Direct in situ sequencing

Abstract

Binding complexes comprising detectable nucleotide conjugates having multiple copies of nucleotides attached to a particle are described. The binding complexes allow localization of detectable signals to active regions of biochemical interactions, e.g., sites of protein-protein interactions, protein-nucleic acid interactions, nucleic acid hybridization, or enzymatic reactions, and can be used to identify base incorporation sites in elongating nucleic acid strands during polymerase reactions, and provide improved base discrimination for sequencing and array-based applications.

Description

[0001] Cross-referencing

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 549,891, filed February 5, 2024, and U.S. Provisional Patent Application No. 63 / 652,540, filed May 28, 2024, which are incorporated herein by reference in their entirety for all purposes.

[0003] By referencing and incorporating into the sequence list

[0004] This application is filed together with an electronic sequence list. The sequence list is provided as a file named 52933-761.601-PCT SL.XML, created on February 4, 2025, and is 6.16 kilobytes in size. Information in the electronic sequence list is incorporated in its entirety by reference. Background Technology

[0005] Nucleic acid sequencing can be used to obtain information in a variety of biomedical contexts, including diagnosis, prognosis, biotechnology, and forensic biology. Various sequencing methods have been developed, including Maxam-Gilbert sequencing and chain termination methods, or de novo sequencing (including shotgun sequencing and bridge PCR), or next-generation methods (including polymerase cloning sequencing, 454 pyrosequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, HeliScope single-molecule sequencing, SMRT® sequencing, etc.). Despite the progress made in DNA sequencing, many challenges remain unresolved. Summary of the Invention

[0006] The aspects disclosed herein provide a method for in situ identification of ribonucleic acid (RNA), the method comprising: (a) providing permeabilized cells or tissues containing RNA; (b) guiding a probe into the cells or tissues under conditions sufficient to couple an RNA-binding sequence to at least a portion of the RNA, wherein the probe comprises (i) an RNA-binding sequence and (ii) an index sequence; (c) amplifying the index sequence to generate one or more amplicons, wherein the one or more amplicons comprise multiple copies of the index sequence or a derivative thereof; (d) guiding a primer nucleic acid sequence into the cells or tissues under conditions sufficient to bind a primer nucleic acid sequence to a portion of the one or more amplicons to generate a primer-binding index sequence; (e) guiding the cells or tissues with a nucleotide conjugate comprising: (i) a detectable marker and (ii) one or more nucleotide portions complementary to the nucleotides of the primer-binding index sequence or a derivative thereof; and (f) detecting the binding complex within the cells or tissues, thereby identifying RNA. In some embodiments, the method does not include performing a reverse transcription reaction of RNA. In some embodiments, the method further includes providing the cells in (a). In some embodiments, the cells are cancer cells. In some embodiments, the method further includes providing the tissue in (a). In some embodiments, the tissue is formalin-fixed paraffin-embedded tissue. In some embodiments, the tissue is freshly frozen tissue. In some embodiments, the cells or tissue are extracted from a subject. In some embodiments, the subject is a human subject. In some embodiments, the index sequence comprises nucleic acids. In some embodiments, the index sequence is 4 to 50 nucleotides in length. In some embodiments, at least a portion of the index sequence or a derivative thereof recognizes a primer nucleic acid sequence. In some embodiments, the primer nucleic acid sequence comprises a blocking group. In some embodiments, the blocking group inhibits the incorporation of another nucleotide into the primer nucleic acid sequence. In some embodiments, the blocking group comprises a 3'-O-azido group, a 3'-O-azidomethyl group, a 3'-O-alkylhydroxyamino group, a 3'-thiophosphate group, a 3'-O-malonyl group, or a 3'-O-benzyl group. In some embodiments, the primer nucleic acid sequence does not include a 3' hydroxyl group. In some embodiments, the primer nucleic acid sequence binds to at least a portion of the index sequence. In some embodiments, the primer nucleic acid sequence binds to at least a portion of the inverse complementary sequence of the index sequence. In some embodiments, the primer-bound index sequence comprises a double-stranded nucleic acid. In some embodiments, the primer-bound index sequence comprises a single-stranded nucleic acid. In some embodiments, cells or tissues are immobilized on the surface of a substrate. In some embodiments, the substrate has a water contact angle of less than or equal to 45 degrees. In some embodiments, the substrate is the inner surface of a flow cell.In some embodiments, the inner surface of the flow cell comprises one or more hydrophilic polymer layers. In some embodiments, the one or more hydrophilic polymer layers comprise a branched polymer. In some embodiments, the one or more hydrophilic polymer layers comprise a polymer containing polyethylene glycol. In some embodiments, the nucleotide conjugate comprises a common core. In some embodiments, one or more nucleotide moieties are coupled to the common core. In some embodiments, the common core comprises a polymer, micelles, liposomes, microparticles, nanoparticles, or quantum dots. In some embodiments, the polymer comprises a protein. In some embodiments, in (e), the nucleotide conjugate is included in a mixture of multiple nucleotide conjugates, wherein each of the multiple nucleotide conjugates comprises nucleotide moieties of different types from each other. In some embodiments, the different types of nucleotide moieties comprise at least three different types of nucleotide moieties. In some embodiments, one or more nucleotide moieties do not contain a blocking group coupled thereto. In some embodiments, detection in (f) comprises imaging the cells or tissue using one or more image sensors. In some embodiments, the one or more image sensors comprise a photodetector array. In some embodiments, the method further comprises washing the cells or tissue after (f) to remove the nucleotide conjugate from the cells or tissue. In some embodiments, the method further includes repeating (e) and (f) with a second nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a second nucleotide in an index sequence or derivative thereof that binds to a primer. In some embodiments, the detectable label comprises a fluorophore. In some embodiments, the detectable label comprises a quantum dot. In some embodiments, conditions sufficient to couple the target-binding sequence to at least a portion of the target in (b) include an incubation temperature of about 20 to 45°C. In some embodiments, conditions sufficient to couple the target-binding sequence to at least a portion of the target in (b) include a salt. In some embodiments, the RNA is messenger RNA. In some embodiments, the RNA is microRNA. In some embodiments, the RNA is ribosomal RNA. In some embodiments, the RNA is transfer RNA. In some embodiments, the RNA is transfer messenger RNA. In some embodiments, the RNA contains a modification. In some embodiments, the modification is 5' phosphorylation. In some embodiments, the modification is internucleotide bonding. In some embodiments, the probe comprises a nucleic acid. In some embodiments, the nucleic acid comprises deoxyribonucleic acid (DNA). In some embodiments, the nucleic acid comprises RNA. In some embodiments, the RNA-binding sequence comprises a nucleic acid. In some embodiments, the nucleic acid includes deoxyribonucleic acid (DNA). In some embodiments, the nucleic acid comprises RNA. In some embodiments, the probe is a lock probe. In some embodiments, the method further includes, prior to (c), using a ligase to attach a first end of the probe to a second end of the probe.In some embodiments, the ligase is T4 ligase. In some embodiments, the ligase is SplintR ligase. In some embodiments, the amplification reaction in (c) includes rolling circle amplification using a polymerase. In some embodiments, the polymerase is Phi29 polymerase. In some embodiments, the cells or tissue are permeabilized using a solvent. In some embodiments, the solvent is methanol. In some embodiments, the solvent is acetone. In some embodiments, the cells or tissue are permeabilized using a detergent. In some embodiments, the detergent is a saponin. In some embodiments, the detergent is Triton X-100.

