Classifications

• • 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)
  • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• • 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)
  • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

• DNA polymerases are classified into Families A, B, C, D, X, Y and RT according to their amino acid sequences. DNA polymerases have several properties that contribute to their replicative fidelity. For example, some DNA polymerases of Families A, B, and D have proofreading 3' to 5' (3 '-5') exonuclease activity. When a DNA polymerase incorporates an incorrect or modified nucleotide, for example, in a primer strand, it detects structural perturbations caused by mispairing or nucleotide modification and transfers the primer strand from the polymerase domain to the 3 '-5' exonuclease active site.
• DNA polymerases restrict access to their active sites to prevent incorporation of ribonucleotides.
• Families A, B, X, Y, and reverse transcriptases have a steric gate that excludes rNTPs from the active site by a steric clash between a bulky amino acid side chain in the steric gate and the 2’-OH of such rNTPs. Reducing the size of the side chain at the steric gate position allows DNA polymerases so modified to incorporate a single rNTP as efficiently as dNTP.
• Polymerases with such modified steric gates have been extensively employed in molecular biology applications such as single-molecule sequencing, sequencing by synthesis, and single nucleotide polymorphism (SNP) detection.
• SNP single nucleotide polymorphism
• Family D DNA polymerases have properties that differ from some or all other polymerases. These distinguishing properties may include one or more of the following: (a) having little to no strand displacement activity, (b) reading through uracils encountered in a template strand and continue polymerization, (c) synthesizing DNA at a slower rate, (d) binding other replication enzymes, including core replisome proteins (e.g., mini-chromosome maintenance (MCM) helicase, DNA ligase, the archaeal Cdc45 protein (GAN) and the processivity factor PCNA), and (e) activity in the presence of amphidicolin.
• MCM mini-chromosome maintenance
• GAN archaeal Cdc45 protein
• PCNA processivity factor
• PolD is a heterodimeric enzyme consisting of a large 5 ’-3’ polymerase subunit and a small MRE11-like 3'-5' exonuclease subunit. The activity of each subunit requires the other subunit to be present.
• Family D DNA polymerases preferentially incorporate dNTPs over rNTPs. The molecular basis for this selectivity has not yet been identified, which may have limited the use of wild type Family D polymerases.
• a variant Family D polymerase may comprise, for example, (a) an amino acid sequence comprising at least two (or all three) of (optionally, in order from amino- to carboxy terminus and/or otherwise corresponding to) (i) a sequence having at least 85% identity to positions 355-362 of SEQ ID NO: 1, (ii) a sequence having at least 85% identity to positions 808-816 of SEQ ID NO: 1, and (iii) a sequence having at least 80% identity to positions 824-828 of SEQ ID NO: 1; and (b) a substitution at a position corresponding to (i) position 930, 931, or 932 of SEQ ID NO: 1, (ii) position 822, 954, 959, or 963 of SEQ ID NO: 3, or (iii) position 13, 203
• a variant Family D polymerase may comprise, in some embodiments, (a) an amino acid sequence comprising at least two (or all three) of (optionally, in order from amino- to carboxy terminus and/or otherwise corresponding to) (i) a sequence having at least 85% identity to positions 355-362 of SEQ ID NO: 1, (ii) a sequence having at least 85% identity to positions 808- 816 of SEQ ID NO: 1, and (iii) a sequence having at least 80% identity to positions 824-828 of SEQ ID NO: 1; and (b)(i) a substitution at a position corresponding to position 930, 931, or 932 of SEQ ID NO: 1, (ii) any amino acid other than tyrosine at one or more positions corresponding to position 13, 203, 822, 885, 890, 954, 984, 1020, 1026, and/or 1165 of SEQ ID NO: 1, (iii) any amino acid other than lysine at one or more positions corresponding to position 392,
• a variant Family D polymerase may comprise (a) an amino acid sequence comprising at least two sequences selected from (i) a sequence having at least 85% identity to positions 355-362 of SEQ ID NO: 1, (ii) a sequence having at least 85% identity to positions 808-816 of SEQ ID NO: 1, and (iii) a sequence having at least 80% identity to positions 824-828 of SEQ ID NO: 1; and (b) any amino acid other than (i) tyrosine at one or more positions corresponding to position 13, 203, 822, 885, 890, 954, 984, 1020, 1026, and/or 1165 of SEQ ID NO: 1, (ii) lysine at one or more positions corresponding to position 392, 398 and/or 959 of SEQ ID NO: 1, (iii) cystine at a position corresponding to position 963 of SEQ ID NO: 1, (iv) phenylalanine at one or more positions corresponding to position 452 and/or 9
• a variant Family D polymerase may comprise, for example, (a) an amino acid sequence comprising at least one (and any two of up to all six) of (optionally, in order from amino- to carboxy terminus and/or otherwise corresponding to) (i) a sequence having at least 90% identity to positions 145-169 of SEQ ID NO: 3 or 4, (ii) a sequence having at least 90% identity to positions 245-271 of SEQ ID NO: 3 or 4, (iii) a sequence having at least 90% identity to positions 354-364 of SEQ ID NO: 3 or 4, (iv) a sequence having at least 90% identity to positions 651-679 of SEQ ID NO: 3 or 4, (v) a sequence having at least 90% identity to positions 821-841 of SEQ ID NO: 3 or 4, and (vi) a sequence having at least 80% identity to positions 947-963 of SEQ ID NO: 3 or 4; and (b) a substitution at a position corresponding to (i) position 930, 931, or 932 of SEQ ID NO: 1, (
• a variant Family D polymerase having such a substitution may comprise, for example, a substitution corresponding to position 13, 203, 822, 885, 890, 980, 984, 1020, 1026, or 1165 of SEQ ID NO: 4.
• a variant Family D polymerase having from one to six of the foregoing sequences may further comprise a sequence having at least 80% identity to positions 808-816 of SEQ ID NO: 4.
• a variant Family D polymerase may comprise a sequence identical to positions 927-930 of SEQ ID NO: 1 and/or positions 932-934 of SEQ ID NO: 1 at, for example, corresponding positions along its length.
• a variant DNA polymerase comprising a substitution at a position corresponding to position 931 of SEQ ID NO: 1 may be selected from an alanine substitution, an aspartate substitution, a cysteine substitution, a glutamate substitution, a glutamine substitution, a glycine substitution, an isoleucine substitution, a leucine substitution, a methionine substitution, a phenylalanine substitution, a proline substitution, a serine substitution, a threonine substitution, and a valine substitution.
