabilized to facilitate transfer of the extension products to the capture probes on the array.
  • the permeabilizing includes the use of an organic solvent (e.g., acetone, ethanol, and methanol), a detergent (e.g., saponin, Triton X-100TM, Tween-20TM, or sodium dodecyl sulfate (SDS)), an enzyme (an endopeptidase, an exopeptidase, a protease), or combinations thereof.
  • an organic solvent e.g., acetone, ethanol, and methanol
  • a detergent e.g., saponin, Triton X-100TM, Tween-20TM, or sodium dodecyl sulfate (SDS)
  • an enzyme an endopeptidase, an exopeptidase, a protease
  • the permeabilizing includes the use of an endopeptidase, a protease, SDS, polyethylene glycol tert-octylphenyl ether, polysorbate 80, and polysorbate 20, N-lauroylsarcosine sodium salt solution, saponin, 38 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Triton X-100TM, Tween-20TM, or combinations thereof.
  • the endopeptidase is pepsin.
  • the endopeptidase is Proteinase K.
  • Array-based spatial analysis methods involve the transfer of one or more analytes from a biological sample to an array of features on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of the analytes within the biological sample.
  • a “capture probe” refers to any molecule capable of capturing (directly or indirectly) and/or labelling an analyte (e.g., an analyte of interest) in a biological sample.
  • the capture probe is a nucleic acid or a polypeptide.
  • the capture probe includes a barcode (e.g., a spatial barcode and/or a unique molecular identifier (UMI)) and a capture domain).
  • UMI unique molecular identifier
  • the capture probe includes a homopolymer sequence, such as a poly(T) sequence.
  • a capture probe can include a cleavage domain and/or a functional domain (e.g., a primer-binding site, such as for next- generation sequencing (NGS)).
  • NGS next- generation sequencing
  • the biological sample is mounted on a first substrate and the substrate comprising the array of capture probes is a second substrate.
  • one or more analytes or analyte derivatives e.g., intermediate agents; e.g., ligation products
  • the release and migration of the analytes or analyte derivatives to the second substrate comprising the array of capture probes occurs in a manner that preserves the original spatial context of the analytes in the biological sample.
  • FIG.1A shows an exemplary sandwiching process 100 where a first substrate (e.g., slide 103), including a biological sample 102, and a second substrate (e.g., array slide 104 including an array having spatially barcoded capture probes 106) are brought into proximity with one another.
  • a first substrate e.g., slide 103
  • a second substrate e.g., array slide 104 including an array having spatially barcoded capture probes 106
  • a liquid reagent drop (e.g., permeabilization solution 105) is introduced on the second substrate in proximity to the capture probes 106 and in between the biological sample 102 and the second substrate (e.g., slide 104 including an array having spatially barcoded capture probes 106).
  • the permeabilization solution 105 may release analytes or analyte derivatives (e.g., intermediate agents; e.g., ligation products) that can be captured by the capture probes of the array 106.
  • the first substrate is aligned with the second substrate, such that at least a portion of the biological sample is aligned with at least a portion of the capture probes (e.g., aligned in a sandwich configuration).
  • the second substrate e.g., array slide 104 is in an inferior position to the first substrate (e.g., slide 103).
  • the first substrate e.g., slide 103
  • the second substrate e.g., slide 104
  • a reagent medium 105 within a gap between the first substrate (e.g., slide 103) and the second substrate (e.g., slide 104) creates a liquid interface between the two substrates.
  • the reagent medium may be a permeabilization solution which permeabilizes and/or digests the biological sample 102.
  • the reagent medium is not a permeabilization solution.
  • analytes e.g., mRNA transcripts
  • analyte derivatives e.g., intermediate agents; e.g., ligation products
  • release from the biological sample and actively or passively migrate (e.g., diffuse) across the gap toward the capture probes on the array 106.
  • migration of the analyte or analyte derivative (e.g., intermediate agent; e.g., ligation product) from the biological sample is performed actively (e.g., electrophoretic, by applying an electric field to promote migration).
  • electrophoretic by applying an electric field to promote migration.
  • one or more spacers 110 may be positioned between the first substrate (e.g., slide 103) and the second substrate (e.g., array slide 104 including spatially barcoded capture probes 106).
  • the one or more spacers 110 may be configured to maintain a separation distance between the first substrate and the second substrate. While the one or more spacers 110 is shown as disposed on the second substrate, the spacer may additionally or alternatively be disposed on the first substrate.
  • the one or more spacers 110 is configured to maintain a separation distance between first and second substrates that is between about 2 microns and 1 mm (e.g., between about 2 microns and 800 microns, between about 2 microns and 700 microns, between about 2 microns and 600 microns, between about 2 microns and 500 microns, between about 2 microns and 400 microns, between about 2 microns and 300 microns, between about 2 microns and 200 microns, between about 2 microns and 100 microns, between about 2 microns and 25 microns, or between about 2 microns and 10 microns), measured in a direction orthogonal to the surface of first substrate that supports the biological sample.
  • a separation distance between first and second substrates that is between about 2 microns and 1 mm (e.g., between about 2 microns and 800 microns, between about 2 microns and 700 microns, between about 2 microns and 600 microns, between about 2 microns and
  • the separation distance is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 microns. In some embodiments, the separation distance is less than 50 microns. In some embodiments, the separation distance is less than 25 microns. In some embodiments, the separation distance is less than 20 microns. The separation distance may include a distance of at least 2 ⁇ m.
  • FIG.1B shows a fully formed sandwich configuration 125 creating a chamber 150 formed from the one or more spacers 110, the first substrate (e.g., the slide 103), and the second substrate (e.g., the slide 104 including an array 106 having spatially barcoded capture probes) in accordance with some example implementations.
  • the liquid reagent e.g., the permeabilization solution 105 fills the volume of the chamber 150 and may create a permeabilization buffer that allows analytes (e.g., mRNA transcripts and/or other molecules) or analyte derivatives (e.g., intermediate agents; e.g., ligation products) to diffuse from the biological sample 102 toward the capture probes of the second substrate (e.g., slide 104).
  • analytes e.g., mRNA transcripts and/or other molecules
  • analyte derivatives e.g., intermediate agents; e.g., ligation products
  • flow of the permeabilization buffer may deflect transcripts and/or molecules from the biological sample 102 and may affect diffusive transfer of analytes or analyte derivatives (e.g., intermediate agents; e.g., ligation products) for spatial analysis.
  • analytes or analyte derivatives e.g., intermediate agents; e.g., ligation products
  • a partially or 41 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC fully sealed chamber 150 resulting from the one or more spacers 110, the first substrate, and the second substrate may reduce or prevent flow from undesirable convective movement of transcripts and/or molecules over the diffusive transfer from the biological sample 102 to the capture probes.
  • the sandwiching process methods described above can be implemented using a variety of hardware components.
  • the sandwiching process methods can be implemented using a sample holder (also referred to herein as a support device, a sample handling apparatus, and an array alignment device). Further details on support devices, sample holders, sample handling apparatuses, or systems for implementing a sandwiching process are described in, e.g., US. Patent Application Pub. No.2021/0189475, and PCT Publ. No. WO 2022/061152 A2, each of which are incorporated by reference in their entirety.
  • the sample holder can include a first member including a first retaining mechanism configured to retain a first substrate comprising a biological sample.
  • the first retaining mechanism can be configured to retain the first substrate disposed in a first plane.
  • the sample holder can further include a second member including a second retaining mechanism configured to retain a second substrate disposed in a second plane.
  • the sample holder can further include an alignment mechanism connected to one or both of the first member and the second member.
  • the alignment mechanism can be configured to align the first and second members along the first plane and/or the second plane such that the sample contacts at least a portion of the reagent medium when the first and second members are aligned and within a threshold distance along an axis orthogonal to the second plane.
  • the adjustment mechanism may be configured to move the second member along the axis orthogonal to the second plane and/or move the first member along an axis orthogonal to the first plane.
  • the adjustment mechanism includes a linear actuator.
  • the linear actuator is configured to move the second member along an axis orthogonal to the plane of the first member and/or the second member.
  • the linear actuator is configured to move the first member along an axis orthogonal to the plane of the first member and/or the second member.
  • the linear actuator is configured to move the first member, the second member, or both the first member and the second member at a velocity of at least 0.1 mm/sec.
  • FIG.2A is a perspective view of an example sample handling apparatus 200 in a closed position in accordance with some example implementations.
  • the sample handling apparatus 200 includes a first member 204, a second member 210, optionally an image capture device 220, a first substrate 206, optionally a hinge 215, and optionally a mirror 216.
  • FIG.2B is a perspective view of the example sample handling apparatus 200 in an open position in accordance with some example implementations.
  • the sample handling apparatus 200 includes one or more first retaining mechanisms 208 configured to retain one or more first substrates 206.
  • the first member 204 is configured to retain two first substrates 206, however the first member 204 may be configured to retain more or fewer first substrates 206.
  • the first substrate 206 and/or the second substrate 212 may be loaded and positioned within the sample handling apparatus 200 such as within the first member 204 and the second member 210, respectively.
  • the hinge 215 may allow the first member 204 to close over the second member 210 and form a sandwich configuration.
  • an adjustment mechanism of the sample handling apparatus 200 may actuate the first member 204 and/or the second member 210 to form the sandwich configuration for the permeabilization step (e.g., bringing the first substrate 206 and the second substrate 212 closer to each other and within a threshold distance for the sandwich configuration).
  • the adjustment mechanism may be configured to control a speed, an angle, a force, or the like of the sandwich configuration.
  • the biological sample (e.g., sample 102 from FIG.1A) may be aligned within the first member 204 (e.g., via the first retaining mechanism 208) prior to closing the first member 204 such that a desired region of interest of the sample is aligned with the 43 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC barcoded array of the second substrate (e.g., the slide 104 from FIG.1A), e.g., when the first and second substrates are aligned in the sandwich configuration.
  • Such alignment may be accomplished manually (e.g., by a user) or automatically (e.g., via an automated alignment mechanism).
  • spacers may be applied to the first substrate 206 and/or the second substrate 212 to maintain a minimum spacing between the first substrate 206 and the second substrate 212 during sandwiching.
  • the permeabilization solution e.g., permeabilization solution 305
  • the first member 204 may then close over the second member 210 and form the sandwich configuration.
  • Analytes or analyte derivatives e.g., intermediate agents; e.g., ligation products
  • the image capture device 220 may capture images of the overlap area between the biological sample and the capture probes on the array 106. If more than one first substrates 206 and/or second substrates 212 are present within the sample handling apparatus 200, the image capture device 220 may be configured to capture one or more images of one or more overlap areas. [000184] Provided herein are methods for delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate.
  • FIGs.3A-3C depict a side view and a top view of an exemplary angled closure workflow 300 for sandwiching a first substrate (e.g., slide 303) having a biological sample 302 and a second substrate (e.g., slide 304 having capture probes 306) in accordance with some exemplary implementations.
  • FIG.3A depicts the first substrate (e.g., the slide 303 including a biological sample 302) angled over (superior to) the second substrate (e.g., slide 304).
  • reagent medium e.g., permeabilization solution
  • FIG.3A depicts the reagent medium on the right hand side of side view, it should be understood that such depiction is not meant to be limiting as to the location of the reagent medium on the spacer.
  • FIG.3B shows that as the first substrate lowers, and/or as the second substrate rises, the dropped side of the first substrate (e.g., a side of the slide 303 angled toward the second substrate) may contact the reagent medium 305.
  • the dropped side of the first substrate may urge 44 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC the reagent medium 305 toward the opposite direction (e.g., towards an opposite side of the spacer 310, towards an opposite side of the first substrate relative to the dropped side).
  • the reagent medium 305 may be urged from right to left as the sandwich is formed.
  • the first substrate and/or the second substrate are further moved to achieve an approximately parallel arrangement of the first substrate and the second substrate.
  • FIG.3C depicts a full closure of the sandwich between the first substrate and the second substrate with the spacer 310 contacting both the first substrate and the second substrate and maintaining a separation distance and optionally the approximately parallel arrangement between the two substrates.
  • the spacer 310 fully encloses and surrounds the biological sample 302 and the capture probes 306, and the spacer 310 form the sides of chamber 350 which holds a volume of the reagent medium 305.
  • Another method is 47 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC to cleave spatially-barcoded capture probes from an array and promote the spatially-barcoded capture probes towards and/or into or onto the biological sample.
  • a second type of capture probe associated with the feature includes the spatial barcode 702 in combination with a random N-mer capture domain 704 for gDNA analysis.
  • a third type of capture probe associated with the feature includes the spatial barcode 702 in combination with a capture domain complementary to the analyte capture agent of interest 705.
  • a fourth type of capture probe associated with the feature includes the spatial barcode 702 in combination with a capture probe that can specifically bind a nucleic acid molecule 706 that can function in a 51 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC CRISPR assay (e.g., CRISPR/Cas9).
  • a perturbation agent can be a small molecule, an antibody, a drug, an aptamer, a miRNA, a physical environmental (e.g., temperature change), or any other known perturbation agents.
  • the functional sequences can generally be selected for compatibility with any of a variety of different sequencing systems, e.g., Ion Torrent Proton or PGM, Illumina ® sequencing instruments, PacBio, Oxford Nanopore, etc., and the requirements thereof. In some embodiments, functional sequences can be selected for compatibility with non-commercialized sequencing systems.
  • FIG.8 depicts an exemplary arrangement of barcoded features within an array.
  • FIG.8 shows (L) a slide including six spatially-barcoded arrays, (C) an enlarged schematic of one of the six spatially-barcoded arrays, showing a grid of barcoded features in 52 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC relation to a biological sample, and (R) an enlarged schematic of one section of an array, showing the specific identification of multiple features within the array (labelled as ID578, ID579, ID560, etc.).
  • one of the oligonucleotides includes at least two ribonucleic acid bases at the 3’ end and/or the other oligonucleotide includes a phosphorylated nucleotide at the 5’ end.
  • one of the two oligonucleotides includes a capture binding capture domain (e.g., a poly(A) sequence, a non-homopolymeric sequence).
  • a ligase e.g., a T4 RNA ligase (Rnl2), a PBCV-1 DNA Ligase or Chorella virus DNA Ligase, a single-stranded DNA ligase, or a T4 DNA ligase
  • a ligase e.g., a T4 RNA ligase (Rnl2), a PBCV-1 DNA Ligase or Chorella virus DNA Ligase, a single-stranded DNA ligase, or a T4 DNA ligase
  • the two oligonucleotides hybridize to 53 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC sequences that are not adjacent to one another.
  • hybridization of the two oligonucleotides creates a gap between the hybridized oligonucleotides.
  • a polymerase e.g., a DNA polymerase
  • the ligation product is released from the analyte.
  • the ligation product is released using an endonuclease (e.g., RNAse H).
  • the ligation product is removed using heat.
  • the ligation product is removed using KOH.
  • the released ligation product can then be captured by capture probes (e.g., instead of direct capture of an analyte) on an array, optionally amplified, and sequenced, thus determining the location and optionally the abundance of the analyte in the biological sample.
  • capture probes e.g., instead of direct capture of an analyte
  • FIG.9A A non-limiting example of templated ligation methods disclosed herein is depicted in FIG.9A.
  • a biological sample is contacted with a substrate including a plurality of capture probes and contacted with (a) a first probe 901 having a target-hybridization sequence 903 and a primer sequence 902 and (b) a second probe 904 having a target-hybridization sequence 905 and a capture domain (e.g., a poly-A sequence) 906, the first probe 901 and a second probe 904 hybridize 910 to an analyte 907.
  • a ligase 921 ligates 920 the first probe to the second probe thereby generating a ligation product 922.
  • the capture probe can also include a unique molecular identifier (UMI) 9007, a spatial barcode 9008, a functional sequence 9009, and a cleavage domain 9010.
  • UMI unique molecular identifier
  • methods provided herein include permeabilization of the biological sample such that the capture probe can more easily bind to the captured ligated probe (i.e., compared to no permeabilization).
  • RT reverse transcription
  • the cDNA can then be enzymatically fragmented and size-selected in order to optimize the cDNA amplicon size.
  • P59016, i59017, i79018, and P79019 and can be used as sample indexes, and TruSeqTM Read 2 can be added via End Repair, A-tailing, Adaptor Ligation, and PCR.
  • the cDNA fragments can then be sequenced using paired-end sequencing using TruSeqTM Read 1 and TruSeqTM Read 2 as sequencing primer sites.
  • detection of one or more analytes e.g., protein analytes
  • analyte capture agents e.g., protein analytes
  • an “analyte capture agent” refers to an agent that interacts with an analyte (e.g., an analyte in a biological sample) and with a capture probe (e.g., a capture probe attached to a substrate or a feature) to identify the analyte.
  • the analyte capture agent includes: (i) an analyte binding moiety (e.g., that binds to an analyte), for example, an antibody or antigen-binding fragment thereof; (ii) analyte binding moiety barcode; and (iii) an analyte capture sequence.
  • analyte binding moiety barcode refers to a barcode that is associated with or otherwise identifies the analyte binding moiety.
  • analyte capture sequence refers to a region or moiety configured to hybridize to, bind to, couple to, or otherwise interact with a capture domain of a capture probe.
  • an analyte binding moiety barcode (or portion thereof) may be able to be removed (e.g., cleaved) from the analyte capture agent. Additional description of analyte capture agents can be found in Section (II)(b)(ix) of PCT Publication No.
  • FIG.10 is a schematic diagram of an exemplary analyte capture agent 1002 comprised of an analyte-binding moiety 1004 and an analyte-binding moiety barcode domain 1008.
  • the exemplary analyte -binding moiety 1004 is a molecule capable of binding to an analyte 1006 and the analyte capture agent is capable of interacting with a spatially-barcoded capture probe.
  • the analyte -binding moiety can bind to the analyte 1006 with high affinity and/or with high specificity.
  • the analyte capture agent can include an analyte-binding moiety barcode domain 1008, a nucleotide sequence (e.g., an oligonucleotide), which can hybridize to at least a portion or an entirety of a capture domain of a capture probe.
  • the analyte-binding moiety barcode domain 1008 can comprise an analyte binding moiety barcode and a capture handle sequence described herein.
  • the analyte-binding moiety 1004 can include a polypeptide and/or an aptamer.
  • FIG.11 is a schematic diagram depicting an exemplary interaction between a feature- immobilized capture probe 1124 and an analyte capture agent 1126.
  • the feature-immobilized capture probe 1124 can include a spatial barcode 1108 as well as functional sequences 1106 and UMI 1110, as described elsewhere herein.
  • the capture probe can be affixed 1104 to a feature (e.g., bead) or array 1102.
  • the capture probe can also include a capture domain 1112 that is capable of binding to an analyte capture agent 1126.
  • the analyte capture agent 1126 can include a functional sequence 1118, analyte binding moiety barcode 1116, and a capture handle sequence 1114 that is capable of binding to the capture domain 1112 of the capture probe 1124.
  • the analyte capture agent can also include a linker 1120 that allows the capture agent barcode domain 1116 to couple to the analyte binding moiety 1122.
  • specific capture probes and the analytes they capture are associated with specific locations in an array of features on a substrate.
  • specific spatial barcodes can be associated with specific array 56 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC locations prior to array fabrication, and the sequences of the spatial barcodes can be stored (e.g., in a database) along with specific array location information, so that each spatial barcode uniquely maps to a particular array location.
  • specific spatial barcodes can be deposited at predetermined locations in an array of features during fabrication such that at each location, only one type of spatial barcode is present so that spatial barcodes are uniquely associated with a single feature of the array.
  • the arrays can be decoded using any of the methods described herein so that spatial barcodes are uniquely associated with array feature locations, and this mapping can be stored as described above.
  • sequence information is obtained for capture probes and/or analytes during analysis of spatial information, the locations of the capture probes and/or analytes can be determined by referring to the stored information that uniquely associates each spatial barcode with an array feature location.
  • Each array feature location represents a position relative to a coordinate reference point (e.g., an array location, a fiducial marker) for the array. Accordingly, each feature location has an “address” or location in the coordinate space of the array.
  • a coordinate reference point e.g., an array location, a fiducial marker
  • each feature location has an “address” or location in the coordinate space of the array.
  • spatial analysis can be performed using dedicated hardware and/or software, such as any of the systems described in Sections (II)(e)(ii) and/or (V) of PCT Publication No. WO2020/176788 and/or U.S.
  • Suitable systems for performing spatial analysis can include components such as a chamber (e.g., a flow cell or sealable, fluid-tight chamber) for containing a biological sample.
  • a chamber e.g., a flow cell or sealable, fluid-tight chamber
  • the biological sample can be mounted for example, in a biological sample holder.
  • One or more fluid chambers can be connected to the chamber and/or the sample holder via fluid conduits, and fluids can be delivered into the chamber and/or sample holder via fluidic pumps, vacuum sources, or other devices coupled to the fluid conduits that create a pressure gradient to drive fluid flow.
  • One or more valves can also be connected to fluid conduits to regulate the flow of reagents from reservoirs to the chamber and/or sample holder.
  • the systems can optionally include a control unit that includes one or more electronic processors, an input interface, an output interface (such as a display), and a storage unit (e.g., a solid state storage medium such as, but not limited to, a magnetic, optical, or other solid state, persistent, writeable and/or re-writeable storage medium).
  • the control unit can optionally be connected to one or more remote devices via a network.
  • the control unit (and components thereof) can generally perform any of the steps and functions described herein. Where the system is connected to a remote device, the remote device (or devices) can perform any of the steps or features described herein.
  • the systems can optionally include one or more detectors (e.g., CCD, CMOS) used to capture images.
  • the systems can also optionally include one or more light sources (e.g., LED-based, diode-based, lasers) for illuminating a sample, a substrate with features, analytes from a biological sample captured on a substrate, and various control and calibration media.
  • the systems can optionally include software instructions encoded and/or implemented in one or more of tangible storage media and hardware components such as application specific integrated circuits.
  • the software instructions when executed by a control unit (and in particular, an electronic processor) or an integrated circuit, can cause the control unit, integrated circuit, or other component executing the software instructions to perform any of the method steps or functions described herein.
  • the systems described herein can detect (e.g., register an image) the biological sample on the array.
  • Exemplary methods to detect the biological sample on an array 58 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC are described in PCT Publication No. WO2021/102003 and/or U.S. Patent Application Publication No.2021/0150707, each of which is incorporated herein by reference in their entireties.
  • the biological sample Prior to transferring analytes from the biological sample to the array of features on the substrate, the biological sample can be aligned with the array.
  • Alignment of a biological sample and an array of features including capture probes can facilitate spatial analysis, which can be used to detect differences in analyte presence and/or level within different positions in the biological sample, for example, to generate a three-dimensional map of the analyte presence and/or level.
