EP4680764A2 - Assay für rekombinase-zugängliches chromatin sowie zugehörige zusammensetzungen und verfahren - Google Patents

Assay für rekombinase-zugängliches chromatin sowie zugehörige zusammensetzungen und verfahren

Info

Publication number: EP4680764A2
Authority: EP; European Patent Office
Prior art keywords: nucleic acid; dna sequence; target dna; biological sample; target
Prior art date: 2023-03-17
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.): Pending

Application number

EP24717528.4A

Other languages

English (en)

French (fr)

Inventor

Joseph M. Beechem

David Ruff

Lidan WU

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Bruker Spatial Biology Inc

Original Assignee

Bruker Spatial Biology Inc

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2023-03-17

Filing date

2024-03-15

Publication date

2026-01-21

2024-03-15 Application filed by Bruker Spatial Biology Inc filed Critical Bruker Spatial Biology Inc

2026-01-21 Publication of EP4680764A2 publication Critical patent/EP4680764A2/de

Status Pending legal-status Critical Current

Links

Classifications

- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions

Definitions

the present disclosure provides for the first time, loci-specific methods of amplifying and sequencing double-stranded DNA molecules under conditions that can preserve one or more of tissue morphology, chromatin patterns and RNA molecule integrity, thereby detecting the presence and abundance of said double-stranded DNA molecules and/or sequences.
the present disclosure provides a method for sequencing at least one target DNA sequence in a biological sample, the method comprising: a 1 ) contacting the biological sample with a solution comprising a plurality of a first recombinase proteins; a plurality of a second recombinase proteins; a plurality of single-stranded DNA binding proteins; and a plurality of primer pairs, wherein individual primer pairs comprise a first primer and a second primer; wherein the first recombinase proteins, second recombinase proteins, single-stranded DNA binding proteins and primer pairs interact with at least one double- stranded DNA molecule comprising the at least one target DNA sequence; b 1 ) performing at least one amplification reaction using the primer pairs that interact with the at least one double- stranded DNA molecule, thereby producing a plurality of a
individual primers in the primer pairs comprise: target binding domain that binds to one strand of the at least one double-stranded DNA molecule comprising the at least one target DNA sequence; and at least one tail domain, optionally wherein the plurality of primer pairs comprises at least two species of primer pairs, wherein the first primer and second primers of distinct primer probe species comprise unique target binding domains, thereby allowing for sequencing of at least two target DNA sequences in the biological sample.
the at least one tail domain comprises at least one primer binding site, and optionally wherein the primer binding site is suitable for sequencing library preparation.
a single oligonucleotide comprises the first primer and the second primer.
sequencing the amplification products produced in step (b1) comprises preparing a sequencing library using the amplification products, or next-generation sequencing (NGS).
performing at least one amplification reaction comprises: contacting the biological sample with a plurality of strand displacing polymerases, optionally wherein the strand displacing polymerases are Bsu polymerases; or optionally contacting the biological sample with at least one crowding agent, optionally wherein the at least one crowding agent is selected from a polyethylene glycol, dextran and Ficoll.
a method for determining the abundance and spatial position of at least one target DNA sequence in a biological sample comprising: a1) contacting the biological sample with a solution comprising: a plurality of a first recombinase proteins; a plurality of a second recombinase proteins; a plurality of single- stranded DNA binding proteins; and a plurality of primers; wherein the first recombinase proteins, second recombinase proteins, single-stranded DNA binding proteins and primers interact with at least one double-stranded DNA molecule comprising the at least one target DNA sequence, thereby exposing the at least one target DNA sequence; b 1 ) contacting the biological sample with a plurality of nucleic acid probes thereby binding a nucleic acid probe to the exposed at least one target DNA sequence, wherein the nu
detecting the ligated probes using RCA comprises a rolling circle amplification step prior to a detection step, optionally wherein the detection step comprises identifying concatemers produced by the RCA by hybridization or sequencing.
the method further comprises a cleavage step to release the ligated probe or concatemer produced by RCA, optionally wherein the cleavage comprises a light-activatable cleavage.
the method further comprises determining the abundance and/or spatial position of the at least one target DNA sequence based on the ligated probes detected in step (d 1 ).
detecting the ligated probes using RCA comprises: optionally, treating the biological sample to produce an acrylamide gel matrix; i) amplifying the ligated nucleic acid probes by contacting the biological sample with a plurality of RCA polymerases; a plurality of RCA primers; and a plurality of dNTPs, wherein the plurality of dNTPs optionally comprise a plurality of fixable nucleotides, optionally wherein the fixable 3 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) nucleotides comprise aminoallyl-dUTPs; ii) treating the biological sample with paraformaldehyde or other reactive NH2 modifying agent to crosslink to amplification products produced in step (i) to the biological sample directly or the acrylamide gel matrix produced in step (i); and iii) contacting the biological sample with a plurality of reporter probes, wherein the reporter probes
a method for determining the abundance and spatial position of at least one target DNA sequence in a biological sample comprising: a 1 ) contacting the biological sample with a solution comprising: a plurality of a first recombinase proteins; a plurality of a second recombinase proteins; a plurality of single- stranded DNA binding proteins; and a plurality of primers; wherein the first recombinase proteins, second recombinase proteins, single-stranded DNA binding proteins and primers interact with at least one double-stranded DNA molecule comprising the at least one target DNA sequence, thereby exposing the at least one target DNA sequence; b1) contacting the biological sample with a plurality of nucleic acid probe pairs, thereby binding a nucleic acid probe pair to the exposed at least one target DNA sequence, wherein the nucleic acid probe pair comprises a first nucleic acid probe and second nucleic acid probe, wherein
the barcode domains of the nucleic acid probes comprise at least two, or at least three, or at least four attachment positions
the method further comprises, after step (f 1 ) and prior to step (g 1 ): (f 2 ) removing the detectable labels of the bound reporter probes; and (f 3 ) repeating steps (e 1 ) – (f 2 ) until each attachment position in the barcode domains of the first nucleic acid probes and/or second nucleic acid probes bound to the target DNA sequences in the biological sample have been bound to a reporter probe comprising at least one detectable label; and wherein step (g 1 ) comprises determining the abundance and spatial position of the target DNA sequence in the biological sample based on the sequence in which the detectable labels were recorded.
the barcode domains of the nucleic acid probes comprise at least two, or at least three, or at least four attachment positions
the method further comprises, after step (g1) and prior to step (h1): (g2) removing the detectable labels of the bound reporter probes; and (g 3 ) repeating steps (f 1 ) – (g 2 ) until each attachment position in the barcode domains of the first nucleic acid probes and/or second nucleic acid probes bound to the target DNA sequences in the biological sample have been bound to a reporter probe comprising at least one detectable label; and wherein step (h 1 ) comprises determining the abundance and spatial position of the target DNA sequence in the biological sample based on the sequence in which the detectable labels were recorded.
the target binding domains are single-stranded polynucleotides comprising a nucleic acid sequence that is complementary to the target DNA sequence, optionally wherein the target binding domains are about 35 to about 40 nucleotides in length, and optionally wherein the target binding domains comprise D-DNA;
the barcode domains are a single-stranded polynucleotide comprising at least one attachment region, optionally wherein each attachment region comprises about one attachment sequence, optionally wherein each of the attachment sequences is about 14 nucleotides in length, optionally wherein the sequences of each of the attachment sequences are different, and optionally wherein the barcode domain comprises L-DNA; and/or the reporter probes comprise a primary nucleic acid molecule comprising a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the second domain of the primary nucleic acid molecule is hybridized to six secondary nucleic acid molecules, wherein individual secondary nucleic acid molecules, where
the method further comprises e1) determining the abundance and spatial position of the at least one target DNA sequence in the biological sample based at least in part on the detectable labels that were recorded in step (d1).
the target DNA sequence is located within a region of open chromatin of genomic DNA.
the target DNA sequence comprises a single nucleotide variant of interest.
the plurality of nucleic acid probes comprises at least two species of nucleic acid probes, wherein the two species of nucleic acid probes comprise unique target binding domains that bind to different target DNA sequences, 7 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) thereby allowing for the determination of the abundance and spatial position of at least two target DNA sequences in the biological sample.
the interaction of the first recombinase proteins, second recombinase proteins, single-stranded DNA binding proteins and primers with the at least one double-stranded DNA molecule results in the denaturing of at least a portion of the double-stranded DNA molecule comprising the target DNA sequence, thereby allowing for the hybridization of a nucleic acid probe pair to the target DNA sequence.
the plurality of primers comprise one species of primer; two species of primers; at least two species of primers; or at least three species of primers.
the first primer and the second primer of individual primer pairs bind to the at least one double-stranded DNA molecule within about 50 nucleotides of the target DNA sequence; about 100 nucleotides of the target DNA sequence; about 250 nucleotides of the target DNA sequence; about 500 nucleotides of the target DNA sequence; about 750 nucleotides of the target DNA sequence; or about 1000 nucleotides of the target DNA sequence.
the method further comprises removing unbound primer pairs.
the biological sample is a tissue sample.
the tissue sample is a fresh frozen tissue sample; or a fixed tissue sample, optionally wherein the fixed tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample.
FFPE formalin-fixed, paraffin-embedded
the first recombinase proteins and the second recombinase proteins comprise the same species of recombinase proteins.
the first recombinase proteins and the second recombinase proteins comprise different species of recombinase proteins.
the first recombinase proteins comprise T4 uvsX recombinase proteins.
the second recombinase proteins comprise T4 uvsY recombinase proteins.
the single-stranded DNA binding proteins comprise T4 Gene 32 Proteins.
provided herein is a system or apparatus for performing the method of any one of the above aspects or embodiments. [0035] Any of the above aspects or aspects described herein can be combined with any other aspect. 8 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) [0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
FIGs.1A, 1B and 1C are schematic diagrams of existing methods of probing double- stranded DNA.
FIGs.2A, 2B and 2C are exemplary schematics of methods of the present disclosure that expose a target DNA sequence within a double-stranded DNA molecule.
FIGs.3A and 3B are images of gel analysis of amplification reactions performed using the methods of the present disclosure to amplify specific gene fragments.
FIGs. 4A, 4B, 4C and 4D are exemplary schematics of methods of the present disclosure that expose a target DNA sequence within a double-stranded DNA molecule and detect the target DNA sequence, e.g., following rolling circle amplification (RCA). 9 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) [0043]
FIGs.5A – 5B are schematic diagrams of exemplary nucleic acid probes of the present disclosure.
the barcode domain of the nucleic acid probe comprises four attachment positions and two target binding domains (FIG. 5A) or a single target binding domain (FIG.5B).
FIG.6 is a schematic diagram of an exemplary reporter probe of the present disclosure.
FIGs.7A, 7B, 7C, 7D, 7E, 7F, 7G and 7H are exemplary schematics of the steps of a method of detecting the abundance and spatial location of more than one species of target DNA sequence in a biological sample.
FIG.8A and 8B are exemplary schematics of the use of one or two barcode domains following the ligation of nucleic acid probe pairs in the methods of the present disclosure.
FIGs.9A and 9B are exemplary schematics showing the use of the methods of the present disclosure for the detection of specific SNVs.
FIG.10 is an exemplary schematic showing the use of the methods of the present disclosure for the detection of specific SNVs.
FIG.11 is an exemplary schematic showing the use of the methods of the present disclosure for the detection of specific SNVs.
DETAILED DESCRIPTION [0050] The present disclosure provides methods for sequencing at least one target DNA sequence in a biological sample. The preceding methods can be multiplexed to sequence a plurality of different target DNA sequences (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more different target DNA sequences).
the present disclosure provides methods for determining the abundance and/or spatial position of at least one target DNA sequence in a biological sample. These preceding methods can also be combined with existing methods to concurrently detect RNA molecules and/or protein molecules, in addition to the target DNA sequence(s).
the present disclosure also provides nucleic acid probes and kits for use in the methods described herein.
Recombinase-based methods of the present disclosure are based on, inter alia, the surprising discovery that recombinase proteins, optionally in combination with single-stranded DNA binding proteins, can be used to one or more “insert” nucleic acid primers into double- stranded DNA molecules within a biological sample such that the one or more nucleic acid 10 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) primers bind to the individual strands of the double-stranded DNA molecules. These bound primers can then be utilized with various further applications.
DNA within a cell e.g., genomic DNA and mitochondrial DNA
genomic DNA and mitochondrial DNA exist primarily as double-stranded molecules, which precludes the binding of single-stranded nucleic acid primers that are typically used to amplify other nucleic acid species such as RNA (see FIG.1A).