• a substitution at a position corresponding to position 931 may be alanine.
• a variant Family D polymerase may comprise a sequence having at least 90%, at least 95% or at least 98% identity (but in each case, less than 100% identity) to SEQ ID NO: 1.
• a variant Family D polymerase may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity (but in each case, less than 100% identity) to SEQ ID NO: 2, 3, or 4.
• a variant Family D polymerase may comprise a sequence having (a) at least 70%, at least 75%, or at least 80%, at least 85% identity (but in each case, less than 100% identity) to SEQ ID NO: 2, 3, or 4 and (b) at least one (and any two of up to all six) of (optionally, in order from amino- to carboxy terminus and/or otherwise corresponding to) (i) a sequence having at least 90% identity to positions 145-169 of SEQ ID NO: 3 or 4, (ii) a sequence having at least 90% identity to positions 245-271 of SEQ ID NO: 3 or 4, (iii) a sequence having at least 90% identity to positions 354-364 of SEQ ID NO: 3 or 4, (iv) a sequence having at least 90% identity to positions 651-679 of SEQ ID NO: 3 or 4, (v) a sequence having at least 90% identity to positions 821-841 of SEQ ID NO: 3 or 4, and (vi) a sequence having at least 80% identity to positions 947-963 of SEQ ID NO:
• a variant Family D polymerase having such a substitution may comprise, for example, a substitution corresponding to position 13, 203, 822, 885, 890, 980, 984, 1020, 1026, or 1165 of SEQ ID NO: 4.
• a variant Family D polymerase having from one to six of the foregoing sequences may further comprise a sequence having at least 80% identity to positions 808-816 of SEQ ID NO:
• a composition comprising a variant Family D polymerase may further include one or more substrates, intermediates, and/or products of such polymerase.
• a composition may comprise one or more ribonucleoside triphosphates, one or more deoxyribonucleoside triphosphates, or one or more ribonucleoside triphosphates and one or more deoxyribonucleoside triphosphates.
• a composition may comprise one or more modified nucleotides (e.g., modified nucleotides selected from the group consisting of dideoxynucleoside triphosphates, acyclic- nucleoside triphosphates, 3'-0-azidomethyl-ddNTPs, 3'-0-amino-ddNTPs, 3 ’-OH unblocked dNTPs, biotin-deoxyuridine triphosphates, rNTP, 3'-dNTP, 2'-amino-NTP, 2'-azido-NTP, and 2'- Omethyl-NTP).
• a product of a variant Family D polymerase included in a composition or arising from a method disclosed herein may comprise, for example, a polynucleotide having at least one ribonucleotide.
• a variant Family D polymerase may have or lack an exonuclease subunit (DPI). If present, the exonuclease subunit may have or lack exonuclease activity (e.g ., 3’ to 5’ exonuclease activity). For example, an exonuclease subunit may comprise one or more substitutions that reduce or eliminate exonuclease activity relative to the corresponding wild type sequence.
• a composition may comprise a fusion protein comprising a variant Family D polymerase and any desired polypeptide.
• a fusion protein may comprise a polypeptide selected from a DNA- binding peptide or protein, a maltose binding domain, a chitin binding domain, or a SNAP-Tag®.
• a method may comprise contacting a variant Family D polymerase with a template polynucleotide (e.g., a DNA template) deoxyribonucleoside triphosphates, and optionally a primer having a sequence complementary to at least a portion of the sequence of the template under suitable conditions (e.g., time, temperature, pH) to produce at least one polynucleotide copy of the template.
• a method may further include contacting a variant Family D polymerase with one or more ribonucleoside triphosphates and/or one or more modified nucleotides.
• a template polynucleotide may comprise at least one ribonucleotide.
• a template may comprise a uracil.
• a polynucleotide copy produced upon or following contact with a variant Family D polymerase may comprise a deoxyribonucleotide at positions complementary to any template positions occupied by a ribonucleotide.
• a DNA template comprising one uracil contacted with a variant Family D polymerase may give rise to a polynucleotide copy of the template, the copy comprising an adenine at the position that is complementary to the uracil.
• methods for filling a gap in a double- stranded polynucleotide are also provided.
• a method may comprise contacting a variant Family D polymerase and one or more deoxyribonucleoside triphosphates with a double-stranded polynucleotide comprising a gap on one strand that is at least one nucleotide in length to produce a product polynucleotide that has no such gap.
• kits including variant Family D polymerases.
• a kit may comprise a variant Family D polymerase and one or more ribonucleoside triphosphates, one or more deoxyribonucleoside triphosphates, one or more modified nucleotides, one or more primers, one or more adapters, one or more buffering agents, or combinations thereof.
• FIGURE 1 shows examples of Family D polymerase sequences.
• SEQ ID NO: 1 contains Family D polymerase consensus sequences identified according to Example 1, in which each X independently can be any amino acid, except SEQ ID NO:l residues X927, which is G or T, X928, which is L, M, or T, X929, which is A or S, X933, which is S, F, or C, and X934, which is G, A, C, or V.
• SEQ ID NOS: 2-3 contain Family D polymerase consensus sequence identified according to Example 1, in which each X independently can be any amino acid.
• SEQ ID NOS: 4 and 5 are Family D polymerase sequences of Euryarchaeota (9°N) and Pyrococcus abyssi, respectively.
• FIGURE 2 shows that Family D polymerase variants incorporated rNTPs relative to dNTPs more efficiently than wild type PolD.
• FIGURE 3 shows that additional Family D polymerase variants incorporated rNTPs relative to dNTPs more efficiently than wild type PolD.
• FIGURE 4 shows the ability of variant Family D polymerases have to incorporate rNTPs successively.
• FIGURE 4A is a sketch illustrating the reaction and FIGURE 4B illustrates data obtained in accordance with Example 5.
• the wild type polD shown incorporates one rNTP, while a polD H931A incorporates up to four successive rNTPs. As controls both polD and pol H931A incorporate successive dNTPs.
• compositions, methods and kits are provided here that improve among other things, the synthesis of DNA that contains modified nucleotides such as ribonucleotides using a class of polymerases that do not belong to the DNA polymerase A or B families.
• Family D polymerases preferentially incorporate deoxyribonucleoside triphosphates relative to larger ribonucleoside triphosphates, which may be attributed to a steric gate motif limiting substrate access to the catalytic site.