[0007] The aspects disclosed herein provide a method for in situ identification of peptides, the method comprising: (a) providing permeabilized cells or tissue, wherein the cells or tissue contain a peptide; (b) guiding a peptide-binding probe to the cells or tissue under conditions sufficient to conjugate the peptide to the peptide, wherein the peptide-binding probe comprises a peptide-binding moiety and an oligonucleotide conjugated thereto; (c) guiding a probe to the cells or tissue under conditions sufficient to conjugate an oligonucleotide-binding probe to at least a portion of the oligonucleotide of the peptide-binding probe, wherein the probe comprises (i) an oligonucleotide-binding probe and (ii) an index sequence; and (d) amplifying the index sequence to generate one or more amplicons, wherein the one or more amplicons contain the index sequence or a derivative thereof. (e) guiding the primer nucleic acid sequence to cells or tissue under conditions sufficient to bind the primer nucleic acid sequence to at least a portion of one or more amplicons to produce a primer-binding index sequence; (f) guiding cells or tissue with a nucleotide conjugate comprising: (i) a detectable marker and (ii) one or more nucleotide portions complementary to the primer-binding index sequence or one or more amplicons thereof; and (g) detecting the binding complex within the cells or tissue to identify the polypeptide. In some embodiments, the method further includes providing the cells in (a). In some embodiments, the cells are cancer cells. In some embodiments, the method further includes providing the tissue in (a). In some embodiments, the tissue is formalin-fixed paraffin-embedded tissue. In some embodiments, the tissue is freshly frozen tissue. In some embodiments, the cells or tissue are extracted from a subject. In some embodiments, the subject is a human subject. In some embodiments, the index sequence comprises nucleic acid. In some embodiments, the length of the index sequence is 4 to 50 nucleotides. In some embodiments, at least a portion of the index sequence or a derivative thereof recognizes the primer nucleic acid sequence. In some embodiments, the primer nucleic acid sequence includes a blocking group. In some embodiments, the blocking group inhibits the incorporation of another nucleotide into the primer nucleic acid sequence. In some embodiments, the blocking group includes a 3'-O-azido group, a 3'-O-azidomethyl group, a 3'-O-alkylhydroxyamino group, a 3'-thiophosphate group, a 3'-O-malonyl group, or a 3'-O-benzyl group. In some embodiments, the primer nucleic acid sequence does not include a 3' hydroxyl group. In some embodiments, the primer nucleic acid sequence binds to at least a portion of the index sequence. In some embodiments, the primer nucleic acid sequence binds to at least a portion of the inverse complementary sequence of the index sequence. In some embodiments, the primer-bound index sequence comprises a double-stranded nucleic acid. In some embodiments, the primer-bound index sequence comprises a single-stranded nucleic acid. In some embodiments, cells or tissues are fixed to the surface of a substrate.In some embodiments, the surface of the substrate has a water contact angle of less than or equal to 45 degrees. In some embodiments, the surface of the substrate is the inner surface of a flow cell. In some embodiments, the inner surface of the flow cell comprises one or more hydrophilic polymer layers. In some embodiments, the one or more hydrophilic polymer layers comprise a branched polymer. In some embodiments, the one or more hydrophilic polymer layers comprise a polymer containing polyethylene glycol. In some embodiments, the nucleotide conjugate comprises a common core. In some embodiments, one or more nucleotide moieties are coupled to the common core. In some embodiments, the common core comprises a polymer, micelles, liposomes, microparticles, nanoparticles, or quantum dots. In some embodiments, the polymer comprises a protein. In some embodiments, in (e), the nucleotide conjugate is included in a mixture of multiple nucleotide conjugates, wherein each of the multiple nucleotide conjugates comprises nucleotide moieties of different types from each other. In some embodiments, the different types of nucleotide moieties comprise at least three different types of nucleotide moieties. In some embodiments, one or more nucleotide moieties do not contain a blocking group coupled thereto. In some embodiments, detection in (f) comprises imaging cells or tissue using one or more image sensors. In some embodiments, the one or more image sensors comprise a photodetector array. In some embodiments, the method further comprises washing the cells or tissue after (f) to remove the nucleotide conjugate from the cells or tissue. In some embodiments, the method further includes repeating (e) and (f) with a second nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a second nucleotide in an index sequence or derivative thereof that binds to a primer. In some embodiments, the detectable label comprises a fluorophore. In some embodiments, the detectable label comprises a quantum dot. In some embodiments, conditions sufficient to couple the target-binding sequence to at least a portion of the target in (b) include an incubation temperature of about 20 to 45°C. In some embodiments, conditions sufficient to couple the target-binding sequence to at least a portion of the target in (b) include a salt. In some embodiments, the polypeptide comprises a protein. In some embodiments, the protein is a cell surface protein. In some embodiments, the protein is an intracellular protein. In some embodiments, the protein is a signal transduction protein. In some embodiments, the polypeptide comprises a protein complex. In some embodiments, the polypeptide-binding portion comprises an antibody or antibody fragment. In some embodiments, the polypeptide-binding portion comprises an aptamer. In some embodiments, the polypeptide-binding portion comprises a nanobody. In some embodiments, the oligonucleotide comprises DNA. In some embodiments, the oligonucleotide is 4 to 50 nucleotides in length. In some embodiments, the polypeptide-binding portion is covalently attached to the oligonucleotide. In some embodiments, the polypeptide-binding portion is non-covalently attached to the oligonucleotide. In some embodiments, the probe comprises a nucleic acid.In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises RNA. In some embodiments, the probe is a lock probe. In some embodiments, prior to (d), a ligase is used to ligate a first end of the probe to a second end. In some embodiments, the ligase is a T4 ligase. In some embodiments, the ligase is a SplintR ligase. In some embodiments, the amplification reaction in (d) includes a rolling circle amplification reaction using a polymerase. In some embodiments, the polymerase is a phi29 polymerase. In some embodiments, the cells or tissue are permeabilized using a solvent. In some embodiments, the solvent is methanol. In some embodiments, the solvent is acetone. In some embodiments, the cells or tissue are permeabilized using a detergent. In some embodiments, the detergent is a saponin. In some embodiments, the detergent is Triton X-100.