  • Exemplary methods to generate a two- and/or three-dimensional map of the analyte presence and/or level are described in PCT Publication No. WO2020/053655 and spatial analysis methods are generally described in PCT Publication No. WO2021/102039 and/or U.S. Patent Application Publication No.2021/0155982, each of which is incorporated herein by reference in their entireties.
  • a map of analyte presence and/or level can be aligned to an image of a biological sample using one or more fiducial markers, e.g., objects placed in the field of view of an imaging system which appear in the image produced, as described in the Substrate Attributes Section, Control Slide for Imaging Section of PCT Publication Nos. WO2020/123320, WO 2021/102005, and/or U.S. Patent Application Publication No.2021/0158522, each of which is incorporated herein by reference in their entireties.
  • fiducial markers e.g., objects placed in the field of view of an imaging system which appear in the image produced, as described in the Substrate Attributes Section, Control Slide for Imaging Section of PCT Publication Nos. WO2020/123320, WO 2021/102005, and/or U.S. Patent Application Publication No.2021/0158522, each of which is incorporated herein by reference in their entireties.
  • Fiducial markers can be used as a point of reference or measurement scale for alignment (e.g., to align a sample and an array, to align two substrates, to determine a location of a sample or array on a substrate relative to a fiducial marker) and/or for quantitative measurements of sizes and/or distances.
  • Reverse transcriptases or reverse transcription (RT) enzymes are RNA-dependent DNA polymerases, typically used to create a copy of an RNA sequence thereby generating a cDNA molecule. Reverse transcription is initiated by hybridization of a priming sequence to an RNA molecule which is extended by a reverse transcription enzyme in a template directed fashion.
  • a reverse transcription enzyme adds a plurality of non-template nucleotides to a nucleotide strand, thereby producing complementary deoxyribonucleic acid (cDNA) molecules.
  • the resultant cDNA can then be dehybridized from the template RNA molecule in any number of ways as 59 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC known in the art.
  • Engineered and/or recombinant are used interchangeably with respect to reverse transcriptase (RT) variant and/or fusion RT.
  • a DNA binding domain is a protein, or a defined region of a protein, that binds to a nucleic acid in a sequence-independent matter. For example, binding of the protein to DNA does not exhibit any preference for a particular sequence.
  • the DNA binding domain may be single or double stranded.
  • the AT-rich interaction domain can be a 62 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC KRPR* repeat, or SEQ ID NO: 21.
  • the AT-rich interaction domain can be a K/RKRGRPKK repeat, or SEQ ID NO: 16.
  • the AT-rich interaction domain can be a mammalian high mobility group I protein (HMG-I, or a-protein) AT-rich interaction domain or SEQ ID NO: 15.
  • the AT-rich interaction domain can comprise a Drosophila melanogaster D1 protein AT-rich interaction domain-consensus domain or SEQ ID NO: 16.
  • the tag is a hexahistidine tag.
  • the tag is selected from a small ubiquitin-like modifier tag (SUMO), a VariFlex C-Terminal solubility enhancement tag, a short peptide C-terminal tag, Thioredoxin (Trx) tag, Solubility-enhancer peptide sequences (SET) tag, IgG domain B1 of Protein G (GB1) tag, IgG repeat domain ZZ of Protein A (ZZ) tag, Solubility enhancing Ubiquitous Tag (SNUT tag), Seventeen kilodalton protein (Skp tag), Phage T7 protein kinase (T7PK) tag, E.
  • SUMO small ubiquitin-like modifier tag
  • Trx VariFlex C-Terminal solubility enhancement tag
  • SET Solubility-enhancer peptide sequences
  • IgG domain B1 of Protein G GB1
  • ZZ Solubility enhancing Ubiquitous Tag
  • the affinity tag may include, but is not limited to, albumin binding protein (ABP), AU1 epitope, AU5 epitope, T7-tag, V5-tag, B-tag, Chloramphenicol Acetyl Transferase (CAT), Dihydrofolate reductase (DHFR), AviTag, Calmodulin-tag, polyglutamate tag, E-tag, FLAG-tag, HA-tag, Myc-tag, NE-tag, S-tag, SBP-tag, Doftag 1, Softag 3, Spot-tag, tetracysteine (TC) tag, Ty tag, VSV-tag, Xpress tag, biotin carboxyl carrier protein 70 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC (BCCP), green fluorescent protein tag, HaloTag, Nus-tag, thioredoxin-tag, Fc-tag, cellulose binding domain, chitin binding protein (ABP),
  • the protease cleavage sequence is recognized by a protease including, but not limited to, alanine carboxypeptidase, Armillaria mellea astacin, bacterial leucyl aminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosol alanyl aminopeptidase, elastase, endoproteinase Arg-C, enterokinase, gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase, human rhinovirus 3C protease, hypodermin C, Iga-specific serine endopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC, lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methionyl aminopeptidase, myxobacter
  • the protease cleavage sequence is a thrombin cleavage sequence.
  • D. Reverse Transcriptase Polypeptides Reverse transcriptases or reverse transcription enzymes are known in the art to perform a reverse transcription reaction. As used herein, “Reverse transcriptase” and “reverse transcription enzyme” are synonymous. Reverse transcription is initiated by hybridization of a priming sequence to an RNA molecule which is extended by an engineered reverse transcription 71 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC enzyme in a template directed fashion.
  • a reverse transcription enzyme adds a plurality of non- template oligonucleotides to a nucleotide strand.
  • the reverse transcription reaction can produce single stranded complementary deoxyribonucleic acid (cDNA) molecules each having a molecular tag on a 5’ end thereof, followed by amplification of cDNA to produce a double stranded DNA having the molecular tag on the 5’ end and a 3’ end of the double stranded DNA.
  • cDNA complementary deoxyribonucleic acid
  • wild-type refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source.
  • the amino acid sequence set forth in SEQ ID NO: 7 is a wild-type MMLV amino acid sequence.
  • the RT polypeptide can comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.
  • the RT polypeptide can comprise an amino acid sequence that is 90-99.99% identical to SEQ ID NO: 1.
  • the RT polypeptide can comprise an amino acid sequence that is 92-99.99% identical to SEQ ID NO: 1.
  • the RT polypeptide can comprise an amino acid sequence that is 93-99.99% identical to SEQ ID NO: 1.
  • the RT polypeptide can comprise an amino acid sequence that is 94-99.99% identical to SEQ ID NO: 1. In some embodiments, the RT polypeptide can comprise an amino acid sequence that is 95-99.99% identical to SEQ ID NO: 1. In some embodiments, the RT polypeptide can comprise an amino acid sequence that is 96-99.99% identical to SEQ ID NO: 1. In some embodiments, the RT polypeptide can comprise an amino acid sequence that is 97-99.99% identical to SEQ ID NO: 1. In some embodiments, the RT polypeptide can comprise an amino acid sequence that is 98-99.99% identical to SEQ ID NO: 1.
  • the RT polypeptide can comprise an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 1.
  • the amino acid variation are at any one position or combination thereof as identified in an alignment of SEQ ID NO: 1 to any one of the RT polypeptide sequences in Table 1, or Table 2.
  • the RT polypeptide can comprise the amino acid sequence set forth in SEQ ID NO: 7.
  • the engineered reverse transcriptase can exhibit an altered reverse 72 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC transcriptase activity as compared to a reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 1 or 7.
  • the RT polypeptide can be a variant MMLV reverse-transcriptase having one or more mutations.
  • the RT polypeptide contemplated by the present disclosure can comprise a combination of mutations in the amino acid sequence of either the wild-type MMLV (SEQ ID NO 7 or 178) or in a MMLV variant (SEQ ID NO: 1, 143 or 179).
  • the amino acid sequence of the RT polypeptide sequence contemplated by the present disclosure can be at least 90% identical to SEQ ID NO: 1 or 143.
  • the amino acid sequence of the RT polypeptide sequence can be about 90% to about 99.99% identical to SEQ ID NO: 1 or 143, about 92% to about 99.99% identical to SEQ ID NO: 1 or 143, about 93% to about 99.99% identical to SEQ ID NO: 1 or 143, about 94% to about 99.99% identical to SEQ ID NO: 1 or 143, about 95% to about 99.99% identical to SEQ ID NO: 1 or 143, about 96% to about 99.99% identical to SEQ ID NO: 1 or 143, about 97% to about 99.99% identical to SEQ ID NO: 1 or 143, or about 98% to about 99.99% identical to SEQ ID NO: 1 or 143.
  • the amino acid sequence of the RT polypeptide sequence can be about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% identical to SEQ ID NO: 1 or 143.
  • the present disclosure relates to engineered RT polypeptides or recombinant RT polypeptide comprising a wild-type RT or modified reverse transcriptases that comprise one or more (e.g., one, two, three, four, five, ten, twelve, fifteen, twenty, etc.) amino 73 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC acid changes. These amino acid changes render the reverse transcriptase more efficient for nucleic acid synthesis (e.g., single cell profiling assay) requiring very small volume, as compared to an unmutated or an unmodified reverse transcriptase.
  • nucleic acid synthesis e.g., single cell profiling assay
  • amino acids identified may be deleted and/or replaced with one or a number of amino acid residues.
  • any one or more of the amino acids may be substituted with any one or more amino acid residues such as Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and/or Val.
  • the RT polypeptide described herein comprises the amino acid sequence of SEQ ID NO:7, and comprises a combination of mutations selected from E69K, L139P, E302R, T306K, W313F, T330P, or N454K; and one or more of M39V, P47L, M66L, F155Y, D200N, D200E, H204R, G429S, L435G, L435K, P448A, D449G, H503V, D524N, T542D, E545G, D583N, H594Q, L603W, L603F, E607K, E607G, P627S, H634Y, H638G, A644V, D653H, K658R or L671P.
  • the engineered polypeptide can comprise the amino acid sequence of SEQ ID NO:7, and comprises a combination of mutations selected from E69K, L139P, D200N, E302R, T306K, W313F, T330P, L435G, P448A, D449G, N454K, D524N, or L603W, and E607K and one or more of M39V, P47L, M66L, F155Y, H204R, G429S, H503V, T542D, E545G, D583N, H594Q, P627S, H634Y, H638G, A644V, D653H, K658R or L671P.
  • the RT polypeptide sequence can comprise an amino acid sequence that is at least 95% identical to SEQ ID NO:1, 7, or 179, and a combination of mutations indexed to SEQ ID NO:7 or 178 selected from a combination of variants consisting of a T542D mutation, a D583N mutation, an E607G mutation, an A644V mutation, a D653H mutation, and a K658R mutation.
  • the RT polypeptide sequence can comprise an amino acid sequence that is at least 95% identical to SEQ ID NO:1, 7, or 179, and a combination of mutations indexed to SEQ ID NO:7 or 178 selected from a combination of variants consisting of an E545G mutation, a D583N mutation, an H594Q mutation, an L603F mutation, and a S679P mutation.
  • the RT polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from: SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ 74 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,
  • the RT polypeptide can comprise SEQ ID NO: 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 141, 143, 145, 147, 149, 151, 157, 159, 172, or 173.
  • the RT polypeptide can comprise an amino acid sequence listed in Table 1 or 2.
  • the amino acid sequence of the RT polypeptide can also comprise E69K, L139P, D200N, E302R, T306K, W313F, T330P, N454K, H503V, D524N, L603W, E607K, and H634Y.
  • the amino acid sequence of the RT polypeptide comprises a combination of mutations selected from: M66L and L435G; M39V, M66L, and L435K; M39V and L435K; M66L, L435G, P448A and D449G; M39V, M66L, L435G, P448A and D449G; or M66L.
  • the amino acid sequence of the RT polypeptide comprises E69K, L139P, D200N, E302R, T306K, W313F, T330P, L435G, P448A, D449G, N454K, D524N, L603W, and E607K; and further comprises a combination of mutations selected from M66L; M66L and H503V; M66L and H634Y; and M66L, H503V, or H634Y.
  • the amino acid sequence of the RT polypeptide comprises M39V, E69K, L139P, a D200 mutation, E302R, T306K, W313F, T330P, G429S, P448A, a D449 mutation, L435K, N454K, a L603 mutation, a E607 mutation, and L671P and further comprises a second combination of mutations selected from D524N, T542D, P627S, A644V, D653H, or K658R mutation.
  • the D200 mutation is a D200N mutation
  • the D449 mutation is a D449G
  • the L603 mutation is an L603W
  • the E607 mutation is an E607G mutation.
  • the amino acid sequence of the RT polypeptide comprises M39V, E69K, L139P, a D200 mutation, E302R, T306K, W313F, T330P, G429S, P448A, a D449 mutation, L435K, N454K, a L603 mutation, a E607 mutation, and L671P and further comprises D524N, T542D, A644V, D653H, an R650H and K658R.
  • the D200 mutation is a D200N mutation
  • the D449 mutation is a D449G mutation
  • the L603 mutation is an L603F mutation
  • the E607 mutation is an E607K mutation.
  • the amino acid sequence of the RT polypeptide comprises M39V, E69K, L139P, a D200 mutation, E302R, T306K, W313F, T330P, G429S, P448A, a D449 mutation, L435K, N454K, a L603 mutation, a E607 mutation, and L671P and further comprises D524N, T542D, A644V, D653H, and K658R.
  • the D200 mutation is a D200N mutation
  • the D449 mutation is a D449E mutation
  • the L603 mutation is an L603W mutation
  • the E607 mutation is an E607G mutation.
  • the amino acid sequence of the RT polypeptide comprises M39V, E69K, L139P, a D200 mutation, E302R, T306K, W313F, T330P, G429S, P448A, a D449 mutation, L435K, N454K, a L603 mutation, a E607 mutation, and L671P and further comprises H204R, D524N, T542D, P627S, D583N, A644V, D653H and K658R.
  • the D200 mutation is a D200E mutation
  • the D449 mutation is a D449G mutation
  • the L603 mutation is an L603W mutation
  • the E607 mutation is an E607G mutation.
  • the amino acid sequence of the RT polypeptide comprises M39V, E69K, L139P, a D200 mutation, E302R, T306K, W313F, T330P, G429S, P448A, a D449 mutation, L435K, N454K, a L603 mutation, a E607 mutation, and L671P and further comprises H204R, E545G, D583N, and H594Q.
  • the D200 mutation is a D200E mutation
  • the D449 mutation is a D449G mutation
  • the L603 mutation is an L603F mutation
  • the E607 mutation is an E607K mutation.
  • the amino acid sequence of the RT polypeptide comprises M39V, E69K, L139P, a D200 mutation, E302R, T306K, W313F, T330P, G429S, P448A, a D449 mutation, L435K, N454K, a L603 mutation, a E607 mutation, and L671P and further comprises P47L, D524N, T542D, D583N, P627S, A644V, D653H, and K658R.
  • the D200 mutation is a D200N mutation
  • the D449 mutation is a D449G mutation
  • the L603 mutation is an L603W mutation
  • the E607 mutation is an E607G mutation.
  • the RT polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 and the amino acid sequence of the engineered reverse transcriptase comprises at least one mutation indexed to SEQ ID NO:7 selected from a M17 mutation; an A32 mutation, a M44 mutation, a M39 mutation, a K47 mutation, a P51 mutation, an M66 mutation, an S67 mutation, an E69 mutation, a L72 mutation, a W94 mutation, a K103 mutation, an R110 mutation, a P117 mutation, an L139 mutation, an F155 mutation, an N178 mutation, an E179 mutation
  • the RT polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 and the amino acid sequence of the engineered reverse transcriptase comprises an M39 mutation, a K47 mutation, an L435 mutation, a D449 mutation, a D524 mutation, an E607 mutation, a D653 mutation, and an L671 mutation as indexed to SEQ ID NO:7 and comprising at least one mutation indexed to SEQ ID NO:7 selected from a M17 mutation; an A32 mutation, a M44 mutation, a M39V mutation, a P51 mutation, an M66 mutation, an S67 mutation, an E69 mutation, a L72 mutation, a W94 mutation, a K103 mutation, an R110 mutation, a P117 mutation, an L139 mutation, an F155 mutation, an N178 mutation, an E179 mutation, a T197 mutation, a D200 mutation, an E201 mutation, an H204 mutation, a Q221 mutation, a V2
  • the engineered RT polypeptide exhibits an altered reverse transcriptase related activity when compared to a reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 1.
  • an RT polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.
  • the engineered reverse transcriptase exhibits an altered reverse transcriptase related activity as compared to a reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 1.
  • the RT polypeptide comprises a combination of mutations indexed to SEQ ID NO:7 selected from: (i) an E69K mutation, an E302R mutation, a T306K mutation, a W313F mutation, a L435G mutation, or an N454K mutation, and comprising at least one mutation selected from an M39V mutation, an M66L mutation, an L139P mutation, an F155Y mutation, a D200N mutation, an E201Q mutation, a T287A mutation, a T330P mutation, an R411F mutation, a P448A mutation, a D449G mutation, an H503V mutation, an H594K mutation, L603W mutation, an E607K mutation, an H634Y mutation, a G637R mutation and an H638G mutation; (ii) an L139P mutation, a D200N mutation, a T330P mutation, an L603W mutation, or an E607K mutation, and comprising at least
  • the RT polypeptide comprises an amino acid sequence that is at least 95% identical to a reverse transcriptase having the amino acid sequence set forth in SEQ ID NO:1.
  • the engineered reverse transcription enzyme comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 and has at least one mutation selected from the group consisting of an M39V mutation, a P47L mutation, M66L mutation, an E69K mutation, an L139P mutation, a D200N mutation, an H204R mutation, an E302R mutation, a T306K mutation, a W313F mutation, a T330P mutation, an L435G mutation, a G429S mutation, an L435K mutation, a P448A mutation, a D449G mutation, a N454K mutation, an H503V mutation, a D524N mutation, a T542 mutation, an E545G mutation, a D583N mutation, an H594Q mutation
  • the disclosure provides an engineered RT polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:1 and the amino acid sequence of the engineered reverse transcriptase comprises a combination of mutations indexed to SEQ ID NO:7 or 178 selected from the group consisting of (a) an E69K mutation, an L139P mutation, a D200N mutation, an E302R mutation, a T306K mutation, a W313F mutation, a T330P mutation, a N454K mutation, an H503V mutation, a D524N mutation, an L603W mutation, an E607K mutation, and an H634Y mutation; (b) an M66L mutation, an E69K mutation, an L139P mutation, a D200N mutation, an E302R mutation, a T306K mutation, a W313F mutation, a T330P mutation, a N454K mutation, a D524N mutation, an H50
  • the D200 mutation is selected from the group consisting of D200N and D200E.
  • the D449 mutation is selected from the group consisting of D449G an D449E.
  • the L603 mutation is selected from the group consisting of L603W and L603F.
  • the E607 mutation is selected from the group consisting of E607G and E607K.
  • the engineered RT polypeptide further comprises at least one mutation selected from the group consisting of P47L, H204R, D524N, T542D, E545G, D583N, H594Q, P627S, A644V, R650H, D653H, K658R, L671P, and S679P.
  • an engineered reverse transcriptase of the present application has an amino acid sequence that is at least 95% identical to SEQ ID NO:1 and the amino acid sequence of said engineered reverse transcriptase comprises a combination of mutations indexed to SEQ ID NO:7 or 178; and the amino acid sequence of said engineered reverse transcriptase comprises a combination of mutations selected from the group consisting of: an E69K mutation, an L139P mutation, a D200N mutation, an E302R mutation, a T306K mutation, a W313F mutation, a T330P mutation, a N454K mutation, an H503V mutation, a D524N mutation, an L603W mutation, an E607K mutation, and an H634Y mutation and further comprising a second combination of mutations selected from the group consisting of: (a) an M66L mutation and an L534G mutation, (b) an M39V mutation, an M66L mutation and an L435K mutation, (
  • an engineered reverse transcriptase of the present application has an amino acid sequence that is at least 95% identical to SEQ ID NO:1 and the amino acid sequence of the engineered reverse transcriptase comprises a combination of mutations selected from the group consisting of: an M39V mutation, an E69K mutation, an L139P mutation, a D200 mutation, an E302R mutation, a T306K mutation, a W313F mutation, a T330P mutation, a 80 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC G429S mutation a P448A mutation, a D449 mutation, an L435K mutation, a N454K mutation, an L603 mutation, an E607 mutation, and an L671P mutation and further comprising a second combination of mutations selected from the group consisting of: (a) a D524N mutation, a T542D mutation, an A644
  • the P47 mutation is a P47L mutation
  • the D200 mutation is a D200N mutation
  • the D449 mutation is a D449G mutation
  • the L603 mutation is an L603W mutation
  • the E607 mutation is an E607G mutation
  • the P627 mutation is a P627S mutation.
  • a variant may comprise a first combination of mutations or alterations and may comprise an additional or second combination of mutations.
  • a first combination of mutations or alterations may include, but is not limited to, a combination of: (1) a M39 mutation, a K47 mutation, an L435 mutation, a D449 mutation, a D524 mutation, an E607 mutation, a D653 mutation and an L671 mutation; (2) an M39V mutation, a K47 mutation, an L435K mutation, a D449G mutation, a D524N mutation, an E607 mutation, a D653 mutation and an L671 mutation; (3) an M39 mutation, an M66 mutation, an E302 mutation, a T306 mutation, an L435 mutation, a D449 mutation, a D524 mutation, an E607 mutation, a D653 mutation and an L671 mutation; (4) an M39 mutation, an M66 mutation, an E302 (K or R) mutation
  • the second combination of mutations in a first engineered reverse transcriptase may comprise either a different set of mutations or a partially different second set of mutations as in a second engineered reverse transcriptase.
  • a second combination of mutations or alterations may include but is not limited to: (a) one or more mutations selected from an M17 mutation; an A32 mutation, a M44 mutation, a P51 mutation, an M66 mutation, an S67 mutation, an E69 mutation, a L72 mutation, a W94 mutation, a K103 mutation, an R110 mutation, a P117 mutation, an L139 mutation, an F155 mutation, an N178 mutation, an E179 mutation, a T197 mutation, a D200 mutation, an E201 mutation, an H204 mutation, a Q221 mutation, a V223 mutation, a V238 mutation, a G248 mutation, a T265 mutation, an E268 mutation, an R279 mutation, an R280 mutation, a K284 mutation, a T287 mutation,
  • the second combination of mutations may comprise a group of mutations as described herein and one or more additional mutations.
  • the engineered RT variants of the present disclosure comprise a M39V, M66I, Q91R, I347V, H594Q, or a combination thereof in the RT backbone of SEQ ID NO: 143 or SEQ ID NO: 7.
  • the engineered RT polypeptide comprises: M39V, M66I, Q91R, I347V, and H594Q (SEQ ID NO: 129 , SOLD 034).