• Variant Family D polymerases with modifications in or modifications impacting this steric gate are provided in some embodiments disclosed herein. For example, variant Family D polymerases may have an enhanced or reduced ability to exclude ribonucleotides from the active site.
• Sources of commonly understood terms and symbols may include: standard treatises and texts such as Komberg and Baker, DNA Replication, Second Edition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); Singleton, et ah, Dictionary of Microbiology and Molecular biology, 2d ed., John Wiley and Sons, New York (1994), and Hale & Markham, the Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991) and the like.
• a protein refers to one or more proteins, i.e., a single protein and multiple proteins.
• claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.
• Numeric ranges are inclusive of the numbers defining the range. All numbers should be understood to encompass the midpoint of the integer above and below the integer i.e., the number 2 encompasses 1.5-2.5. The number 2.5 encompasses 2.45-2.55 etc. When sample numerical values are provided, each alone may represent an intermediate value in a range of values and together may represent the extremes of a range unless specified.
• buffering agent refers to an agent that allows a solution to resist changes in pH when acid or alkali is added to the solution.
• suitable non-naturally occurring buffering agents include, for example, any of Tris, HEPES, TAPS, MOPS, tricine, and MES.
• corresponding to refers to positions that lie across from one another when sequences are aligned, e.g., by the BLAST algorithm.
• An amino acid position in a functional or structural motif in one polymerase may correspond to a position within a functionally equivalent functional or structural motif in another polymerase.
• DNA polymerase refers to an enzyme that is capable of replicating DNA and optionally may have exonuclease activity.
• DNA template refers to the DNA strand read by a DNA polymerase and of which a copy is synthesized.
• “Family D polymerase” refers to a heterodimeric archaeal DNA polymerase having a small exonuclease subunit (DPI) and a large polymerase subunit (DP2).
• Family D polymerases may be produced in cells in an immature form and then undergo post-translational processing (e.g., removal of amino-terminal sequences, inteins, or both). Examples of Family D polymerases may include 9°N PolD (Euryarchaeota), Genbank Accession No. KPV61551.1 (Bathyarchaeota), Accession No.
• fusion protein refers to protein composed of a plurality of polypeptide components that are un-joined in their native state.
• Fusion proteins may be a combination of two, three or four or more different proteins.
• the term polypeptide is not intended to be limited to a fusion of two heterologous amino acid sequences.
• a fusion protein may have one or more heterologous domains added to the N-terminus, C-terminus, and or the middle portion of the protein. If two parts of a fusion protein are “heterologous”, they are not part of the same protein in its natural state.
• fusion proteins include a variant Family D polymerase fused to an SS07 DNA binding peptide (see for example, US Patent 6,627,424), a transcription factor (see for example, US patent 10,041,051), a binding protein suitable for immobilization such as maltose binding domain (MBP), a histidine tag (“His-tag”), chitin binding domain (CBD) or a SNAP-Tag® (New England Biolabs, Ipswich, MA (see for example US patents 7,939,284 and 7,888,090)).
• MBP maltose binding domain
• His-tag histidine tag
• CBD chitin binding domain
• SNAP-Tag® New England Biolabs, Ipswich, MA (see for example US patents 7,939,284 and 7,888,090)
• fusion proteins include a heterologous targeting sequence, a linker, an epitope tag, a detectable fusion partner, such as a fluorescent protein, b-galactosidase, luciferase and the functionally similar peptides.
• modified nucleotide refers to a non-canonical nucleotide that may be incorporated into a growing polynucleotide strand.
• modified nucleotides include nucleotide terminators (dideoxy nucleoside triphosphates (ddNTPs) and acyclic-nucleoside triphosphates (acycloNTPs)), reversible nucleotide terminators (3'-0-azidomethyl-ddNTPs, 3'-0- amino-ddNTPs, and 3 ’-OH unblocked dNTPs), and tagged nucleotides (biotin-deoxyuridine triphosphates (biotin-dUTPs)).
• ddNTPs deoxy nucleoside triphosphates
• acycloNTPs acyclic-nucleoside triphosphates
• reversible nucleotide terminators (3'-0-azidomethyl-ddNTPs
• modified nucleotides include nucleotides with any atom or group other than a hydrogen at the 2' position (e.g ., rATP, 3'-dATP, 2'-amino-ATP, 2'- azido-ATP and 2'- (9-methyl- ATP).
• 3’-OH unblocked dNTPs e.g., Lightning TerminatorsTM (Agilent Technologies, Inc., Houston, Tex.) (Gardner AF, Wang J, Wu W, Karouby J, Li H, Stupi BP, Jack WE, Hersh MN, Metzker ML. (2012) Rapid incorporation kinetics and improved fidelity of a novel class of 3'-OH unblocked reversible terminators. Nucleic Acids Res., 40, 7404-7415)).
• NTP refers to a nucleoside triphosphate including, for example, any deoxyribonucleoside triphosphate (“dNTP”) and any ribonucleoside triphosphate (“rNTP”).
• dNTP deoxyribonucleoside triphosphate
• rNTP ribonucleoside triphosphate
• non-naturally occurring refers to a polynucleotide, polypeptide, carbohydrate, lipid, or composition that does not exist in nature.
• a polynucleotide, polypeptide, carbohydrate, lipid, or composition may differ from naturally occurring polynucleotides polypeptides, carbohydrates, lipids, or compositions in one or more respects.
• a polymer e.g ., a polynucleotide, polypeptide, or carbohydrate
• the component building blocks e.g., nucleotide sequence, amino acid sequence, or sugar molecules.
• a polymer may differ from a naturally occurring polymer with respect to the molecule(s) to which it is linked.
• a “non-naturally occurring” protein may differ from naturally occurring proteins in its secondary, tertiary, or quaternary structure, by having a chemical bond (e.g., a covalent bond including a peptide bond, a phosphate bond, a disulfide bond, an ester bond, and ether bond, and others) to a polypeptide (e.g., a fusion protein), a lipid, a carbohydrate, or any other molecule.
• a chemical bond e.g., a covalent bond including a peptide bond, a phosphate bond, a disulfide bond, an ester bond, and ether bond, and others
• a “non-naturally occurring” polynucleotide or nucleic acid may contain one or more other modifications (e.g., an added label or other moiety) to the 5’- end, the 3’ end, and/or between the 5’- and 3 ’-ends (e.g., methylation) of the nucleic acid.