[0008] The aspects disclosed herein provide a method for in situ identification of DNA, the method comprising: (a) providing permeabilized cells or tissues, wherein the cells or tissues contain DNA, wherein the DNA is not cDNA; (b) guiding a probe into the cells or tissues under conditions sufficient to couple a DNA-binding sequence to at least a portion of the DNA, wherein the probe comprises (i) a DNA-binding sequence and (ii) an index sequence; (c) amplifying the index sequence to generate one or more amplicons, wherein the one or more amplicons comprise multiple copies of the index sequence or a derivative thereof; (d) guiding a primer nucleic acid sequence into the cells or tissues under conditions sufficient to bind a primer nucleic acid sequence to at least a portion of the one or more amplicons to generate a primer-binding index sequence; (e) guiding the cells or tissues with a nucleotide conjugate comprising: (i) a detectable marker and (ii) one or more nucleotide portions complementary to the nucleotides of the primer-binding index sequence or a derivative thereof; and (f) detecting the binding complex within the cells or tissues, thereby identifying the DNA. In some embodiments, the method further includes providing the cells in (a). In some embodiments, the cells are cancer cells. In some embodiments, the method further includes providing the tissue in (a). In some embodiments, the tissue is formalin-fixed paraffin-embedded tissue. In some embodiments, the tissue is freshly frozen tissue. In some embodiments, the cells or tissue are extracted from a subject. In some embodiments, the subject is a human subject. In some embodiments, the index sequence comprises nucleic acid. In some embodiments, the index sequence is 4 to 50 nucleotides in length. In some embodiments, at least a portion of the index sequence or a derivative thereof recognizes a primer nucleic acid sequence. In some embodiments, the primer nucleic acid sequence comprises a blocking group. In some embodiments, the blocking group inhibits the incorporation of another nucleotide into the primer nucleic acid sequence. In some embodiments, the blocking group comprises a 3'-O-azido group, a 3'-O-azidomethyl group, a 3'-O-alkylhydroxyamino group, a 3'-thiophosphate group, a 3'-O-malonyl group, or a 3'-O-benzyl group. In some embodiments, the primer nucleic acid sequence does not include a 3' hydroxyl group. In some embodiments, the primer nucleic acid sequence binds to at least a portion of the index sequence. In some embodiments, the primer nucleic acid sequence binds to at least a portion of the inverse complementary sequence of the index sequence. In some embodiments, the primer-binding index sequence comprises a double-stranded nucleic acid. In some embodiments, the primer-binding index sequence comprises a single-stranded nucleic acid. In some embodiments, cells or tissues are immobilized on the surface of a substrate. In some embodiments, the surface of the substrate has a water contact angle of less than or equal to 45 degrees. In some embodiments, the surface of the substrate is the inner surface of a flow cell.In some embodiments, the inner surface of the flow cell comprises one or more hydrophilic polymer layers. In some embodiments, the one or more hydrophilic polymer layers comprise a branched polymer. In some embodiments, the one or more hydrophilic polymer layers comprise a polymer containing polyethylene glycol. In some embodiments, the nucleotide conjugate comprises a common core. In some embodiments, one or more nucleotide moieties are coupled to the common core. In some embodiments, the common core comprises a polymer, micelles, liposomes, microparticles, nanoparticles, or quantum dots. In some embodiments, the polymer comprises a protein. In some embodiments, wherein in (e) the nucleotide conjugate is included in a mixture of multiple nucleotide conjugates, wherein each of the multiple nucleotide conjugates comprises nucleotide moieties of different types from each other. In some embodiments, the different types of nucleotide moieties comprise at least three different types of nucleotide moieties. In some embodiments, one or more nucleotide moieties do not contain a blocking group coupled thereto. In some embodiments, detection in (f) comprises imaging the cells or tissue using one or more image sensors. In some embodiments, the one or more image sensors comprise a photodetector array. In some embodiments, the method further comprises washing the cells or tissue after (f) to remove the nucleotide conjugate from the cells or tissue. In some embodiments, the method further includes repeating (e) and (f) with a second nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a second nucleotide in an index sequence or derivative thereof that binds to a primer. In some embodiments, the detectable label comprises a fluorophore. In some embodiments, the detectable label comprises a quantum dot. In some embodiments, conditions sufficient to couple the target-binding sequence to at least a portion of the target in (b) include an incubation temperature of about 20 to 45°C. In some embodiments, conditions sufficient to couple the target-binding sequence to at least a portion of the target in (b) include a salt. In some embodiments, the DNA contains a modification. In some embodiments, the modification is a methylation modification. In some embodiments, the modification is a phosphorylation modification. In some embodiments, the probe comprises a nucleic acid. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises RNA. In some embodiments, the DNA-binding sequence comprises a nucleic acid. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises RNA. In some embodiments, the probe is a lock probe. In some embodiments, the method further includes, prior to (c), ligating a first end of the probe. In some embodiments, the ligase is a T4 ligase. In some embodiments, the ligase is a SplintR ligase. In some embodiments, the amplification reaction in (c) includes rolling circle amplification using a polymerase. In some embodiments, the polymerase is phi29 polymerase. In some embodiments, the cells or tissues are permeabilized using a solvent.In some embodiments, the solvent is methanol. In some embodiments, the solvent is acetone. In some embodiments, cells or tissues are permeated with a detergent. In some embodiments, the detergent is a saponin. In some embodiments, the detergent is Triton X-100.