  • the engineered RT variants of the present disclosure comprise M39V, T542D, D583N, E607G, A644V, D653H, K658R, L671P, or a combination thereof in the RT backbone of SEQ ID NO: 143 or SEQ ID NO: 7.
  • the engineered RT polypeptide comprises: M39V, T542D, D583N, E607G, A644V, D653H, K658R, and L671P (SEQ ID NO: 111, SOLD 025).
  • the engineered RT polypeptide comprises: M39V, M66I, Q91R, I347V, H594Q, or a combination thereof, and optionally M39V, M66I, Q91R, I347V, H594Q, or the combination thereof (substituted) in the RT sequence of SEQ ID NO: 143 (SEQ ID NO: 129, SOLD 034).
  • the engineered RT polypeptide comprises: M39V, T542D, D583N, E607G, A644V, D653H, K658R, L671P, or a combination thereof, and optionally M39V, T542D, D583N, E607G, A644V, D653H, K658R, L671P, or the combination thereof substituted in the RT sequence of SEQ ID NO: 143 (or SEQ ID NO: 7) (SEQ ID NO: 111, SOLD 025).
  • the engineered RT polypeptide comprises SOLD 33 VDG or SEQ ID NO: 173.
  • the engineered RT polypeptide described herein comprises an amino acid sequence that is at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 27-61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 141, 143, 145, 147, 149, 151, 157, 159, 172, and 173.
  • the RT polypeptide is 42B L (SEQ ID NO: 145), 50A+G (SEQ ID NO: 147), SOLD 022 (SEQ ID NO: 105), SOLD 023 (SEQ ID NO: 107), SOLD 025 (SEQ ID NO: 111), SOLD 031 (SEQ ID NO: 123), SOLD 033 (SEQ ID NO: 127), SOLD 034 (SEQ ID NO: 129), SOLD 035 (SEQ ID NO: 131), SOLD 001 (SEQ ID NO: 65), SOLD 33 VDG (SEQ ID NO: 173), or an RT polypeptide set forth in SEQ ID NO: 143, or SEQ ID NO: 172.
  • E. Engineered DAT1 Reverse Transcriptases [000309]
  • One aspect of the present disclosure provides an engineered RT polypeptide comprising an amino acid sequence that is at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence of an RT disclosed in Table 1, or Table 2, and a DNA binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 3, 5, 6, 8, and 9.
  • Another aspect of the present disclosure provides an engineered RT polypeptide comprising an amino acid sequence that is at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence of SEQ ID NOs: 27-61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 141, 143, 145, 147, 149, 151, 157, 159, 172, or 173; and a DNA binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 3, 5, 6, 8, and 9.
  • Another aspect of the present disclosure provides an engineered RT polypeptide comprising an amino acid sequence that is at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence of SEQ ID NOs: 27-61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 141, 143, 145, 147, 149, 151, 157, 159, 172, or 173; and a DNA binding domain comprising an amino acid sequence selected
  • an engineered RT polypeptide comprising an amino acid sequence of an RT disclosed in Table 1 or Table 2; and an amino acid sequence of DNA binding domain disclosed in Table 1.
  • the engineered RT polypeptide described herein comprises the amino acid sequence of any one of SEQ ID NO: 174-188.
  • the engineered RT polypeptide described herein can comprise 42B L RT (SEQ ID NO: 145) operably linked to a full length DAT1 at the N-terminus.
  • the engineered RT can comprise the amino acid sequence of SEQ ID NO: 183.
  • the engineered RT polypeptide described herein can comprise 42B L RT operably linked to a full length DAT1 at the C-terminus.
  • the engineered RT can comprise the amino acid sequence of SEQ ID NO: 184.
  • DAT1(90) can be fused to any reverse transcriptase enzymes described herein, such as variants of MMLV reverse transcriptases disclosed in Table 1 or Table 2. While the majority of engineered RT polypeptide embodiments disclosed herein are N-terminus fusion proteins, the present disclosure also contemplates engineered RT polypeptides where the DNA binding domain, e.g., DAT1 or variant thereof is operably linked to the C-terminus of any RT polypeptide described herein.
  • the present disclosure provides any combination of DNA binding domain and RT polypeptide described in Table 1 or Table 2.
  • Non-exhaustive list of possible engineered RT polypeptides or recombinant RT proteins can comprise reverse transcriptases fused to full length DAT protein, DAT1 truncated to 36 amino acids, or DAT1(90) homologs.
  • the present disclosure provides an engineered RT comprising the amino acid sequence of SEQ ID NO: 178 (e.g., N-DAT-9042B).
  • the engineered RT can comprise the amino acid sequence of SEQ ID NO: 179 (e.g., N-DAT-9050A+ G).
  • the engineered RT can comprise the amino acid sequence of SEQ ID NO: 180 (e.g., N-DAT-90 SOLD 33 VDG).
  • the engineered RT can comprise the amino acid sequence of SEQ ID NO: 176 (e.g., N-DAT-90 SOLD 01).
  • the engineered RT can comprise the amino acid sequence of SEQ ID NO: 177 (e.g., C-DAT-90 SOLD 01).
  • an engineered RT polypeptide comprising N-DAT-9042B (SEQ ID NO: 178) and/or N-DAT-9050A+ G (SEQ ID NO: 178) were shown to enhance the sensitivity of the 5’ single cell assay as shown by enhanced transcript capture.
  • the engineered RT polypeptide described herein comprises 42B L RT (SEQ ID NO: 145) operably linked to an N-terminal truncated variant of DAT1 (D90) comprising the first 90 amino acids of the full length DAT1 at the C-terminus.
  • the engineered RT can comprise the amino acid sequence of SEQ ID NO: 174.
  • the variant can have at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% more or at least 2-fold, 3- fold, 4-fold, 5-fold, or 10-fold or more activity than the wild-type or known variant.
  • an engineered reverse transcription enzyme of the current application may exhibit an altered base-biased template switching activity such as an increased base-biased template switching activity, decreased base- biased template switching activity or an altered base-bias to the template switching activity.
  • the engineered RT polypeptide or the recombinant RT protein described herein exhibits increased template switching (TS) efficiency, increased processivity efficiency, increased binding affinity, increased transcription efficiency, increased chemical tolerance, improved ability to yield mitochondrial unique molecular identity (UMI) counts, improved ability to yield ribosomal unique molecular identity (UMI) counts, longer shelf life, higher strand displacement, higher end-to-end template jumping, or any combination thereof, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • TS template switching
  • UMI mitochondrial unique molecular identity
  • UMI ribosomal unique molecular identity
  • variants comprising a M39V or a M66L mutation that do not exhibit altered performance in the 5’ GEM assay may exhibit an altered processivity, an altered kd or both.
  • L435K mutants may improve thermostability in the presence of primer template.
  • L435K variants may exhibit a thermal denaturation profile similar to that of the wild-type protein.
  • L435K, P448 and D449 are residues in the connection domain; altering these residues may result in increased conformational flexibility. Additionally, the connection domain is thought to impact the conformational flexibility of the RNAse H domain. H503 and H634 occur within the RNAse H domain.
  • the H503V and H634Y variants may impact primer-template contacting, processivity or both primer-template contacting and processivity.
  • Some variants share the following alterations: (a) the combination of variants consisting of a T542D mutation, a D583N mutation, an E607G mutation, an A644V mutation, a D653H mutation, and a K658R mutation.
  • Some variants share the following alterations: (b) the combination of variants consisting of an E545G mutation, a D583N mutation, an H594Q mutation, an L603F mutation, and a S679P mutation.
  • These variants may further comprise additional alterations that may affect one or more reverse transcriptase related activities.
  • the combination of variants consisting of a T542D mutation, a D583N mutation, an E607G mutation, an A644V mutation, a D653H mutation, and a K658R mutation and the combination of variants consisting of an E545G mutation, a D583N mutation, an H594Q mutation, an L603F mutation and a S679P mutation may exhibit an altered RNAse H activity.
  • RNase H activity [000340]
  • the engineered reverse transcriptase polypeptide or recombinant RT protein is engineered to have reduced and/or abolished RNase activity.
  • engineered reverse transcriptases or recombinant RT proteins of the disclosure are preferably modified or mutated such that the transcription efficiency of the engineered RT polypeptide or recombinant RT protein is increased or enhanced.
  • engineered reverse transcription polypeptide or recombinant RT proteins of the present disclosure may have an unexpectedly greater ability to associate or bind to full-length transcripts (e.g., in T-cell receptor paired transcriptional profiling), as compared to that exhibited by an enzyme having the amino acid sequence set forth in SEQ ID NO:1 or non-DAT1 engineered RT.
  • An altered transcription efficiency may be an increased transcription efficiency or a decreased transcription efficiency as compared to the transcription efficiency of a reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 1.
  • Altered transcription efficiency may be at least .1X, 0.2X, 0.3X, 0.4X, 0.5X, 0.6X, 0.7X, 0.8X, 0.9X, 1X, 1.5X, 2X, 2.5X, 3X, 3.5X, 4X, 4.5X, 5X, 5.5X, 6X, 6.5X, 7X, 7.5X, 8X, 8.5X, 9X, 10X, 15X, 20X, 25X or at least 30X greater than the transcription efficiency of a reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 1, 143, 145, or 172 or non-DAT1 engineered RT.
  • Transcription efficiency may be calculated as the sum of the area under the curve for the elongation, elongation plus tail, incomplete template switching (TSO) and complete template switching (TSO) regions over the total area under the curve for all products. Transcription efficiency reflects all those products for which transcription was successfully completed. Template switching oligonucleotide efficiency may be calculated as the area under the curve for the complete template switching region over the total area under the curve for all full-length products.
  • An engineered reverse transcriptase may have an increased transcription efficiency, an increased TSO efficiency or both an increased transcription efficiency and an increased TSO efficiency. 4.
  • the engineered reverse transcriptase polypeptide or recombinant RT protein described herein possesses one or more of the following characteristics when compared to a wild-type polymerase and/or reverse transcriptase: increased thermostability; increased thermoreactivity; increased resistance to reverse transcriptase inhibitors; increased ability to reverse transcribe difficult templates; increased speed; increased processivity; increased specificity; enhanced polymerization activity; increased sensitivity, or any combination thereof.
  • Processivity is defined as the ability of a polymerase or reverse transcriptase to carry out continuous nucleic acid synthesis on a template nucleic acid without frequent dissociation.
  • DNA polymerase or reverse transcriptase alone produces short DNA product strand per binding event.
  • Most DNA polymerases or reverse transcriptases are intrinsically low-processivity enzymes. The low processivity of DNA polymerase or reverse transcriptase alone is insufficient for the timely replication of a large genome.
  • the polymerization activity of the engineered reverse transcriptase polypeptide or recombinant RT protein as described herein is enhanced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 90%, or about 100% as compared to the wild-type reverse transcriptase.
  • the engineered reverse transcriptase enzyme or engineered reverse transcriptase polypeptide or recombinant RT protein reverse transcribes a RNA molecule having at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 nucleotides.
  • the engineered reverse transcriptase polypeptide or recombinant RT protein reverse transcribes a RNA molecule that is at least about 1kb, at least about 2kb, at least about 3kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10kb, at least about 11 kb, at least about 12 kb, at least about 13 kb, at least about 14kb, or at least about 15 kb.
  • the engineered reverse transcriptase polypeptide or recombinant RT protein reverse transcribes a RNA molecule that is at least about 7kb or at least about 8kb.
  • the increase in thermoreactivity, resistance to reverse transcriptase inhibitors, ability to reverse transcribe difficult templates, speed, processivity, specificity, or sensitivity of the engineered reverse transcriptase polypeptide or recombinant RT protein as described herein has is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 90%, or about 100% as compared to the wild-type polymerase.
  • the enhanced reverse transcriptase activity is an increased binding affinity and template switching efficiency as compared to a reverse transcriptase having the amino acid sequence set forth in SEQ ID NO:1, 143, 145, or 172 or non-DAT1 engineered RT.
  • the enhanced reverse transcriptase activity is an enhanced processivity as compared to the processivity of a reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 1, 143, 145, or 172 or non-DAT1 engineered RT.
  • Processivity relates to a reverse transcriptase’s ability to remain associated with the template while incorporating nucleotides.
  • Measurements of processivity may include but are not limited to the number of nucleotides incorporated in a single binding event of a reverse transcriptase molecule.
  • Processivity also relates to the affinity of the enzyme for the substrate; thus, an enzyme with increased processivity may be more resistant to the presence of an inhibitor.
  • NUCLEIC ACIDS AND EXPRESSION VECTORS A. Nucleic Acids [000356]
  • One aspect of the present disclosure provides an isolated nucleic acid molecule encoding the engineered reverse transcriptase, the recombinant RT protein or derivatives thereof as described herein.
  • One aspect of the present disclosure provides an isolated nucleic acid molecule encoding any of the engineered RT polypeptides or the recombinant RT protein described herein.
  • the engineered reverse transcriptase polypeptide or the recombinant RT protein disclosed herein can been coded by a nucleic acid set forth herein or readily derived in light of polypeptide information provided herein and known in the art.
  • the isolated nucleic acid molecule encoding the RT polypeptide can comprise a sequence selected from SEQ ID NO; 25; SEQ ID NO: 136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:167, SEQ ID NO:169, or SEQ ID NO: 171; or a non- limiting embodiment of a nucleic acid sequence of Table 1 or Table 2.
  • the reverse transcriptase polypeptides, or the DNA binding domains need not be encoded by any specific nucleic acid exemplified herein.
  • redundancy in the genetic code allows for variations in nucleotide codon sequences that nevertheless encode the same amino acid.
  • engineered polymerases of the present disclosure can be produced from nucleic acid sequences that are different from those set forth herein, for example, being codon optimized for a particular expression system. Codon optimization can be carried out, for example, as set forth in Athey et al., BMC Bioinformatics, 18:391-401 (2017).
  • Wild type nucleic acids may be isolated from naturally occurring sources to be used as starting material to generate novel polymerases.
  • nomenclature and the laboratory procedures in recombinant DNA technology described below are those well-known and commonly employed in the art. Standard techniques for cloning, DNA and RNA isolation, amplification and purification are known.
  • enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases are the 97 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC like are performed according to the manufacturer's specifications.
  • nucleic acids e.g., RT or DNA binding domain
  • the isolation of nucleic acids may be accomplished by a variety of techniques.
  • the nucleic acids of the present disclosure can be generated from the wild type sequences.
  • the wild type sequences are altered to create modified sequences.
  • Wild type molecules e.g., RT or DNA binding domain
  • Exemplary modification methods are site-directed mutagenesis, point mismatch repair, or oligonucleotide-directed mutagenesis.
  • a “vector” refers to a polynucleotide, which when independent of the host chromosome, is capable replication in a host organism.
  • Preferred vectors include plasmids and typically have an origin of replication.
  • Vectors can comprise, e.g., transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular nucleic acid.
  • the polymerases of the present disclosure can be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeasts, filamentous fungi, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines.
  • E. coli E. coli
  • yeasts yeasts
  • filamentous fungi various higher eukaryotic cells
  • COS COS
  • CHO and HeLa cells lines and myeloma cell lines eukaryotic cells
  • Techniques for gene expression in microorganisms are described in, for example, Smith, Gene Expression in Recombinant Microorganisms (Bioprocess Technology, Vol.22), Marcel Dekker, 1994.
  • bacteria examples include, but are not limited to, Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus.
  • Filamentous fungi that are useful as expression hosts include, for example, the following genera: Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Mucor, Cochliobolus, and Pyricularia.
  • yeast Synthesis of heterologous proteins in yeast is 98 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC well known and described in the literature.
  • C. Host cells Another aspect of the present disclosure provides a host cell transfected with the expression vector comprising the isolated nucleic acid encoding the engineered reverse transcriptase as described herein.
  • Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.
  • vectors include Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp series plasmids) and pGPD-2.
  • Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus.
  • exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • the engineered reverse transcriptase or a derivative thereof can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity purification columns, column chromatography, gel electrophoresis and the like. Substantially pure compositions of at least about 90 to about 95% homogeneity are preferred, and about 98 to about 99% or more homogeneity are most preferred. Once purified, partially or to homogeneity as desired, the polypeptides may then be used (e.g., as immunogens for antibody production).
  • the nucleic acids that encode the engineered reverse transcriptase or derivatives thereof can also include a coding sequence for an epitope or “tag” for which an affinity binding reagent is available.
  • suitable epitopes include the myc and V-5 reporter genes; expression vectors useful for recombinant production of fusion polypeptides having these epitopes are commercially available (e.g., Invitrogen (Carlsbad Calif.) vectors pcDNA3.1/Myc-His and 99 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC pcDNA3.1/V5-His are suitable for expression in mammalian cells).
  • Additional expression vectors suitable for attaching a tag to the fusion proteins of the disclosure, and corresponding detection systems are known to those of skill in the art as described herein, and several are commercially available (e.g., FLAG′′ (Kodak, Rochester N.Y.).
  • FLAG′′ Kodak, Rochester N.Y.
  • Another example of a suitable tag is a polyhistidine sequence, which is capable of binding to metal chelate affinity ligands. Typically, six adjacent histidines are used (6His-tag, his-tag), although one can use more or less than six.
  • Suitable metal chelate affinity ligands that can serve as the binding moiety for a polyhistidine tag include nitrilo-tri-acetic acid (NTA).
  • the engineered reverse transcriptase or derivatives thereof may possess a conformation substantially different than the native conformations of the constituent polypeptides. In this case, it may be necessary or desirable to denature and reduce the engineered reverse transcriptase or a derivative thereof and cause the engineered reverse transcriptase or a derivative thereof to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re- folding are well known to those of skill in the art. V.
  • compositions comprising a variety of components in various combinations needed for nucleic acid amplification using the engineered RT polypeptides or recombinant proteins disclosed herein.
  • One aspect of the present disclosure provides a composition comprising any of the recombinant RT proteins described herein.
  • One aspect of the present disclosure provides a composition comprising any of the engineered RT polypeptides described herein.
  • One aspect of the present disclosure provides a composition comprising any of the engineered RT polypeptides described herein.
  • composition comprising any of the expression vectors described herein.
  • any one of the compositions described herein further comprise a buffer.
  • the compositions are formulated by admixing one or more engineered reverse transcriptase polypeptides or recombinant RT proteins, 100 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC or derivatives thereof of the present disclosure in a buffered salt solution.
  • One or more DNA polymerases and/or one or more nucleotides, and/or one or more primers may optionally be added to create the compositions of the invention.
  • compositions can be used in the methods disclosed herein to produce, analyze, quantitate and otherwise manipulate nucleic acid molecules (e.g., using reverse transcription or one-step RT-PCR procedures).
  • the engineered reverse transcriptases or the recombinant RT proteins disclosed herein are provided at working concentrations (e.g., 1 ⁇ ) in stable buffered salt solutions.
  • working concentration means the concentration of an enzyme (e.g., engineered reverse transcriptase or the recombinant RT protein) that is at or near the optimal concentration used in a solution to perform a particular function such as reverse transcription of nucleic acids.
  • Such compositions can also be formulated as concentrated stock solutions (e.g., 2 ⁇ , 3 ⁇ , 4 ⁇ , 5 ⁇ , 6 ⁇ , 10 ⁇ , etc.). In some embodiments, having the composition as a concentrated (e.g., 5x) stock solution allows a greater amount of nucleic acid sample to be added (such as, for example, when the compositions are used for nucleic acid synthesis).
  • the water used in forming the compositions of the present invention is preferably distilled, deionized and sterile filtered (through a 0.1-0.2 micrometer filter) and is free of contamination by DNase and RNase enzymes.
  • Such water is available commercially, for example from Life Technologies (Carlsbad, Calif.) or may be made as needed according to methods well known to those skilled in the art.
  • METHODS FOR USING ENGINEERED REVERSE TRANSCRIPTASES [000373]
  • the engineered reverse transcriptases of the present disclosure may be used in any application in which a reverse transcriptase with the indicated altered activity is desired.
  • One aspect of the present disclosure provides a method for performing a reverse transcription reaction for generating a nucleic acid product from an RNA template using an engineered reverse transcriptase or recombinant RT protein described herein.
  • the engineered reverse transcriptases or recombinant RT protein of the present application may be used in any application in which a reverse transcriptase with the indicated altered activity is desired. Methods of using reverse transcriptases are known in the art. One skilled in the art may select any of the engineered reverse transcriptases disclosed herein.
  • One aspect of the present disclosure provides a method for performing a reverse transcription reaction for generating a nucleic acid product from an RNA template comprising contacting under suitable conditions a biological sample or extract thereof with an engineered RT polypeptide, or a recombinant RT protein described herein.
  • the cell can be fixed.
  • the cell can be permeabilized.
  • the cell can be permeabilized and fixed.
  • the cell is a cell bead.
  • the cell bead is fixed.
  • the nucleus is permeabilized or fixed.
  • the nucleus is permeabilized and fixed.
  • the biological sample comprises a suitable cellular preparation selected from cell populations and/or single cells.
  • the biological sample comprises a tissue. 102 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC [000376]
  • the reverse transcription reaction is part of a single cell RNA sequencing assay.
  • the single cell RNA sequencing assay further comprises, prior to the reverse transcription, partitioning the cell, the cell bead, or the nucleus into a partition.
  • the single cell RNA sequencing assay further comprises, after the reverse transcription reaction, hybridizing the nucleic acid product to an oligonucleotide molecule comprising a partition-specific barcode.
  • the reverse transcription reaction is part of a spatial RNA sequencing assay.
  • the engineered RT polypeptide or the recombinant RT protein enhances template switching (TS) efficiency, processivity efficiency, binding affinity, transcription efficiency, chemical tolerance, ability to yield mitochondrial unique molecular identity (UMI) counts, ability to yield ribosomal unique molecular identity (UMI) counts, strand displacement, end-to-end template jumping, or any combination thereof, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • TS template switching
  • UMI mitochondrial unique molecular identity
  • UMI ribosomal unique molecular identity
  • the engineered RT polypeptide or the recombinant RT protein enhances at least two or more of template switching (TS) efficiency, processivity efficiency, binding affinity, transcription efficiency, chemical tolerance, ability to yield mitochondrial unique molecular identity (UMI) counts, strand displacement, end-to-end template jumping, or ability to yield ribosomal unique molecular identity (UMI) counts, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • TS template switching
  • UMI mitochondrial unique molecular identity
  • UMI ribosomal unique molecular identity
  • the engineered RT polypeptide or the recombinant RT protein can comprise a DNA binding domain comprising an amino acid sequence selected from SEQ ID NO:2, 3, 5, 6, 8, 9, or 11-24; and an amino acid sequence selected from SEQ ID NOs: 27-61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 141, 143, 145, 147, 149, 151, 157, 159, 172, or 173.