• a “non-naturally occurring” composition may differ from naturally occurring compositions in one or more of the following respects: (a) having components that are not combined in nature, (b) having components in concentrations not found in nature, (c) omitting one or components otherwise found in naturally occurring compositions, (d) having a form not found in nature, e.g., dried, freeze dried, crystalline, aqueous, and (e) having one or more additional components beyond those found in nature (e.g., buffering agents, a detergent, a dye, a solvent or a preservative).
• All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
• polynucleotide copy refers to the product of polymerization activity of a DNA polymerase.
• a polynucleotide copy may comprise deoxyribonucleotides with or without ribonucleotides.
• position refers to the place such amino acid occupies in the primary sequence of a peptide or polypeptide numbered from its amino terminus to its carboxy terminus.
• substitution at a position in a comparator amino acid sequence refers to any difference at that position relative to the corresponding position in a reference sequence, including a deletion, an insertion, and a different amino acid, where the comparator and reference sequences are at least 80% identical to each other.
• a substitution in a comparator sequence in addition to being different than the reference sequence, may differ from all corresponding positions in naturally-occurring sequences that are at least 80% identical to the comparator sequence.
• variant Family D polymerase refers to a non-naturally occurring archaeal Family D DNA polymerase that has an amino acid sequence that is less than 100% identical to the amino acid sequence of a naturally occurring DNA polymerase from archaea or has a non-naturally occurring chemical modification (e.g ., a polypeptide fused to its amino terminal or carboxy terminal end or other chemical modification).
• a variant amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of a naturally occurring Family D polymerase without being 100% identical to any known naturally occurring polymerase. Sequence differences may include insertions, deletions and substitutions of one or more amino acids.
• a variant Family D polymerase may have a small exonuclease subunit (DPI) and a large polymerase subunit (DP2).
• DPI small exonuclease subunit
• DP2 large polymerase subunit
• a variant Family D polymerase may have exonuclease activity or may be exonuclease deficient, where exonuclease activity is 5 ’-3’.
• Exonuclease deficient variants may have one or more amino acid substitutions in the small subunit (DPI) including, for example, substitutions at positions corresponding to D507A, H554A, or both D507A and H554A of 9°N (SEQ ID NO: 6).
• a variant Family D polymerase lacking exonuclease activity may comprise only such portion of the DPI subunit as necessary to support catalytic activity of the polymerase (DP2) subunit.
• Variant Family D polymerases are provided here that differ from wild type Family D DNA polymerases in their abilities to incorporate rNTPs.
• the incorporation ratio of adenosine triphosphate to deoxyadenosine triphosphate (rA:dA) may be at least 0.19, less than 0.26 (or less than wild type), more than 0.26 (or more than wild type), at least 0.28, at least 0.3, at least 0.4, at least 0.5, at least 0.8, or at least about.0.9.
• Use of wild type Family D polymerases may have been limited in the past, in part, by their rNTP/dNTP selectivity.
• Family D DNA polymerases may have little to no strand displacement activity. When polD encounters a downstream strand of DNA hybridized to the template strand during synthesis, it pauses rather than displacing the hybridized strand. Accordingly, polD may be used in applications for which strand displacement is not desired or required, including, for example, gap-filling applications. While Family B polymerases stall when they encounter a uracil in the template strand, variant Family D polymerases are able to read through and continue polymerization. Accordingly, variant Family D polymerases could be useful for sequencing polynucleotides that have or have the potential to have one or more deaminated cytosines.
• variant Family D polymerases may synthesize DNA at a slower rate than many other polymerases.
• DNA sequencing by synthesis applications typically require use of a reversible terminator. While faster reaction rates are commonly preferred, the slower rate of variant Family D polymerases could be optimized for real time detection of base incorporation in the growing DNA strand.
• a fusion protein may comprise a variant Family D polymerase and a replisome protein.
• Variant Family D polymerases may bind other replication enzymes, including core replisome proteins, for example, mini-chromosome maintenance (MCM1 and MCM2) helicase, DNA ligase, the archaeal Cdc45 protein (GAN), Cdc6 and the processivity factor PCNA, whereas Family B DNA polymerases may bind MCM2 and single- stranded binding protein RPA3.
• Combining a replisome protein and a variant Family D polymerase in a fusion protein may enhance the activity of one or both proteins (e.g., by making more efficient use of substrates, improving the quantity /purity of products, and/or facilitate delivery of both molecules to a desired site. Binding or fusing polD with accessory proteins may enable coupling polD synthesis with other activities such as MCM helicase DNA unwinding, ligation of nicks, or increases in processivity with PCNA clamps.
• Aphidicolin is an antibiotic that acts on Family B polymerases to reversibly inhibit eukaryotic DNA replication.
• Variant Family D polymerases like PolD itself, may remain active, polymerizing DNA in the presence of aphidicolin. Accordingly, where both polB and a variant Family D polymerase are present, but polD activity is preferred or required, a reaction may be performed in the presence of aphidicolin.
• Variant Family D polymerases may have an amino acid sequence comprising one or more of the consensus domains shown in Table 1, each of which may have the indicated number of substitutions. Each of these domains corresponds to the indicated portion of wild type Family D polymerase (e.g., Euryarchaeota 9°N PolD-L; SEQ ID NO: 4) and one or more of the degenerate sequences disclosed herein (SEQ ID NOS: 1-3). Variant Family D polymerases have at least one substitution relative to wild type Family D polymerase (e.g ., in domain Gl, G2, G3, or G4) and are not naturally occurring.
• wild type Family D polymerase e.g., Euryarchaeota 9°N PolD-L; SEQ ID NO: 4
• variant Family D polymerases have at least one substitution relative to wild type Family D polymerase (e.g ., in domain Gl, G2, G3, or G4) and are not naturally occurring.
• a variant Family D polymerase may have an amino acid sequence comprising domains as shown in Table 2 with domain C referring to either of domains Cl and C2, domain F referring to either of domains FI and F2, and domain G referring to any of domains Gl, G2, G3, and G4 of Table 1.
• Each domain when present, may appear in alphabetical order from the amino terminal end to the carboxy terminal end of a variant Family D polymerase as shown in Table 2 and further may appear in a position corresponding to the position of the comparable domain in a wild type Family D polymerase.
• Table 2 Variant Family D Polymerase Domains
• variant Family D polymerases have been engineered and tested for their ability to preferentially incorporate ribonucleotides into polynucleotides.