[0009] The aspects disclosed herein provide a method for in situ identification of ribonucleic acid (RNA), the method comprising: (a) providing permeabilized cells or tissue, wherein the cells or tissue contain ribonucleic acid; (b) guiding a nucleic acid molecule into the cells or tissue under conditions sufficient to couple a target-binding sequence to at least a portion of the ribonucleic acid, wherein the nucleic acid molecule contains a target-binding sequence, wherein the nucleic acid molecule further contains an index sequence; (c) guiding a primer nucleic acid sequence into the cells or tissue under conditions sufficient to bind a primer nucleic acid sequence to at least a portion of one or more amplicones to generate a primer-binding index sequence; (d) guiding the cells or tissue with a nucleotide conjugate comprising: (i) a detectable marker and (ii) one or more nucleotide portions complementary to the nucleotides in the primer-binding index sequence or a derivative thereof; and (e) detecting the binding complex within the cells or tissue to identify the ribonucleic acid. In some embodiments, the method further comprises providing the cells in (a). In some embodiments, the cells are cancer cells. In some embodiments, the method further includes providing the tissue in (a). In some embodiments, the tissue is formalin-fixed paraffin-embedded tissue. In some embodiments, the tissue is freshly frozen tissue. In some embodiments, cells or tissue are extracted from a subject. In some embodiments, the subject is a human subject. In some embodiments, the ribonucleic acid (RNA) is messenger RNA. In some embodiments, the RNA is microRNA. In some embodiments, the RNA is ribosomal RNA. In some embodiments, the RNA is transfer RNA. In some embodiments, the RNA is transfer messenger RNA. In some embodiments, the RNA contains a modification. In some embodiments, the modification includes a methyl group. In some embodiments, the nucleic acid molecule is deoxyribonucleic acid (DNA). In some embodiments, the length of the nucleic acid molecule is about 10 to 80 nucleotides. In some embodiments, the length of the nucleic acid molecule is about 20 to 60 nucleotides. In some embodiments, the length of the nucleic acid molecule is about 30 to 50 nucleotides. In some embodiments, the nucleic acid molecule contains a modification. In some embodiments, the modification is 5' phosphorylation. In some embodiments, the modification is internucleotide bonding. In some embodiments, the target binding sequence is about 5 to 25 nucleotides long. In some embodiments, the method further includes introducing the index-binding molecule under conditions sufficient to couple the index-binding molecule to the index sequence. In some embodiments, the index-binding molecule comprises a nucleic acid. In some embodiments, the index-binding molecule is a lock-lock probe. In some embodiments, the method further includes ligating the index-binding molecule using a ligase. In some embodiments, the ligase is a T4 ligase. In some embodiments, the ligase is a SplintR ligase.In some embodiments, the method further includes amplifying an index sequence. In some embodiments, amplification includes rolling circle amplification using a polymerase. In some embodiments, the polymerase is PHI29 polymerase. In some embodiments, amplification generates multiple copies of the index sequence or an inverse complementary sequence of the index sequence. In some embodiments, the conditions sufficient in (b) to couple the target binding sequence to at least a portion of the ribonucleic acid include a salt. In some embodiments, the conditions sufficient in (b) to couple the target binding sequence to at least a portion of the ribonucleic acid include an incubation temperature of about 20 to 45°C. In some embodiments, the conditions sufficient in (b) to couple the target binding sequence to at least a portion of the ribonucleic acid include a liquid-dissociating reagent. In some embodiments, the index sequence is uniquely associated with the ribonucleic acid. In some embodiments, the index sequence is associated with the ribonucleic acid and at least one other analyte. In some embodiments, the other analyte is a nucleic acid. In some embodiments, the nucleic acid is ribonucleic acid. In some embodiments, the nucleic acid is deoxyribonucleic acid. In some embodiments, the other analyte is a polypeptide. In some embodiments, the polypeptide is a protein. In some embodiments, the index sequence is 4 to 50 nucleotides in length. In some embodiments, at least a portion of the index sequence recognizes a primer nucleic acid sequence. In some embodiments, the primer nucleic acid sequence includes a blocking group. In some embodiments, the blocking group inhibits the incorporation of another nucleotide into the primer nucleic acid sequence. In some embodiments, the blocking group includes a 3'-O-azido group, a 3'-O-azidomethyl group, a 3'-O-alkylhydroxyamino group, a 3'-thiophosphate group, a 3'-O-malonyl group, or a 3'-O-benzyl group. In some embodiments, the primer nucleic acid sequence does not include a 3' hydroxyl group. In some embodiments, the primer nucleic acid sequence binds to at least a portion of an index sequence. In some embodiments, the primer nucleic acid sequence binds to at least a portion of the inverse complementary sequence of the index sequence. In some embodiments, the primer-bound index sequence comprises a double-stranded nucleic acid. In some embodiments, the primer-bound index sequence comprises a single-stranded nucleic acid. In some embodiments, cells or tissues are fixed to a surface of a substrate. In some embodiments, the surface has a water contact angle of less than or equal to 45 degrees. In some embodiments, the surface is the inner surface of a flow cell. In some embodiments, the inner surface of the flow cell comprises one or more hydrophilic polymer layers. In some embodiments, the one or more hydrophilic polymer layers comprise a branched polymer. In some embodiments, the one or more hydrophilic polymer layers comprise a polymer containing polyethylene glycol. In some embodiments, the nucleotide conjugate includes a common core. In some embodiments, one or more nucleotide moieties are coupled to the common core. In some embodiments, the common core includes a polymer, micelles, liposomes, microparticles, nanoparticles, or quantum dots. In some embodiments, the polymer includes a protein.In some embodiments, in step (d), the nucleotide conjugate is included in a mixture of multiple nucleotide conjugates, each of which comprises a nucleotide moiety of a different type from each other. In some embodiments, the different types of nucleotide moiety moiety comprise at least three different types of nucleotide moiety moiety. In some embodiments, one or more nucleotide moiety moiety does not contain a blocking group coupled thereto. In some embodiments, detection in step (e) includes imaging the cell or tissue using one or more image sensors. In some embodiments, the one or more image sensors comprise a photodetector array. In some embodiments, the method further includes washing the cell or tissue after step (e) to remove the nucleotide conjugate from the cell or tissue. In some embodiments, the method further includes repeating steps (d) and (e) with a second nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moiety moiety complementary to a second nucleotide in an index sequence or derivative thereof that binds to a primer. In some embodiments, the detectable label comprises a fluorophore. In some embodiments, the detectable label comprises a quantum dot.