FIG.1A is an exemplary schematic that shows a double-stranded DNA molecule that includes a target DNA sequence of interest (shown as Target DNA sequence #1). Nucleic acid primers that are complementary to the target DNA sequence are unable to bind because they are physically excluded by the double-stranded structure of the DNA molecule.
genomic DNA is typically found wrapped around octamers of histones into nucleosomes.
the DNA that is wrapped around these histones is typically inaccessible to proteins such as transcriptase proteins, which precludes the expression of genes on these pieces of DNA. This is typically referred to as a “closed” chromatin state or as “heterochromatin” (see FIG.1C).
other parts of the DNA are not wrapped around the histones, rendering them accessible to proteins, allowing for the expression of genes on these pieces of DNA.
Histones and chromatin are continually remodeled within the cell, meaning the same piece of DNA comprising a certain gene can transition from being located within an open chromatin area to a closed chromatin area, based on a variety of factors including cell cycle, environmental cues and disease states. Accordingly, probing what specific stretches of DNA are open or closed at a given moment in time can give insight into cellular states, including disease states. Thus, there is a need to be able to specifically probe open vs. closed states of chromatin.
the present disclosure uses recombinase proteins, optionally in combination with single-stranded DNA binding proteins, to target specific regions of double-stranded DNA for denaturation and subsequent invasion of the double-stranded DNA by nucleic acid primers.
this targeted denaturation/probe invasion is accomplished in the methods of the present disclosure by treating a biological sample with a combination of one or more pluralities of recombinase proteins, one or more pluralities of single-stranded DNA binding proteins, and one or more plurality of primers that target the recombinase proteins to predetermined DNA sequences.
treating a biological sample with one or more pluralities of recombinase proteins, one or more pluralities of single-stranded DNA binding proteins and one or more pluralities of nucleic acid primer pairs allows for the recombinase proteins, single-stranded DNA proteins and primers to interact with at least one double-stranded DNA molecule to insert one or more primer pairs into the double-stranded DNA molecule, thereby allowing the target DNA sequence to be amplified.
the interaction of the one or more pluralities of recombinase proteins, one or more pluralities of single-stranded DNA binding proteins and one or more pluralities of nucleic acid primer pairs with the at least one double-stranded DNA molecule results in the denaturing of at least a portion of the double-stranded DNA molecule, thereby allowing for the hybridization of the nucleic acid primer pairs to the target DNA sequence.
the primers can be designed to bind within a certain distance of a target DNA sequence to allow targeted denaturation of double-stranded DNA molecules at or near the locations that include the target DNA sequences.
the methods of the present disclosure provide, for the first time, the ability to specifically target regions of double- stranded DNA molecules (e.g., genomic DNA molecules) for denaturation and subsequent binding of nucleic acid primer pairs for amplification.
double-stranded DNA molecules e.g., genomic DNA molecules
existing methods such Assay for Transposase-Accessible Chromatin with high throughput sequencing (hereafter referred to as “ATAC-Seq”) are not sequence specific and therefore indiscriminately sample 12 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) all accessible double-stranded DNA in a cell.
the methods of the present disclosure increase the sensitivity, specificity and accuracy of detection by reducing the potential for nonspecific binding of nucleic acid primers to non-targeted regions.
a single species of recombinase proteins is used in the methods of the present disclosure.
a combination of species of recombinase proteins are used in the methods of the present disclosure.
a combination of recombinase proteins can comprise a first recombinase protein and a second recombinase protein.
the tissue sample is contacted with a primer pair comprising a first primer and second primer that target the recombinase proteins to portions of a double- stranded DNA molecule that flank the target DNA sequence, thereby allowing the recombinase proteins, in combination with the single-stranded DNA binding proteins, to denature said portions.
FIG.2A shows a schematic diagram in which a primer pair is used in combination with one or more recombinase species, single-stranded DNA binding proteins and ATP to selectively denature a portion of a double-stranded DNA molecule comprising a target DNA sequence.
a target DNA sequence (Target DNA sequence #1) is located within a double-stranded DNA molecule and is flanked by primer binding site #1 and primer binding site #2.
the sample is treated with a combination of recombinases (e.g., UvsX, UvsY, etc.), a primer species specific to primer binding site #1, a primer species specific to primer binding site #2, single-stranded DNA binding proteins (e.g., T4 gene 32) and ATP.
the primers and recombinases bind to create primer-recombinase complexes. These complexes then invade the double-stranded DNA molecule, locally denaturing the double-stranded DNA around primer binding sites to create “bubble” structures.
a primer species or primer pair may bind sequentially to the recombinase to form a complex, wherein the complex binds to the target DNA sequence.
a primer species or primer pair and recombinase may bind individually to the target DNA sequence.
the sample can be further treated 13 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) with a strand-displacing polymerase (e.g., Bsu polymerase), dNTPs and single-stranded DNA binding proteins to allow for extension of the bound primers.
a strand-displacing polymerase e.g., Bsu polymerase
dNTPs single-stranded DNA binding proteins
the strand-displacing polymerase will continue to extend the primer until it reaches the “bubble” structure formed by the other primer-recombinase complex bound to the double-stranded DNA molecule.
the recombinase- based methods of the present disclosure also allow for the preservation of native chromatin states. As shown in FIG.2C, the recombinase proteins can mediate the localized denaturation of specific regions with open DNA, allowing these regions to be interrogated using the probes of the present disclosure.
the recombinase protein cannot mediate the localized denaturation of specific regions of closed DNA, meaning those regions will not be rendered accessible to the probes of the present disclosure. Accordingly, the absence of signal from said probes can inform the user of the method that this region of DNA is located within a closed DNA.
the tissue sample is contacted with a single primer species that targets the recombinase proteins to the portion of a double-stranded DNA molecule that includes the target DNA sequence, thereby allowing the recombinase proteins, in combination with the single-stranded DNA binding proteins, to denature said portion.
FIG.4A shows a schematic diagram in which a single primer species is used in combination with one or more recombinase species, single-stranded DNA binding proteins and ATP to selectively denature a portion of a double-stranded DNA molecule comprising a target DNA sequence.
a target DNA sequence (Target DNA sequence #1) is located within a double-stranded DNA molecule.
the sample is treated with a combination of recombinases (e.g., UvsX, UvsY, etc.), a single primer species specific to target DNA sequence #1, single-stranded DNA binding proteins (e.g., T4 gene 32) and ATP.
the single primer species and recombinases bind to create primer-recombinase complexes. These complexes then invade the double-stranded DNA molecule, locally denaturing the double-stranded DNA around the target DNA sequence to create a “bubble” structure. However, this structure is inherently unstable, as indicated by the double-headed arrow. The single-stranded DNA binding proteins bind to this bubble structure to stabilize it in an ATP-dependent manner. 14 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) [0066] As would be readily appreciated by the skilled artisan, a primer species may bind sequentially to the recombinase to form a complex, wherein the complex binds to the target DNA sequence.
a primer species and recombinase may bind individually to the target DNA sequence.
the tissue sample is contacted with a two primer species that targets the recombinase proteins to the portion of a double-stranded DNA molecule that includes the target DNA sequence, thereby allowing the recombinase proteins, in combination with the single-stranded DNA binding proteins, to denature said portion.
FIG. 4B shows a schematic diagram in which a two primer species are used in combination with one or more recombinase species and single-stranded DNA binding proteins to selectively denature a portion of a double-stranded DNA molecule comprising a target DNA sequence.
the nucleic acid probe pairs themselves can be used to target the recombinase proteins to allow for denaturation of a double-stranded DNA molecule comprising the target DNA sequence (see FIG.4C).
the bound nucleic acid probe pairs can be further amplified, thereby allowing identification of probe pairs in a spatially resolved manner via either in situ or off-sample readout.
the recombinase- based methods of the present disclosure also allow for the preservation of native chromatin states. As shown in FIG.4D, the recombinase proteins can mediate the localized denaturation of specific regions with open DNA, allowing these regions to be interrogated using the probes of the present disclosure. In contrast, the recombinase protein cannot mediate the localized denaturation of specific regions of closed DNA, meaning those regions will not be rendered accessible to the probes of the present disclosure.
the present disclosure provides methods for sequencing at least one target DNA sequence in a biological sample, the method comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; 15 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of a primer pairs, wherein each primer pair comprises a first primer and a second primer; wherein the first recombinase proteins, second recombinase proteins, single-stranded DNA binding proteins and primer pairs interact with at least one double-stranded DNA molecule comprising the at least one target DNA
sequencing the amplification products produced in step (b 1 ) comprises preparing a sequencing library using the amplification products. As would be appreciated by the skilled artisan, any sequence library preparation technique that is known in the art can be used to prepare a sequencing using the amplification products produced by the methods of the present disclosure.
sequencing the amplification products produced in step (b 1 ) comprises next-generation sequencing.
performing at least one amplification reaction can comprise contacting the biological sample with a plurality of strand displacing polymerases.
performing at least one amplification reaction can comprises contacting the biological sample with at least one crowding agent.
the methods of the present disclosure can further comprise after step (a 1 ) and prior to step (b 1 ), removing unbound primer pairs.
the present disclosure provides methods for determining the presence of at least one target DNA sequence in a biological sample, the methods comprising: 16 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of primers; wherein the first recombinase proteins, second recombinase proteins, single- stranded DNA binding proteins and primers interact with at least one double-stranded DNA molecule comprising the at least one target DNA sequence, thereby exposing the at least one target DNA sequence; b 1 ) contacting the biological sample with a plurality of nucleic acid probes thereby binding a nucleic acid probes thereby binding a nucleic acid
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins. 17 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) [0081]
the preceding method can further comprise: e1) determining the presence of the at least one target DNA sequence based on the ligated probes amplified at step (d 1 ).
the present disclosure provides methods for determining the abundance and/or spatial position of at least one target DNA sequence in a biological sample, the methods comprising: a 1 ) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of primers; wherein the first recombinase proteins, second recombinase proteins, single- stranded DNA binding proteins and primers interact with at least one double-stranded DNA molecule comprising the at least one target DNA sequence, thereby exposing the at least one target DNA sequence; b 1 ) contacting the biological sample with a plurality of nucleic acid probes thereby binding a nucleic acid probe to the exposed at least one target DNA sequence, wherein the nucleic acid probes comprise:
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the preceding method can further comprise: e 1 ) determining the abundance and/or spatial position of the at least one target DNA sequence based on the ligated probes amplified at step (d1).
the present disclosure provides methods for determining the presence of at least one target DNA sequence in a biological sample, the methods comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of nucleic acid probes; wherein the nucleic acid probes comprise: i) a first target binding domain that binds to a first portion of the at least one target DNA sequence and that is located at one terminus of the nucleic acid probes; ii) a second target binding domain that binds to a second portion of the at least one target DNA sequence and that is located at the other terminus of the nucleic acid probes; and iii) a barcode domain specific for the at least one target DNA sequence, wherein the first portion of the
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the preceding method can further comprise: d 1 ) determining the presence of the at least one target DNA sequence based on the ligated probes amplified at step (c1).
the present disclosure provides methods for determining the abundance and/or spatial position of at least one target DNA sequence in a biological sample, the methods comprising: a 1 ) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of nucleic acid probes; wherein the nucleic acid probes comprise: i) a first target binding domain that binds to a first portion of the at least one target DNA sequence and that is located at one terminus of the nucleic acid probes; ii) a second target binding domain that binds to a second portion of the at least one target DNA sequence and that is located at the other terminus of the nucleic acid probes; and iii) a barcode domain specific for the at least one target DNA sequence,
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the preceding method can further comprise: d 1 ) determining the abundance and/or spatial position of the at least one target DNA sequence based on the ligated probes amplified at step (c 1 ).
the ligation of target binding domains of a single nucleic acid probe that bound immediately adjacent to each other results in the circularization of the nucleic acid probes that are bound to the target DNA sequences. Accordingly, following ligation, these probes can be referred to as “circularized nucleic acid probes.”
Detecting ligated probes using rolling circle amplification (RCA) can be accomplished using standard RCA detection methods known in the art. Such methods include, but are not limited to, the methods described in Larsson et al.
RCA is an amplification protocol that uses RCA- compatible polymerases to amplify circularized nucleic acids (e.g., the ligated nucleic acid 21 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) probes in the methods of the present disclosure).