• a variant Family D polymerase may have an amino acid sequence comprising one or more substitutions at one or more positions selected from positions corresponding to positions 145-169, 245-271, 336, 354-364, 368, 392, 395, 398, 452-455, 651-679, 808-816, 821-841, 918-943 (e.g., 930, 931, and 932), 947-964 (e.g., 954, 959, and 963), 966, and 1020 of SEQ ID NO: 4.
• a variant Family D polymerase may have an amino acid sequence comprising one or more sequences having (a) at least 90% identity to domain A, B, D or F2, (b) at least 85% identity to domain E or H, (c) at least 80% identity to domain Cl, C2, or FI, (d) up to 97% identity to Gl, G2, G3, or G4, in some embodiments.
• a variant DNA polymerase may have an amino acid sequence comprising one or more substitutions at one or more positions selected from positions corresponding to positions 13, 203, 822, 885, 890, 980, 984, 1020, 1026, or 1165 of SEQ ID NO: 4.
• variant Family D polymerases may have a substitution at a position corresponding to position 931 of SEQ ID NO: 1, 2, 3, or 4 or one or more substitutions in the triplet Pro-His-Thr (SEQ ID NO: 15) corresponding to positions 930-932 of SEQ ID NO:l, 2, 3, or 4 (domain G4).
• a variant Family D polymerase comprises at least one substitution in Gl, G2, G3, or G4.
• Such variants may further include a non-naturally occurring sequence having at least 80%, 85%, 90%, 95% or 97% or 100% sequence identity with one or more of the other sequence motifs described in Table 1 and Fig. 1.
• a variant Family D polymerase may have an amino acid sequence comprising a sequence having (a) at least 85% identity to domain Cl or C2, (b) at least 85% identity to domain E, (c) at least 80% identity to domain FI or F2, and (d) up to 97% identity to domain Gl, G2, G3, or G4 (e.g., Table 2, numbers 4, 61, 69, 72, 79, 89, 103, 105, 108, 110, 113, 120, 124, 125, 127, 130, and 131).
• a variant Family D polymerase may have an amino acid sequence comprising, for example, (a) a sequence having up to 97% identity to domain Gl, G2, G3, or G4, and (b) a sequence having a sequence having (i) at least 90% identity to domain A (e.g., Table 2, number 5), (ii) at least 90% identity to domain B (e.g., Table 2, number 6), (iii) at least 80% identity to domain Cl or C2 (e.g., Table 2, number 7), (iv) at least 90% identity to domain D (e.g., Table 2, number 8), (v) at least 85% identity to domain E (e.g., Table 2, number 9), (vi) at least 90% identity to domain FI or F2 (e.g., Table 2, number 10), (vii) at least 85% identity to domain H (e.g., Table 2, number 11), or any combinations thereof (e.g., Table 2, numbers 12-17, 20-23, 30, 31, 33, 34, 40-42, 48, 50-52, 60,
• a variant Family D polymerase may comprise a substitution at a position corresponding to position 931 of any of SEQ ID NOS: 1-4.
• Substitutions at position 931 may include H931D, H931E, H931C, H931L, H931I, H931F, H931G, H931T, H931V, H931Q, H931M, H931P, H931S, and H931A, may be limited to H931D and H931E (e.g., where a rA:dA ratio below wild type is desired), or may be limited to H931C, H931L, H931I, H931F, H931G, H931T, H931V, H931Q, H931M, H931P, H931S, and H931A (e.g., where a rA:dA ratio above wild type is desired).
• a variant Family D polymerase may have one or more substitutions at one or more positions corresponding to any of the positions of the Pyrococcus abyssi Family D polymerase disclosed in Table 1 of U.S. Provisional Application No. 62/976,039 filed February 13, 2020 (e.g., positions 106-161, 243-267, 326-330, 361-365, 385-397, 441-451, 657-667, 822-829, 919-928, 940-962, and 981-997 of SEQ ID NO:5 or positions 109-164, 246-270, 332-336, 367-371, 391- 403, 447-447, 665-675, 830-837, 927-936, 948-970, 989-1005 of SEQ ID NO:4).
• a variant Family D polymerase may have any desired length.
• a variant Family D polymerase may be shorter (e.g., up to 10% shorter) or longer (e.g., up to 10% longer) than a wild-type sequence.
• a variant Family D polymerase may have the same or about the same length as a wild-type family D polymerase (e.g., 1250-1300 amino acids).
• a Family D polymerase in some embodiments, may have fewer (e.g., 1-20 fewer) or may have more (e.g., 1-20 more) amino acids than SEQ ID NO: 1, 2, 3, or 4.
• a variant Family D polymerase may have an amino acid sequence having at least 90% identity to SEQ ID NOS: 1-4.
• a variant Family D polymerase having a substitution at a position corresponding to position 931 of any of SEQ ID NOS: 1-4 may comprise a sequence having at least 90% identity to any of SEQ ID NOS: 1-4, at least 95% identity to any of SEQ ID NOS: 1-4, at least 98% identity to any of SEQ ID NOS: 1-4, at least 99% identity to any of SEQ ID NOS: 1-4, or at least 99.5% identity to any of SEQ ID NOS: 1-4, in each case, including all substitutions in the percent identity calculation.
• Variant Family D polymerases may have improved properties including, for example, an increased ability to incorporate ribonucleotides, increased replicative fidelity, or both. Additional properties may include, for example, no strand displacement activity, no aphidicolin sensitivity, ability to read through uracil on a template strand, slower rate of DNA synthesis relative to other families of polymerases, ability to bind other replication enzymes, and combinations thereof.
• a variant Family D polymerase may be included in a composition with other materials.
• a composition may comprise, for example, a variant Family D polymerase and one or more of a buffering agent, a crowding agent (such as polyethylene glycol included in a storage or reaction mixture), a single strand binding protein or portions thereof, an unwinding agent (e.g ., a helicase), a detergent (e.g., a nonionic, cationic, anionic or zwitterionic detergent), an additive (e.g., albumin), glycerol, salt (e.g. KC1), EDTA, a dye, a reaction enhancer or inhibitor, an oxidizing agent, a reducing agent, a solvent and/or a preservative.
• a buffering agent such as polyethylene glycol included in a storage or reaction mixture
• a crowding agent such as polyethylene glycol included in a storage or reaction mixture
• a single strand binding protein or portions thereof an unwinding agent (e
• the buffering agent combined with additional reagents that are standard in the art may be formulated for storage of a variant Family D polymerase and/or for the desired reaction mixture.