[0010] The aspects disclosed herein provide a system for in situ identification of RNA, the system comprising: a computing device including one or more computer processors, an operating system configured to execute executable instructions, a memory, and a computer program including instructions comprising any of the methods described herein. In some embodiments, the system further comprises RNA.

[0011] The aspects disclosed herein provide a system for in-situ identification of peptides, the system comprising: a computing device including one or more computer processors, an operating system configured to execute executable instructions, a memory, and a computer program including instructions comprising any of the methods described herein. In some embodiments, the system further comprises a peptide. In some embodiments, the system further comprises a peptide-binding probe.

[0012] The aspects disclosed herein provide a system for in situ identification of DNA, the system comprising: a computing device including one or more computer processors, an operating system configured to execute executable instructions, a memory, and a computer program including instructions comprising any of the methods described herein. In some embodiments, the system further comprises DNA. In some embodiments, the system further comprises cells. In some embodiments, the cells are cancer cells. In some embodiments, the system further comprises tissue. In some embodiments, the tissue is formalin-fixed paraffin-embedded tissue. In some embodiments, the tissue is fresh-frozen tissue. In some embodiments, the cells or tissue are extracted from a subject. In some embodiments, the subject is a human subject. In some embodiments, the system further comprises a probe. In some embodiments, the probe is a lock probe. In some embodiments, the lock probe comprises a nucleic acid. In some embodiments, the system further comprises a primer nucleic acid molecule sequence. In some embodiments, the system further comprises a ligase. In some embodiments, the ligase is a T4 ligase. In some embodiments, the ligase is a SplintR ligase. In some embodiments, the system further comprises a polymerase. In some embodiments, the polymerase is a Phi29 polymerase. In some embodiments, the system further comprises a salt. In some embodiments, the salt comprises NaCl, MgCl2, or CaCl2. In some embodiments, the system further comprises a solvent. In some embodiments, the solvent is a polar solvent. In some embodiments, the solvent is methanol. In some embodiments, the solvent is acetone. In some embodiments, the system further includes a detergent. In some embodiments, the detergent is a saponin. In some embodiments, the detergent is Triton X-100. In some embodiments, the system further includes a substrate. In some embodiments, the substrate is the inner surface of the flow cell. In some embodiments, one or more computer processors include a central processing unit. In some embodiments, one or more computer processors include a graphics processing unit. In some embodiments, one or more computer processors include a general-purpose processing unit. In some embodiments, one or more processors include a field-programmable gate array. In some embodiments, the system further includes a fluid system. In some embodiments, the fluid system is configured to provide fluid flow control capability for delivering reagents to a sample in the flow cell.