  • the amino acid sequence of the engineered RT polypeptide or the recombinant RT protein comprises an amino acid sequence having at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 174-188.
  • the engineered RT polypeptide or the recombinant RT protein can comprise M39V, M66I, Q91R, I347V, and H594Q substitution in SEQ ID NO: 143.
  • the engineered RT polypeptide or the recombinant RT protein can comprise SEQ ID NO: 129 (SOLD 034).
  • the engineered RT polypeptide or the recombinant RT protein can comprise M39V, T542D, D583N, E607G, A644V, D653H, K658R, and/or L671P in SEQ ID NO: 143.
  • the engineered RT polypeptide or the recombinant RT protein can comprise SEQ ID NO: 111 (SOLD 025).
  • the engineered RT or the recombinant RT protein can comprise a M39V, M66I, Q91R, I347V, and/or H594Q in SEQ ID NO: 143.
  • the engineered RT polypeptide or the recombinant RT protein can comprise an amino acid sequence that is at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical to an amino acid sequence disclosed in Table 1 or Table 2.
  • One aspect of the present disclosure provides a method of using the engineered RT polypeptide, or the recombinant RT protein described herein, the method comprising contacting the engineered RT polypeptide or the recombinant RT protein with a nucleic acid template under suitable conditions to produce a polymerized nucleic acid product.
  • the nucleic acid template comprises an RNA, a DNA, or a nucleic acid comprising an unnatural nucleotide.
  • the nucleic acid template comprises an RNA.
  • the engineered reverse transcriptase comprises an M39 mutation, a K47 mutation, an L435 mutation, a D449 mutation, a D524 mutation, an E607 mutation, a D653 mutation and an L671 mutation in SEQ ID NO:7.
  • the engineered reverse transcriptase comprises a mutation selected from a K13 mutation, a K13L mutation, a D36 mutation, an N37 mutation, a V2 mutation, a D36L mutation, an insertion, and a combination thereof.
  • the engineered reverse transcriptases or the recombinant RT proteins, or derivatives thereof of the present disclosure are used in reverse transcription reactions, such as RT-PCR, or other known reactions in the art where nucleic acids, for example RNA molecules, are reverse transcribed using a reverse transcriptase.
  • the engineered reverse transcriptase, the recombinant RT protein or a derivative thereof as described herein may be used to make nucleic acid molecules from one or more templates.
  • Such methods can comprise mixing one or more nucleic acid templates (e.g., RNA, such as non-coding RNA (ncRNA), messenger RNA (mRNA), micro RNA (miRNA), and small interfering RNA (siRNA) molecules) with one or more of the engineered reverse transcriptases of the disclosure and incubating the mixture under conditions sufficient to generate one or more nucleic acid molecules complementary to all or a portion of the one or more nucleic acid templates.
  • RNA such as non-coding RNA (ncRNA), messenger RNA (mRNA), micro RNA (miRNA), and small interfering RNA (siRNA) molecules
  • ncRNA non-coding RNA
  • mRNA messenger RNA
  • miRNA micro RNA
  • siRNA small interfering RNA
  • the method of using the engineered reverse transcriptase, or the recombinant RT protein or a derivative thereof as described herein comprises the amplification of one or more nucleic acid molecules comprising mixing one or more nucleic acid templates with one of the engineered reverse transcriptase polypeptide or recombinant RT 105 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC proteins or a derivative thereof of the disclosure, and incubating the mixture under conditions sufficient to amplify one or more nucleic acid molecules complementary to all or a portion of the one or more nucleic acid templates.
  • the method may comprise the use of one or more DNA polymerases and may be employed as in standard reverse transcription-polymerase chain reaction (RT-PCR) reactions.
  • RT-PCR reverse transcription-polymerase chain reaction
  • the method of using the engineered reverse transcriptase, recombinant RT protein or a derivative thereof as described herein may be one-step (e.g., one- step RT-PCR) or two-step (e.g., two-step RT-PCR) reactions.
  • the one-step RT-PCR type reactions may be accomplished in one tube thereby lowering the possibility of contamination.
  • Such one-step reactions can comprise (a) mixing a nucleic acid template (e.g., mRNA) with one or more engineered reverse transcriptase polypeptides or recombinant RT proteins or derivatives thereof of the present disclosure and one or more polymerases and (b) incubating the mixture under conditions sufficient to amplify a nucleic acid molecule complementary to all or a portion of the template.
  • a nucleic acid template e.g., mRNA
  • engineered reverse transcriptase polypeptides or recombinant RT proteins or derivatives thereof of the present disclosure e.g., RNA
  • Such methods can comprise (a) mixing a nucleic acid template (e.g., mRNA) with an engineered reverse transcriptase polypeptide or a recombinant RT protein or a derivative thereof of the present disclosure, (b) incubating the mixture under conditions sufficient to make a nucleic acid molecule (e.g., a DNA molecule) complementary to all or a portion of the template, (c) mixing the nucleic acid molecule with one or more DNA polymerases and (d) incubating the mixture of step (c) under conditions sufficient to amplify the nucleic acid molecule.
  • a nucleic acid template e.g., mRNA
  • an engineered reverse transcriptase polypeptide or a recombinant RT protein or a derivative thereof of the present disclosure incubating the mixture under conditions sufficient to make a nucleic acid molecule (e.g., a DNA molecule) complementary to all or a portion of the template
  • a nucleic acid molecule
  • a combination of DNA polymerases and the engineered reverse transcriptase polypeptide or recombinant RT protein or a derivative thereof of the present disclosure may be used.
  • Amplification methods which may be used in accordance with the present invention (e.g., using one or more engineered reverse transcriptase polypeptides or recombinant RT proteins or derivatives thereof of the present disclosure) include PCR, Isothermal Amplification, Strand Displacement Amplification (SDA), and Nucleic Acid Sequence-Based Amplification (NASBA); as well as more complex PCR-based nucleic acid fingerprinting techniques such as Random Amplified Polymorphic DNA (RAPD) analysis, Arbitrarily Primed PCR (AP-PCR) 106 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC DNA Amplification Fingerprinting (DAF); microsatellite PCR; Directed Amplification of Minisatellite-region DNA (DAVID); digital droplet PCT (ddPCR) and Amplification Fragment Length Polymorphism (AFLP) analysis.
  • RAPD Random Amplified Polymorphic DNA
  • the engineered reverse transcriptase disclosed herein may be used in methods of amplifying or sequencing a nucleic acid molecule comprising one or more polymerase chain reactions (PCRs), such as any of the PCR- based methods described above.
  • PCRs polymerase chain reactions
  • Methods of producing an engineered reverse transcriptase, an engineered reverse transcriptase or a derivative thereof of the present disclosure are known to those of skill in the art of molecular biology or molecular genetics.
  • nucleic acids encoding the wild-type polymerase or nucleic acid binding domains can be generated using routine techniques in the field of recombinant genetics.
  • nucleic Acid Sample Processing Another aspect of the present disclosure provides a nucleic acid extension method comprising contacting a target nucleic acid molecule with an engineered reverse transcriptase or a recombinant RT protein and a plurality of nucleic acid barcoded molecules comprising a barcode sequence, and incubating the target nucleic acid, the engineered reverse transcriptase or the recombinant RT protein and barcoded molecules under conditions in which the barcoded molecules are extended by the engineered reverse transcriptase or the recombinant RT protein.
  • the engineered reverse transcriptase or the recombinant RT protein comprises the amino acid sequence of an engineered RT or an recombinant RT protein described herein or a derivatives thereof.
  • the target nucleic acid hybridizes to one of the plurality of barcoded molecules and the hybridized barcoded molecule is extended by the engineered reverse transcriptase or the recombinant RT protein described herein.
  • the novel engineered reverse transcriptase polypeptide or the recombinant RT protein described herein can be used to generate a Single Cell 3' (SC-3') and/or 5’ (SC-5') gene expression libraries.
  • the SC-3' and SC-5' assays are similar but capture different ends of the polyadenylated transcript in the final library. Both solutions use poly-dT primer for reverse transcription).
  • the poly-dT sequence is located on the gel bead oligo.
  • the SC-5' assay (FIGs 14A) the poly-dT is supplied as an RT primer.
  • a template switching oligo (TSO) is used in both assays to reverse transcribe the full-length transcript.
  • transcripts are randomly fragmented under conditions that favor 300-400 bp length fragments. Downstream of fragmentation, only transcripts containing both (1) a 10x Barcode and (2) an Illumina ® Read 2 adaptor, which is ligated on to the cDNA after fragmentation, can be amplified during the Sample Index PCR.
  • the nucleic acid is a ribonucleic acid (RNA) molecule; and the engineered reverse transcriptase polypeptide or recombinant RT protein reverse transcribes the RNA molecule thereby generating a first strand cDNA.
  • a first strand cDNA reaction can be optionally performed using template switching oligonucleotides.
  • a template switching oligonucleotide can hybridize to a poly(C) tail added to a 3’ end of the cDNA by the engineered reverse transcriptase polypeptide or recombinant RT protein described herein.
  • the original mRNA template and template switching oligonucleotide can then be denatured from the cDNA and a barcoded capture probe can then hybridize with the cDNA and a complement of the cDNA can be generated.
  • the first strand cDNA can then be purified and collected for downstream amplification steps.
  • the first strand cDNA can be amplified using PCR, where the forward and reverse primers flank the spatial barcode and target analyte regions of interest, generating a library associated with a particular spatial barcode.
  • the cDNA comprises a sequencing by synthesis (SBS) primer sequence.
  • the library amplicons are sequenced and analyzed to decode spatial information.
  • a reverse transcription reaction introduces a barcode.
  • the barcoding reaction is an enzymatic reaction.
  • the barcoding reaction is a reverse transcription amplification reaction that generates complementary deoxyribonucleic acid (cDNA) molecules upon reverse transcription of ribonucleic acid (RNA) molecules of the cell.
  • RNA molecules are released from the cell.
  • the RNA molecules are released from the cell by lysing the cell.
  • the RNA molecules are released from the cell by permeabilizing the cell, or a tissue which comprises a plurality of the same and/or different cell types.
  • the RNA molecules are messenger RNA (mRNA).
  • a reverse transcription reaction using the engineered reverse transcriptase, the recombinant RT protein or derivative thereof of the present disclosure is initiated at the point of hybridization of the capture sequences to the RNA molecules, with the capture probe being extended by the engineered reverse transcriptase polypeptide or recombinant RT protein of the present disclosure in a template directed fashion using the hybridized mRNA as a template.
  • the recombinant RT protein or the engineered RT polypeptide can exhibit increased transcript capture during amplification.
  • the DNA binding domain of the engineered Rt polypeptide or recombinant RT protein can enhance the hybridization of a transcript and a primer during a nucleic acid amplification process.
  • the primer can comprise a poly-dT or a poly(dT)VN sequence and a non-poly(dT) sequence; and the transcript can comprise a poly-dA sequence.
  • the DNA binding domain e.g., DAT1 or variant thereof
  • the primer is a barcoded molecule.
  • the reverse transcription reaction produces single stranded cDNA molecules each having a molecular tag and barcode associated with the cDNA, followed by amplification of cDNA to produce a double stranded cDNA that includes the sequences of the barcoded molecules.
  • the plurality of nucleic acid barcoded molecules comprise an oligo(dT) sequence.
  • the engineered reverse transcriptase polypeptide or recombinant RT protein reverse transcribes the mRNA molecule into a complementary DNA molecule using the mRNA hybridized to the oligo(dT) sequence of the nucleic acid barcoded 109 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC molecules as a template, and the nucleic acid binding domain binds and stabilizes the mRNA- oligo(dT) hybrid during the reverse transcription.
  • the engineered reverse transcriptase polypeptide or recombinant RT protein as described herein further amplifies the complementary DNA molecule comprising the barcode sequence, thereby generating an amplified DNA product comprising the barcode sequence, molecular tag sequence, or complements thereof.
  • the method can comprise a second nucleic acid molecule comprising an oligo(dT) sequence.
  • the plurality of nucleic acid barcoded molecules comprise an oligo(dT) sequence; and the nucleic acid binding domain of the engineered reverse transcriptase polypeptide or recombinant RT protein binds and stabilizes the mRNA-Oligo(dT) hybrid, while the polymerase domain of the engineered reverse transcriptase polypeptide or recombinant RT protein reverse transcribes the mRNA molecule using the second nucleic acid molecule comprising the oligo(dT) sequence, thereby generating a complementary DNA molecule.
  • the engineered reverse transcriptase polypeptide or recombinant RT protein further amplifies the complementary DNA molecule, thereby generating an amplified DNA product comprising a barcode sequence.
  • the nucleic acid extension method comprises a cell, a population of cells, or a tissue and the template nucleic acid molecule is from the cell, population of cells or the tissue.
  • the molecular tags are coupled to priming sequences and the barcoding reaction is initiated by hybridization of the priming sequences to the RNA molecules.
  • each priming sequence comprises a random N-mer sequence.
  • the random N-mer sequence is complementary to a 3’ sequence of a ribonucleic acid molecule of the cell.
  • the random N-mer sequence comprises a poly- dT sequence having a length of at least 5 bases.
  • the random N-mer sequence comprises a poly-dT sequence having a length of at least 10 bases.
  • the barcoding reaction is performed by extending the priming sequences in a template directed fashion using reagents for reverse transcription.
  • the reagents for reverse transcription comprise a reverse transcription enzyme 110 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC (e.g., engineered RT polypeptide or recombinant RT protein), a buffer and a mixture of nucleotides.
  • the reverse transcription enzyme adds a plurality of non- template oligonucleotides upon reverse transcription of a ribonucleic acid molecule.
  • the reverse transcription enzyme is an engineered RT polypeptide or recombinant RT protein as disclosed herein.
  • the barcoding reaction produces single stranded complementary deoxyribonucleic acid (cDNA) molecules each having a molecular tag from said molecular tags on a 5’ end thereof, followed by amplification of cDNA to produce a double stranded cDNA having the molecular tag on the 5’ end and a 3’ end of the double stranded cDNA.
  • cDNA complementary deoxyribonucleic acid
  • a molecular tag which comprises a barcode plus additional functional sequences, or only additional functional sequences is further included into a cDNA molecule generated during a reverse transcription reaction.
  • the reagents for reverse transcription comprise a reverse transcription enzyme (e.g., the engineered reverse transcriptase or the recombinant RT protein described herein), a buffer, and a mixture of nucleotides.
  • the reverse transcription enzyme adds a plurality of non- template oligonucleotides upon reverse transcription of a ribonucleic acid molecule from the nucleic acid molecules.
  • the reverse transcription enzyme is an engineered reverse transcriptase or a recombinant RT protein as disclosed herein.
  • the present disclosure provides methods that utilize the engineered reverse transcriptase polypeptides or the recombinant RT protein described herein for nucleic acid sample processing.
  • the method comprises contacting a template ribonucleic acid (RNA) molecule with an engineered reverse transcriptase to reverse transcribe the RNA molecule to a complementary DNA (cDNA) molecule.
  • the contacting step may be in the presence of a plurality of nucleic acid barcode molecules, wherein each nucleic acid barcode molecule comprises a barcode sequence.
  • the nucleic acid barcode molecule may comprise a sequence configured to couple to a template RNA molecule.
  • RNA molecules include, without limitation, an oligo(dT) sequence, a random N-mer primer, or a target-specific primer.
  • the nucleic acid barcode molecule may comprise a template switching sequence. 111 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC [000413]
  • the RNA molecule is a messenger RNA (mRNA) molecule.
  • the contacting step provides conditions suitable to allow the engineered reverse transcriptase to: (i) transcribe the mRNA molecule into the cDNA molecule with the oligo(dT) sequence and/or (ii) perform a template switching reaction, thereby generating the cDNA molecule which comprises the barcode sequence, or a derivative thereof.
  • the contacting step may occur in (i) a partition having a reaction volume (e.g., as further described herein and see e.g., US Patent Nos.10,400,280 and 10,323,278, each of which is incorporated herein by reference in its entirety); (ii) in a bulk reaction where the reaction components (e.g., template RNA and engineered reverse transcriptase) are in solution; or (iii) on a nucleic acid array (see e.g., US Patent Nos.10,480,022 and 10,030,261 as well as WO/2020/047005 and WO/2020/047010, each of which is incorporated herein by reference in its entirety).
  • a reaction volume e.g., as further described herein and see e.g., US Patent Nos.10,400,280 and 10,323,278, each of which is incorporated herein by reference in its entirety
  • the reaction components e.g., template RNA and engineered reverse transcriptase
  • the reverse transcription reaction may occur in a tissue (e.g., in situ reverse transcription), on a template that is associated with a sequence on a substrate, such as practiced in spatial transcriptomics, or further in a RT-PCR or other reverse transcription reaction in vitro on a purified target, partially purified target or unpurified target as found for example in a cellular lysate.
  • tissue e.g., in situ reverse transcription
  • RT-PCR reverse transcription reaction in vitro on a purified target, partially purified target or unpurified target as found for example in a cellular lysate.
  • assays involving nucleic acid sample processing may include, but are not limited to, single-cell transcription profiling, single-cell sequence analysis, immune profiling of individual T and B cells, single-cell chromatin accessibility analysis (e.g., ATAC seq analysis), single cell processing and analysis, paired single cell TCR sequencing, paired TCR ⁇ and TCR ⁇ .
  • exemplary assays may be carried out using commercially available systems for encapsulating biological samples, gel beads, barcodes, and/or other compounds/materials in droplets, such as The Chromium System (10X Genomics, Pleasanton CA USA).
  • Engineered RT polypeptide or recombinant RT protein may be used in methods of profiling a T-Cell receptor (TCR).
  • TCR T-Cell receptor
  • the poly-dT sequence may be extended in a reverse transcription reaction using the mRNA as a template to produce a cDNA transcript complementary to the mRNA and also includes sequence of a barcode oligonucleotide.
  • Terminal transferase activity of the reverse transcriptase can add additional bases to the cDNA transcript (e.g., polyC).
  • the switch oligo may then hybridize with the additional bases added to the cDNA 112 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC transcript and facilitate template switching.
  • a sequence complementary to the switch oligo sequence can then be incorporated into the cDNA transcript via extension of the cDNA transcript using the switch oligo as a template.
  • all the cDNA transcripts of the individual mRNA molecules include a common barcode sequence.
  • the transcripts made from different mRNA molecules within a given partition will vary at this unique sequence. As described elsewhere herein, this provides a quantification feature that can be identifiable even following any subsequent amplification of the contents of a given partition, e.g., the number of unique segments associated with a common barcode can be indicative of the quantity of mRNA originating from a single partition, and thus, a single cell.
  • the cDNA transcript may then be amplified with PCR primers.
  • the amplified product may then be purified (e.g., via solid phase reversible immobilization (SPRI)).
  • SPRI solid phase reversible immobilization
  • the amplified product can be ligated to additional functional sequences, and further amplified (e.g., via PCR).
  • the functional sequences may include a sequencer specific flow cell attachment sequence such as but not limited to., a P7 sequence for Illumina ® sequencing systems, as well as functional sequence, which may include a sequencing primer binding site, e.g., for a R2 primer for Illumina ® sequencing systems, as well as functional sequence, which may include a sample index, e.g., an i7 sample index sequence for Illumina ® sequencing systems.
  • a sequencer specific flow cell attachment sequence such as but not limited to., a P7 sequence for Illumina ® sequencing systems, as well as functional sequence, which may include a sequencing primer binding site, e.g., for a R2 primer for Illumina ® sequencing systems, as well as functional sequence, which may include a sample index, e.g., an i7 sample index sequence for Illumina ® sequencing systems.
  • the present disclosure provides novel engineered reverse transcriptase 113 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC polypeptide or recombinant RT proteins that function efficiently in high throughput amplification reaction assays that require reaction volumes of less than about 1 nanoliter.
  • the method comprises providing a reaction volume which comprises an engineered reverse transcriptase and a template ribonucleic acid (RNA) molecule.
  • the contacting occurs in a reaction volume, which may be less than 1 nanoliter, less than 750 picoliters, or less than 500 picoliters.
  • the reaction volume is present in a partition, such as a droplet or well (including a microwell or a nanowell).
  • the engineered reverse transcriptases, the recombinant RT protein, or derivatives thereof as described herein are used in a reaction volume less than about 1 nanoliter (nL).
  • the engineered reverse transcriptases, the recombinant RT proteins, or derivatives thereof, as described herein are used in a reaction volume that is less than about 500 picoliter (pL).
  • the reaction volume is contained within a partition. In some embodiments, the reaction volume is contained within a droplet. In some embodiments, the reaction volume is contained within a droplet in an emulsion. In some embodiments, the reaction volume is contained within a droplet emulsion having a reaction volume of less than about 1 nL. In some embodiments, the reaction volume is contained within a droplet emulsion having a reaction volume of less than about 500 pL. [000421] In some embodiments, the reaction volume is contained within a well. In some embodiments, the reaction volume is contained within a well having a reaction volume less than about 1 nL. In some embodiments, the reaction volume is contained within a well.
  • the reaction volume is contained within a well having a reaction volume less than about 500 pL. In some embodiments, the reaction volume is contained within a well in an array of wells having an extracted nucleic acid molecule, and the template nucleic acid molecule is the extracted nucleic acid molecule. In some embodiments, the reaction volume is contained within a well in an array of wells having a cell comprising a template nucleic acid molecule, and where the template nucleic acid molecule is released from the cell. [000422] In another embodiment, a method comprises providing a reaction volume, which comprises an engineered reverse transcriptase and a template ribonucleic acid (RNA) molecule and is considered a “low volume reaction”.
  • RNA ribonucleic acid
  • the reaction volume may comprise a plurality of nucleic acid barcode molecules, and each nucleic acid barcode molecule comprises a barcode 114 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC sequence.
  • the contacting occurs in a reaction volume, a low volume reaction, which may be less than 1 nanoliter, less than 750 picoliters, or less than 500 picoliters.
  • the reaction volume is present in a partition, such as a droplet or well (including a microwell or a nanowell). 3.
  • the barcoding reaction produces single stranded complementary deoxyribonucleic acid (cDNA) molecules each having a molecular tag on a 5’ end thereof, followed by amplification of the cDNA to produce a double stranded DNA having the molecular tag on the 5’ end and a 3’ end of the double stranded DNA.
  • the molecular tags e.g., barcode oligonucleotides
  • the UMIs are oligonucleotides.
  • the molecular tags are coupled to priming sequences.
  • each of the priming sequences comprises a random N-mer sequence.
  • the random N-mer sequence is complementary to a 3’ sequence of the RNA molecules.