the amplification produces single-strand, linear concatenated copies of the circular sequence.
detecting ligated probes using RCA can comprise treating the biological sample to produce an acrylamide gel matrix.
detecting ligated probes using RCA can comprise amplifying the ligated nucleic acid probes by contacting the biological sample with: a plurality of RCA polymerases; a plurality of RCA primers; and a plurality of dNTPs.
the plurality of dNTPs can comprise a plurality of aminoallyl-dUTPs.
the RCA can further comprise treating the biological sample with paraformaldehyde to crosslink to amplification products produced in step (ii) to an acrylamide gel matrix.
detecting ligated probes using RCA comprises fixing, e.g., cross- linking, the nucleic acids, e.g., concatemer, to the extracellular matrix of the biological sample directly.
fixative e.g., formamide, or other reactive NH2 modifying agent as known in the art (e.g., Label It® Reagent from MirusBio®).
detecting ligated probes using RCA can comprise amplifying the ligated nucleic acid probes by contacting the biological sample with a plurality of reporter probes, wherein the reporter probes bind to the barcode domain of the amplified nucleic acid probes, and wherein the reporter probe comprises at least one detectable label.
detecting ligated probes using RCA can comprise: i) treating the biological sample to produce an acrylamide gel matrix; ii) amplifying the ligated nucleic acid probes by contacting the biological sample with: a plurality of RCA polymerases; a plurality of RCA primers; and a plurality of dNTPs, wherein the plurality of dNTPs comprise a plurality of aminoallyl-dUTPs; iii) treating the biological sample with paraformaldehyde to crosslink to amplification products produced in step (ii) to the acrylamide gel matrix produced in step (i); iv) contacting the biological sample with a plurality of reporter probes, wherein the reporter probes bind to the barcode domain of the amplified nucleic acid probes, and wherein the reporter probes comprise at least one detectable label.
detecting ligated probes using RCA does not comprising treating the biological sample to produce the acrylamide gel matrix. 22 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) [00101]
RCA polymerases can be selected from phi29 polymerase and T7 DNA polymerase.
detecting ligated probes using RCA comprises cleavage of the probes or RCA concatemers. This cleavage releases part of the nucleic acid from the biological sample, at a selected time, and thus allows detection and/or identification of the released probes or RCA concatemers.
cleavage is performed after recording the original spatial location of the probe or concatemer in the biological sample.
the cleavage is light-activatable.
the light-activatable cleavage is achieved via photocleavable linker within nucleic acid probes or RCA concatemer.
the light-activatable cleavage is achieved via light-activatable caged chelator (e.g,, DMNP-EDTA, DMNP-EGTA, etc.), which releases bivalent cations that could fragment the nucleic acid when present at high local concentrations.
the light-activatable cleavage is achieved via light-activatable caged chelator and cofactor-dependent nuclease enzymes (e.g., DNase I, RNase H, and restriction endonucleases), whose activity in nucleic acid cleavage depends on the local concentration of bivalent cation cofactor and thus could be modulated by light via photo-caged chelator.
cofactor-dependent nuclease enzymes e.g., DNase I, RNase H, and restriction endonucleases
the detection and identification of the released probes or RCA concatemer is achieved via sequencing methods (e.g., NGS sequencing or sequencing-by-ligation method) or hybridization-based methods (e.g., microarray or fluorescent in situ hybridization).
the present disclosure provides methods of determining the presence of at least one target DNA sequence in a biological sample, the method comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of primers; wherein the first recombinase proteins, second recombinase proteins, single-stranded DNA binding proteins and primers interact with at least one double-stranded DNA molecule comprising the at least one target DNA sequence, thereby exposing the at least one target DNA sequence; 23 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) b1) contacting the biological sample with a plurality of nucleic acid probe pairs,
the preceding method can further comprise: g1) determining the presence of the at least one target DNA sequence in the biological sample based on the detectable labels that were recorded in step (f 1 ).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the present disclosure provides methods for determining the abundance and/or spatial position of at least one target DNA sequence in a biological sample, the method comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of primers; wherein the first recombinase proteins, second recombinase proteins, single-stranded DNA binding proteins and primers interact with at least one double-stranded DNA molecule comprising the at least one target DNA sequence, thereby exposing the at least one target DNA sequence; b 1 ) contacting the biological sample with a plurality of nucleic acid probe pairs, thereby binding a nucleic acid probe pair to the exposed at least one target DNA sequence, wherein the nucleic acid probe pair comprises a first
the preceding method can further comprise: g1) determining the abundance and/or spatial position of the at least one target DNA sequence in the biological sample based on the detectable labels that were recorded in step (f 1 ).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the barcode domains of the first nucleic acid probes and/or the second nucleic acid probes can comprise at least two, or at least three, or at least four attachment positions
the method further comprises, after step (f1) and prior to step (g1): (f 2 ) removing the detectable labels of the bound reporter probes; and (f 3 ) repeating steps (e 1 ) – (f 2 ) until each attachment position in the barcode domains of the first nucleic acid probes and/or second nucleic acid probes bound to the target DNA sequences in the biological sample have been bound to a reporter probe comprising at least one detectable label; and 26 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) wherein step (g1) comprises determining the presence, abundance and/or spatial position of the target DNA sequence in the biological sample based on the sequence in which the detectable labels were recorded.
the preceding method can further comprise: f1) determining the presence of the at least one target DNA sequence in the biological sample based on the detectable labels that were recorded in step (e 1 ).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the present disclosure provides, in some aspects, methods for determining the abundance and/or spatial position of at least one target DNA sequence in a biological sample, the method comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of nucleic acid probe pairs, wherein the nucleic acid probe pair comprises a first nucleic acid probe and second nucleic acid probe, wherein the first nucleic acid probe comprises: i) a target binding domain that binds to a first portion of the at least one target DNA sequence; and ii) a barcode domain specific for the at least one target DNA sequence, wherein the barcode domain comprises at least one attachment position
the preceding method can further comprise: f1) determining the abundance and/or spatial position of the at least one target DNA sequence in the biological sample based on the detectable labels that were recorded in step (e 1 ).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the barcode domains of the first nucleic acid probes and/or the second nucleic acid probes can comprise at least two, or at least three, or at least four attachment positions
the method further comprises, after step (e1) and prior to step (f1): (e 2 ) removing the detectable labels of the bound reporter probes; and (e 3 ) repeating steps (d 1 ) – (e 2 ) until each attachment position in the barcode domains of the first nucleic acid probes and/or second nucleic acid probes bound to the target DNA sequences in the biological sample have been bound to a reporter probe comprising at least one detectable label; and wherein step (f1) comprises determining the presence, abundance, and/or spatial position of the target DNA sequence in the biological sample based on the sequence in which the detectable labels were recorded.
the present disclosure provides, in some aspects, methods for determining the presence of at least one target DNA sequence in a biological sample, the method comprising: a 1 ) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of nucleic acid probe pairs, 32 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) wherein the nucleic acid probe pair comprises a first nucleic acid probe and second nucleic acid probe, wherein the first nucleic acid probe comprises: i) a target binding domain that binds to a first portion of the at least one target DNA sequence; wherein the second nucleic acid probe comprises: i) a target binding domain that binds to a second portion of the at least one target DNA
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the preceding method can further comprise: e 1 ) determining the presence of the at least one target DNA sequence in the biological sample based on the ligated probes that were detected in step (d1).
the present disclosure provides, in some aspects, methods for determining the abundance and/or spatial position of at least one target DNA sequence in a biological sample, the method comprising: 33 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of nucleic acid probe pairs, wherein the nucleic acid probe pair comprises a first nucleic acid probe and second nucleic acid probe, wherein the first nucleic acid probe comprises: i) a target binding domain that binds to a first portion of the at least one target DNA sequence; wherein the second nucleic acid probe comprises: i) a target binding domain that binds to a second portion of the at least
the preceding method can further comprise: e 1 ) determining the abundance and/or spatial position of the at least one target DNA sequence in the biological sample based on the ligated probes that were detected (d1).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
34 299255491 Attorney Docket No: NATE-055/01WO (321329-2902)
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
determining the presence, abundance, and/or spatial position of the at least one target DNA sequence in the biological sample based on the detectable labels that were recorded in step (e1), step (f1), or step (g1) of the methods of the present disclosure comprises determining the presence, abundance, and/or spatial position of the at least one target DNA sequence based on the identity of the detectable labels recorded in step (e1), step (f1), or step (g1) (e.g., in aspects wherein the detectable labels are fluorescent labels, the identity can correspond the color of the detectable label) and/or the order in which the specific detectable labels appeared.
the present disclosure provides methods of determining the abundance and/or spatial position of at least one target DNA sequence in a biological sample, the method comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of primers; wherein the first recombinase proteins, second recombinase proteins, single-stranded DNA binding proteins and primers interact with at least one double-stranded DNA molecule comprising the at least one target DNA sequence, thereby exposing the at least one target DNA sequence; b 1 ) contacting the biological sample with a plurality of nucleic acid probe pairs, thereby binding a nucleic acid probe pair to the exposed at least one target DNA sequence, wherein the nucleic acid probe pair comprises a first
the present disclosure provides methods of determining the presence of at least one target DNA sequence in a biological sample, the method comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of primers; wherein the first recombinase proteins, second recombinase proteins, single-stranded DNA binding proteins and primers interact with at least one double-stranded DNA molecule comprising the at least one target DNA sequence, thereby exposing the at least one target DNA sequence; b 1 ) contacting the biological sample with a plurality of nucleic acid probe pairs, thereby binding a nucleic acid probe pair to the exposed at least one target DNA sequence, wherein the nucleic acid probe pair comprises a first nucleic acid probe
the preceding method can further comprise: g1) determining the presence of the at least one target DNA sequence in the biological sample based on the ligated probes that were detected in step (f 1 ).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the preceding method can further comprise: g 1 ) determining the abundance and/or spatial position of the at least one target DNA sequence in the biological sample based on the ligated probes detected in step (f1).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the present disclosure provides methods of determining the presence of at least one target DNA sequence in a biological sample, the method comprising: 41 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of nucleic acid probe pairs; wherein the nucleic acid probe pair comprises a first nucleic acid probe and second nucleic acid probe, wherein the first nucleic acid probe comprises: i) a target binding domain that binds to a first portion of the at least one target DNA sequence, wherein the target binding domain of the first nucleic acid probe comprises at least one uracil residue; and optionally ii) a barcode domain specific for
the preceding method can further comprise: g1) determining the presence of the at least one target DNA sequence in the biological sample based on the detectable labels that were recorded in step (f 1 ).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the present disclosure provides methods of determining the abundance and/or spatial position of at least one target DNA sequence in a biological sample, the method comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of nucleic acid probe pairs; wherein the nucleic acid probe pair comprises a first nucleic acid probe and second nucleic acid probe, wherein the first nucleic acid probe comprises: i) a target binding domain that binds to a first portion of the at least one target DNA sequence, wherein the target binding domain of the first nucleic acid probe comprises at least one uracil residue; and optionally 43 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) ii) a
the preceding method can further comprise: g1) determining the abundance and/or spatial position of the at least one target DNA sequence in the biological sample based on the detectable labels that were recorded in step (f 1 ).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the barcode domains of the nucleic acid probes can comprise at least two, or at least three, or at least four attachment positions, wherein the method further comprises, after step (f 1 ) and prior to step (g 1 ): (f2) removing the detectable labels of the bound reporter probes; and (f3) repeating steps (e1) – (f2) until each attachment position in the barcode domains of the first nucleic acid probes and/or second nucleic acid probes bound to the target DNA sequences in the biological sample have been bound to a reporter probe comprising at least one detectable label; and wherein step (g 1 ) comprises determining the abundance and spatial position of the target DNA sequence in the biological sample based on the sequence in which the detectable labels were recorded.