• the formulation for storage of the polymerase variant or for an amplification reaction may be the same or different.
• a variant Family D polymerase may be prepared for storage as a reagent or included in a mastermix for storage.
• a polymerase may be included in a reaction buffer or mastermix.
• the reaction mix and the storage mix may be the same or different.
• a storage mix may be a concentrated form of the reagent for dilution into a reaction mix.
• the polymerase variant may be in a lyophilized form.
• a polymerase variant may be in a master mix containing deoxyribose nucleoside triphosphates (e.g., one, two, three or all four of dATP, dTTP, dGTP and dCTP and/or one or more modified dNTPs) and, optionally, ribonucleoside triphosphates.
• concentration of the one or more labeled NTP in a composition may be in the range of 10 nM to 200 mM.
• the molar ratio of a labeled NTP to the corresponding unlabeled version of the same NTP e.g .
• biotin-dCTP to dCTP in the composition may be in the range of 1:1000 to 1000:1, e.g., 1:100 to 100:1 or 1:10 to 10:1.
• the molar ratio of the labeled dNTP to the corresponding unlabeled dNTP (e.g. biotin- dCTP to dCTP) in the nucleotide mix may be in the range of 1:1000 to 1:100, 1:100 to 1:10, 1:10 to 1:1, 1:1 to 1:10, 1:10 to 1:100, or 1:100 to 100:1000.
• the composition may optionally comprise primers.
• the primers may be partially or complete random.
• the primers may be exonuclease-resistant.
• primers may comprise one or more chemical modifications, for example, phosphorothioate modifications.
• the chemical modifications on the primers may occur at 3 ’-terminal or both of 3’ and 5’ terminals of the primers.
• the chemical modifications on the primers may further occur at one or more non-terminal positions of the primers.
• a variant Family D polymerase or, optionally, a composition comprising a variant Family D polymerase may be prepared in any desired form. Examples include a liquid or an emulsion. Alternatively, the variant or composition containing the variant Family D polymerase may be formulated as a solid, granule, lyophilized state, gel, pellet, or powder. A variant may be immobilized on a solid surface such as a matrix, bead, paper, plastic, resin, column, chip, microfluidic device or other instrument platform where the variant Family D polymerase adheres directly or via an affinity binding domain.
• a variant Family D polymerase may be included in a fusion protein with an affinity binding domain such as a maltose binding protein, a chitin binding domain, a his tag, a self-labeling protein tag (a SNAP-Tag®, New England Biolabs), biotin or combinations thereof.
• An affinity binding domain may be associated with a variant Family D polymerase non-co valently such as via an antibody.
• a composition comprising a variant Family D polymerase may further comprise a second polymerase wherein the second polymerase differs from the variant Family D polymerase.
• the present disclosure further relates to methods of using variant Family D polymerases.
• methods of polynucleotide synthesis including amplification (e.g., isothermal amplification, helicase dependent amplification, rolling circle amplification (RCA), multiple strand displacement amplification (MDA), whole genome amplification), quantitative amplification, and sequencing by synthesis.
• amplification e.g., isothermal amplification, helicase dependent amplification, rolling circle amplification (RCA), multiple strand displacement amplification (MDA), whole genome amplification
• MDA multiple strand displacement amplification
• sequencing by synthesis e.g., sequencing by synthesis.
• a method of synthesizing a copy of a polynucleotide template may comprise contacting the polynucleotide template with (a) a variant Family D polymerase, (b) dNTPs and at least one of an rNTP and a modified NTP, and (c) optionally, at least one primer having a sequence complementary to at least a portion of the sequence of the polynucleotide template for a time and at a temperature to produce the copy of the polynucleotide template.
• the need for a primer may be obviated where the template polynucleotide is hybridized to a complementary strand.
• a method of polynucleotide synthesis may include combining a polynucleotide template (e.g ., a target DNA) to be amplified with a variant Family D polymerase, dNTPs, one or more rNTPs, and optionally, one or more primers to produce a reaction mixture; and incubating the reaction mixture to synthesize one or more copies of the template.
• a polynucleotide template may comprise one or more uracils (e.g., cytosines that have been oxidized to uracil). Synthesized fragments may be used for any analytical purpose (e.g., sequencing), synthetic purpose (e.g., cloning), or diagnostic purpose (e.g., single nucleotide polymorphism (SNP) detection).
• a method of filling in a gap in an otherwise double- stranded DNA molecule comprises contacting the DNA molecule with (a) a variant Family D polymerase, (b) dNTPs, and (c) optionally, rNTPs for a time and at a temperature to fill in the gap.
• a variant Family D polymerase may be used to synthesize synthetic molecules containing modified nucleotides.
• a variant Family D polymerase may be used to synthesize modified nucleic aptamers for use as inhibitors or therapeutics.
• a method may comprise contacting an aptamer template polynucleotide with a variant Family D polymerase, dNTPs, and at least one rNTP. Aptamers synthesized by variant Family D polymerase with partial or total substitutions with ribonucleotides increase the chemical diversity and functionality of the aptamer molecule.
• a variant Family D polymerase may be used to synthesize synthetic molecules containing modified nucleotides.
• a variant Family D polymerase may be used to synthesize modified nucleic aptamers for use as inhibitors or therapeutics.
• a method may comprise contacting an aptamer template polynucleotide with a variant Family D polymerase, dNTPs, and at least one modified NTP. Aptamers synthesized by variant Family D polymerase with partial or total substitutions with 2'-modified nucleotides increase the chemical diversity and functionality of the aptamer molecule.
• Variant Family D polymerases may be used to site-specifically label DNA by incorporation of a nucleotide containing a 2’ modification, for example 2’-azido-ATP.
• This 2’ modification could be a molecular handle which allows for reactivity of the nucleotide, and in turn, DNA into which it has been incorporated.
• a modified nucleotide could be incorporated into double- stranded polynucleotide at a primer/template junction, or at a nick or gap site by incubating (a) a variant Family D polymerase, (b) a DNA molecule, and (c) a modified nucleotide for a time and temperature to allow for incorporation of the modified nucleotide into a synthetized copy of the DNA.
• the molecular handle (i.e. azido group) present on the modified nucleotide can be a reactive group that allows for DNA labeling, i.e. click-chemistry (Nat Chem Biol. 2017 Sep 19; 13(10): 1057. doi: 10.1038/nchembio.2482. PMID: 28926550).