  • the priming sequence comprises a poly-dT sequence having a length of at least 5 bases.
  • the priming sequence comprises a poly-dT sequence having a length of at least 10 bases (SEQ ID NO: 4).
  • the priming sequence comprises a poly-dT sequence having a length of at least 5 bases, at least 6 bases, at least 7 bases, at least 8 bases, at least 9 bases, at least 10 bases.
  • UMIs Unique molecular identifiers
  • nucleic acid sequences are assigned or associated with individual cells or populations of cells, in order to tag or label the cell’s components (and as a result, its characteristics).
  • UMIs Unique molecular identifiers
  • These unique molecular identifiers may be used to attribute the cell’s components and characteristics to an individual cell or group of cells, additionally to be used as a method for counting the individual cells or groups of cells by their incorporation.
  • the unique molecular identifiers are provided in the form of nucleic acid molecules (e.g., oligonucleotides) that comprise nucleic acid barcode sequences that may be attached to or otherwise associated with the nucleic acid contents of individual cell, or to other components of the cell, and particularly to fragments of those nucleic acids.
  • the nucleic acid molecule can, and do have differing barcode sequences, or at least represent a large number of 115 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC different barcode sequences across all of the partitions in a given analysis.
  • nucleic acid barcode sequences can include from about 6 to about 20 or more nucleotides within the sequence of the nucleic acid molecules (e.g., oligonucleotides).
  • the nucleic acid barcode sequences can include from about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides.
  • the length of a barcode sequence may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer.
  • the length of a barcode sequence may be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence may be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides may be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they may be separated into two or more separate subsequences that are separated by 1 or more nucleotides. In some cases, separated barcode subsequences can be from about 4 to about 16 nucleotides in length.
  • the barcode subsequence may be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence may be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence may be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.
  • the resulting population of partitions can also include a diverse barcode library that may include at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences.
  • each partition of the population can include at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least 116 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules and in some cases at least about 1 billion nucleic acid molecules.
  • the enhanced reverse transcriptase activity of the engineered reverse transcriptase disclosed herein is an enhanced ability to yield mitochondrial UMI counts as compared to a reverse transcriptase having the amino acid sequence set forth in SEQ ID NO:1 or 15. In some embodiments, the enhanced reverse transcriptase activity is an enhanced ability to yield increased ribosomal UMI counts as compared to a reverse transcriptase having the amino acid sequence set forth in SEQ ID NO:1 or 15.
  • Read counting and UMI counting are the principal gene expression quantification schemes used in single-cell RNA-sequencing (scRNA- seq) analysis, as such with increased ribosomal UMI counts sensitivity and accuracy increases for a scRNA-seq assay in determining transcriptome profiles for any given cell, group of cells or tissues. Numerous metrics can be used for quality control of single-cell RNA-sequencing, including percent of reads mapping to ribosomal genes, percent of reads mapping to mitochondrial genes, total number of UMIs detected, or number of features to which 50% of the reads map.
  • the number of different UMIs can be indicative of the quantity of mRNA originating from a given partition, and thus from the cell.
  • the transcripts can be amplified, purified and sequenced to identify the sequence of the cDNA transcript of the mRNA, as well as to sequence the barcode segment and the UMI segment. While a poly-dT primer sequence is described, other targeted or random primer sequences may also be used in priming the reverse transcription reaction.
  • the nucleic acid molecules bound to the bead may be used to hybridize and capture the mRNA on the solid phase of the bead, for example, in order to facilitate the separation of the RNA from other cell contents.
  • certain reverse transcriptase enzymes may increase UMI reads from genes of a desired length or length of interest.
  • the desired length of genes may be selected from lengths comprising less than 500 nucleotides, between 500 and 1000 nucleotides, between 1000 and 1500 nucleotides and greater than 1500 nucleotides.
  • a reverse transcriptase may preferentially increase UMI reads from genes of one length range. It is 117 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC recognized that an engineered reverse transcriptase may perform similarly, differently or comparably in a 3’-reverse transcription assay or a 5’-reverse transcription assay. It is similarly recognized that an engineered reverse transcriptase may preferentially increase UMI reads from a length of genes in a 3’-reverse transcription assay than in a 5’-reverse transcription assay. 4.
  • the engineered reverse transcriptases or the recombinant RT protein of the present disclosure may be suitable for use in methods in which a cell can be co-partitioned along with a nucleic acid barcode molecule bearing bead.
  • the nucleic acid barcode molecules can be released from the bead in the partition.
  • the poly-dT poly-deoxythymine, also referred to as oligo (dT)
  • dT oligo
  • Reverse transcription results in a cDNA transcript of the mRNA, but that transcript includes each of the sequence segments of the nucleic acid molecule.
  • the nucleic acid molecule comprises an anchoring sequence, it may be more likely hybridize to and prime reverse transcription at the sequence end of the poly-A tail of the mRNA.
  • all of the cDNA transcripts of the individual mRNA molecules may include a common barcode sequence segment.
  • the transcripts made from the different mRNA molecules within a given partition may vary at the unique UMI segment.
  • the number of different UMIs can be indicative of the quantity of mRNA originating from a given partition, and thus from the cell.
  • the transcripts can be amplified, cleaned up and sequenced to identify the sequence of the cDNA transcript of the mRNA, as well as to sequence the barcode segment and the UMI segment. While a poly-dT primer sequence is described, other targeted or random priming sequences may also be used in priming the reverse transcription reaction.
  • the plurality of nucleic acid barcoded molecules are attached to a support (e.g., a particle, a slide, a chip, a bead, etc.).
  • the support is selected from an array, a bead, a gel bead, a microparticle, and a polymer.
  • the nucleic acid barcoded molecules attached to a support comprise molecular tags (UMIs), primer sequences, capture sequences, 118 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC cleavage sequences, or additional functional sequences.
  • UMIs molecular tags
  • the support is a gel bead.
  • the nucleic acid barcoded molecules are releasably attached to the gel bead.
  • the gel bead comprises a polyacrylamide polymer.
  • a cross-section of the gel bead is less than about 100 ⁇ m. In some embodiments, a cross-section of a gel bead is less than about 60 ⁇ m. In some embodiments, a cross-section of a gel bead is less than about 50 ⁇ m. In some embodiments, a cross-section of a gel bead is less than about 40 ⁇ m.
  • a cross-section of a gel bead is less than about 100 ⁇ m, less than about 99 ⁇ m, less than about 98 ⁇ m, less than about 97 ⁇ m, less than about 96 ⁇ m, less than about 95 ⁇ m, less than about 94 ⁇ m, less than about 93 ⁇ m, less than about 92 ⁇ m, less than about 91 ⁇ m, less than about 90 ⁇ m, less than about 89 ⁇ m, less than about 88 ⁇ m, less than about 87 ⁇ m, less than about 86 ⁇ m, less than about 85 ⁇ m, less than about 84 ⁇ m, less than about 83 ⁇ m, less than about 82 ⁇ m, less than about 81 ⁇ m, less than about 80 ⁇ m, less than about 79 ⁇ m, less than about 78 ⁇ m, less than about 77 ⁇ m, less than about 76 ⁇ m, less than about 75 ⁇ m, less than about 74 ⁇ m, less than about 82
  • nucleic acid molecules e.g., oligonucleotides
  • Functionalization of beads for attachment of nucleic acid molecules may be achieved through a wide range of different approaches, including activation of chemical groups within a polymer, incorporation of active or activatable functional groups in the polymer structure, or attachment at the pre-polymer or monomer stage in bead production.
  • precursors e.g., monomers, cross-linkers
  • precursors e.g., monomers, cross-linkers
  • precursors e.g., monomers, cross-linkers
  • bead may comprise acrydite moieties, such that when a bead is generated, the bead also comprises acrydite moieties.
  • the acrydite moieties can be attached to a nucleic acid molecule (e.g., oligonucleotide), which may include a priming sequence (e.g., a primer for amplifying target nucleic acids, random primer, primer sequence for messenger RNA) and/or one or more barcode sequences.
  • the one more barcode sequences may include sequences that are the same for all nucleic acid molecules coupled to a given bead and/or sequences that are different across 119 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC all nucleic acid molecules coupled to the given bead.
  • the nucleic acid molecule may be incorporated into the bead.
  • the nucleic acid molecule can comprise a functional sequence, for example, for attachment to a sequencing flow cell, such as, for example, a P5 sequence for Illumina ® sequencing.
  • the nucleic acid molecule or derivative thereof e.g., oligonucleotide or polynucleotide generated from the nucleic acid molecule
  • can comprise another functional sequence such as, for example, a P7 sequence for attachment to a sequencing flow cell for Illumina ® sequencing.
  • the nucleic acid molecule can comprise a barcode sequence.
  • the primer can comprise a unique molecular identifier (UMI).
  • the primer can comprise an R1 sequence for use in Illumina ® sequencing workflows. In some cases, the primer can comprise an R2 sequence for use in Illumina ® sequencing workflows.
  • nucleic acid molecules e.g., oligonucleotides, polynucleotides, etc.
  • examples of such nucleic acid molecules e.g., oligonucleotides, polynucleotides, etc.
  • uses thereof as may be used with compositions, devices, methods and systems of the present disclosure, are provided in U.S. Patent Pub. Nos.2014/0378345 and 2015/0376609, each of which is entirely incorporated herein by reference.
  • the present invention is not limited as to a composition of any nucleic acid molecule or derivative thereof, or any particular sequencing platform and these characterizations serve as examples only which may be useful in a reverse transcription workflow.
  • a cell in operation, can be co-partitioned along with a barcode bearing bead.
  • the barcoded nucleic acid molecules affixed to a bead can be released from the bead in the partition.
  • the poly-dT (poly-deoxythymine, also referred to as oligo (dT)) segment of one of the released nucleic acid molecules can hybridize to (e.g., capture)_the poly-A tail of a mRNA molecule.
  • Reverse transcription may result in a cDNA transcript of the mRNA which cDNA transcript also includes each of the sequence segments of the nucleic acid molecule.
  • the nucleic acid molecule comprises additional functional sequences (e.g., capture domains, primer domains, UMIs, barcodes, etc.), it can hybridize to and prime reverse transcription of the mRNA using the hybridized mRNA as a template.
  • additional functional sequences e.g., capture domains, primer domains, UMIs, barcodes, etc.
  • all of the cDNA transcripts of the individual mRNA molecules may include a common barcode sequence.
  • the transcripts made from the different mRNA molecules within a given partition may vary with respect to unique molecular 120 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC identifying sequences (e.g., UMIs).
  • the number of different UMIs can be indicative of the quantity of mRNA originating from a given partition, and thus from the cell.
  • the transcripts can be amplified and sequenced to identify the sequence of the original mRNA captured template, as well as the sequence of the associated barcode and UMI. While a poly-dT capture sequence is described, other targeted or random capture sequences may also be used in capture or hybridize to a template for initiating the reverse transcription reaction.
  • the poly-dT segment may be extended in a reverse transcription reaction using the mRNA as a template to produce a cDNA transcript complementary to the mRNA and also includes sequence segments of a barcode oligonucleotide.
  • Terminal transferase activity of the reverse transcriptase can add additional bases to the cDNA transcript (e.g., polyC).
  • the switch oligo may then hybridize with the additional bases added to the cDNA transcript and facilitate template switching.
  • a sequence complementary to the switch oligo sequence can then be incorporated into the cDNA transcript via extension of the cDNA transcript using the switch oligo as a template.
  • all the cDNA transcripts of the individual mRNA molecules include a common barcode sequence segment.
  • the transcripts made from different mRNA molecules within a given partition will vary at this unique sequence. As described elsewhere herein, this provides a quantification feature that can be identifiable even following any subsequent amplification of the contents of a given partition, e.g., the number of unique segments associated with a common barcode can be indicative of the quantity of mRNA originating from a single partition, and thus, a single cell.
  • the cDNA transcript may then be amplified with PCR primers.
  • the amplified product may then be purified (e.g., via solid phase reversible immobilization (SPRI)).
  • the amplified product may be sheared, ligated to additional functional sequences, and further amplified (e.g., via PCR).
  • Any of the engineered RT enzymes of the present disclosure including without limitation any of the enzymes comprising the amino acid sequence and/or non-limiting embodiment of nucleic acid sequences shown in Table 1, or Table 2, could be analyzed in any suitable assay, including without limitation the assays described herein.
  • Assays include without limitation 5’ gene expression analyses, with or without VDJ analysis, 3’ gene expression 121 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC analysis, epigenetic analysis, or multiomic analyses.
  • the sample can comprise a cell, a cell bead, a permeabilized cell, or a nucleus.
  • the cell can be fixed.
  • the cell can be permeabilized.
  • the cell can be permeabilized and fixed.
  • the cell bead is fixed.
  • the nucleus is permeabilized or fixed.
  • the nucleus is permeabilized and fixed.
  • the biological sample comprises a suitable cellular preparation selected from cell populations and/or single cells.
  • the biological sample comprises a tissue.
  • the sample can also be a suitable cellular preparation selected from cell populations and/or single cells.
  • the sample can be a tissue.
  • the sample comprises cells in suspension, fresh cells, or fixed cells.
  • the sample can also comprise cells and tissues immobilized on various solid surfaces.
  • the reverse transcription reaction described herein is part of a single cell RNA sequencing assay.
  • the single cell RNA sequencing assay further comprises, prior to the reverse transcription, partitioning the cell, the cell bead, or the nucleus into a partition. In one embodiment, the single cell RNA sequencing assay further comprises, after the reverse transcription reaction, hybridizing the nucleic acid product to an oligonucleotide molecule comprising a partition-specific barcode.
  • the reverse transcription reaction described herein is part of a spatial RNA sequencing assay.
  • the sample is a fresh tissue. In some embodiments, the sample is a frozen sample. In some embodiments, the sample was previously frozen.
  • the sample is a formalin-fixed, or paraffin embedded (FFPE) sample.
  • FFPE formalin-fixed, or paraffin embedded
  • 10X Genomics Ref.: 100-165501PC samples generally are heavily cross-linked and fragmented, and therefore this type of sample allows for limited RNA recovery using conventional detection techniques.
  • methods of targeted RNA capture provided herein are less affected by RNA degradation associated with FFPE fixation than other methods (e.g., methods that take advantage of oligo-dT capture and reverse transcription of mRNA).
  • methods provided herein enable sensitive measurement of specific genes of interest that otherwise might be missed with a whole transcriptomic approach.
  • a biological sample e.g., tissue section
  • methanol stained with hematoxylin and eosin
  • fixing, staining, and imaging occurs before one or more oligonucleotide probes are hybridized to the sample.
  • a destaining step e.g., a hematoxylin and eosin destaining step
  • destaining can be performed by performing one or more (e.g., one, two, three, four, or five) washing steps (e.g., one or more (e.g., one, two, three, four, or five) washing steps performed using a buffer including HCl).
  • the images can be used to map spatial gene expression patterns back to the biological sample.
  • a permeabilization enzyme can be used to permeabilize the biological sample directly on the slide.
  • the methods of targeted RNA capture as disclosed herein include hybridization of multiple probe oligonucleotides. In some embodiments, the methods include 2, 3, 4, or more probe oligonucleotides that hybridize to one or more analytes of interest.
  • the methods include two probe oligonucleotides.
  • the probe oligonucleotide includes sequences complementary that are complementary or substantially complementary to an analyte.
  • the probe oligonucleotide includes a sequence that is complementary or substantially complementary to an analyte (e.g., an mRNA of interest (e.g., to a portion of the sequence of an mRNA of interest)).
  • analyte e.g., an mRNA of interest (e.g., to a portion of the sequence of an mRNA of interest)
  • a method of analyzing a sample comprising a nucleic acid molecule may comprise providing a plurality of nucleic acid molecules (e.g., RNA molecules), where each nucleic acid molecule comprises a first target region (e.g., a sequence that is 3′ of a target sequence or a sequence that is 5′ of a target sequence) and a second target region (e.g., a 123 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC sequence that is 5′ of a target sequence or a sequence that is 3′ of a target sequence), a plurality of first probe oligonucleotides, and a plurality of second probe oligonucleotides.
  • a first target region e.g., a sequence that is 3′ of a target sequence or a sequence that is 5′ of a target sequence
  • a second target region e.g., a 123 4876-6828-
  • the templated ligation methods that allow for targeted RNA capture as provided herein include a first probe oligonucleotide and a second probe oligonucleotide.
  • the first and second probe oligonucleotides each include sequences that are substantially complementary to the sequence of an analyte of interest.
  • the first and/or second probe oligonucleotide is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a sequence in an analyte.
  • the first probe oligonucleotide and the second probe oligonucleotide hybridize to adjacent sequences on an analyte.
  • the first and/or second probe as disclosed herein includes one of at least two ribonucleic acid bases at the 3′ end; a functional sequence; a phosphorylated nucleotide at the 5′ end; and/or a capture probe binding domain.
  • the functional sequence is a primer sequence.
  • the capture probe binding domain is a sequence that is complementary to a particular capture domain present in a capture probe.
  • the capture probe binding domain includes a poly(A) sequence.
  • the capture probe binding domain includes a poly-uridine sequence, a poly-thymidine sequence, or both.
  • the capture probe binding domain includes a random sequence (e.g., a random hexamer or octamer). In some embodiments, the capture probe binding domain is complementary to a capture domain in a capture probe that detects a particular target(s) of interest. [000451] In some embodiments, a capture probe binding domain blocking moiety that interacts with the capture probe binding domain is provided. In some instances, the capture probe binding domain blocking moiety includes a nucleic acid sequence. In some instances, the capture probe binding domain blocking moiety is a DNA oligonucleotide. In some instances, the capture probe binding domain blocking moiety is an RNA oligonucleotide.
  • a capture probe binding domain blocking moiety includes a sequence that is complementary or substantially complementary to a capture probe binding domain. In some embodiments, a capture probe binding domain blocking moiety prevents the capture probe binding domain from binding 124 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC the capture probe when present. In some embodiments, a capture probe binding domain blocking moiety is removed prior to binding the capture probe binding domain (e.g., present in a ligated probe) to a capture probe. In some embodiments, a capture probe binding domain blocking moiety comprises a poly-uridine sequence, a poly-thymidine sequence, or both.
  • the first probe oligonucleotide hybridizes to an analyte.
  • the second probe oligonucleotide hybridizes to an analyte.
  • both the first probe oligonucleotide and the second probe oligonucleotide hybridize to an analyte. Hybridization can occur at a target having a sequence that is 100% complementary to the probe oligonucleotide(s).
  • hybridization can occur at a target having a sequence that is at least (e.g., at least about) 80%, at least (e.g., at least about) 85%, at least (e.g., at least about) 90%, at least (e.g., at least about) 95%, at least (e.g., at least about) 96%, at least (e.g., at least about) 97%, at least (e.g., at least about) 98%, or at least (e.g., at least about) 99% complementary to the probe oligonucleotide(s).
  • the first probe oligonucleotide is extended.
  • the second probe oligonucleotide is extended. Extending probes can be accomplished using any method disclosed herein.
  • a polymerase e.g., a DNA polymerase
  • methods disclosed herein include a wash step. In some instances, the wash step occurs after hybridizing the first and the second probe oligonucleotides.
  • the wash step removes any unbound oligonucleotides and can be performed using any technique or solution disclosed herein or known in the art. In some embodiments, multiple wash steps are performed to remove unbound oligonucleotides.
  • the probe oligonucleotides e.g., first and the second probe oligonucleotides
  • the probe oligonucleotides are ligated together, creating a single ligated probe that is complementary to the analyte. Ligation can be performed enzymatically or chemically, as described herein.
  • the engineered RT polypeptide or the recombinant RT protein enhances template switching (TS) efficiency, processivity efficiency, binding affinity, transcription efficiency, chemical tolerance, ability to yield mitochondrial unique molecular identity (UMI) counts, ability to yield ribosomal unique molecular identity (UMI) counts, strand displacement, end-to-end template jumping, or any combination thereof, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • TS template switching
  • UMI mitochondrial unique molecular identity
  • UMI ribosomal unique molecular identity
  • the engineered RT polypeptide or the recombinant RT protein enhances at least two or more of template switching (TS) efficiency, processivity efficiency, binding affinity, transcription efficiency, chemical tolerance, ability to yield mitochondrial unique molecular identity (UMI) counts, strand displacement, end-to-end template jumping, or ability to yield ribosomal unique molecular identity (UMI) counts, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • TS template switching
  • UMI mitochondrial unique molecular identity
  • UMI ribosomal unique molecular identity
  • the recombinant RT protein or the engineered RT enhances transcript capture during amplification, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • the DNA binding domain of the recombinant RT protein or the engineered RT enhances the hybridization of a transcript and a primer during the amplification process, when compared to an RT polypeptide or a recombinant RT protein lacking a conjugated DNA binding domain.
  • Engineered reverse transcriptase polypeptides or recombinant RT proteins described herein may be used in methods of a T-Cell receptor (TCR) and a B-cell receptor (BRC) profiling.
  • TCR T-Cell receptor
  • BRC B-cell receptor
  • an engineered reverse transcriptase is used in methods including but not limited to processing of a TCR from an individual T cell(s) or groups of T cell(s), determining the nucleotide sequence of the TCR(s) of T cell(s), and obtaining TCR repertoire profile.
  • a nucleic acid barcode sequence is appended to a nucleic acid molecule encoding for a TCR (e.g.
  • a TCR such as a TCR ⁇ and/or a TCR ⁇ mRNA
  • a barcoded nucleic acid molecule may serve as a template, such as a template polynucleotide, that can be further processed (e.g., amplified) and sequenced to obtain the target nucleic acid sequence.
  • a barcoded nucleic acid molecule may be further processed (e.g., amplified) and sequenced to obtain the nucleic acid sequence of the TCR.
  • TCR is a molecule found on the surface of T cells. Typically binding of the TCR by an antigenic molecule results in cell activation and response. The TCR is a heterodimer composed of two different protein chains. In many T cells, these two proteins are alpha ( ⁇ ) and beta ( ⁇ ) chains.
  • T cells these two proteins are gamma ( ⁇ ) and delta ( ⁇ ) chains.
  • the ratio of TCRs comprised of ⁇ / ⁇ chains versus ⁇ / ⁇ chains may change during a diseased state such as cancer, tumor, infectious disease, inflammatory disease or autoimmune disease.
  • Engagement of the TCR with a peptide-MHC activates a T cell through a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.
  • Each of the two chains of a TCR contains multiple copies of gene segments- a variable ‘V’ gene segment, a diversity ‘D’ segment and a joining ‘J’ segment.
  • the TCR alpha chain is generated by recombination of V and J segments, while the beta chain is generated by recombination of V, D and J segments.
  • generation of the TCR gamma chain involves recombination of V and J segments.