the present disclosure provides methods of determining the presence of at least one target DNA sequence in a biological sample, the method comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of nucleic acid probe pairs; wherein the nucleic acid probe pair comprises a first nucleic acid probe and second nucleic acid probe, wherein the first nucleic acid probe comprises: i) a target binding domain that binds to a first portion of the at least one target DNA sequence, wherein the target binding domain of the first nucleic acid probe comprises at least one uracil residue; wherein the second nucleic acid probe comprises: 45 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) i)
the preceding method can further comprise: f1) determining the presence of the at least one target DNA sequence in the biological sample based on the ligated probes detected in step (e 1 ).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the present disclosure provides methods of determining the abundance and/or spatial position of at least one target DNA sequence in a biological sample, the method comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and 46 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) iv) a plurality of nucleic acid probe pairs; wherein the nucleic acid probe pair comprises a first nucleic acid probe and second nucleic acid probe, wherein the first nucleic acid probe comprises: i) a target binding domain that binds to a first portion of the at least one target DNA sequence, wherein the target binding domain of the first nucleic acid probe comprises at least one uracil residue wherein the second nucleic acid probe comprises
the preceding method can further comprise: f 1 ) determining the abundance and/or spatial position of the at least one target DNA sequence in the biological sample based on the detectable labels that were recorded in step (e 1 ).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins. 47 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) [00171]
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the steps of contacting the biological sample with a plurality of ligases, followed by contacting the biological sample with a plurality of uracil-DNA glycosylase enzymes, followed by heating the biological sample to a temperature sufficient to unbind any second nucleic acid probes that were not ligated to first nucleic acid probe means that only first nucleic acid probes and second nucleic acid probes that successfully bind immediately adjacent to each other on exposed target DNA sequences will be subsequently detected using the reporter probes, as only these probes would be successfully ligated.
the use of the ligation, uracil-DNA glycosylase enzyme treatment, and heating steps can allow for an “error-checking mechanism” by removing probes that are not bound adjacent to each other, and therefore most likely represent off- target binding of one probe in a probe pair.
This error-checking mechanism can be used to increase the accuracy, specificity and/or sensitivity of the methods of the present disclosure.
the use of the ligation, uracil-DNA glycosylase (UDG) enzyme treatment, and heating steps can allow the probing of a single-nucleotide variant (SNV), as shown in FIG.10.
FIG.10 shows a non-limiting example wherein the first NA probe has a target binding domain comprising multiple uracil residues and the second NA probe has a target binding domain with a melting temperature (Tm) of approximately 45°C.
Tm melting temperature
NA probe 2 with its “new” “combined” target binding domain will stay bound after heating the sample to approximately 55°C to 60°C.
the probe pair does not match the SNV, no ligation could occur, meaning no “combined” target binding domain would be formed.
the second nucleic acid probe will be removed when the sample is heated to approximately 55°C to 60°C.
Single-stranded DNA binding proteins and recombinase proteins are not shown in FIG.10, for clarity.
the use of ligation can allow the probing of a single-nucleotide variant (SNV) by creating longer, and thus more stable, hybridized probe regions.
SNV single-nucleotide variant
FIG.11 shows a non-limiting example wherein the first NA probe has a target binding domain specific for region “1A” of a target DNA sequencing comprising an SNV and 48 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) a second NA probe is specific for a region “1B” of the target DNA sequence.
the double- stranded DNA molecule comprising the target DNA sequence is treated with a combination of the two NA probes, recombinase proteins, single-stranded DNA binding proteins, ATP, NTPs, and DNA ligase.
a probe pair properly matches an SNV, then ligation is successful. If ligation is successful, a longer hybridized region that is more stable and thus less easily removed is created.
the present disclosure provides methods of determining the presence of at least one target DNA sequence in a biological sample, the methods comprising: a 1 ) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of primers; wherein the first recombinase proteins, second recombinase proteins, single-stranded DNA binding proteins and primers interact with at least one double- stranded DNA molecule comprising the at least one target DNA sequence, thereby exposing the at least one target DNA sequence; b1) contacting the biological sample with a plurality of nucleic acid probes
the preceding method can further comprise: e1) determining the presence of the at least one target DNA sequence in the biological sample based at least in part on the detectable labels that were recorded in step (d 1 ).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the present disclosure provides methods of determining the abundance and/or spatial position of at least one target DNA sequence in a biological sample, the methods comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of primers; wherein the first recombinase proteins, second recombinase proteins, single-stranded DNA binding proteins and primers interact with at least one double- stranded DNA molecule comprising the at least one target DNA sequence, thereby exposing the at least one target DNA sequence; b 1 ) contacting the biological sample with a plurality of nucleic acid probes thereby binding a nucleic acid probe to the exposed at least one target DNA sequence, wherein the nucleic acid probes comprise: i)
the preceding method can further comprise: e1) determining the abundance and/or spatial position of the at least one target DNA sequence in the biological sample based at least in part on the detectable labels that were recorded in step (d 1 ).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the barcode domains of the nucleic acid probes comprise at least two, or at least three, or at least four attachment positions
the method can further comprise, after step (d 1 ) (and optionally prior to step (e 1 )): (d2) removing the detectable labels of the bound reporter probes; and (d3) repeating steps (c1) – (d2) until each attachment position in the barcode domains of the nucleic acid probes bound to the target DNA sequences in the biological sample have been bound to a reporter probe comprising at least one detectable label; and wherein step (e 1 ) comprises determining the abundance and spatial position of the target DNA sequence in the biological sample based at least in part on the sequence in which the detectable labels were recorded.
the present disclosure provides methods of determining the presence of at least one target DNA sequence in a biological sample, the methods comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of nucleic acid probes, wherein the nucleic acid probes comprise: a target binding domain that binds to the at least one target DNA sequence; and a barcode domain specific for the at least one target DNA sequence, wherein the barcode domain comprises at least one attachment position; wherein the first recombinase proteins, second recombinase proteins, single- stranded DNA binding proteins and nucleic acid probes interact with at least one 51 299255491 Attorney Docket No: NATE-055/01WO (321329-290
the preceding method can further comprise: d1) determining the presence of the at least one target DNA sequence in the biological sample based at least in part on the detectable labels that were recorded in step (c 1 ).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the present disclosure provides methods of determining the abundance and/or spatial position of at least one target DNA sequence in a biological sample, the methods comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of nucleic acid probes, wherein the nucleic acid probes comprise: a target binding domain that binds to the at least one target DNA sequence; and a barcode domain specific for the at least one target DNA sequence, wherein the barcode domain comprises at least one attachment position; wherein the first recombinase proteins, second recombinase proteins, single- stranded DNA binding proteins and nucleic acid probes interact with at least one 52 299255491 Attorney Docket No: NATE-055/01WO (
the preceding method can further comprise: d1) determining the abundance and/or spatial position of the at least one target DNA sequence in the biological sample based at least in part on the detectable labels that were recorded in step (c 1 ).
the first recombinase proteins and second recombinase proteins are the same, e.g., comprise a single species of recombinase proteins.
the first recombinase proteins and second recombinase proteins are different, e.g., comprise a combination of species of recombinase proteins.
the barcode domains of the nucleic acid probes comprise at least two, or at least three, or at least four attachment positions
the method can further comprise, after step (c 1 ) (and optionally prior to step (d 1 )): (c2) removing the detectable labels of the bound reporter probes; and (c 3 ) repeating steps (b 1 ) – (c 2 ) until each attachment position in the barcode domains of the nucleic acid probes bound to the target DNA sequences in the biological sample have been bound to a reporter probe comprising at least one detectable label; and wherein step (d 1 ) comprises determining the abundance and spatial position of the target DNA sequence in the biological sample based at least in part on the sequence in which the detectable labels were recorded.
determining the presence, abundance, and/or spatial position of the at least one target DNA sequence in the biological sample based at least in part on the detectable labels that were recorded in step (c1) or step (d1) comprises determining the presence, abundance, and/or spatial position of the at least one target DNA sequence based at least in part on the identity of the detectable labels recorded in step (c 1 ) or step (d 1 ) (e.g., in aspects wherein the detectable labels are fluorescent labels, the identity can correspond the 53 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) color of the detectable label) and/or the order in which the specific detectable labels appeared.
determining the presence, abundance, and/or spatial position of the at least one target DNA sequence in the biological sample based on the ligated probes detected step (c1) or step (d1) of the methods of the present disclosure comprises determining the presence, abundance, and/or spatial position of the at least one target DNA sequence based on the identity of the detectable labels recorded when performing RCA (e.g., in aspects wherein the detectable labels are fluorescent labels, the identity can correspond the color of the detectable label) and/or the order in which the specific detectable labels appeared.
determining the presence, abundance, and/or spatial position of the at least one target DNA sequence in the biological sample based on the ligated probes or detectable labels of the reporter probes can comprise the use of a computer and an accompanying program.
Said program can contain a list of patterns of detectable labels that correspond specific DNA sequences that are to be detected. For example, a detectable label pattern of “Green-Blue-Red” can corresponding to a first target DNA sequence and a detectable label pattern of “Red-Yellow-Red” can correspond to a second target DNA sequence. In some aspects, the pattern could be a single detectable label.
a detectable label of “Green” can correspond to a first target DNA sequence and a detectable label of “Red” can correspond to a second target DNA sequence.
a detectable label of “Green” can correspond to a first target DNA sequence and a detectable label of “Red” can correspond to a second target DNA sequence.
the plurality of nucleic acid probes comprises at least two species of nucleic acid probes, wherein the two species of nucleic acid probes comprise unique target binding domains that bind to different target DNA sequences, thereby allowing for the determination of the presence, abundance, and/or spatial position of at least two target DNA sequences in a biological sample.
nucleic acid probes comprising barcode domains, in combination with labeled-reporter probes, for detecting one or more specific nucleic acid sequences are further detailed in PCT Application Publication No.
each species of target DNA sequence that is to be detected in a biological sample is assigned a predetermined and unique nucleic acid probe that comprises: a) two target binding domains that are complementary to that specific portions of the target nucleic acid (i.e., that is designed such that it only hybridizes to that specific species of target nucleic acid); and b) a unique barcode domain comprising a unique nucleic acid sequence that is specific to that species of target DNA sequence.
the unique nucleic acid sequence of the barcode domain is designed such that a specific reporter probe with a specific detectable label will bind to the barcode domain, thereby allowing detection.
the unique nucleic acid sequence of the barcode domain is designed such that a specific sequence of reporter probes of the present disclosure will bind sequentially to the different attachment regions in the barcode domain, thereby creating a “order of detectable labels” which is specific to that species of target nucleic acid.
FIGs.7A-7H A schematic of a non-limiting example of these methods is shown in FIGs.7A-7H, which shows the detection of two different species of target DNA sequence in a biological sample using the nucleic acid probes of the present disclosure and reporter probes of the present disclosure.
the method begins in FIG.7A with a biological sample that comprises two copies of target DNA sequence #1 (one in the upper left part of the sample and one in the lower right part of the sample) and one copy of target DNA sequence #2 (in the upper right part of the biological sample).
the biological sample is contacted with a plurality of probes of the present disclosure.
the nucleic acid (NA) probes with target binding domains that are complementary to target DNA sequence #1 hybridize to target nucleic acid #1
NA probes with target binding domains that are complementary to target nucleic acid #2 hybridize to target DNA sequence #2.
a third type of probe which has a target binding domain complementary to a third type of target DNA sequence does not hybridize within the biological sample, because the biological sample does not contain the third type of target DNA sequence.
the non-hybridized NA probes are optionally washed off of the biological sample.
the biological sample is contacted with a plurality of reporter probes comprising detectable labels.
the detectable labels are fluorescent labels.
NA probe type #1 is designed such that the first attachment region hybridizes to a reporter probe with a red fluorescent label and NA 55 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) probe type #2 is designed such that the first attachment region hybridizes to a reporter probe with a green-fluorescent label.