• the incorporation of modified nucleotides by a variant Family D polymerase could deem the newly synthesized molecule resistant to exonucleases, and therefore protect the molecule from degradation in vivo.
• variant Family D polymerases with the ability to readily incorporate ribonucleotides during synthesis may be used in RNA sequencing.
• variant Family D polymerase synthesizes DNA molecules with partial ribonucleotide substitution by replacing a single dN with a rN.
• a variant Family D polymerase is contacted with an RNA template and dA, rATP, dCTP, dGTP and dTTP so synthetic products contain.
• dA, rA, dC, dG, dT are examples of synthetic products contain.
• C produces molecules with dA, dC, rC, dG, dT
• G produces molecules with dA, dC, dG, rG, dT
• T produces molecules with dA, dC, dG, dT, rU.
• produced polynucleotide molecules are treated with alkaline or RNaseH2 to cleave at ribonucleotide sites, resulting in a population of products, each terminating with a rNTP. Products are separated by electrophoresis and analyzed to determine the sequence pattern.
• variant Family D polymerase may be used to create polynucleotides resistant to one or more restriction enzymes. Restriction enzymes recognize and cleave specific DNA sequences. Modifying that recognition sequence (e.g., by methylating cytosine in the recognition sequence) may block restriction enzyme cleavage.
• a method for producing a restriction enzyme resistant polynucleotide may comprise contacting a template polynucleotide having a recognition sequence for a restriction enzyme with a variant Family D polymerase, deoxyribonucleotides as dNTPs and a ribonucleotide as rNTP to produce a polynucleotide having a modified recognition sequence for the restriction enzyme, the modified recognition sequence comprising the ribonucleotide.
• Incorporation of rNTPs by variant Family D polymerases may modify restriction enzyme recognition sequences from DNA to partially or fully ribosubstituted molecules that may block restriction enzyme cleavage. This technique could enable selective restriction enzyme cleavage of certain polynucleotides in populations of molecules.
• Variant Family D polymerase can also introduce modifications to enable site-specific cleavage.
• a template polynucleotide comprising a primer binding sequence and a target nucleotide adjacent to the 5’ end of the primer binding sequence, may be contacted with a primer having 5’ end, a 3’ end, and a sequence complementary to the primer binding sequence, a variant Family D polymerase, and a ribonucleotide as rNTP complementary to the target nucleotide to form a reaction mixture and produce an extended primer comprising the ribonucleotide at its 3’ end.
• the reaction mixture may be contacted with dNTPs to further extend the extended primer.
• Variant Family D polymerases may have an even higher capacity to discriminate against rNTP compared to wild type.
• the incorporation ratio of adenosine to adenine (rA:dA) may be less than 0.25, less than 0.24, less than 0.23, less than 0.22, less than 0.21, or less than 0.20.
• a variant Family D polymerase may comprise an aspartate or glutamate substitution at the position corresponding to position 931 of SEQ ID NOS:l, 2, 3 or 4.
• Products produced by variant Family D polymerases e.g ., comprising H931D and H931E substitutions
• products produced by variant Family D polymerases may include fewer rNMPs than products produced by wild type polymerases or other variant Family D polymerases.
• kits including a variant Family D DNA polymerase as described herein.
• a kit may include a variant Family D DNA polymerase and dNTPs, rNTPs, primers, other enzymes (e.g., other polymerases, enzymes other than polymerases, or both), buffering agents, or combinations thereof.
• a variant Family D polymerase may be included in a storage buffer (e.g., comprising glycerol and a buffering agent).
• a kit may include a reaction buffer which may be in concentrated form, and the buffer may contain additives (e.g. glycerol), salt (e.g. KC1), reducing agent, EDTA or detergents, among others.
• a kit comprising dNTPs may include one, two, three of all four of dATP, dTTP, dGTP and dCTP.
• a kit comprising rNTPs may include one, two, three of all four of rATP, rUTP, rGTP and rCTP.
• a kit may further comprise one or more modified nucleotides.
• the kit may optionally comprise one or more primers (random primers, bump primers, exonuclease-resistant primers, chemically-modified primers, custom sequence primers, or combinations thereof).
• One or more components of a kit may be included in one container for a single step reaction, or one or more components may be contained in one container, but separated from other components for sequential use or parallel use. The contents of a kit may be formulated for use in a desired method or process.
• a kit contains: (i) a variant Family D polymerase; and (ii) a buffer.
• the variant polymerase may have a lyophilized form or may be included in a buffer ( e.g ., a storage buffer or a reaction buffer in concentrated form).
• the kit may contain the variant polymerase in a mastermix suitable for receiving and amplifying a template nucleic acid.
• the DNA polymerase may be a purified enzyme so as to contain substantially no DNA or RNA and no nucleases.
• the reaction buffer in (ii) and/or storage buffers containing the DNA polymerase in (i) may include non-ionic, ionic e.g. anionic or zwitterionic surfactants and crowding agents.
• a kit may further include one or more dNTPs including dNTPs with large adducts such as a fluorescent-label or biotin-modified nucleotide, or a methylated nucleotide or other modified nucleotide.
• the kit may include the DNA polymerase and reaction buffer in a single tube or in different tubes.
• Steric gate motifs of wild type Family D polymerases from representative members of the archaeal phyla were aligned using the Multiple Sequence Alignment Tool Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).
• Table 3 shows that the Pro-His-Thr triplet (SEQ ID NO: 15) is conserved among Euryarchaeota, Bathyarchaeota, Thaumarchaeota, Aigarchaeota, Korarchaeota, Odinarchaeota, Lokiarchaeota, Thorarchaeota and Heimdallarchaeota.
• Invariant motif amino acids P930 and T932 are underlined and invariant steric gate amino acid H931 is shown in bold.
• a steric gate amino acid will block ribonucleotide incorporation at low ribonucleotide concentrations. Altering steric gate amino acid may reduce discrimination against ribonucleotides at low concentration.
• Family D DNA polymerases the position of the rNTP 2'-OH in the active site had not been identified. To identify the determinants of ribonucleotide discrimination in Family D DNA polymerases, active site amino acid variants were screened for increased incorporation at low ribonucleotide (1 mM) concentration.
• a nucleotide incorporation assay was performed to determine the ratio of rATP to dATP incorporated by wild type (H931) and variant Family D polymerases (H931X).