  • Generation of the TCR delta chain occurs by recombination of V, D and J gene segments. The intersection of these specific regions (V and J for the alpha or gamma chain, or V,D, J for the beta or delta chain) corresponds to the CDR3 region involved in antigen-MHC recognition.
  • Complementarity determining regions e.g., CDR1, CDR2 and CDR3 or hypervariable regions are sequences in the variable domains of antigen receptors (e.g., T cell receptor and immunoglobulin) that can complement an antigen.
  • antigen receptors e.g., T cell receptor and immunoglobulin
  • Most of the diversity of CDRs is found in CDR3, with the diversity being generated by somatic recombination events during the development of T lymphocytes.
  • CDR3 which is encoded by the junctional region 127 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC between the V and J or D and J genes, is highly variable.
  • CDR3 is often used as a region of interest to determine T cell clonotypes, a unique nucleotide sequence that arises during the gene rearrangement process, as it is highly unlikely that two T cells will express the same CDR3 nucleotide sequence unless they are derived from the same clonally expanded T cell.
  • TCR gene sequences may include, but are not limited to, sequences of various T cell receptor alpha variable genes (TRAV genes), T cell receptor alpha joining genes (TRAJ genes), T cell receptor alpha constant genes (TRAC genes), T cell receptor beta variable genes (TRBV genes), T cell receptor beta diversity genes (TRBD genes), T cell receptor beta joining genes (TRBJ genes), T cell receptor gamma variable genes (TRGV genes), T cell receptor gamma joining genes (TRGJ genes), T cell receptor gamma constant genes (TRGC genes), T cell receptor delta variable genes (TRDV genes), T cell receptor delta diversity genes (TRDD genes), T cell receptor delta joining genes (TRDJ genes) and T cell receptor delta constant genes (TRDC genes).
  • TRAV genes T cell receptor alpha variable genes
  • TRAJ genes T cell receptor alpha joining genes
  • TRBV genes T cell receptor beta variable genes
  • TRBD genes T cell receptor beta diversity genes
  • TRBJ genes T cell receptor beta joining genes
  • TRGV genes T cell receptor gamma variable genes
  • kits comprising the engineered reverse transcriptase polypeptide or recombinant RT protein, the DNA binding domains or a derivative thereof as described herein.
  • the kit comprises one or more of a vector, a nucleotide, a buffer, a composition, a salt, and/or instructions.
  • a kit may comprise an engineered reverse transcriptase polypeptide or recombinant RT protein or a derivative thereof for use in reverse transcription or amplification of a nucleic acid molecule.
  • a kit may be used for single cell profiling of the transcriptome.
  • a kit may be used for spatial transcriptomics methods and assays.
  • a kit may be used for in situ methods and assays.
  • the kit may include suitable reaction buffers, dNTPs, one or more primers, one or more control reagents, or any other reagents disclosed for performing the methods of the present disclosure.
  • the engineered reverse transcriptase polypeptide or recombinant RT protein or a derivative thereof, reaction buffer, and dNTPs may be provided separately or may be provided together in a master mix solution.
  • the master mix is present at a concentration at least two times the working concentration indicated in instructions for use in an extension reaction. In other cases, the master mix may be present at a concentration at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times, the working concentration indicated.
  • the primer in the kits may be a poly-dT primer, a random N-mer primer, or a target-specific primer.
  • the kits may further include one, two, three, four, five or more, up to all of partitioning fluids, including both aqueous buffers and non-aqueous partitioning fluids or oils, nucleic acid barcode capture probes that are releasably associated with beads, as described herein, microfluidic devices, reagents for disrupting cells, reagents for amplifying nucleic acids, as well as instructions for using any of the foregoing in the methods described herein.
  • the instructions for using any of the methods are generally recorded on a suitable recording medium (e.g., printed on a substrate such as paper or plastic), or available in a digital format.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging).
  • the instructions may be present as an electronic storage data file present on a suitable computer readable storage medium.
  • the actual instructions may not be present in the kit but means for obtaining the instructions from a remote source, e.g., via the internet, may be provided.
  • Kits according to this aspect of the present disclosure comprise a carrier means, such as a box, carton, tube or the like, having in close confinement therein one or more container means, such as vials, tubes, ampoules, bottles and the like, wherein a first container means contains one or more of the engineered reverse transcriptase polypeptide or recombinant RT proteins or derivatives thereof of the present disclosure having reverse transcriptase activity.
  • kits of the 129 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC disclosure can also comprise (in the same or separate containers) one or more DNA polymerases, a suitable buffer, one or more nucleotides and/or one or more primers.
  • the kits of the disclosure can also comprise one or more hosts or cells including those that are competent to take up nucleic acids (e.g., DNA molecules including vectors).
  • Preferred hosts may include chemically competent or electrocompetent bacteria such as E. coli (including DH5, DH5 ⁇ , DH10B, HB101, Top 10, and other K-12 strains as well as E. coli B and E. coli W strains).
  • E. coli including DH5, DH5 ⁇ , DH10B, HB101, Top 10, and other K-12 strains as well as E. coli B and E. coli W strains).
  • kits of the disclosure can include one or more components (in mixtures or separately) including one or more engineered reverse transcriptase polypeptides or recombinant RT proteins or derivatives thereof having reverse transcriptase activity of the disclosure, one or more nucleotides (one or more of which may be labeled, e.g., fluorescently labeled) used for synthesis of a nucleic acid molecule, and/or one or more primers (e.g., oligo(dT) for reverse transcription, randomers for extension reactions, etc.).
  • Such kits can comprise one or more DNA polymerases.
  • the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ⁇ up to 10%, up to ⁇ 5%, or up to ⁇ 1%.
  • the term “about” or “approximately” as used herein means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.
  • “about” can mean within an acceptable standard deviation, per the practice in the art.
  • “about” can mean a range of up to ⁇ 20%, preferably up to ⁇ 10%, more preferably up to ⁇ 5%, and more preferably still up to ⁇ 1% of a given value.
  • the term can mean within an order of magnitude, preferably within 2-fold, of a value.
  • Analyte is intended a biological molecule. Analytes include but are not limited to a DNA analyte, an RNA analyte, an oligonucleotide, a reporter molecule, a reporter molecule configured to directly couple to a protein, a reporter molecule configured to indirectly couple to a protein, a reporter molecule configured to directly couple to a metabolite, and a reporter molecule configured to indirectly couple to a metabolite. [000482]
  • Adaptor(s),” “Adapter(s)” and “Tag(s)” may be used synonymously.
  • Barcoded nucleic acid molecule generally refers to a nucleic acid molecule that results from, for example, the processing of a nucleic acid barcoded molecule with a nucleic acid sequence (e.g., nucleic acid sequence complementary to a nucleic acid primer sequence encompassed by the nucleic acid barcoded molecule).
  • the nucleic acid sequence may be a targeted sequence or a non-targeted sequence.
  • the nucleic acid barcoded 132 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC molecule may be coupled to or attached to the nucleic acid molecule comprising the nucleic acid sequence.
  • a nucleic acid barcoded molecule described herein may be hybridized to an analyte (e.g., a messenger RNA (mRNA) molecule) of a cell. Reverse transcription can generate a barcoded nucleic acid molecule that has a sequence corresponding to the nucleic acid sequence of the mRNA and the barcode sequence (or a reverse complement thereof).
  • analyte e.g., a messenger RNA (mRNA) molecule
  • Reverse transcription can generate a barcoded nucleic acid molecule that has a sequence corresponding to the nucleic acid sequence of the mRNA and the barcode sequence (or a reverse complement thereof).
  • the processing of the nucleic acid molecule comprising the nucleic acid sequence, the nucleic acid barcoded molecule, or both can include a nucleic acid reaction, such as, in non-limiting examples, reverse transcription, nucleic acid extension, ligation, etc.
  • the nucleic acid reaction may be performed prior to, during, or following barcoding of the nucleic acid sequence to generate the barcoded nucleic acid molecule.
  • the nucleic acid molecule comprising the nucleic acid sequence may be subjected to reverse transcription and then be attached to the nucleic acid barcoded molecule to generate the barcoded nucleic acid molecule, or the nucleic acid molecule comprising the nucleic acid sequence may be attached to the nucleic acid barcoded molecule and subjected to a nucleic acid reaction (e.g., extension, ligation) to generate the barcoded nucleic acid molecule.
  • a barcoded nucleic acid molecule may serve as a template, such as a template polynucleotide, that can be further processed (e.g., amplified) and sequenced to obtain the target nucleic acid sequence.
  • a barcoded nucleic acid molecule may be further processed (e.g., amplified) and sequenced to obtain the nucleic acid sequence of the nucleic acid molecule (e.g., mRNA).
  • a nucleic acid barcoded molecule of a plurality of nucleic acid molecules may be used to generate a “barcoded nucleic acid molecule.”
  • a barcoded molecule comprises a different reporter barcode sequence that identifies a second analyte.
  • a different reporter barcode sequence or an analyte-specific barcode sequence may identify a protein, a lipid, a metabolite or other second analyte.
  • Barcoded nucleic acids may be generated (e.g., via a nucleic acid reaction, such as nucleic acid extension or ligation) from the constructs described in FIG.12.
  • capture handle sequence may then be hybridized to complementary sequence, such as capture sequence 1223 to generate (e.g., via a nucleic acid reaction, such as nucleic acid extension or ligation) a barcoded nucleic acid molecule comprising cell (e.g., partition specific) barcode sequence 1222 (or a reverse complement thereof) and reporter barcode sequence 1222 (or a 133 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC reverse complement thereof).
  • capture handle sequence 4323 comprises a sequence complementary to a template switching oligonucleotide on the capture sequence 1223.
  • the nucleic acid barcoded molecule 1290 e.g., partition-specific barcoded molecule
  • UMI not shown
  • Barcoded nucleic acid molecules can then be optionally processed as described elsewhere herein, e.g., to amplify the molecules and/or append sequencing platform specific sequences to the fragments. See, e.g., U.S. Pat. Pub.2018/0105808, which is hereby entirely incorporated by reference for all purposes. Barcoded nucleic acid molecules, or derivatives generated therefrom, can then be sequenced on a suitable sequencing platform.
  • analysis of multiple analytes may be performed.
  • analysis of an analyte e.g., a nucleic acid, a polypeptide, a carbohydrate, a lipid, a glycan, a glycan motif, a metabolite, a protein, etc.
  • an analyte e.g., a nucleic acid, a polypeptide, a carbohydrate, a lipid, a glycan, a glycan motif, a metabolite, a protein, etc.
  • a nucleic acid barcoded molecule 1290 e.g., partition specific barcoded molecule
  • nucleic acid barcoded molecule 1290 is attached to a support 1230 (e.g., a bead, such as a gel bead), such as those described elsewhere herein.
  • a support 1230 e.g., a bead, such as a gel bead
  • nucleic acid barcoded molecule 1290 may be attached to support 1230 via a releasable linkage 1240 (e.g., comprising a labile bond), such as those described elsewhere herein.
  • Nucleic acid barcoded molecule 1290 may comprise a functional sequence 1221 and optionally comprise other additional sequences, for example, a barcode sequence 1222 (e.g., common barcode, partition-specific barcode, or other functional sequences described elsewhere herein), and/or a UMI sequence (not shown).
  • the nucleic acid barcoded molecule 1290 may comprise a capture sequence 1223 that may be complementary to another nucleic acid sequence, such that it may hybridize to a particular sequence, e.g., capture handle sequence 1223.
  • capture sequence 1223 may comprise a poly-T sequence and may be used to hybridize to mRNA.
  • nucleic acid barcoded molecule 1290 comprises capture sequence 1223 complementary to a sequence of RNA molecule 1260 from a cell.
  • capture sequence 1223 comprises a sequence specific for an RNA molecule.
  • Capture sequence 1223 may comprise a known or targeted sequence or a random sequence.
  • a nucleic acid extension reaction may be 134 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC performed, thereby generating a barcoded nucleic acid product comprising capture sequence 12323, the functional sequence 1221, barcode sequence 1222, any other functional sequence, and a sequence corresponding to the RNA molecule 1260.
  • capture sequence 1223 may be complementary to an overhang sequence or an adapter sequence that has been appended to an analyte. Any suitable agent may degrade beads. Suitable agents may include, but are not limited to, changes in temperature, changes in pH, reduction, oxidation and exposure to water or other aqueous solutions.
  • a cell that is bound to labelling agent which is conjugated to oligonucleotide and support 1230 e.g., a bead, such as a gel bead
  • oligonucleotide and support 1230 e.g., a bead, such as a gel bead
  • nucleic acid barcoded molecule 1290 is partitioned into a partition amongst a plurality of partitions (e.g., a droplet of a droplet emulsion, a well of a microwell array, a fixed cell and/or nucleus, a fixed and permeabilized cell and/or nucleus).
  • the term “Bead,” as used herein, generally refers to a particle. The bead may be a solid or semi-solid particle.
  • the bead may be a gel bead.
  • the gel bead may include a polymer matrix (e.g., matrix formed by polymerization or cross-linking).
  • the polymer matrix may include one or more polymers (e.g., polymers having different functional groups or repeat units). Polymers in the polymer matrix may be randomly arranged, such as in random copolymers, and/or have ordered structures, such as in block copolymers. Cross-linking can be via covalent, ionic, or inductive, interactions, or physical entanglement.
  • the bead may be a macromolecule.
  • the bead may be formed of nucleic acid molecules bound together.
  • the bead may be formed via covalent or non-covalent assembly of molecules (e.g., macromolecules), such as monomers or polymers. Such polymers or monomers may be natural or synthetic. Such polymers or monomers may be or include, for example, nucleic acid molecules (e.g., DNA or RNA).
  • the bead may be formed of a polymeric material.
  • the bead may be magnetic or non-magnetic.
  • the bead may be rigid.
  • the bead may be flexible and/or compressible.
  • the bead may be disruptable or dissolvable.
  • the bead may be a solid particle (e.g., a metal-based particle including but not limited to iron oxide, gold or silver) covered with a coating comprising one or more polymers.
  • each when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every 135 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.
  • Efficiency in the context of a nucleic acid modifying enzyme of this invention refers to the ability of the enzyme to perform its catalytic function under specific reaction conditions. Typically, “efficiency” as defined herein is indicated by the amount of product generated under given reaction conditions.
  • the term “Enhances” in the context of an enzyme refers to improving the activity of the enzyme, i.e., increasing the amount of product per unit enzyme per unit time.
  • the term “Fidelity” refers to the accuracy of polymerization, or the ability of the reverse transcriptase to discriminate correct from incorrect substrates, (e.g., nucleotides) when synthesizing nucleic acid molecules which are complementary to a template.
  • % homology refers to the level of nucleic acid or amino acid sequence identity between the nucleic acid sequence that encodes any one of the inventive polypeptides (e.g., variant reverse transcriptases) or the inventive polypeptide's amino acid sequence, when aligned using a sequence alignment program.
  • the term “Identical” in the context of two nucleic acids or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence, as measured using a sequence comparison algorithms. Sequence comparison algorithms are known to those skill in the art. See. e.g., ebi.ac.uk/Tools/msa/clustalo/. [000497] As used herein, the term “Inhibitor resistance” refers to the ability of a reverse transcriptase to perform reverse transcription in the presence of a compound, chemical, protein, buffer, etc. that is typically inhibitory to the reverse transcriptase (prevents or inhibits reverse transcriptase activity).
  • the term “Low volume reaction” means a reaction volume less than 1 nanoliter, less than 750 picoliters, or less than 500 picoliters.
  • the term “Molecular tag,” as used herein, generally refers to a molecule capable of binding to a macromolecular constituent.
  • the molecular tag may bind to the macromolecular constituent with high affinity.
  • the molecular tag may bind to the macromolecular constituent with high specificity.
  • the molecular tag may comprise a nucleotide sequence.
  • the molecular tag may comprise a nucleic acid sequence.
  • the nucleic acid sequence may be at least a portion or an entirety of the molecular tag.
  • the molecular tag may be a nucleic acid molecule or may be part of a nucleic acid molecule.
  • the molecular tag may be an oligonucleotide or a polypeptide.
  • the molecular tag may comprise a DNA aptamer.
  • the molecular tag may be or comprise a primer.
  • the molecular tag may be, or comprise, a protein.
  • the molecular tag may comprise a polypeptide.
  • the molecular tag may be a barcode. [000500]
  • the term “mutation” or “mutant” or “variant“ indicates a change or changes introduced in a wild-type DNA sequence or a wildtype amino acid sequence.
  • mutations or variants include, but are not limited to, substitutions, insertions, deletions, and point mutations. Mutations can be made either at the nucleic acid level or at the amino acid level.
  • the term “Operably linked” or “conjugated” or “fusion” means that, in relation to the engineered RT polypeptide or the recombinant RT protein sequence, there are one or more sequences at the N or C terminus that, when transcribed and translated, create additional polypeptides in association with the enzyme amino acid sequence, thereby created a conjugation or fusion of one or more polypeptides from one expression vector.
  • Partition refers to a space or volume that may be suitable to contain one or more species or conduct one or more reactions.
  • a partition may be a physical compartment, such as a droplet, well or a fixed and/or permeabilized cell and/or nucleus.
  • the partition may isolate space or volume from another space or volume.
  • the droplet may be a first phase (e.g., aqueous phase) in a second phase (e.g., oil) immiscible with the first phase.
  • the droplet may be a first phase in a second phase that does not phase separate from the first phase, such as, for example, a capsule or liposome in an aqueous phase.
  • a partition may comprise one or more other (inner) partitions.
  • a partition may be a virtual compartment that can be defined and identified by an index (e.g., indexed libraries) across 137 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC multiple and/or remote physical compartments.
  • a physical compartment may comprise a plurality of virtual compartments.
  • partitions systems and methods for partitioning of one or more particles (such as, but not limited to, biological particles, macromolecular constituents of biological particles, beads, reagents, etc.) into discrete compartments or partitions (referred to interchangeably here as partitions), wherein each partition maintains separation of its own content from the contents of other partitions are known in the art. See for example US 2020/0032335, herein incorporated by reference in its entirety.
  • the partition can be a droplet in an emulsion.
  • a partition may comprise one or more other partitions.
  • a “plurality of nucleic acid barcoded molecules” may comprise at least about 500 nucleic acid barcoded molecules, at least about 1,000 nucleic acid barcoded molecules, at least about 5,000 nucleic acid barcoded molecules, at least about 10,000 nucleic acid barcoded molecules, at least about 50,000 nucleic acid barcoded molecules, at least about 100,000 nucleic acid barcoded molecules, at least about 500,000 nucleic acid barcoded molecules, at least about 1,000,000 barcoded molecules, at least about 5,000,000 nucleic acid barcoded molecules, at least about 10,000,000 nucleic acid barcoded molecules, at least about 100,000,000 nucleic acid barcoded molecules, at least about 1,000,000,000 nucleic acid barcoded molecules.
  • a plurality of nucleic acid barcoded molecules comprise a partition-specific barcode sequence.
  • Each of the plurality of nucleic acid barcoded molecules may include an identifier sequence separate from the partition-specific barcode sequence, where the identifier sequence is different for each nucleic acid partition-specific barcoded molecule of the plurality of nucleic acid partition specific barcoded molecules.
  • an identifier sequence is a unique molecular identifier (UMI) as described elsewhere herein.
  • UMI sequences can uniquely identify a particular nucleic acid molecule that is barcoded, which may be identifying particular nucleic acid molecules that are analyzed, counting particular nucleic acid molecules that are analyzed, etc.
  • each of the plurality of nucleic acid barcoded molecules can comprise the partition specific barcode sequence and the bead can 138 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC be from plurality of beads, such as a population of barcoded beads.
  • Each of the partition specific barcode sequences can be different from partition specific barcode sequences of nucleic acid barcoded molecules of other beads of the plurality of beads. Where this is the case, a population of barcoded beads, with each bead comprising a different partition specific barcode sequence can be analyzed.
  • the term “Processivity” refers to the ability of a reverse transcriptase to continuously extend a primer without disassociating from the nucleic acid template.
  • the length of a template a reverse transcriptase or polymerase is capable of replicating can also be used to describe the processivity of that reverse transcriptase or polymerase.
  • “Processivity” refers to the ability of a polymerase to remain bound to the template or substrate and perform DNA synthesis. Processivity is measured by the number of catalytic events that take place per binding event.
  • Reverse transcriptase activity indicates the capability of an enzyme to synthesize a DNA strand (that is, complementary DNA or cDNA) using RNA as a template. Reverse transcriptase activity may be measured by incubating an enzyme in the presence of an RNA template and deoxynucleotides, in the presence of an appropriate buffer, under appropriate conditions, for example as described in the Example below.
  • RT activity comprises the engineered RT fusion protein described herein or the engineered RT variant described herein.
  • Reverse transcriptase RT is used in its broadest sense to refer to any enzyme that exhibits reverse transcription activity as measured by methods disclosed herein or known in the art.
  • a "reverse transcriptase” of the present invention therefore, includes reverse transcriptases from retroviruses, other viruses, as well as a DNA polymerase exhibiting 139 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC reverse transcriptase activity, such as Tth DNA polymerase, Taq DNA polymerase, Tne DNA polymerase, Tma DNA polymerase, etc.
  • RT from retroviruses include, but are not limited to, Moloney Murine Leukemia Virus (M-MLV) RT, Human Immunodeficiency Virus (HIV) RT, Avian Sarcoma-Leukosis Virus (ASLV) RT, Rous Sarcoma Virus (RSV) RT, Avian Myeloblastosis Virus (AMV) RT, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV RT, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV RT, Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A RT, Avian Sarcoma Virus UR2 Helper Virus UR2AV RT, Avian Sarcoma Virus Y73 Helper Virus YAV RT, Rous Associated Virus (RAV) RT, and Myeloblastosis Associated Virus (MAV)
  • Patent Application 2003/0198944 (hereby incorporated by reference in its entirety). For review, see e.g., Levin, 1997, Cell, 88:5-8; Brosius et al.51995, Virus Genes 11:163-79.
  • Known reverse transcriptases from viruses require a primer to synthesize a DNA transcript from an RNA template.
  • Reverse transcriptase has been used primarily to transcribe RNA into cDNA, which can then be cloned into a vector for further manipulation or used in various amplification methods such as polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), or self-sustained sequence replication (3SR).
  • PCR polymerase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • TMA transcription mediated amplification
  • 3SR self-sustained sequence replication
  • sample generally refers to a biological sample of a subject.
  • the biological sample may comprise any number of macromolecules, for example, cellular macromolecules.
  • the sample may be a cell sample.
  • the sample may be a cell line or cell culture sample.
  • the sample can include one or more cells.
  • the sample can include one or more microbes.