probe type #2 is designed such that the first attachment region hybridizes to a reporter probe with a green-fluorescent label.
FIG.7C the identity and spatial location of the detectable labels of the hybridized reporter probes are recorded. Accordingly, during the first round of imaging, a red label was detected in “Location 1”, a green label was detected in “Location 2” and a red label was detected in “Location 3”.
the detectable labels are removed from the hybridized reporter probes.
the reporter probes comprise photocleavable moieties that can be cleaved by illumination with UV light, which releases the detectable labels, which are subsequently washed away.
the biological sample is contacted with a second plurality of reporter probes comprising detectable labels.
the barcode domain of NA probe type #1 is designed such that the second attachment region hybridizes to a reporter probe with a yellow fluorescent label
MA probe type #2 is designed such that the second attachment region hybridizes to a reporter probe with a red fluorescent label.
a seventh step as shown in FIG.7F, the identity and spatial location of the detectable labels of the hybridized reporter probes are recorded. Accordingly, during the second round of imaging, a yellow label was detected in Location 1, a red label was detected in Location 2 and a yellow label was detected in Location 3.
the detectable labels are removed from the hybridized reporter probes by UV-induced cleavage of photocleavable moieties within the reporter probes. [00208] These steps are repeated until each of the attachment regions in each NA probe has been bound by a reporter probe, and the identity of the detectable label of the reporter probe has been recorded.
a “order of detectable labels” will have been recorded at each location of interest.
the order of detectable labels at Location 1 and Location 3 was red-yellow-green- red and the order of detectable labels at Location 2 was green-red-yellow-yellow.
red-yellow-green-red is specific to target DNA sequence #1
green-red-yellow-yellow is specific to target DNA sequence #2
the method has allowed for the identification of two copies of target DNA sequence #1 in the biological sample, with one of the copies being present at Location 1 and one of the copies being present at Location 3, and the identification of one copy of target DNA sequence #2 at Location 2.
FIGs.7A-7H display only one nucleic acid probe of a possible nucleic acid probe pair that have bound to a target DNA sequence and that have been ligated together. From the description of the methods presented herein, it is apparent that a ligated probe pair may be bound to a target DNA sequence and thus two barcode domains will be indirectly bound to the target DNA sequence via the ligated target binding domains of the ligated probes at a given time. This situation is shown in the exemplary schematic presented in FIG.8A.
the target DNA sequence can be probed (e.g., assessed for its presence, signal intensity, abundance and/or spatial location) using the first barcode domain (as shown in Option #1 presented in FIG.8B), the second barcode domain (as shown in Option #2 in FIG. 8B) or both the first and the second barcode domain (as shown in Option #3 in FIG.8B).
Single-stranded DNA binding proteins and recombinase proteins are not shown in FIGs.8A- 8B for clarity.
both of the barcode domains can allow for an “error-checking mechanism”, i.e., the presence of both barcode domains must be verified within the same location of the tissue sample to confirm that both probes of a probe pair were bound to the target DNA sequence and were successfully ligated. If only one barcode is identified at a given location, that barcode identification can be labeled off-target binding of one of the probes of the probe pair.
This error-checking mechanism can be used to increase the accuracy, specificity and/or sensitivity of the methods of the present disclosure.
the use of both of the barcode domains can allow for an increase in detection signal such that it exceeds the background threshold.
the methods of the present disclosure can be used to determine the spatial abundance of at least about 10, or at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or at least about 100, or at least about 110, or at least about 120, or at least about 130, or at least about 140, or at least about 150, or at least about 160, or at least about 170, or at least about 180, or at least about 190, or at least about 200, or at least about 210, or at least about 220, or at least about 240, or at least about 250, or at least about 260, or at least about 270, or at least about 280, or at least about 290, or at least about 300, or at least about 500, or at least about 1,000, or at least about 10,000, or at least about 100,000
the methods of the present disclosure can be used to determine the spatial abundance of about 10, or about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100, or about 110, or about 120, or about 130, or about 140, or about 150, or about 160, or about 170, or about 180, or about 190, or about 200, or about 210, or about 220, or about 240, or about 250, or about 260, or about 270, or about 280, or about 290, or about 300, or about 500, or about 1,000, or about 10,000, or about 100,000, or about 1,000,000 different species of target nucleic acids within a biological sample [00214] In some aspects, the methods of the present disclosure can further comprise after step (
the methods of the present disclosure can further comprise after step (b1) and prior to step (c1), removing unbound reporter probes.
the unbound reporter probes can be removed by washing.
the methods of the present disclosure can further comprise, prior to the addition of reporter probes, removing unbound nucleic acid probes from the sample.
the unbound nucleic acid probes can be removed by washing.
the methods of the present disclosure can further comprise contacting a biological sample with ATP. In a non-limiting example, when a biological sample is contacted with a plurality of single-stranded DNA binding proteins, the biological sample can be concurrently contacted with ATP.
a primer can comprise a target binding domain that binds to one strand of the at least one double-stranded DNA molecule comprising the at least one target DNA sequence.
a primer can comprise at least on tail domain.
a primer can comprise: i) a target binding domain that binds to one strand of the at least one double-stranded DNA molecule comprising the at least one target DNA sequence; and ii) at least one tail domain.
a tail domain does not hybridize to DNA molecule comprising the at least one target DNA sequence.
a tail domain comprises at least one primer binding site.
a primer binding site is suitable for sequencing library preparation.
a primer can be a single stranded polynucleotide.
a primer can be about 10 nucleotides in length, about 15 nucleotides in length, about 20 nucleotides in length, about 25 nucleotides in length, or about 30 nucleotides in length, about 35 nucleotides in length, about 40 nucleotides in length, 45 nucleotides in length, about 50 nucleotide in length, about 55 nucleotides in length, about 60 nucleotides in length, about 65 nucleotides in length, about 70 nucleotides in length, about 75 nucleotides in length, about 80 nucleotides in length, about 85 nucleotides in length, about 90 nucleotides in length, about 95 nucleotides in length or about 100 nucleotides in length.
a primer can comprise a nucleic acid sequence that is completely or partially complementary to the target DNA sequence to which the primer corresponds (i.e., the target DNA sequence that the primer is being used to direct the recombinase proteins to).
a primer can comprise a nucleic acid sequence that is completely or partially complementary to the sequence which is complementary to the target DNA sequence to which the primer corresponds.
a primer can comprise a nucleic acid sequence that is complementary to a sequence within a double-stranded DNA molecule that comprises the target DNA sequence to which the primer corresponds such that the primer binds within about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 450 nucleotides, about 500 nucleotides, about 550 nucleotides, about 600 nucleotides, about 650 nucleotides, about 700 nucleotides, about 750 nucleotides, about 800 nucleotides, about 850 nucleotides, about 900 nucleotides, about 950 nucleo
a primer can bind to the same strand as the target DNA sequence to which the primer corresponds. In some aspects, a primer can bind to the opposing stand of the target DNA sequence to which the primer corresponds.
a modified primer or primer pair is used in the methods of the disclosure. Modified primers can include modified residues (e.g., 2′-O-methyl RNA or abasic residues, or light-activatable moieties including 6-nitropiperonyloxymethyl (NPOM)-caged nucleic acid). Such modified primers may reduce sequencing artifacts, improving accuracy, or allow additional control in downstream reactions of the methods described herein.
the plurality of primer pairs can comprise at least two species of primer pairs, wherein the first primer and second primers of distinct primer probe species comprise unique target binding domains, thereby allowing for sequencing of at least two target DNA sequences in the biological sample. That is, the use of 60 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) multiple species of primer pairs can allow the methods of the present disclosure to be multiplexed such that more than one target DNA sequence can be sequenced.
the plurality of primer pairs can comprise at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about 10, at least about 25, at least about 50, at least about 100, at least about 250, at least about 500, at least about 750 or at least about 1000 species of primer pairs, wherein the first primer and second primers of distinct primer probe species comprise unique target binding domains.
the plurality of primer pairs can comprise at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750 or at least 1000 species of primer pairs, wherein the first primer and second primers of distinct primer probe species comprise unique target binding domains.
the methods of the present disclosure can be multiplexed to sequence any number of target nucleic acids at any number of locations within a biological sample.
the methods of the present disclosure can be used to sequence at least about 10, or at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or at least about 100, or at least about 110, or at least about 120, or at least about 130, or at least about 140, or at least about 150, or at least about 160, or at least about 170, or at least about 180, or at least about 190, or at least about 200, or at least about 210, or at least about 220, or at least about 240, or at least about 250, or at least about 260, or at least about 270, or at least about 280, or at least about 290, or at least about 300, or at least about 500, or at least about 1,000, or at least about 10,000, or at least about 100,000, or at least about 1,000,000 different species of target nucleic acids within a biological sample.
the methods of the present disclosure can be multiplexed to sequence any number of target nucleic acids at any number of locations within a biological sample.
the methods of the present disclosure can be used to sequence about 10, or about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100, or about 110, or about 120, or about 130, or about 140, or about 150, or about 160, or about 170, or about 180, or about 190, or about 200, or about 210, or about 220, or about 240, or about 250, or about 260, or about 270, or about 280, or about 290, or about 300, or about 500, or about 1,000, or about 10,000, or about 100,000, or about 1,000,000 different species of target nucleic acids within a biological sample 61 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) [00235]
the concentration of the first primer and the second primer are about the same when the tissue sample is treated with the primers.
“symmetric amplification” of a region of a double-stranded DNA molecule can be accomplished, i.e., both strands of the double-stranded DNA molecule will be amplified to approximately the same amount.
a first primer in a primer pair can be present at a concentration that is greater than the concentration of the second primer in the primer pair.
the concentration of the first primer is at least about ten times, or at least about 100 times, or at least about 1000 times greater than the concentration of the second primer.
“asymmetric amplification” of a region of a double-stranded DNA molecule can be accomplished, i.e., one strand of the double-stranded DNA molecule will be amplified more than the opposite strand.
recombinase proteins can be selected from T4 uvsX protein, T4 uvsY protein, recA protein, recF protein, recO protein, recR protein, RadA protein, RadB protein, Rad51 protein, RuvA protein, RuvB protein, RuvC protein and RecG protein or combinations thereof.
recombinase proteins of the present disclosure comprise a single species of recombinase proteins.
recombinase proteins of the present disclosure comprise a combination of species of recombinase proteins, e.g., more than one species of recombinase protein.
a T4 bacteriophage uvsX protein can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 1, or a biologically active portion thereof.
a T4 bacteriophage uvsY protein can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 62 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 2, or a biologically active portion thereof.
single-stranded DNA binding proteins can be selected from T4 bacteriophage Gene 32 protein and E. coli Single-Stranded Binding (SSB) protein.
a T4 bacteriophage Gene 32 protein can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 3, or a biologically active portion thereof.
a plurality of a first recombinase proteins can be a plurality of T4 bacteriophage uvsX proteins
a plurality of a second recombinase proteins can be a plurality of T4 bacteriophage uvsY proteins
a plurality of single-stranded DNA binding proteins can be a plurality of T4 bacteriophage Gene 32 proteins.
any derivatives and functional analogs of a recombinase protein above may also function itself as a recombinase protein, and is contemplated for use in the methods of the present disclosure.
a peptide comprising residues 193 to 212 of E. coli recA can mediate pairing of single stranded oligonucleotides, that is, retaining certain functional aspects of the recombination properties of recA, may be used in the methods of the disclosure.
Polymerases [00246] In some aspects of the methods of the present disclosure, strand displacing polymerases can be selected from Bsu polymerases and phi29 polymerases.
a Bsu polymerase can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 4, or a biologically active portion thereof.
Crowding Agents [00248] In some aspects of the methods of the present disclosure, a crowding agent may be used. Crowding agents can be selected from polyethylene glycol, dextran, Ficoll, at least one inert protein, at least one polysaccharide (e.g., methyl cellulose) and any combination thereof.
a ligase may be used.
Ligases can be selected from SplintR ligase and T4 DNA ligase, and variants thereof.
5′ exonucleases [00250] In some aspects of the methods of the present disclosure, a 5′ exonuclease can be used.5′ exonucleases can be selected from T7 exonuclease and T5 exonuclease, and variants thereof.
a 3′ exonuclease can be used.
a 3′ exonuclease can be selected from Exonuclease III and variants thereof.
Uracil-DNA glycosylase enzymes [00252] In some aspects of the methods of the present disclosure, a Uracil-DNA glycosylase enzyme can be used.
a Uracil-DNA glycosylase enzyme can be selected from E. coli uracil- DNA glycosylase and variants thereof.