• a primer- template was used to monitor rATP and dATP incorporation and was prepared by annealing the 50mer 5’-FAM primer (10 mM) (5'-FAM-AGT GAA TTC GAG CTC GGT ACC CGG GGA TCC TCT AGA GTC GAC CTG CAG GT-3' (SEQ ID NO: 29)) to the 67mer template (15 mM) (5'-AAG CAC GAA AGC AGG GTA CCT GCA GGT CGA CTC TAG AGG ATC CCC GGG TAC CGA GCT CGA ATT CAC T-3' (SEQ ID NO: 30)) in IX
• ThermoPol buffer (20 mM Tris- HC1, 10 mM (NH 4 )2S0 4 , 10 mM KC1, 2 m
• a 10 pi aliquot of polD (SEQ ID NO: 4) or H931X DNA was prepared by mixing ThermoPol buffer (IX final concentration), primer/template DNA (40 nM final concentration) and polD- (2 pi of heat lysate containing over-expressed polD or H931X).
• a second 10 m ⁇ aliquot was prepared by mixing ThermoPol buffer (IX final concentration) and dATP or ATP (2 mM final concentration).
• the polD/DNA and dATP or ATP aliquots were mixed (20 pL reaction) and placed at 65 °C for 10 min, after which 20 uL of 50 mM EDTA were added.
• a negative control reaction was performed in which 10 uL of lx Thermopol buffer was added to the polD/DNA mixture instead of nucleotides.
• Reaction products were separated by capillary electrophoresis using a 3730x1 Genetic Analyzer (Applied Biosystems) and fluorescent peaks were analyzed using Peak Scanner software version 1.0 (Applied Biosystems). The percentage of ribonucleotide incorporation was divided by the percentage of deoxyribonucleotide incorporation to obtain the rA:dA ratio. All assays were performed in triplicate to ensure experiment reproducibility.
• Results are shown in FIGURE 2, in which it can be seen that wild type polD incorporated rATP and dATP at a ratio of 0.26 and variant Family D polymerases incorporated rATP and dATP at a ratio ranging from 0.2 to 0.92 with polD H931A having the highest ratio of rATP to dATP incorporation.
• a nucleotide incorporation assay was performed to determine the ability of polD and polD H931Ato incorporate successive ribonucleotides into a primer/template (FIGURES 4A and 4B).
• the 50mer DNA primer/ 67mer DNA template was created as described above.
• a construct with a 50 nucleotide RNA primer (same sequence) was also annealed to the 67mer DNA template as described above.
• a 20 uL reaction containing 20 nM nucleotide construct, 100 nM polD or polD H931A, and 1 uM dNTPs or rNTPs in lx Thermopol buffer was placed at 65 °C for 15 mins. The reaction was quenched with equal volume of 50 mM EDTA and analyzed by capillary electrophoresis.
• FIGURE 4 illustrates the ability of variant Family D polymerases have to incorporate successive dNTPs and rNTPs.
• Figure 4A is a sketch illustrating binding of a variant Family D polymerase to a template strand (lower strand) and a primer (upper strand) having a fluorescent label. In the presence of NTPs and dNTPs, incubation at an appropriate temperature for an appropriate time results in extension of the primer.
• the traces shown on the left side of FIGURE 4B show that both polD and polD H931A can readily incorporate successive dNTPs onto DNA and RNA templates. However, the traces on the right show that variant polD H931 A can incorporate 4 successive ribonucleotides onto a DNA template, while polD can only incorporate a single ribonucleotide onto a DNA primer.
• EXAMPLE 5 Enhanced incorporation of 2'-modified nucleotides by variant Family D polymerase
• Modified nucleotides rATP, 3'-dATP (Cordycepin), 2'-amino-ATP, 2'-azido-ATP and 2'- (9-methyl-ATP were from Trilink Biotechnologies.
• the primer-template used to monitor modified nucleotide incorporation was prepared by annealing the 50mer 5’-FAM primer (10 mM) (5'-FAM-AGT GAA TTC GAG CTC GGT ACC CGG GGA TCC TCT AGA GTC GAC CTG CAG GT-3' (SEQ ID NO: 29)) to the 62mer Template (15 pM) (5'-AAG CAC GAA AGC AGG GTA CCT GCA GGT CGA CTC TAG AGG ATC CCC GGG TAC CGA GCT CGA ATT CAC T-3' (SEQ ID NO: 30)) in IX ThermoPol buffer (20 mM Tris-HCl, 10 mM (NH 4 ) 2 S0 4 , 10 m
• a 100 pi reaction mix was prepared containing IX ThermoPol Buffer, 10 nM primer-template, 25 nM wild type Family D DNA polymerase (polD) or variant Family D DNA polymerase (polD/H931A) and 1 pM modified nucleotide. Reactions were incubated for 1 minute at 65°C and 10 pi aliquots were removed and mixed with 50 mM EDTA to halt the reaction. Reaction products were separated by capillary electrophoresis using a 3730x1 Genetic Analyzer (Applied Biosystems) and fluorescent peaks were analyzed using Peak Scanner software version 1.0 (Applied Biosystems). The concentration of product (51 nt DNA with a FAM label) was graphed as a function of time (Table).
• PolD incorporated rATP, 3'-dATP, 2'-amino-ATP, 2'-azido-ATP and 2'- (9-methyl- ATP poorly (Table 4, middle column).
• PolD/H931A incorporated 2'-modified nucleotides rATP, 3'- dATP, 2'-amino-ATP, 2'-azido-ATP and 2'- (9-methyl- ATP with higher yield than polD (Table 4, right column).
• EXAMPLE 6 Enhanced incorporation of modified nucleotides by variant Family D polymerase Additional variant positions with the potential to impact activity and/or selectivity of a variant Family D polymerase were prepared as summarized in Table 5.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Wood Science & Technology (AREA)
• Molecular Biology (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• Genetics & Genomics (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Biomedical Technology (AREA)
• Medicinal Chemistry (AREA)
• Enzymes And Modification Thereof (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=74859517

Family Applications (1)

Country Status (2)

Families Citing this family (1)

Family Cites Families (5)

• 2021
  • 2021-02-12 EP EP21710368.8A patent/EP4103701A1/de active Pending
  • 2021-02-12 WO PCT/US2021/017956 patent/WO2021163561A1/en not_active Ceased

Also Published As

Similar Documents

Legal Events