  • the biological sample may be a nucleic acid sample or protein sample.
  • the biological sample may also be a carbohydrate sample or a lipid sample.
  • the biological sample may be derived from another sample.
  • the sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate.
  • the sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample.
  • the sample may be a skin sample.
  • the sample may be a cheek swab.
  • the sample may be a plasma or serum sample.
  • the sample may be a cell-free or cell free sample.
  • a cell-free sample may include extracellular polynucleotides. Extracellular polynucleotides may be isolated from a bodily sample that may be selected from blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears.
  • Sequequencing generally refers to methods and technologies for determining the sequence of nucleotide bases in one or more polynucleotides. Any method of sequencing known in the art may be used to evaluate the products of a reaction performed by an engineered reverse transcriptase of the current application. Sequencing can be performed by various systems currently available, such as, without limitation, a sequencing system by Illumina ® , Pacific Biosciences (PacBio ® ), Oxford Nanopore ® , or Life Technologies (Ion Torrent ® ).
  • sequencing may be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR), or isothermal amplification.
  • PCR polymerase chain reaction
  • a read may include a string of nucleic acid bases corresponding to a sequence of a nucleic acid molecule that has been sequenced.
  • systems and methods provided herein may be used with proteomic information.
  • substantially complementary means that a first sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-40, 40-60, 60-100, or more nucleotides, or that the two sequences hybridize under stringent hybridization conditions.
  • Substantially complementary also means that a sequence in one strand is not completely and/or perfectly complementary to a sequence in an opposing strand, but that sufficient bonding occurs between bases on the two strands to form a stable hybrid complex in set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequences and standard mathematical calculations known to those skilled in the art.
  • the term “Subject,” as used herein, generally refers to an animal, such as a mammal (e.g., human) or avian (e.g., bird), or other organism, such as a plant.
  • the subject can be a vertebrate, a mammal, a rodent (e.g., a mouse), a primate, a simian or a human.
  • Animals may include, but are not limited to, farm animals, sport animals, and pets.
  • a subject can be a healthy or asymptomatic individual, an individual that has or is suspected of having a disease (e.g., cancer) or a pre-disposition to the disease, and/or an individual that is in need of therapy or suspected of needing therapy.
  • a subject can be a patient.
  • a subject can be a microorganism or microbe (e.g., bacteria, fungi, archaea, viruses).
  • Thermoreactivity refers to the ability of a reverse transcriptase to exhibit enzyme activity at elevated temperatures.
  • “Thermostability” or “thermostable” refers to the ability of a reverse transcriptase to withstand exposure to elevated temperatures, but not necessarily show activity at such elevated temperatures.
  • thermostable reverse transcriptase or polymerase refers to any enzyme that catalyzes polynucleotide synthesis by addition of nucleotide units to a nucleotide chain using DNA or RNA as a template and has an optimal activity at a temperature above 53° C.
  • unique molecular identifier As used herein, the terms “Unique molecular identifier”, “Unique molecular identifying sequence”, “UMI” and “UMI sequence” are used synonymously.
  • Individual barcoded molecules may comprise a common barcode sequence such as a partition specific sequence or a spatial array where every capture probe has a unique barcode sequence.
  • flanking sequence is intended a nucleic acid sequence capable of binding to an analyte.
  • Variant means a protein which is derived from a precursor protein (such as the native protein, for example MMLV native protein as set forth in SEQ ID NO:7) by addition of one or more amino acids to either or both the C- and N-terminal end, substitution of one or more amino acids at one or a number of different sites in the amino acid sequence, deletion of one or more amino acids at either or both ends of the protein or at one or more sites in the amino acid sequence, or addition of a fusion domain.
  • SEQ ID NO:1 is a variant of MMLV.
  • an enzyme variant is preferably achieved by modifying a DNA sequence which encodes for the wild-type protein, transformation of that DNA sequence into a suitable host, and expression of the modified DNA sequence to form the derivative enzyme.
  • a variant reverse transcriptase of the invention includes a protein comprising altered amino acid sequences in comparison with a precursor enzyme amino acid sequence wherein the variant reverse transcriptase retains the characteristic enzymatic nature of the precursor enzyme but which may have altered properties in some specific aspect.
  • an engineered reverse transcriptase variant may have an altered pH optimum or increased temperature stability but may retain its characteristic transcriptase activity.
  • a “Variant” may have at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to a polypeptide sequence when optimally aligned for comparison.
  • Percent identity may pertain to the percent identity of the DNA binding domain or the engineered reverse transcriptase portion of an engineered reverse transcriptase.
  • a variant residue position is described in relation to the wild-type or precursor amino acid sequence set forth in SEQ ID NO:7; the amino acid position is indexed to SEQ ID NO:7.
  • a fusion variant comprises at least one fusion domain selected from DNA binding domains described elsewhere herein.
  • a protein having a certain percent (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of sequence identity with another sequence means that, when aligned, that percentage of bases or amino acid residues are the same in comparing the two sequences.
  • This alignment and the percent homology or identity can be determined using any suitable software program known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al., eds., 1987, Supplement 30, section 7.7.18.
  • Representative programs include the Vector NTI AdvanceTM 9.0 (Invitrogen Corp. Carlsbad, CA), GCG Pileup, FASTA (Pearson et al. (1988) Proc. Natl Acad. ScL USA 85:2444-2448), and BLAST (BLAST Manual, Altschul et al., Nat’l Cent. Biotechnol. Inf., Nat’l Lib. Med. (NCIB NLM NIH), Bethesda, Md., and Altschul et al., (1997) Nucleic Acids Res.25:3389-3402) programs.
  • Another typical alignment program is ALIGN Plus (Scientific and Educational Software, PA), generally using default parameters.
  • sequence alignment software programs that find use are the TFASTA Data Searching Program available in the Sequence Software Package Version 6.0 (Genetics Computer Group, University of Wisconsin, Madison, WI and CLC Main Workbench (Qiagen) Version 20.0. The present disclosure is not limited to the software being used to align two or more sequences.
  • WT Wild-type or “WT” refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring 143 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC source.
  • amino acid sequence set forth in SEQ ID NO:7 is a WT Murine Moloney Leukemia Virus (MMLV) sequence (Genbank NP_955591.1 p80 RT).
  • MMLV Murine Moloney Leukemia Virus
  • Genbank NP_955591.1 p80 RT WT Murine Moloney Leukemia Virus
  • nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole. EXAMPLES [000525] It will be understood that the reference to the below examples is for illustration purposes only and do not limit the scope of the claims.
  • Exemplary engineered RTs comprising a MMLV RT variant (42BL) operably linked N-terminally to a DNA binding protein derived from a budding yeast DAT1 were generated.
  • engineered RT comprising C-terminal fusions can also be constructed. Dat from different budding yeasts that contains at least three repeated pentads of G-R-K-P-G can be used.
  • DAT1 N-terminal fusion protein was generated; and an DAT1C-terminal fusion protein was generated.
  • the DAT1 fusion proteins are produced with an N-terminal 6x His Tag and thrombin cleavage site.
  • the 6x His Tag was used for purification purposes and removed by thrombin cleavage.
  • Exemplary engineered RTs e.g., variant MMLV
  • RT operably linked N-terminally to a truncated DAT1 molecule comprising 90 amino acid was also generated. While the majority of exemplary engineered RT tested below are of N-terminal fusions, C-terminal RT fusions can also be constructed.
  • homologs of DAT1 from other organisms can also be used to generate the engineered reverse transcriptase described herein as shown by N-DAT1-TL-QID04042BL; N-DAT1-TL-XP36142BL; N-DAT1-TL- XP55842BL; or N-DAT1-TL-XP68342BL.
  • DAT1(90) can be fused to other reverse transcriptase enzymes, such as other variants of MMLV reverse transcriptases as shown by N-DAT-9042B; N-DAT-9050A+ G; N-DAT-90 SOLD 33 VDG; N-DAT-90 SOLD 01; C-DAT-90 SOLD 01.
  • Example 1 Capillary Electrophoresis Assay Validation [000531] Reverse transcription and sequencing reactions were prepared. The reaction volume was 50 ⁇ l and reactions contained 5’-end labeled FAM Reverse Transcriptase primer 2, RT Reagent B (Chromium Next GEM Single Cell Reagent, 10X Genomics), RNA template (RNA Temp 2), template switching oligo 1 (TSO1), and the indicated engineered reverse transcriptase.
  • Table 3A Capillary Electrophoresis Assay Reactants R k Fi l 145 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC
  • Table 3A Capillary Electrophoresis Assay Reactants Reagent Stock Final on 5’ kit (10X Genomics, Inc), except the reverse transcriptase was altered for a particular reaction. Stock concentrations and final concentrations in the reactions are shown in Tables 3A-B. Variations of the assay stock concentrations and final concentrations in the reactions shown in Table 4 were used. The reactions included stoichiometrically equal amounts of enzyme and template for single turnover conditions.
  • Samples were loaded on a SeqstudioTM (Thermo Fisher Scientific) and fragment analysis by capillary electrophoresis was carried out with the appropriate dye channels and size standards.
  • the assay was validated with synthetically sized oligonucleotides and with a transcription positive, template switching null engineered reverse transcriptase and a transcription positive, template switching positive reverse transcriptase (Enzyme Mix C,).
  • the GEM-U reagent approximates the formulation of the actual reagent mixture in a GEM assay 146 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC when the contents of the Z1 and Z2 channels are mixed.
  • Capillary Electrophoresis Assay Reactants are disclosed in Table 1A, Capillary Electrophoresis Assay template, Primer and TSO sequences are shown in Table 4A.
  • Reverse transcription and sequencing reactions were also prepared using GAPDH or GRCh38 as a template. The reaction volume was 50 ⁇ l; reactions contained 5’-end labeled GAPDH or GRCh38 primer, GEM-U reagent, RNA template (GAPDH or GRCh38 template), template switching oligo 1 (TSO1), and the indicated engineered reverse transcriptase. Stock concentrations and final concentrations in the reactions are shown in Table 4B. The reactions included stoichiometrically equal amounts of enzyme and template for single turnover conditions.
  • reaction volume was 50 ⁇ l; reactions contained 5’-end labeled GAPDH primer, GEM-U reagent, RNA template (GAPDH template), template switching oligo 1 (TSO1), and the indicated engineered reverse transcriptase(s).
  • the final concentrations in the reactions are shown in Table 4B.
  • the reaction buffer was SOP for SC-5’ and the reaction time was 45 minutes.
  • Tables 4A-B show Capillary Electrophoresis (CE) Assay Reactants and Template, Primer and TSO sequences (SEQ ID NOS:173, 175, 176, respectively in order of appearance.)
  • Table 4A Capillary Electrophoresis Assay Template, Primer and TSO sequences 147 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC
  • Table 4A Capillary Electrophoresis Assay Template, Primer and TSO sequences Reagent Stock Final Volume [000539] Several mutants were constructed using a Q5 mutagenesis kit (NEB) with mutagenic primers per manufacturing instructions.
  • NEB Q5 mutagenesis kit
  • Amplification conditions were an initial denaturation at 95°C for 2.5 minutes, 30 cycles of denature (95°C, 30 sec), a 45 sec gradient annealing and extension at 72°C for 6 minutes, 35 sec, followed by a final extension at 72°C for 2 minutes.
  • Amplification reactions with multiple annealing gradient temperatures (65.2°C, 67°C, 68.5°C and 69.6°C) were performed.
  • Amplification products were evaluated on a 1.2% agarose E-Gel using SYBR-Safe. Products were pooled prior to clean-up. Cloning and expression were performed in the Acella cell line from EdgeBio (San Jose, CA). Cells were selected on LB-Kanamycin plates.
  • DAT1 N- terminal and C-terminal fusions to an engineered reverse transcriptase having the amino acid sequence set forth in SEQ ID NO:1 were obtained by screening of bacterial colonies. The sequences of the fusion proteins were confirmed using method known in the art. 148 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC
  • Example 3 Single Cell Sensitivity and Mapping [000542]
  • PBMCs peripheral blood monocytes
  • C57B/L6 mouse peripheral blood monocyte cells
  • Results from engineered reverse transcriptase were compared to results obtained from a commercially available engineered MMLV or MMLV variants (SOLD 001 (SEQ ID NO: 65); and SOLD 33 VDG (SEQ ID NO: 173).
  • SOLD 001 SEQ ID NO: 65
  • SOLD 33 VDG SEQ ID NO: 173
  • FIG.20 and FIG.21 show that DAT1 in combination with reverse transcriptase (MMLV, 42B, or other 42B variant thereof (e.g., SOLD 001 or SOLD 033 VDG)), either fused at the N-terminus or C-terminus of the RT improved GEX sensitivity even at low sequencing depth. Improvement was observed with both gene expression, which was increased by up to ⁇ 37% and UMI , which was increased by up to 13% as captured at 20k rrpc. Gains were even more significant at higher read depth.
  • MMLV, 42B, or other 42B variant thereof e.g., SOLD 001 or SOLD 033 VDG
  • the engineered RT disclosed herein exhibited large change in differential gene expression in single cell assays.
  • the engineered RT molecules comprising DAT1 or variant thereof disclosed herein picked-up to about 5000 additional genes when compared to a non-DAT1 RT (e.g., 42B).
  • FIGs.20-26 These engineered RT polypeptide also exhibited increase in median UMI counts per spot and median gene counts per spot in spatial assay.
  • the engineered RT molecules gave decrease in fraction of reads mapped to exons with gain in fraction mapped to introns. See e.g., FIG.21. A performance difference between FPLC and plate purified proteins was performed.
  • RT enzymes tested included 42B (SEQ ID NO: 1, SEQ ID NO: 143, or SEQ ID NO: 172), 50A+G (Table 2; SEQ ID NO: 147), 42B_L (Table 2; SEQ ID NO: 145).
  • FIGs.22A-B show the relative differences in performance of the engineered RT polypeptides or control non-DAT RT compared to control RT (42B).
  • the median genes and UMIs/cell at 50k raw-reads per cell were used to compare the sensitivity of three reverse transcriptases with and without the DAT fusion domain.42B-NDAT showed 43.29% (Median genes/cell) and 39.80% (median UMIs/cell) enhancement over 42B alone.
  • 42BL-CDAT showed 30.07% (Median genes/cell) and 23.76% (median UMIs/cell) enhancement over 42B alone.
  • 42BL-NDAT showed 47.40% (Median genes/cell) and 41.32% (median UMIs/cell) enhancement over 42B alone.
  • 50A+G-NDAT showed 45.00% (Median genes/cell) and 40.63% (median UMIs/cell) enhancement over 42B alone.
  • non-DAT RT, 42 B L only showed 7.25% (Median genes/cell) and 13.91% (median UMIs/cell) enhancement over 42B alone.
  • the non-DAT RT, 50A+G only showed 28.66% (Median genes/cell) and 38.01% (median UMIs/cell) enhancement over 42B alone.
  • both a C-terminal fusion and an N-terminal fusion of DAT to 42BL increased median genes per cell and median UMIs per cell in the single cell assays.
  • the C- terminal fusion increased median genes per cell by 21% compared to 42BL (3032 versus 2500) and median UMIs per cell by 9 % (8976 versus 8262) as compared to 42BL.
  • the N-terminal fusion increased median genes per cell by 37% (3436 versus 2500) and median UMIs per cell by 24%.
  • the 50A+G-DAT fusion showed a 13 % increase in genes per cell (3380 versus 2999) and a 2% increase in UMIs per cell (10200 versus 10010).
  • each DAT RT 150 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC fusion tested demonstrated improved quality metrics in a transcriptomics assay as compared to a matched control including the same RT but lacking the DAT domain.
  • These results showed clear performance gains in engineered RT polypeptides comprising a DAT fusion on either the N-terminal or C-terminal domain.
  • results also demonstrated the enhanced sensitivity of a single cell assay using an engineered reverse transcriptase polypeptide or an engineered recombinant reverse transcriptase comprising DAT1 described herein as shown by the median genes identified per cell (Median genes/cell) or the median UMIs identified per cell (median UMIs/cell).
  • an engineered RT of the present disclosure significantly improved the RT sensitivity when compared to a non-DAT1 RT; and an engineered reverse transcriptase comprising a DAT1 binding domain further significantly increased the gain in sensitivity of the engineered reverse transcriptase described herein.
  • FIG.23A The performance of various engineered RT disclosed herein were also analyzed at maximum normalization depth using the median genes/cell (FIG.23A) or the median UMIs/Cell (FIG.23B) to compare the generated library complexity of three reverse transcriptases (42B, 42B L, and 50A+G) with (N-DAT or C-DAT) and without the DAT domain.
  • FIGs.23 C-D show saturation curves of the median genes (FIG.23C) and counts/cell (FIG.27C) as a function of read depth, which further demonstrate that the median genes and counts/cell were higher using the engineered RT with a DAT DNA binding domain when compared to MMLV variants lacking the DAT DNA binding domain.
  • FIG.23 demonstrates a clear benefit of using the DAT DNA binding domain in a Single Cell 5’ (SC-5’) gene expression assay.
  • SC-5 Single Cell 5’
  • FIGs.24A-F and FIGs.25A-F showed significant levels of differential gene expression with 42B L-CDAT, 42B L-NDAT, 50A+G-NDAT.
  • FIGs.24A-F shows the differential gene expression of some engineered RT comprising DAT1 at the N- terminus.
  • FIGs.24A, C, and E feature scatter plots showing gene expression correlation of three reverse transcriptases with and without the DAT fusion domain.
  • FIGs.24B, D, and F feature 151 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC volcano plots showing the number of differentially expressed genes between three reverse transcriptases with and without the DAT fusion domain.
  • FIGs.25A-F illustrate the differential gene expression of some engineered RT comprising DAT1 at the C-terminus.
  • FIGs.25A, C, and E feature scatter plots showing gene expression correlation comparison of engineered RT comprising N- Terminal DAT Domain and C- Terminal DAT Domain.
  • FIGs.25B, D, and F feature volcano plots comparing the number of differentially expressed genes between various RT. Gains in performance, were still present, but not as significant with a C-terminal fusion.
  • the C-DAT terminal fusion did not perform as well as the N-terminal DAT fusion, but both N-terminal DAT fusion and C-DAT terminal fusion showed performance gains when compared to the RT backbone (42B L; SEQ ID NO: 145) alone.
  • the conditions become more similar in complexity (42B L - NDAT > 42B L - CDAT > 42B L, see FIGs 22-23), there are lower levels of DEGs and the feature scatter plots begin to correlate better.
  • FIGs.26A-D show graphs illustrating the performance comparison of the impact of the DAT fusion domain across three reverse transcriptase backbones based on median genes (FIG.26A) and UMIs/cell at maximum normalization depth (FIG.26B), gene expression correlation (FIG.26C), and differential gene expression (FIG.26D).
  • the aggregated metrics comparing the performance among 42B, 42B L, and 50A+G backbones with and without the DAT fusion showed a clear performance benefit from the DAT domain, such as e.g., enhanced sensitivity. Therefore, the 42B L-DAT1 fusion RT significantly improve in single cell assay performance.
  • Example 5 Assays for analyses of Engineered RT polypeptides
  • Any of the engineered RT enzymes of the invention including without limitation any of the enzymes described in Table 1, or Table 2 could be analyzed in any suitable assay, including without limitation the assays described herein.
  • Assays include without limitation 5’ gene expression analyses, with or without VDJ analysis, 3’ gene expression analysis, epigenetic analysis, or multiomic analyses.
  • experiments are carried out as found in the manufacturer’s instructions for the Chromium Single Cell 5’ Gene Expression 152 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Assay kit (10X Genomics); Chromium Single Cell 3’ Gene Expression Assay kit (10X Genomics), including any of multiomic extensions or applications.
  • Example 6 Single Cell 3’ and 5’ cDNA Yields [000556]
  • PBMCs peripheral blood monocytes
  • Emulsion droplets can contain gel beads with either barcoded poly-dT primer sequences (3’ configuration) or barcoded template switch oligo sequences (5’ configuration) that also include a UMI and Illumina ® Read 1 sequence.
  • the poly-dT primer hybridizes to the poly-A tail of the cellular mRNA, which is extended by the reverse transcriptase. Once the end of the template is reached, the reverse transcriptase will exhibit terminal transferase activity to add an overhang of three non-templated deoxycytidines (CCC) to the 3’ end of the synthesized cDNA.
  • CCC non-templated deoxycytidines
  • the CCC overhang will hybridize to the 3 riboguanosines (rGrGrG) present on the 3’ end of the template switch oligo, allowing the reverse transcriptase to “switch” templates and continue synthesis to the 5’ end of the template switch oligo.
  • the barcode and UMI will allow either the 3’ or 5’-end of the mRNA molecule to be identified in the final sequencing library.
  • cDNA was then amplified via PCR, purified with a 0.6x SPRI, and quantified with an Agilent Bioanalyzer using the DNA High Sensitivity Kit. The cDNA yield (ng) was then obtained.
  • PBMCs peripheral blood monocytes
  • Either 10 ⁇ L of the amplified cDNA (3’ conditions) or 20 ⁇ L containing a maximum of 50 ng of amplified cDNA (5’ conditions) can then be fragmented and A-tailed, cleaned with a double-sided SPRI (0.6x/0.8x), ligated to functional adaptors with an Illumina ® Read 2 sequence, cleaned with a 0.8x SPRI, and then can be further amplified with sample indexing primers that include the P5 and P7 priming sites and the i5 and i7 sample indexes.
  • the amplification product can be cleaned up with a double-side (0.6x/0.8x) SPRI, and the average size can be determined with an Agilent Bioanalyzer using the DNA High Sensitivity Kit.
  • the material can then be quantified by qPCR 153 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC and pooled for next generation sequencing on an Illumina ® Novaseq targeting a sequencing depth of at least 50,000 reads per cell and using the following run parameters (Read 1: 28 cycles, i7 Index: 10 cycles, i5 Index: 10 cycles, Read 2: 90 cycles). Data can be collected, demultiplexed, and processed. Standard quality metrics were obtained. [000558] Generally, the single cell 5’ reactions use less enzyme and TSO oligo than the single cell 3’ reactions.
  • the 5’ TSO oligo is also twice the length of the 3’ TSO oligo with varied sequence context due to the presence of the UMI and the barcode.
  • the single cell 5’ reaction conditions are generally considered a more stringent test of performance than the 3’ single cell reaction conditions.
  • FIG.21 summarizes results from a series of experiments using the 5’ reaction conditions. The figure summarizes metrics of the 5’ single cell experiments, including 20k read metrics, 50K read metrics and reads mapped to the transcriptome.