Target DNA Sequences [00253] In some aspects, a target DNA sequence to be probed using the methods of the present disclosure can be located within a genomic DNA molecule.
a target DNA sequenced to be probed using the methods of the present disclosure can be located within a mitochondrial DNA molecule. [00255] In some aspects, a target DNA sequence to be probed using the methods of the present disclosure can be located within a region of open chromatin of genomic DNA. [00256] In some aspects, a target DNA sequence to be probed using the methods of the present disclosure can be located within a viral double-stranded DNA molecule. [00257] In some aspects, a target DNA sequence can comprise a single nucleotide variant of interest.
a biological sample can be a tissue sample. In some aspects a tissue sample can be a fresh frozen tissue sample.
a tissue can be a fixed tissue sample.
a fixed tissue sample can be a formalin-fixed, paraffin-embedded (FFPE) tissue sample.
FFPE formalin-fixed, paraffin-embedded
a tissue sample can be a cell culture sample. 64 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) [00259]
a biological sample can be an FFPE microtome section that is at least about 1 ⁇ m, or at least about 2 ⁇ m, or at least about 3 ⁇ m, or at least about 4 ⁇ m, or at least about 5 ⁇ m, or at least about 6 ⁇ m, or at least about 7 ⁇ m, or at least about 8 ⁇ m, or at least about 9 ⁇ m, or at least about 10 ⁇ m thick.
the biological sample is an FFPE microtome section that is at least about 5 ⁇ m thick.
a biological sample can be an FFPE microtome section that is about 1 ⁇ m, or about 2 ⁇ m, or about 3 ⁇ m, or about 4 ⁇ m, or about 5 ⁇ m, or about 6 ⁇ m, or about 7 ⁇ m, or about 8 ⁇ m, or about 9 ⁇ m, or about 10 ⁇ m thick.
the biological sample is an FFPE microtome section that is about 5 ⁇ m thick.
the biological sample can be a tissue sample from any organ.
the biological sample is a tissue sample from the Intestine, Embryo, Brain, Spleen, Eye, Retina, Liver, Kidney, Breast, Throat, Colon, Lung, Prostate, Lymph node, Tonsil, Pancreas, Cervix, Head, Neck, Liver, Skin, Nevus, Placenta or any other organ.
the biological sample can comprise non-cancerous cells.
the biological sample can comprise cancerous cells.
the biological sample can comprise a combination of both non-cancerous cells and cancerous cells.
the cancerous cells can be from a carcinoma, lymphoma, blastoma, sarcoma, leukemia and germ cell tumors.
the cancerous cells can be from a adrenocortical carcinoma, bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoid neoplasm diffuse large B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, kidney chromophobe, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, acute myeloid leukemia, brain lower grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic
cancers include breast cancer, lung cancer, lymphoma, melanoma, liver cancer, colorectal cancer, ovarian cancer, bladder cancer, renal cancer or gastric cancer.
Further examples of cancer include neuroendocrine cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, thyroid cancer, endometrial cancer, biliary cancer, esophageal cancer, anal cancer, salivary, cancer, vulvar cancer, cervical cancer, Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML), Adrenal gland tumors, Anal cancer, Bile duct cancer, Bladder cancer, Bone 65 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) cancer, Bowel cancer, Brain tumors, Breast cancer, Cancer of unknown primary (CUP), Cancer spread to bone, Cancer spread to brain, Cancer spread to liver, Cancer spread to lung, Carcinoid, Cervical cancer, Children’s cancers, Chronic lymphocytic leukemia (
Retinoblastoma Salivary gland cancer, Secondary’ cancer, Signet cell cancer, Skin cancer, Small bowel cancer, Soft tissue sarcoma, Stomach cancer, T cell childhood non Hodgkin lymphoma (NHL), Testicular cancer, Thymus gland cancer, Thyroid cancer, Tongue cancer, Tonsil cancer, Tumors of the adrenal gland, Uterine cancer. Vaginal cancer, Vulval cancer, Wilms’ tumor, Womb cancer and Gynaecological cancer.
cancer also include, but are not limited to, Hematologic malignancies, Lymphoma, Cutaneous T-cell lymphoma, Peripheral T-cell lymphoma, Hodgkin’s lymphoma, Non-Hodgkin’s lymphoma, Multiple myeloma, Chrome lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, Myelodysplastic syndromes, Myelofibrosis, Biliary tract cancer, Hepatocellular cancer, Colorectal cancer, Breast cancer, Lung cancer, Non-small cell lung cancer, Ovarian cancer, Thyroid Carcinoma, Renal Cell Carcinoma, Pancreatic cancer, Bladder cancer, skin cancer, malignant melanoma, merkel cell carcinoma, Uveal Melanoma or Glioblastoma multiforme.
the biological sample can be derived from any species, including, but not limited to, humans, mice, rats, dogs, cats, sheep, rabbits, cows, goats or any other species.
a biological sample can be derived from a fungi.
a biological sample can be derived from a plant.
the biological sample can be a mounted biological sample.
the mounted biological sample is in a flow cell. 66 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) [00265]
any of the methods of the present disclosure can further comprise morphology scanning of the biological sample.
morphology scanning can be used to determine one or more regions of interest to be imaged.
morphology scanning can be used to identify specific features of the biological sample (e.g., tumorous cells, healthy cells, tumor margins, cellular membranes, cellular nuclei, one or more cellular organelles, vasculature, or any other features known in the art by the skilled artisan).
the specific features of the biological sample can be correlated with the abundance and spatial position of target analytes measured using the methods of the present disclosure.
morphology scanning can be used to determine the boundaries of individual cells within the biological sample. The determination of the boundaries of individual cells is referred to herein as "cell segmentation". Morphology scanning can include the use of fiducial markers.
Morphology scanning can include optical scanning.
the methods of the present disclosure can further comprise staining the biological sample with a membrane specific-fluorescent staining solution and imaging the biological sample to identify the spatial location of cellular membranes within the sample. This staining can be performed at any step in the protocol.
the methods of the present disclosure can further comprise staining the biological sample with a nuclear-specific fluorescent staining solution and imaging the biological sample to identify the spatial location of cellular nuclei in the sample. This staining can be performed at any step in the methods of the present disclosure.
the methods of the present disclosure can further comprise staining the biological sample with a membrane specific-fluorescent staining solution and imaging the biological sample to identify the spatial location of cellular membranes within the sample.
the method can further comprise staining the biological sample with a nuclear-specific fluorescent staining solution and imaging the biological sample to identify the spatial location of cellular nuclei in the sample.
membrane and/or nuclear stains are used to perform morphology scanning on the biological sample.
the membrane and/or nuclear stains can be used to determine one or more regions of interest to be imaged during determination of the abundance and spatial position of target analytes (e.g., target nucleic acid molecules.).
target analytes e.g., target nucleic acid molecules.
the total time to run imaging experiments can be decreased 67 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) by imaging only particular regions of interest, as the total duration of an experiment increases as more areas of the biological sample are imaged.
fiducial markers added to the biological sample can be used to focus the biological sample using methods standard in the art, as would be appreciated by the skilled artisan. Specifically, the fiducial markers can be used to determine the best x, y or z-position for imaging a particular location within the biological sample. In some aspects of the methods of the present disclosure, fiducial markers added to the biological sample can be used to correlate images of the sample obtained at varying steps of the methods.
a biological sample can be pre-treated using standard methods known in the art to allow for the recombinase proteins, single-stranded DNA binding proteins, primers, nucleic acid probes and reporter probes to permeate throughout the sample, including, but not limited to, within individual cells in the sample. That is, a biological sample can be permeabilized using standard methods known in the art prior to performing the methods of the present disclosure.
a biological sample can be treated with proteinase K or other suitable proteinases known in the art.
Sequencing as referred to herein, can be carried out using any suitable “sequencing-by-synthesis” technique, wherein nucleotides are added successively to a free 3' hydroxyl group, resulting in synthesis of a polynucleotide chain in the 5' to 3' direction.
the nature of the nucleotide added is preferably determined after each nucleotide addition.
One sequencing method which can be used in accordance with the present disclosure relies on the use of modified nucleotides that can act as chain terminators.
the modified nucleotide has been incorporated into the growing polynucleotide chain complementary to the region of the template being sequenced there is no free 3'-OH group available to direct further sequence extension and therefore the polymerase cannot add further nucleotides.
the 3' block may be removed to allow addition of the next successive nucleotide.
each of the modified nucleotides has attached a different label, known to correspond to the particular base, to facilitate discrimination between the bases added at each incorporation step.
a separate reaction may be carried out containing each of the modified nucleotides separately.
the modified nucleotides may carry a label to facilitate their detection.
this is a fluorescent label.
Each nucleotide type may carry a different fluorescent label.
the detectable label need not be a fluorescent label. Any label can be used which allows the detection of an incorporated nucleotide.
One method for detecting fluorescently labelled nucleotides comprises using laser light of a wavelength specific for the labelled nucleotides, or the use of other suitable sources of illumination.
the fluorescence from the label on the nucleotide may be detected by a CCD camera or other suitable detection means.
the methods of the present disclosure are not intended to be limited to use of the sequencing method outlined above, as essentially any sequencing methodology which relies on successive incorporation of nucleotides into a polynucleotide chain can be used.
Suitable alternative techniques include, for example, Pyrosequencing, FISSEQ (fluorescent in situ sequencing), MPSS (massively parallel signature sequencing), any next-generation sequencing technique, and sequencing by ligation-based methods.
Nucleic acid probes of the present disclosure Target binding domain
a nucleic acid probe can comprise a first target binding domain that binds to a first portion of a target DNA sequence and that is located at one terminus of the nucleic acid probe; a second target binding domain that binds to a second portion of the target DNA sequence and that is located at the other terminus of the nucleic acid probes; and a barcode domain specific for the target DNA sequence to which the nucleic acid probe binds.
a schematic of this exemplary nucleic acid probe is shown in FIG.5A.
a nucleic acid probe can comprise a target binding domain that binds to a portion of a target DNA sequence and that is located at one terminus of the nucleic acid probe and a barcode domain specific for the target DNA sequence to which the nucleic acid probe binds; such a probe is shown in FIG. 5B.
69 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) [00281]
the first portion of the at least one target DNA sequence and the second portion of the at least one target DNA sequence are immediately adjacent to each other such that the first target binding domain and the second target binding domain of a single nucleic acid probe bind immediately adjacent to each other on exposed target DNA sequences.
a target binding domain can be a single stranded polynucleotide.
a target binding domain can comprise a sequence that is complementary to a target DNA sequence that is to be probed using the methods of the present disclosure.
a target binding domain can be at least about 25 nucleotides in length to at least about 45 nucleotides in length.
a target binding domain in be at least about 35 nucleotides in length to at least about 40 nucleotides in length.
a target binding domain can be about 25 nucleotides to about 45 nucleotides in length.
a target binding domain can be about 35 nucleotides to about 40 nucleotides in length.
a target binding domain can comprise about 20 nucleotides, or about 21 nucleotides, or about 22 nucleotides, or about 23 nucleotides, or about 24 nucleotides, or about 25 nucleotides, or about 26 nucleotides, or about 27 nucleotides, or about 28 nucleotides, or about 29 nucleotides, or about 30 nucleotides, or about 31 nucleotides, or about 32 nucleotides, or about 33 nucleotides, or about 34 nucleotides, or about 35 nucleotides, or about 36 nucleotides, or about 37 nucleotides, or about 38 nucleotides, or about 39 nucleotides, or about 40 nucleotides, or about 41 nucleotides, or about 42 nucleotides, or about 43 nucleotides, or about 45 nucleotides in length.
a target binding domain comprises D-DNA. In some aspects, a target binding domain consists of D-DNA. [00285] In some aspects, a target binding domain can be about 35 nucleotides to about 40 nucleotides in length and comprises D-DNA. In some aspects, a target binding domain can be about 35 nucleotides to about 40 nucleotides in length and consists of D-DNA. Barcode domain [00286] In some aspects, a barcode domain can be a single stranded polynucleotide. [00287] A barcode domain can comprise at least one attachment region.
a barcode domain can comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten attachment regions. [00288] In some aspects, a barcode domain can comprise about 2 attachment regions. [00289] In some aspects, a barcode domain can comprise about 3 attachment regions. 70 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) [00290] In some aspects, a barcode domain can comprise about 4 attachment regions. [00291] An attachment region can comprise at least one nucleic acid sequence that is capable of being reversibly bound by a reporter probe of the present disclosure.
an attachment region of a barcode domain can comprise at least one attachment sequence.
the attachment sequences within a single attachment region can be identical; thus, the reporter probes that bind within that single attachment region will be identical.
the attachment sequences within a single attachment can be different; thus, the reporter probes that bind within that single attachment will be different.
the attachment sequences in each of the different attachment regions can be different; thus, different reporter probes will bind to each attachment region in the barcode domain.
an attachment sequence can be about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides, or about 11 nucleotides, or about 12 nucleotides, or about 13 nucleotides, or about 14 nucleotides, or about 15 nucleotides, or about 16 nucleotides, or about 17 nucleotides, or about 18 nucleotides, or about 19 nucleotides, or about 20 nucleotides in length. In some aspects, an attachment sequence can be about 14 nucleotides in length.