[0013] The aspects disclosed herein provide a system for in situ identification of ribonucleic acid (RNA), the system comprising: a computing device including one or more computer processors, an operating system configured to execute executable instructions, a memory, and a computer program including instructions comprising any of the methods disclosed herein. In some embodiments, the system further includes cells. In some embodiments, the cells are cancer cells. In some embodiments, the system further includes tissue. In some embodiments, the tissue is formalin-fixed paraffin-embedded tissue. In some embodiments, the tissue is fresh-frozen tissue. In some embodiments, the cells or tissue are extracted from a subject. In some embodiments, the subject is a human subject. In some embodiments, the system further includes ribonucleic acid (RNA). In some embodiments, the system further includes nucleic acid molecules. In some embodiments, the system further includes primer nucleic acid sequences. In some embodiments, the system further includes an index-binding molecule. In some embodiments, the index-binding molecule comprises nucleic acid. In some embodiments, the index-binding molecule is a lock probe. In some embodiments, the index-binding molecule is configured to bind to an index sequence. In some embodiments, the system further includes a ligase. In some embodiments, the ligase is a T4 ligase. In some embodiments, the ligase is a SplintR ligase. In some embodiments, the system further includes a polymerase. In some embodiments, the polymerase is a phi29 polymerase. In some embodiments, the system further includes a salt. In some embodiments, the salt includes NaCl, MgCl2, or CaCl2. In some embodiments, the system further includes a solvent. In some embodiments, the solvent is a polar solvent. In some embodiments, the system further includes a liquid-dissociating reagent. In some embodiments, the system further includes a substrate. In some embodiments, the substrate is the inner surface of the flow cell. In some embodiments, one or more computer processors include a central processing unit. In some embodiments, one or more computer processors include a graphics processing unit. In some embodiments, one or more computer processors include a general-purpose processing unit. In some embodiments, one or more processors include a field-programmable gate array (FPGA). In some embodiments, the system further includes a fluid system. In some embodiments, the fluid system is configured to provide fluid flow control capability for delivering reagents to a sample in the flow cell.

[0014] The aspects disclosed herein provide a kit for in situ identification of RNA, the kit comprising: (a) a probe comprising (i) an index sequence and (ii) an RNA-binding sequence, wherein the RNA-binding sequence recognizes at least a portion of a target; (b) a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to the nucleotides of the primer-binding index sequence, wherein the nucleotide conjugate is configured to form a binding complex between the one or more nucleotide moieties and the nucleotides of the primer-binding index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (b) instructions for in situ identification of RNA, wherein the instructions comprise any of the methods disclosed herein.

[0015] The aspects disclosed herein provide a kit for in situ identification of peptides, the kit comprising: (a) a peptide-binding probe comprising (i) a peptide-binding moiety and (ii) an oligonucleotide conjugated thereto; (b) a probe comprising (i) an index sequence and (ii) an oligonucleotide-binding probe, wherein the oligonucleotide-binding probe recognizes at least a portion of the oligonucleotide; (c) a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to the nucleotides of the primer-binding index sequence, wherein the nucleotide conjugate is configured to form a binding complex between the one or more nucleotide moieties and the nucleotides of the primer-binding index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (d) instructions for in situ identification of peptides, wherein the instructions comprise any of the methods disclosed herein.

[0016] The aspects disclosed herein provide a kit for in situ identification of DNA, the kit comprising: (a) a probe comprising (i) an index sequence and (ii) an RNA-binding sequence, wherein the RNA-binding sequence recognizes at least a portion of a target; (b) a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to the nucleotides of the primer-binding index sequence, wherein the nucleotide conjugate is configured to form a binding complex between the one or more nucleotide moieties and the nucleotides of the primer-binding index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (c) instructions for in situ identification of RNA, wherein the instructions comprise any of the methods disclosed herein. In some embodiments, the kit further comprises a primer nucleic acid sequence configured to bind to at least a portion of the index sequence or a derivative thereof to produce a primer-binding index sequence. In some embodiments, the probe is a lock probe. In some embodiments, the lock probe comprises a nucleic acid. In some embodiments, the kit further comprises a ligase. In some embodiments, the ligase is a T4 ligase. In some embodiments, the ligase is a SplintR ligase. In some embodiments, the kit further comprises a polymerase. In some embodiments, the polymerase is PHI29 polymerase. In some embodiments, the kit further includes a buffer. In some embodiments, the buffer contains a salt. In some embodiments, the salt includes NaCl, MgCl2, or CaCl2. In some embodiments, the buffer contains a solvent. In some embodiments, the solvent is polar. In some embodiments, the buffer contains a dissociation reagent. In some embodiments, the kit further includes a solvent. In some embodiments, the solvent is methanol. In some embodiments, the solvent is acetone. In some embodiments, the kit further includes a detergent. In some embodiments, the detergent is a saponin. In some embodiments, the detergent is Triton X-100. In some embodiments, the kit further includes a flow cell. In some embodiments, the flow cell includes a substrate configured to be coupled to cells or tissue.

[0017] The aspects disclosed herein provide a kit for in situ identification of ribonucleic acid (RNA), the kit comprising: (a) a nucleic acid molecule comprising (i) an index sequence and (ii) a target-binding sequence, wherein the target-binding sequence is complementary to at least a portion of a ribonucleic acid sequence; (b) a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to nucleotides in the primer-binding index sequence, wherein the nucleotide conjugate is configured to form a binding complex between one or more nucleotide moieties and nucleotides of the primer-binding index sequence without incorporating one or more nucleotide moieties into the primer nucleic acid sequence; and (c) instructions for in situ identification of RNA, wherein the instructions comprise any of the methods disclosed herein. In some embodiments, the kit further comprises a primer nucleic acid sequence configured to bind to at least a portion of the index sequence or a derivative thereof to produce a primer-binding index sequence. In some embodiments, the kit further comprises an index-binding molecule. In some embodiments, the index-binding molecule comprises a nucleic acid. In some embodiments, the index-binding molecule is a lock probe. In some embodiments, the index-binding molecule is configured to bind to an index sequence. In some embodiments, the kit further comprises a ligase. In some embodiments, the ligase is T4 ligase. In some embodiments, the ligase is SplintR ligase. In some embodiments, the kit further includes a polymerase. In some embodiments, the polymerase is Phi29 polymerase. In some embodiments, the kit further includes a buffer. In some embodiments, the buffer contains a salt. In some embodiments, the salt includes NaCl, MgCl2, or CaCl2. In some embodiments, the buffer contains a solvent. In some embodiments, the solvent is polar. In some embodiments, the buffer contains a dissociation reagent. In some embodiments, the kit further includes a flow cell. In some embodiments, the flow cell includes a substrate configured to be coupled to cells or tissue.