  • the engineered reverse transcriptase variants have the amino acid sequences provided set forth in SEQ ID NO: 65 (SOLD 001), SEQ ID NO: 173 (SOLD 33 VDG), SEQ ID NO: 174 (C-DAT 42BL), and SEQ ID NO: 175 (N-DAT 42BL).
  • the percent indicates the percent change from the results obtained with an engineered reverse transcriptase having the amino acid sequence set forth in SEQ ID NO:1 or 179 (42B).
  • the engineered reverse transcriptase polypeptides showed a significant improvement in sensitivity.
  • FIG.21 shows additional metrics related to results obtained from the indicated engineered reverse transcriptase in single cell 5’ experiments.
  • Immune profiling is an extension of the 5’ chemistry to profile genes specifically for T-cell and/or B-cell receptors in the mRNA pool.
  • Immune profiling are known in the art and generally include additional rounds of PCR on the cDNA with a pool of sequence specific primers to allow for targeted enrichment of T-cell and/or B-cell receptor genes. Immune profiling assays may also detect UMIs for B-cell receptor genes, namely IGH, IGK, and IGL (Immunoglobulin heavy chain (IGH), kappa (IGK), and light (IGL) chain). Immune profiling data is informative for immunology research and is an extension of standard gene expression evaluation. Methods of immune profiling include but are not limited to Chromium Next Gen Single CellTM kits (10X Genomics, Pleasanton CA).
  • Amplified cDNA (2 ⁇ l) from the 5’ configuration of reverse transcription reactions can be subjected to two additional rounds of PCR enrichment with TCR immune profiling, which included a double-sided (0.5x/0.8x) SPRI clean-up between the first and second round of thermal cycling reactions.
  • the amplified products can then be cleaned-up with a subsequent double-sided (0.5x/0.8x) SPRI, fragmented and A-tailed, ligated to functional adaptors with an Illumina ® Read 2 sequence, cleaned up with a 0.8x SPRI, and then further amplified with sample indexing primers that include the P5 and P7 priming sites and the i5 and i7 sample indexes.
  • the amplification product can be cleaned up with a 0.8x SPRI, and average size can be determined with an Agilent Bioanalyzer using the DNA High Sensitivity Kit.
  • the material can then be quantified by qPCR and can be pooled for next generation sequencing on an Illumina ® Novaseq targeting a sequencing depth of at least 5,000 reads per cell and using the following run parameters (Read 1: 28 cycles, i7 Index: 10 cycles, i5 Index: 10 cycles, Read 2: 90 cycles).
  • Data can be collected, demultiplexed, and single-cell V(D)J analysis can be performed. Results that are obtained from engineered reverse transcriptases can be compared to results are obtained from a commercially available enzyme or an RT lacking the DAT 1 DNA binding domain.
  • the percent change in median TRA UMI’s and median TRB UMI’s from mouse and human PBMCs for each RT tested can be shown as a percent change in median IGH, IGK and IGL from mouse PBMC’s. It is expected that the median TRA UMIs and median TRB UMIs obtained with any 155 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC engineered reverse transcriptase described herein h will be greater than those obtained with a commercially available reverse transcriptase or non-DAT RT in both human PBMCs and mouse PBMCs.
  • Example 9 Spatial assays [000565] This example shows various methods for performing spatial analysis using the engineered RT enzymes of the present disclosure.
  • the Example provides an exemplary method for detecting an individual gene expression or globally-expressed RNA transcripts in a fresh or frozen tissue using an engineered RT of SEQ ID NO: 175, which includes N-Terminal fusion of the first 90a.a.
  • N-DAT Dat 1
  • a 42B variant of SEQ ID NO: 1, 143, 145, or 172 compared to a control RT variant (having the same RT enzyme but lacking the N-DAT or SEQ ID NO: _1, 143, 145, or 172) using three different reverse transcription buffer formulations, the commercially available RT reagent, buffer X.4 and buffer X.5.
  • the commercially available RT reagent comprises 2% Glycerol, 50 mM Tris pH 8.3, 3 mM MgCl2, 75.04 mM KCl, 0.5% Supersonic F-108, 0.489 mg/ml BSA, and 3.16 mM dNTPs.
  • Buffer X.4 and buffer X.5 have the same components as the commercial RT reagent with the following differences.
  • Buffer X.4 contains 1.05% Glycerol, 158.3 mM NaCl, 0.579 mg/ml BSA, 4.685 mM dNTPs, 1.5mM dCTP, and 0.775mM GTP.
  • Buffer X.5 contains 133.8 mM NaCl, 1% Supersonic F-108, 3.96 mM dNTPs, 1.31 mM dCTP, and 0.655 mM GTP. [000566] Sample preparation.
  • a formalin-fixed, paraffin-embedded (FFPE) human tonsil sample, a frozen Human tonsil sample, or a fresh Human tonsil sample can be used for the method described herein.
  • Human tonsil FFPE sections on standard slides (for sandwich conditions; FIGs.1A-B, FIGs.2A-B, and FIGs.3A-C) or gene expression (GEx) slides (for non-sandwich control conditions) were deparaffinized, H&E stained, and imaged. After the imaging, the human tonsil sections were hematoxylin-destained with HCL solution. The sections were then decrosslinked by incubating at 70°C for 1 hour in decrosslinking solution.
  • the permeabilization solution was washed out and the human tonsil sample was prepared for analyte capture by adding 0.1X SSC buffer and subjected to a pre-equilibration thermocycling protocol (e.g., lid temperature and pre-equilibrate at 53 °C, reverse transcription at 53 °C for 45 minutes, and then hold at 4 °C).
  • a pre-equilibration thermocycling protocol e.g., lid temperature and pre-equilibrate at 53 °C, reverse transcription at 53 °C for 45 minutes, and then hold at 4 °C.
  • the SSC buffer was removed.
  • a Master Mix comprising a commercially available RT reagent buffer, buffer X.4, or buffer X.5, nuclease-free water, a template switch oligo, a reducing agent, and a reverse transcriptase (control 42B RT, N-DAT-42B RT, or small scale-purified N-DAT- 42B RT) was added to the human tonsil sample and subjected to a thermocycling protocol, which comprised performing a reverse transcription reaction at 53 °C for 45 minutes and hold at 4 °C.
  • a second strand synthesis was performed on the sample by subjecting the sample to a thermocycling protocol, which comprised e.g., pre-equilibrating at 65 °C, synthesizing the second strand at 65 °C for 15 minutes, then holding at 4 °C.
  • the Master Mix reagents were removed from the sample and 0.8M KOH was added and incubated for 5 minutes at room temperature. The KOH was removed and followed by the addition of an elution buffer.
  • a Second Strand Mix including a second strand reagent, a second strand primer, and a second strand-polymerizing enzyme, can be added to the sample and the sample can be sealed and incubated.
  • the reagents can be removed and elution buffer can be added and removed from the sample, and 0.8 M KOH can be added again to the sample and the sample can be incubated for 10 minutes at room temperature. Tris-HCl can be added and the reagents can be mixed. The sample can be transferred to a new tube, vortexed, and placed on ice.
  • cDNA amplification and quality control A qPCR Mix, including nuclease-free water, qPCR Master Mix, and cDNA primers, was prepared and pipetted into wells in a qPCR plate.
  • the sample was incubated 157 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC and thermocycled (e.g., lid temperature at 105 °C for -45-60 minutes; step 1: 98 °C for 3 minutes, step 2: 98 °C for 15 seconds, step 3: 63 °C for 20 seconds, step 4: 72 °C for one minute, step 5: [the number of cycles determined by qPCR Cq Values], step 6: 72 °C for 1 minute, and step 7: hold at 4 °C).
  • the sample can then be stored at 4 °C for up to 72 hours or at -20 °C for up to 1 week or use immediately.
  • a Fragmentation Mix including a fragmentation buffer and a fragmentation enzyme was prepared on ice. Elution buffer and fragmentation mix was added to each sample, mixed, and centrifuged. The sample mix was then placed in a thermocycler and cycled according to a predetermined protocol (e.g., lid temperature at 65 °C for ⁇ 35 minutes, pre-cool block down to 4 °C before fragmentation at 32 °C for 5 minutes, End-repair and A-tailing at 65 °C for 30 minutes and holding at 4 °C).
  • a predetermined protocol e.g., lid temperature at 65 °C for ⁇ 35 minutes, pre-cool block down to 4 °C before fragmentation at 32 °C for 5 minutes, End-repair and A-tailing at 65 °C for 30 minutes and holding at 4 °C.
  • SPRI Cleanup The 0.6X SPRI select Reagent was added to the sample and incubated for 5 minutes at room temperature.
  • the sample was placed on a magnet (e.g., in the high position) until the solution cleared, and the supernatant was transferred to a new tube strip.
  • 0.8X 158 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC SPRI select Reagent was added to the sample, mixed, and incubated for 5 minutes at room temperature.
  • the sample was placed on a magnet (e.g., in the high position) until the solution cleared.
  • the supernatant was removed and 80% ethanol was added to the pellet, incubated for 30 seconds, and washed. The ethanol wash was repeated and the sample placed on a magnet (e.g., in the low position) until the solution cleared.
  • elution buffer was added to the sample, mixed, and incubated for 2 minutes at room temperature.
  • the sample was placed on a magnet (e.g., in the high position) until the solution cleared, and a portion of the sample was moved to a new tube strip.
  • Ligation An Adaptor Ligation Mix, including ligation buffer, DNA ligase, and adaptor oligos, was prepared and centrifuged. The Adaptor Ligation Mix was added to the sample, pipette-mixed, and centrifuged briefly.
  • the average fragment size was determined using a Bioanalyzer trace or an Agilent TapeStation.
  • the library was sequenced using a sequencing platform, for example, MiSeq, NextSeq 500/550, HiSeq 2500, HiSeq 3000/4000, NovaSeq, and iSeq. See, Illumina ® , Indexed Sequencing Overview Guides, February 2018, Document 15057455v04; and Illumina ® Adapter Sequences, May 2019, Document #1000000002694vl 1, each of which is hereby incorporated by reference, for information on P5, P7, i7, i5, TruSeqTM Read 2, indexed sequencing, and other reagents described herein.
  • N-DAT RT variant improved the sensitivity of the spatial assay while maintaining good spatial resolution in human samples.
  • the effect of the 42B RT control variant (SEQ ID NO: 1, 143, 145, or 172) and the N- DAT-42B RT variant were compared using the three buffer types as previously described.
  • the human tonsil samples were permeabilized using a Kryptonite permeabilization buffer comprising 6% Sarkosyl, 1M urea, and 4ug/ul proteinase K.
  • the reverse transcriptase reaction was performed on human tonsil samples were prepared as disclosed above using six different conditions: (1) the 42B RT control variant was used with RT reagent; (2) the 42B RT control variant was used with Buffer X.5; (3) the engineered N-DAT-42B RT variant was used with the RT reagent; (4) the engineered N-DAT-42B RT variant was used with buffer X.4; (5) the engineered N-DAT-42B RT variant was used with buffer X.5; and (6) the small-scale purified engineered N-DAT-42B RT variant was used with buffer X.5. Most of the engineered N-DAT- 42B RT variants tested were purified by FPLC.
  • N-DAT-42B RT variants underwent a small scale purification. RT was performed using a 2 step protocol: 45 min at 53 °C and 30 min at 42 °C. [000580] As shown in FIGs.28A-F, the engineered N-DAT-42B RT variant maintained a good spatial resolution using all three buffers tested.
  • FIGs.28A-F show UMI heat maps showing globally detected gene expression in a human tonsil tissue analyzed using N-DAT-42B RT variant (FIGs.28C-F) or 42B RT variant (FIGs.28A-B) and the three different buffer formulations.
  • the NDAT1 RT variant was either FPLC-purified or underwent a small scale purification (FIG.28F).
  • the global detection of gene expression is shown as a heat map.
  • the RT Reagent (FIG.28A and FIG.28C) showed better mapping metrics when compared to buffer X.4 (FIG.28D), and buffer X.5 (FIG.28B, FIG.28E, and FIG.28F).
  • the commercially available RT reagent comprised 2% Glycerol, 50 mM Tris pH 8.3, 3 mM MgCl2, 75.04 mM KCl, 0.5% Supersonic F-108, 0.489 mg/ml BSA, and 3.16 mM dNTPs.
  • Buffer X.4 and buffer X.5 have the same components as the commercial RT reagent with the following differences.
  • Buffer X.4 contains 1.05% Glycerol, 158.3 mM NaCl, 0.579 mg/ml BSA, 4.685 mM dNTPs, 1.5mM dCTP, and 0.775mM GTP.
  • Buffer X.5 contains 133.8 mM NaCl, 1% Supersonic F-108, 3.96 mM dNTPs, 1.31 mM dCTP, and 0.655 mM GTP.
  • a MMLV RT variant without a DAT fusion domain was used as a control (e.g., a RT variant comprising the amino acid sequence of SEQ ID 160 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC NO: 1, 143, 145, or 172).
  • the NDAT1 RT variant was either FPLC-purified or underwent a small-scale purification (FIG.28F).
  • FIGs.28A-F show that NDAT1 RT variant improved the sensitivity of the assay while maintaining good spatial resolution when compared to the control RT variant, with all three buffer conditions.
  • FIGs.29A-F show that the engineered N-DAT-42B RT also increased the quality and the sensitivity metrics of the spatial assays.
  • FIGs.29A-F show graphs comparing the quality, sensitivity, and detection of gene expression under the same six conditions shown in FIGs.28A- F.
  • fraction reads in spots under tissue (FIG.29A) was substantially the same among the 6 conditions tested.
  • the smallest standard deviation across the six conditions tested was obtained using RT reagent and with the engineered N-DAT-42B RT.
  • FIG.29D Analysis of median UMI counts per spot (30k mapped spot-reads per spot, mapped to GRch38 reference genome assembly) (FIG.29D) showed that median UMI counts per spot were significantly enhanced in all three conditions using an N-DAT RT variant when compared to the three conditions using the RT control variant. Unexpectedly, N-DAT RT in bufferX.4 (N- DAT_RTX4) showed less variability. In addition, the GRch38 median genes per spot (30k mapped spot-reads per spot) (FIG.29E) analysis showed that median genes per spot were significantly enhanced in all samples comprising an N-DAT RT variant when compared to the 42B RT samples regardless of the buffer used.
  • engineered NDAT1 RT variant improved the sensitivity of the spatial assay while maintaining good spatial resolution when compared to the 42B RT variant (FIGs.29D-E).
  • the RT Reagent appeared to show better mapping metrics when compared to Buffer X.4, and Buffer X.5.
  • RT Reagent B with N-DAT at various concentrations and timings can also be tested and will likely show similar results as the RT Reagent. [000582]
  • Assessment of individual gene expression also showed substantially similar results as globally detected gene expression.
  • FIGs.30A-F show UMI heat maps shown as a log10(UMI) 161 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC ranging from cold to hot (0, 0.5, 1.0, 1.5, and 2.0), which illustrate the gene expression of RGS3 in a human tonsil tissue analyzed using N-DAT RT variant (FIGs.30C-F) or 42BRT control variant (FIGs.30A-B) under the three different buffer formulations (RT reagent (FIG.30A and FIG.30C), Buffer X.4 (FIG.30D), and Buffer X.5 (FIG.30B, FIG.30E, and FIG.30F)).
  • N-DAT RT variant FIGs.30C-F
  • 42BRT control variant FIGs.30A-B
  • FIGs.31A-C show UMI heat maps shown as a log10(UMI) ranging from cold to hot (0, 0.5, 1.0, 1.5, 2.0, and 2.5) illustrating the gene expression of KRT5 in a human tonsil tissue analyzed using N-DAT RT variant (FIGs.31C-F) or RT control variant (FIGs.31A-B) using three different buffer formulations (a commercially available RT reagent (FIG.31A and FIG. 31C), Buffer X.4 (FIG.31D), and Buffer X.5 (FIG.31B, FIG.31E, and FIG.31F)).
  • a commercially available RT reagent FIG.31A and FIG. 31C
  • Buffer X.4 FIG.31D
  • Buffer X.5 FIG.31B, FIG.31E, and FIG.31F
  • Example 11 Spatial resolution in non-human/mouse tissue.
  • SD Visium standard definition
  • HD Visium high definition
  • Visium HD slides contain two 6.5 x 6.5 mm Capture Areas with a continuous lawn of oligonucleotides arrayed in ⁇ 11 million 2 x 2 ⁇ m barcoded squares without gaps, achieving single cell–scale spatial resolution.
  • the data are output at 2 ⁇ m, as well as multiple bin sizes.
  • the 8 x 8 ⁇ m bin is the recommended starting point for visualization and analysis. See e.g., 10xgenomics.com/platforms/visium; 10xgenomics.com/products/visium-hd-spatial-gene-expression.
  • the zebrafish samples were permeabilized using a Kryptonite permeabilization buffer comprising 6% Sarkosyl, 1M urea, and 4ug/ul proteinase K.
  • the reverse transcriptase reaction was performed on zebrafish samples using T reagent, the engineered N-DAT-42B RT (NDAT- 42BL) variant, TSO, and dTT.
  • the RT was performed using a 2 step protocol-45 min at 53 °C and 30 min at 42 °C.
  • the single strand synthesis was performed using a control RT (e.g., MMLV TR variant comprising an amino acid sequence of SEQ ID NO: 1, 143, 145, or 172), RT Reagent, and SS Primers.
  • N-DAT fusion did not sufficiently process the synthesis of the second strand cDNA in the Zebrafish tissue. This was 162 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC likely caused by the N-DAT fusion configuration or the RT variant configuration. For example, the configuration may have slowed the enzyme from initiating or continuing the synthesis of the second strand. However, the N-DAT fusion sufficiently process the synthesis of the first strand cDNA in the Zebrafish tissue.
  • FIGs.32A-B show UMI heat maps shown as a count log10 ranging from cold to hot (0.5, 1, 1.5, 2, 2.5, 3, 4, 4.5, and 5), illustrating globally detected gene expression in Zebrafish (non-human or mouse tissue) using a spatial assay with a first slide configuration.
  • FIGs.32C-D show heat maps illustrating individual gene expression.
  • FIG.32C shows Crgm1 gene expression as log normalized per experiment ranging from cold to hot (0.00-5.0).
  • FIG.32D shows KRT5 gene expression as log normalized per experiment ranging from cold to hot (0.0-7.0).
  • FIG.32E shows Rho gene expression as log normalized per experiment ranging from cold to hot (0.0-8.0).
  • Fraction reads mapped to genome are the fraction of reads that mapped to a unique gene in the genome. In general, the read must be consistent with annotated splice junctions and are considered for UMI counting.
  • FIGs.33A-D also show UMI heat maps showing globally detected gene expression (FIG.33A) or individual gene expression (FIGs.33B-C) in Zebrafish (non-human or mouse tissue) analyzed using NDAT1 RT variant (42BL-N-DAT1) and RT reagent buffer under sandwich configuration conditions for a spatial assay with a second slide configuration.
  • FIG. 33B shows Crgm1 gene expression as log normalized per experiment ranging from cold to hot (0.00-4.0).
  • FIG.33C shows KRT5 gene expression as log normalized per experiment ranging from cold to hot (0.0-9.0).
  • FIG.33D shows Rho gene expression as log normalized per experiment ranging from cold to hot (0.0-7.0).
  • the quality, and sensitivity metrics were: fraction reads mapped to genome (0.72).
  • the engineered N-DAT RT variants disclosed herein enhanced the quality and the sensitivity of the spatial assay metrics while maintaining good spatial resolution when compared to a control RT variant.
  • the control RT variant lacked the N-DAT but comprised the same RT backbone as the engineered N-DAT RT variant.
  • the control RT variant consisted of the amino acid sequence of SEQ ID NO: 1, 142, 143, or 172.
  • Table 1 shows listing of non-limiting embodiments of RT enzymes of the present disclosure.
  • Tables 2 and 5 shows additional listing of amino acid and nucleic acid sequences of non-limiting embodiments of the engineered RTs of the present disclosure. Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences S L S P G V K H P K L T 165 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences I K E G E L I A E N A 166 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences aa ga tc tc cc g at c 167 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences g c g c I V P GI I A T T P K W Q Y H P I V P GI I A T P K W Q Y H I V P GI I 168 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences A T P K W Q Y H I P P G P F L P H S G P I P P P G P F L P H S G P 169 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences I V P GI I A T P K T W Q Y H I V P GI I A T P K T W Q Y H V F S F K L 170 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences E V F S F K L E M V F S F K L E N A V F S 171 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences F K L E V F S D C Q G A G A V F S D C Q G A G K 172 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences D V F S F K L E I V P GI I A T P K T W Q Y H I V P GI I A T P 173 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences K T W Q Y H I P P G P F L P H S G P I P P P G P F L P H S G P I V P GI I A 174 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences T P K W Q Y H I V P GI I A T P K T W Q Y H I V P GI I A T P K W Q Y H I V 175 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences P GI I A T P K W Q Y H I V P GI I A T P K W Q Y H I P P G P F L P H S G 176 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences P I P P G P F L P H S G P I V P GI I A T P K W Q Y G I V P G I A T P K 177 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences W Q Y G I V P GI I A T P K W Q Y H I V P G I A T P K W Q Y H I V P GI I A T 178 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences P K W Q Y H L L R L Q E F R L A K Q L L R L Q E F R L A K Q 179 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences I P P G P F L P H S G P S P P P G P F L P H S G P K K P IS K P P 180 4876-6828-0003.1 Foley Ref.: 131488-0215 10X Genomics Ref.: 100-165501PC Table 1.
  • Non-limiting embodiments of RT variants of the invention SEQ ID NO Description Sequences T W Q Y H P 181 4876-6828-0003.1 5 1 C 2 P 0 - 1 8 0 8 5 4 5 1 6 3 1 - 1: 0 . f 0 e 1: R.

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Abstract

La présente invention concerne des transcriptases inverses recombinées (par exemple, des polypeptides de transcriptase inverse modifiés) comportant un ou plusieurs domaines de liaison à l'ADN conjugués à une transcriptase inversée de type sauvage ou à une transcriptase inversée mutante présentant une ou plusieurs activités modifiées liées à la transcriptase inverse, telles que, mais sans s'y limiter, une efficacité de commutation de matrice modifiée, une efficacité de transcription modifiée ou les deux à la fois. La présente invention concerne également des compositions et des kits comportant les transcriptases inverses recombinées (par exemple, des polypeptides de transcriptase inverse modifiés) et des procédés de production, d'amplification ou de séquençage de molécules d'acide nucléique par l'utilisation des transcriptases inverses recombinées.
PCT/US2024/033519 2023-06-13 2024-06-12 Protéines de liaison à l'adn spécifiques d'une séquence Pending WO2024258911A1 (fr)

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