a barcode domain comprises L-DNA. In some aspects, a barcode domain consists of L-DNA. [00295] In some aspects, a barcode domain can comprise about 4 attachment regions, wherein each attachment region comprises about 1 attachment sequence, wherein each attachment sequence is about 14 nucleotides in length, such that the barcode domain is about 56 nucleotides in length, and wherein the nucleic acid sequence of each of the attachment sequences are different, wherein the barcode domain comprises L-DNA.
a barcode domain can comprise about 4 attachment regions, wherein each attachment region comprises about 1 attachment sequence, wherein each attachment sequence is about 14 nucleotides in length, such that the barcode domain is about 56 nucleotides in length, and wherein the nucleic acid sequence of each of the attachment sequences are different, wherein the barcode domain consists of L-DNA.
nucleic acid probe pairs comprising a first nucleic acid probe and a second nucleic acid probe, wherein the first nucleic acid probe comprises: i) a target binding domain that binds to a first portion of the at least one target DNA sequence; and ii) a barcode domain specific for the at least one target DNA sequence, wherein the barcode domain comprises at least one attachment position, and wherein the second nucleic acid probe comprises: i) a target binding domain that binds to a second portion of the at least one target DNA sequence; and ii) a barcode domain specific for the at least one target DNA sequence, wherein the barcode domain comprises at least one attachment position.
the first and second nucleic acid probe of a nucleic acid probe pair comprise different parts of the same oligonucleotide.
the first and second nucleic acid probe may within a padlock oligonucleotide, whose first and second target binding domains are at the ends of the same oligonucleotide.
the first portion of the at least one target DNA sequence and the second portion of the at least one target DNA sequence are immediately adjacent to each other such that the first nucleic acid probe and the second nucleic acid probe bind immediately adjacent to each other on an exposed at least one target DNA sequence.
the two target binding domains within a nucleic acid probe pair can be designed with specific melting temperatures and specific placement of uracil residues for use in the methods of the present disclosure.
Reporter probes of the present disclosure [00300] The present disclosure provides, in some aspects, reporter probes for use in the methods of the present disclosure.
the reporter probes of the present disclosure bind to the attachment sequences within the attachment regions of the barcode domains of the nucleic acid probes of the present disclosure.
the reporter probes comprise at least one detectable label, e.g., a fluorescent moiety, that allows them to be detected in the methods of the present disclosure.
a reporter probe can comprise at least two domains, wherein the first domain hybridizes to an attachment sequence and the second domain comprises at least one detectable label.
a reporter probe can comprise at least about 10, or at least about 15, or at least about 20, or at least about 25, or at least about 30, or at least about 35, or at least 72 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) about 40, or at least about 45, or at least about 50 detectable labels.
a reporter probe can comprise about 10, or about 15, or about 20, or about 25, or about 30, or about 35, or about 40, or about 45, or about 50 detectable labels.
a reporter probe can be pre-assembled prior to being contacted with a biological sample.
a reporter probe can comprise a primary nucleic acid molecule.
a primary nucleic acid molecule can be a single-stranded polynucleotide.
a primary nucleic acid molecule can comprise L-DNA.
a primary nucleic acid molecule can consist of L-DNA.
a primary nucleic acid molecule can comprise at least two domains. In some aspects, the first domain of a primary nucleic acid molecule can hybridize to an attachment sequence in an attachment region of a barcode domain of an nucleic acid probe of the present disclosure.
the second domain of a primary nucleic acid molecule comprises at least one detectable label.
the second domain of a primary nucleic acid molecule can hybridize to at least one secondary nucleic acid molecule.
a primary nucleic acid molecule can hybridize to at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten secondary nucleic acid molecules.
a primary nucleic acid molecule can hybridize to about 6 secondary nucleic acid molecules.
a primary nucleic acid molecule can further comprise a cleavable linker moiety.
the cleavable linker moiety can be located between the first domain and the second domain, such that when the cleavable linker moiety is cleaved, the first domain and the second domain are separated.
the cleavable linker moiety is a photocleavable linker moiety.
the first domain of a primary nucleic acid molecule can be about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides, or about 11 nucleotides, or about 12 nucleotides, or about 13 nucleotides, or about 14 nucleotides, or about 15 nucleotides, or about 16 nucleotides, or about 17 nucleotides, or about 18 nucleotides, or about 19 nucleotides, or about 20 nucleotides in length.
the first domain of a primary nucleic acid molecule can be about 14 nucleotides in length.
the second domain of a primary nucleic acid molecule can be about 75 nucleotides, or about 76 nucleotides, or about 77 nucleotides, or about 78 nucleotides, or 73 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) about 79 nucleotides, or about 80 nucleotides, or about 81 nucleotides, or about 82 nucleotides, or about 83 nucleotides, or about 84 nucleotides, or about 85 nucleotides, or about 86 nucleotides, or about 87 nucleotides, or about 88 nucleotides, or about 89 nucleotides, or about 90 nucleotides in length.
a primary nucleic acid can be about 98 nucleotides in length.
a reporter probe can comprise at least one secondary nucleic acid molecule.
a reporter probe can comprise at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten secondary nucleic acid molecules.
a reporter probe can comprise about six secondary nucleic acid molecules.
a secondary nucleic acid molecule can be a single-stranded polynucleotide.
a secondary nucleic acid molecule can comprise L-DNA.
a secondary nucleic acid molecule can consist of L-DNA.
a secondary nucleic acid molecule can comprise at least two domains.
the first domain of a secondary nucleic acid molecule can hybridize to a primary nucleic acid molecule.
the second domain of a secondary nucleic acid molecule can comprise at least one detectable label.
a secondary nucleic acid molecule can further comprise a cleavable linker moiety.
the cleavable linker moiety can be located between the first domain and the second domain, such that when the cleavable linker moiety is cleaved, the first domain and the second domain of the secondary nucleic acid molecule are separated.
the cleavable linker moiety is a photocleavable linker moiety.
the second domain of a secondary nucleic acid molecule can hybridize to at least one tertiary nucleic acid molecule.
the second domain of a secondary nucleic acid molecule can hybridize to at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at 74 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) least about eight, or at least about nine, or at least about ten tertiary nucleic acid molecules. In some aspects, the second domain of a secondary nucleic acid molecule can hybridize to about five tertiary nucleic acid molecules.
the first domain of a secondary nucleic acid molecule can be about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides, or about 11 nucleotides, or about 12 nucleotides, or about 13 nucleotides, or about 14 nucleotides, or about 15 nucleotides, or about 16 nucleotides, or about 17 nucleotides, or about 18 nucleotides, or about 19 nucleotides, or about 20 nucleotides in length.
a reporter probe can comprise at least one tertiary nucleic acid molecule.
a reporter probe can comprise at least about 20, or at least about 21, or at least about 22, or at least about 23, or at least about 24, or at least about 25, or at least about 26, or at least about 27, or at least about 28, or at least about 29, or at least about 30, or at least about 31, or at least about 32, or at least about 33, or at least about 34, or at least about 35, or at least about 36, or at least about 37, or at least about 38, or at least about 39, or at least about 40 tertiary nucleic acid molecules.
a reporter probe can comprise about 30 tertiary nucleic acid molecules.
a tertiary nucleic acid molecule can comprise a domain that hybridizes to a secondary nucleic acid molecule.
a tertiary nucleic acid molecule can comprise at least one detectable label.
a tertiary nucleic acid molecule can be about 5 nucleotides, or about 6 nucleotides, or about 7 nucleotides, or about 8 nucleotides, or about 9 nucleotides, or about 10 nucleotides, or about 11 nucleotides, or about 12 nucleotides, or about 13 nucleotides, or 75 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) about 14 nucleotides, or about 15 nucleotides, or about 16 nucleotides, or about 17 nucleotides, or about 18 nucleotides, or about 19 nucleotides, or about 20 nucleotides, or about 21 nucleotides, or about 22 nucleotides, or about 23 nucleotides, or about 24 nucleotides, or about 25 nucleotides in length.
a tertiary nucleic acid molecule can be about 15 nucleotides in length.
all of the detectable labels of the reporter probe can have the same emission spectrum.
the detectable labels are fluorescent labels
reporter probes wherein all of the detectable labels have the same emission spectrum can be referred to as “single-color” reporter probes.
the reporter probe can have two or more detectable labels that each have a different emission spectra.
reporter probes that have two or more detectable labels that each have a different emission spectra can be referred to as “multi-color” reporter probes.
the present disclosure provides a reporter probe comprising a primary nucleic acid molecule comprising a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the second domain of the primary nucleic acid molecule is hybridized to about six secondary nucleic acid molecules, wherein each secondary nucleic acid molecule comprises a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the first domain of each of the secondary nucleic acid molecules is hybridized to the second domain of the primary nucleic acid molecule, wherein the second domain of each of the secondary nucleic acid molecules is hybridized to about five tertiary nucleic acid molecules, wherein each of the tertiary nucleic acid molecules comprise at
FIG.6 A schematic of an exemplary reporter probe is shown in FIG.6.
the first domain of the primary nucleic acid molecule is about 14 nucleotides in length
the second domain of the primary nucleic acid molecule is about 84 nucleotides in length
the first domain of the secondary nucleic acid molecules is about 14 nucleotides in length
the second domain of the secondary nucleic acid molecules is about 75 nucleotides in length
each of the tertiary nucleic acid molecules is about 15 nucleotides in length.
the present disclosure provides a reporter probe comprising a primary nucleic acid molecule comprising a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the second domain of the primary nucleic acid molecule is hybridized to about six secondary nucleic acid molecules, wherein each secondary nucleic acid molecule comprises a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the first domain of each of the secondary nucleic acid molecules is hybridized to the second domain of the primary nucleic acid molecule, wherein the second domain of each of the secondary nucleic acid molecules is hybridized to about five tertiary nucleic acid molecules, wherein each of the tertiary nucleic acid molecules comprise at least one detectable label, and wherein the primary nucleic acid molecule, the secondary nucleic acid molecules, the secondary nucleic acid molecules
the first domain of the primary nucleic acid molecule is about 14 nucleotides in length
the second domain of the primary nucleic acid molecule is about 84 nucleotides in length
the first domain of the secondary nucleic acid molecules is about 14 nucleotides in length
the second domain of the secondary nucleic acid molecules is about 75 nucleotides in length
each of the tertiary nucleic acid molecules is about 15 nucleotides in length.
a photocleavable moiety can be cleaved upon exposure to UV light.
the light can be provided by a light source selected from the group consisting of an arc-lamp, a laser, a focused UV light source, and light emitting diode.
a cleavable linker moiety can be stereoisomer or salt thereof
a cleavable linker moiety can be 77 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) stereoisomer or salt thereof.
a cleavable linker moiety can 78 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) [00330] A cleavable linker moiety can [00331] A cleavable linker moiety can [00332] A cleavable linker moiety can [00333] In some aspects, a detectable label can be a fluorescent moiety or a fluorescent label. One of skill in the art can consult references directed to labeling nucleic acids.
fluorescent moieties include, but are not limited to, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansyl chloride, phycocyanin, phycoerythrin and the like.
YFP yellow fluorescent protein
GFP green fluorescent protein
CFP cyan fluorescent protein
RFP red fluorescent protein
umbelliferone fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansyl chloride, phycocyanin, phycoerythrin and the like.
fluorescent label comprises a signaling moiety that conveys information through the fluorescent absorption and/or emission properties of one or more molecules.
fluorescent properties include fluorescence intensity, fluorescence lifetime, emission spectrum characteristics, energy transfer, and the like.
fluorescent nucleotide analogues readily incorporated into nucleotide and/or oligonucleotide sequences include, but are not limited to, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), fluorescein- 12- dUTP, tetramethylrhodamine-6-dUTP, TEXAS REDTM-5-dUTP, CASCADE BLUETM-7- dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHODAMINE GREENTM-5-dUTP, OREGON GREENRTM 488-5-dUTP, TEXAS REDTM- 12-dUTP, BODIPY TM 630/650- 14-dUTP, BODIPY TM 650/665- 14
Nucleic acid could also be stained, a priori, with an intercalating dye such as DAPI, YOYO- 1 , ethidium bromide, cyanine dyes (e.g., SYBR Green) and the like.
an intercalating dye such as DAPI, YOYO- 1 , ethidium bromide, cyanine dyes (e.g., SYBR Green) and the like.
Other fluorophores available for post-synthetic attachment include, but are not limited to, ALEXA FLUORTM 350, ALEXA FLUORTM 405, ALEXA FLUORTM 430, ALEXA FLUORTM 532, ALEXA FLUORTM 546, ALEXA FLUORTM 568, ALEXA FLUORTM 594, ALEXA FLUORTM 647, BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550,
FRET tandem fluorophores can also be used, including, but not limited to, PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, and 680), APC-Alexa dyes and the like.
Metallic silver or gold particles can be used to enhance signal from fluorescently labeled nucleotide and/or oligonucleotide sequences (Lakowicz et al. (2003) BioTechniques 34:62).
Suitable labels for an oligonucleotide sequence can include fluorescein (FAM, FITC), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), phosphor-amino acids (e.g., P-tyr, P-ser, P-thr) and the like.
hapten/antibody pairs can be used for detection, in which each of the antibodies is derivatized with a detectable label: biotin/a-biotin, digoxigenin/a-digoxigenin, dinitrophenol (DNP)/a-DNP, 5-Carboxyfluorescein (FAM)/a-FAM.
detectable labels described herein are spectrally resolvable.