[0018] The aspects disclosed herein provide a method for in situ sequencing, the method comprising: a) providing a biological sample deposited on a solid carrier, wherein the biological sample contains multiple RNAs, the multiple RNAs including at least a first target RNA; b) providing a plurality of locking probes, wherein an individual locking probe among the plurality of locking probes comprises a linear oligonucleotide, the linear oligonucleotide comprising a first binding arm at one end and a second binding arm at the other end, and a universal sequence region between the first binding arm and the second binding arm, wherein the first and second binding arms of the locking probe are capable of partially hybridizing with a target RNA molecule to form an RNA-locking probe complex, wherein the individual locking probe comprises two or more sequences including each of the following: Any one or any combination of: (i) a universal binding site for sequencing primers; (ii) a universal binding site for amplification primers; (iii) a universal binding site for compacted oligonucleotides; and / or (iv) an optional target barcode sequence that uniquely identifies the target RNA; c) contacting a biological sample with a plurality of lock probes, wherein the contact is performed under conditions suitable for moving the plurality of lock probes into the biological sample and suitable for hybridizing individual lock probes with at least a portion of homologous target RNA within the biological sample to form a plurality of RNA-lock probe complexes, wherein the first and second binding arms of individual lock probes hybridize with proximal positions on the target RNA molecule to form circular lock probes, which, upon hybridization... d) A notch or gap exists between the first and second binding arms; d) The biological sample is contacted with a lock-on probe closure reagent, wherein the contact is performed under conditions suitable for moving the lock-on probe closure reagent into the biological sample and suitable for generating multiple covalently closed lock-on probes; e) The biological sample is contacted with a rolling circle amplification reagent, wherein the contact is performed under conditions suitable for moving the rolling circle amplification reagent into the biological sample; f) Rolling circle amplification is performed within the biological sample to generate multiple tandem molecules, wherein the rolling circle amplification reaction is carried out in the presence of multiple compacted oligonucleotides, wherein each compacted oligonucleotide includes a 5' region binding to the first part of the tandem molecule and a 3' region binding to the second part of the same tandem molecule. This pulls the distal portions of the tandem molecules together, causing the tandem molecules to compact to form DNA nanospheres; and g) under conditions suitable for hybridizing multiple universal sequencing primers with their respective universal sequencing primer binding sites on the tandem and performing multiple polymerase-catalyzed sequencing cycles to generate multiple sequencing reads within the biological sample, the multiple tandem molecules within the biological sample are sequenced by contacting the multiple tandem molecules within the biological sample with: (i) multiple universal sequencing primers, (ii) multiple sequencing polymerases, and (iii) multiple detectable labeled multivalent molecules, wherein the individually detectable labeled multivalent molecules contain a nucleus attached to multiple nucleotide arms, wherein the nucleotide arms are attached to nucleotide portions.

[0019] Further aspects and advantages of this disclosure will become apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the disclosure are shown and described. As will be appreciated, this disclosure is capable of other and different embodiments, and certain details thereof can be modified in various obvious ways without departing from this disclosure. Therefore, the drawings and description are to be regarded in an illustrative rather than restrictive manner.

[0020] By incorporating references

[0021] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference. In the event that any publication or patent or patent application incorporated by reference contradicts the disclosure contained herein, this specification is intended to substitute for and / or give precedence to any such contradictory material. Attached Figure Description

[0022] The novel features of the inventive concept are specifically set forth in the appended claims. A better understanding of the features and advantages of the inventive concept will be obtained by referring to the following detailed description of illustrative embodiments, which utilize the principles of the inventive concept and are illustrated in the accompanying drawings:

[0023] Figures 1A to 1H The steps for target nucleic acid sequencing using non-limiting examples of multivalent binding compositions (e.g., nucleotide conjugates) are demonstrated: Figure 1A Non-limiting example 4 of target nucleic acid attachment to a surface is shown; Figure 1B It demonstrates the cloning of target nucleic acids to form amplified target nucleic acid molecular clusters; Figure 1C Non-limiting examples of making a target nucleic acid bind to a primer to produce a primer-bound target nucleic acid are shown; Figure 1D Non-limiting examples of contacting primer-bound target nucleic acids with binding complexes and polymerases to form binding complexes are presented; Figure 1E Non-limiting examples of images of surface-captured bound complexes are shown; Figure 1F A non-limiting example of extending the primer strand by one nucleotide is shown; Figure 1G This demonstrates another non-limiting example of contacting the primer-bound target nucleic acid with the binding complex and polymerase to form a binding complex; and Figure 1H A non-limiting example is shown, illustrating images of the binding complex captured on the surface during subsequent sequencing cycles.

[0024] Figures 2A to 2B Non-limiting examples of using nucleotide conjugates (e.g., detectable nucleotide conjugates) to detect target nucleic acids are presented. Figure 2AThe steps for contacting polymerase and nucleotide conjugates (e.g., detectable nucleotide conjugates) with some nucleic acid molecules are shown; Figure 2B The diagram illustrates the binding complex formed between polymerase, nucleotide conjugate (e.g., detectable nucleotide conjugate), and target nucleic acid molecule.

[0025] Figure 3 A flowchart is shown for sequencing the target nucleic acid and extending the primer chain by adding a single base.

[0026] Figure 4 A flowchart is shown for sequencing target nucleic acids and extending primer chains by incorporating nucleotides into particle-nucleotide conjugates.