“Spectrally resolvable” in reference to a plurality of fluorescent labels means that the fluorescent emission bands of the labels are sufficiently distinct, i.e., sufficiently non-overlapping, that molecular tags to which the respective labels are attached can be distinguished on the basis of the fluorescent signal generated by the respective labels by standard photodetection systems, e.g., employing a system of band pass filters and photomultiplier tubes, or the like, as exemplified by the systems described in U.S. Patent Nos.4,230,558; 4,811,218; or the like, or in Wheeless et al., pgs.21-76, in Flow Cytometry: Instrumentation and Data Analysis (Academic Press, New York, 1985).
Spectrally resolvable organic dyes such as fluorescein, rhodamine, and the like, 81 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) means that wavelength emission maxima are spaced at least 20 nm apart, and in another aspect, at least 40 nm apart.
spectrally resolvable means that wavelength emission maxima are spaced at least 10 nm apart, or at least 15 nm apart.
a kit of the present disclosure can comprise one or more pluralities of recombinase proteins, as described herein.
a kit of the present disclosure can comprise one or more pluralities of single-stranded DNA binding proteins, as described herein.
a kit of the present disclosure can comprise one or more pluralities of primers, as described herein.
a kit of the present disclosure can further comprise a plurality of primer pairs, as described herein.
a kit of the present disclosure can further comprise a plurality of nucleic acid probe pairs.
a kit of the present disclosure can further comprise a plurality of reporter probes of the present disclosure. [00347] In some aspects, a kit of the present disclosure can further comprise a plurality of ligases. [00348] In some aspects, a kit of the present disclosure can further comprise a plurality of uracil-DNA glycosylase enzymes. [00349] In some aspects, a kit of the present disclosure can further comprise a plurality of 5′ exonucleases. [00350] In some aspects, a kit of the present disclosure can further comprise a plurality of 3′ exonucleases.
a kit of the present disclosure can further comprise a plurality of strand displacing polymerases.
a kit of the present disclosure can comprise a system suitable for use in the methods of the present disclosure.
a kit of the present disclosure can comprise an apparatus suitable for use in the methods of the present disclosure.
a method of determining the abundance and spatial position of at least one target DNA sequence in a biological sample comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of primers; wherein the first recombinase proteins, second recombinase proteins, single- stranded DNA binding proteins and primers interact with at least one double-stranded DNA molecule comprising the at least one target DNA sequence, thereby exposing the at least one target DNA sequence; b 1 ) contacting the biological sample with a plurality of nucleic acid probes thereby binding a nucleic acid probe to the exposed at least one target DNA sequence, wherein the nucleic acid probes comprise: i) a target binding domain that binds to the at least
the plurality of nucleic acid probes comprises at least two species of nucleic acid probes, wherein the two species of nucleic acid probes comprise unique target binding domains that bind to different target DNA sequences, thereby allowing for the determination of the abundance and spatial position of at least two target DNA sequences in the biological sample.
the plurality of primers comprise: i) one species of primer; ii) two species of primers; iii) at least two species of primers; or iv) at least three species of primers. 7.
the first recombinase proteins are T4 uvsX recombinase proteins.
the second recombinase proteins are T4 uvsY recombinase proteins.
the single-stranded DNA binding proteins are T4 Gene 32 Proteins.
the plurality of primers comprises at least two species of primers, wherein the at least two species of primers bind to the at least one double-stranded DNA molecule within 500 nucleotides of the target DNA sequence. 11.
the primers are at least 30 nucleotides in length, at least 35 nucleotides in length, at least 40 nucleotides in length or at least 45 nucleotides in length.
the target binding domains are single-stranded polynucleotides comprising a nucleic acid sequence that is complementary to the target DNA sequence, preferably wherein the target binding domains are about 35 to about 40 nucleotides in length, and preferably wherein the target binding domains comprise D-DNA.
the barcode domains are a single-stranded polynucleotide comprising at least one attachment region, preferably wherein each attachment region comprises about one attachment sequence, preferably wherein each of the attachment sequences is about 14 nucleotides in length, preferably wherein the sequences of each of the attachment sequences are different, and preferably wherein the barcode domain comprises L-DNA. 14.
step (d 1 ) the barcode domains of the nucleic acid probes comprise at least two, or at least three, or at least four attachment positions
the method further comprises, after step (d 1 ): (d2) removing the detectable labels of the bound reporter probes; and (d3) repeating steps (c1) – (d2) until each attachment position in the barcode domains of the nucleic acid probes bound to the target DNA sequences in the biological sample have been bound to a reporter probe comprising at least one detectable label; and wherein step (e1) comprises determining the abundance and spatial position of the target DNA sequence in the biological sample based at least in part on the sequence in which the detectable labels were recorded.
the reporter probes comprise: a primary nucleic acid molecule comprising a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the second domain of the primary nucleic acid molecule is hybridized to about six secondary nucleic acid molecules, wherein each secondary nucleic acid molecule comprises a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the first domain of each of the secondary nucleic acid molecules is hybridized to the second domain of the primary nucleic acid molecule, wherein the second domain of each of the secondary nucleic acid molecules is hybridized to about five tertiary nucleic acid molecules, wherein each of the tertiary nucleic acid molecules comprise at least one detectable label, and 85 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) wherein the primary nucleic acid molecule, the secondary nucleic acid molecules, the secondary nucleic acid molecules,
the at least one detectable label is a fluorescent moiety.
the method further comprises, after step (c 1 ) and prior to step (d 1 ), removing unbound reporter probes.
the biological sample is a tissue sample.
the tissue sample is: i) a fresh frozen tissue sample; or ii) a fixed tissue sample, preferably wherein the fixed tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample.
a method for determining the abundance and spatial position of at least one target DNA sequence in a biological sample comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of primers; wherein the first recombinase proteins, second recombinase proteins, single- stranded DNA binding proteins and primers interact with at least one double-stranded DNA molecule comprising the at least one target DNA sequence, thereby exposing the at least one target DNA sequence; b1) contacting the biological sample with a plurality of nucleic acid probe pairs, thereby binding a nucleic acid probe pair to the exposed at least one target DNA sequence, wherein the nucleic acid probe pair comprises a first nucleic acid probe and second nucleic acid probe, where
step (f 1 ) comprises determining the abundance and spatial position of the target DNA sequence in the biological sample based on the sequence in which the detectable labels were recorded.
a method for determining the abundance and spatial position of at least one target DNA sequence in a biological sample comprising: a 1 ) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of primers; wherein the first recombinase proteins, second recombinase proteins, single- stranded DNA binding proteins and primers interact with at least one double-stranded DNA molecule comprising the at least one target DNA sequence, thereby exposing the at least one target DNA sequence; b1) contacting the biological sample with a plurality of nucleic acid probe pairs, thereby binding a nucleic acid probe pair to the exposed at least one
step (g 1 ) and prior to step (h 1 ) the barcode domains of the nucleic acid probes comprise at least two, or at least three, or at least four attachment positions
the method further comprises, after step (g 1 ) and prior to step (h 1 ): (g 2 ) removing the detectable labels of the bound reporter probes; and (g3) repeating steps (f1) – (g2) until each attachment position in the barcode domains of the first nucleic acid probes and/or second nucleic acid probes bound to the target DNA sequences in the biological sample have been bound to a reporter probe comprising at least one detectable label; and wherein step (h1) comprises determining the abundance and spatial position of the target DNA sequence in the biological sample based on the sequence in which the detectable labels were recorded.
the plurality of nucleic acid probe pairs comprises at least two species of nucleic acid probe pairs, wherein the two species of nucleic acid probe pair comprise unique target binding domains that bind to different target DNA sequences, thereby allowing for the determination of the abundance and spatial position of at least two target DNA sequences in the biological sample.
the plurality of primers comprise: i) one species of primer; ii) two species of primers; iii) at least two species of primers; or iv) at least three species of primers.
the first recombinase proteins are T4 uvsX recombinase proteins.
the second recombinase proteins are T4 uvsY recombinase proteins.
the single-stranded DNA binding proteins are T4 Gene 32 Proteins.
the plurality of primers comprises at least two species of primers, wherein the at least two species of primers bind to the at least one double-stranded DNA molecule within 500 nucleotides of the target DNA sequence.
the primers are at least 30 nucleotides in length, at least 35 nucleotides in length, at least 40 nucleotides in length or at least 45 nucleotides in length.
the target binding domains are single-stranded polynucleotides comprising a nucleic acid sequence that is complementary to the target DNA sequence, preferably wherein the target binding domains are about 35 to about 40 nucleotides in length, and preferably wherein the target binding domains comprise D-DNA.
the barcode domains are a single-stranded polynucleotide comprising at least one attachment region, preferably wherein each attachment region comprises about one attachment sequence, preferably wherein each of the attachment sequences is about 14 nucleotides in length, preferably wherein the sequences of each of the attachment sequences are different, and preferably wherein the barcode domain comprises L-DNA. 36.
the reporter probes comprise: a primary nucleic acid molecule comprising a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the second domain of the primary nucleic acid molecule is hybridized to about six secondary nucleic acid molecules, wherein each secondary nucleic acid molecule comprises a first domain, a second domain and a photocleavable linker located between the first domain and the second domain, wherein the first domain of each of the secondary nucleic acid molecules is hybridized to the second domain of the primary nucleic acid molecule, wherein the second domain of each of the secondary nucleic acid molecules is hybridized to about five tertiary nucleic acid molecules, wherein each of the tertiary nucleic acid molecules comprise at least one detectable label, and wherein the primary nucleic acid molecule, the secondary nucleic acid molecules, and the tertiary nucleic acid molecules comprise L-DNA.
the at least one detectable label is a fluorescent moiety. 38. The method of any one of the preceding embodiments, wherein the method further comprises, after step (e 1 ) and prior to step (f 1 ), or after step (f 1 ) and prior to step (g 1 ), removing unbound reporter probes. 39. The method of any one of the preceding embodiments, wherein the biological sample is a tissue sample. 40.
tissue sample is: i) a fresh frozen tissue sample; or 91 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) ii) a fixed tissue sample, preferably wherein the fixed tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample. 41.
FFPE formalin-fixed, paraffin-embedded
a method for determining the abundance and spatial position of at least one target DNA sequence in a biological sample comprising: a1) contacting the biological sample with a solution comprising: i) a plurality of a first recombinase proteins; ii) a plurality of a second recombinase proteins; iii) a plurality of single-stranded DNA binding proteins; and iv) a plurality of primers; wherein the first recombinase proteins, second recombinase proteins, single- stranded DNA binding proteins and primers interact with at least one double-stranded DNA molecule comprising the at least one target DNA sequence, thereby exposing the at least one target DNA sequence; b1) contacting the biological sample with a plurality of nucleic acid probes thereby binding a nucleic acid probe to the exposed at least one target DNA sequence, wherein the nucleic acid probes comprise: i) a first target binding domain that binds to a first portion
detecting the ligated probes using RCA comprises: i) treating the biological sample to produce an acrylamide gel matrix; ii) amplifying the ligated nucleic acid probes by contacting the biological sample with: a plurality of RCA polymerases; a plurality of RCA primers; and a plurality of dNTPs, wherein the plurality of dNTPs comprise a plurality of aminoallyl-dUTPs; iii) treating the biological sample with paraformaldehyde to crosslink to amplification products produced in step (ii) to the acrylamide gel matrix produced in step (i); iv) contacting the biological sample with a plurality of reporter probes, wherein the reporter probes bind to the barcode domain of the amplified nucleic acid probes, and wherein the reporter probe comprises at least one detectable label.
the primers are at least 30 nucleotides in length, at least 35 nucleotides in length, at least 40 nucleotides in length or at least 45 nucleotides in length.
the nucleic acid probes are single-stranded polynucleotides 56.
the at least one detectable label is a fluorescent moiety.
the biological sample is a tissue sample.
the first primer in a primer pair is present at a concentration that is greater than the concentration of the second primer in the primer pair, preferably wherein the concentration of the first primer is at least about ten times, or at least about 100 times, or at least about 1000 times greater than the concentration of the second primer.
the method further comprises, after step (a 1 ) and prior to step (b 1 ), removing unbound primer pairs.
the biological sample is a tissue sample.
the lanes for the gel shown in FIG.3A are as follows: 98 299255491 Attorney Docket No: NATE-055/01WO (321329-2902) [00360]
the lanes for the gel shown in FIG.3B are as follows: [00361] Without wishing to be bound by theory, the results presented in this example demonstrate that the methods of the present disclosure can be used to amplify target sequences from double-stranded DNA molecules, including those present in genomic DNA samples. Equivalents [00362]
the foregoing description has been presented only for the purposes of illustration and is not intended to limit the disclosure to the precise form disclosed. The details of one or more embodiments of the disclosure are set forth in the accompanying description above.

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