[0027] Figures 5A to 5C illustrate non-limiting examples of different configurations of nucleotide conjugates (e.g., detectable nucleotide conjugates): Figure 5A shows nucleotide conjugates (e.g., detectable nucleotide conjugates) with various multi-arm configurations; Figure 5B shows polymer-nucleotide conjugates with polymer branches radiating from the center; and Figure 5C shows nucleotide conjugates (e.g., detectable nucleotide conjugates) with a biotin-binding moiety.

[0028] Figure 6 A general graphical description of the increase in signal intensity observed during the binding, retention and washing, and removal of the multivalent substrate is shown.

[0029] Figures 7A to 7G illustrate multivalent polyethylene glycol (PEG) polymer-nucleotide (base-labeled) conjugates containing 20 nM Klenow polymerase and 2.5 mM Sr +2 Fluorescence images of conjugates containing DNA templates (with G or A as the first base; prepared using rolling circle amplification (RCA)) after contact with the surface of a vector in exposure buffer. These conjugates have an effective nucleotide concentration of 500 nanomolars (nM) and different PEG branch lengths. Images were acquired after washing with an imaging buffer of the same composition as the exposure buffer but lacking nucleotides and polymerase. The inset images show images obtained using multivalent PEG-nucleotide ligands with the following arm lengths: Figure 7A: 1K PEG. Figure 7B: 2K PEG. Figure 7C: 3K PEG. Figure 7D: 5K PEG. Figure 7E: 10K PEG. Figure 7F: 20K PEG. Figure 7G The images shown are obtained using 10K PEG and an inactivated Klenow polymerase containing the mutant D882H. Figure 7H shows an image obtained using 10K PEG and an inactivated Klenow polymerase containing the mutant D882E. Figure 7I shows an image obtained using 10K PEG and an inactivated Klenow polymerase containing the mutant D882A. Figure 7GImages obtained using 10KPEG and active wild-type Klenow polymerase are shown.

[0030] Figure 8 The quantitative representation of fluorescence intensity of the images shown in Figures 7A to 7F is shown, separated by color value, with orange trajectories corresponding to red markers (Cy3 markers; A bases) and blue trajectories corresponding to green markers (Cy5 markers; G bases).

[0031] Figures 9A to 9J show fluorescence images of steps in the sequencing reaction using a multivalent PEG substrate composition. Figure 9A. DNA RCA template (G and A first bases) in a solution containing 20 nM Klenow polymerase and 2.5 mM Sr +2 Red and green fluorescence images of the polyvalent PEG-nucleotide (base-labeled) ligands PB1 (Fig. 9B), PB2 (Fig. 9C), PB3 (Fig. 9D), and PB5 (Fig. 9E) after exposure to 500 nM of base-labeled nucleotides (A-Cy3 and G-Cy5) in exposure buffer. Images were collected after washing with imaging buffer containing the same composition as the exposure buffer but without nucleotides or polymerase. Contrast was scaled to maximize visualization of the weakest signals, but no signal was observed after washing with imaging buffer (a. inset). Figs. 9B-9E: Fluorescence images of polyvalent PEG-nucleotide (base-labeled) ligands PB1 (Fig. 9B), PB2 (Fig. 9C), PB3 (Fig. 9D), and PB5 (Fig. 9E) with an effective nucleotide concentration of 500 nM after mixing in exposure buffer and imaging in imaging buffer as described above. Fig. 9F: Fluorescence image of PB5 at 2.5 μM after mixing in exposure buffer and imaging in imaging buffer as described above. Figures 9G to 9I show fluorescent images of further base differentiation by exposing the binding complex to the inactivating mutant of Klenow polymerase (Figure 9G. D882H; Figure 9H. D882E; Figure 9I. D882A) relative to the wild-type Klenow (control) enzyme (Figure 9J).

[0032] Figures 10A to 10B This demonstrates the effectiveness of multivalent reporter compounds in determining the base sequence of DNA sequences through five sequencing cycles: Figure 10A The template image and expected sequence obtained after each sequencing cycle are shown; and Figure 10B It shows the use of Figure 10A The results of the alignment and sequencing of the images obtained from the image.

[0033] Figure 11 The fluorescence of the multivalent substrate bound to the DNA cluster, as shown in Figure 9, is normalized, and the substrate complex is formed in the presence (condition B) and absence (condition A) of Triton-X100 (0.016%).

[0034] Figure 12Normalized fluorescence intensities of the multivalent substrate and free nucleotides are shown. (Top panel) Two repeats of the multivalent substrate bound to the DNA cluster (conditions A and B) after 1 minute, relative to the binding complex formed using labeled free nucleotides (condition C); (Bottom panel) Fluorescence time-courses of the multivalent substrate complex over a 60-minute period.

[0035] Figure 13 A non-limiting example of a computing device is shown; in this case, the device has one or more processors, memory, storage devices, and network interfaces.

[0036] Figure 14 A non-limiting example of a system that provides web / mobile applications is shown; in this case, the system provides a browser-based and / or native mobile user interface.

[0037] Figure 15 A non-limiting instance of a cloud-based web / mobile application delivery system is shown; in this case, the system includes elastically load-balanced, auto-scaling web server and application server resources, as well as synchronously replicated databases.

[0038] Figure 16 A schematic diagram is shown, comprising a multivalent molecular core attached to multiple nucleotide arms.

[0039] Figure 17 A schematic diagram of a multivalent molecule containing a dendritic nucleus attached to multiple nucleotide arms is shown.

[0040] Figure 18 A schematic diagram is shown of a multivalent molecule comprising a nucleus attached to multiple nucleotide arms, wherein the nucleotide arms contain biotin, spacers, linkers, and nucleotide moieties.

[0041] Figure 19 A schematic diagram of a nucleotide arm is shown, comprising a nuclear attachment portion, a spacer, a linker, and a nucleotide portion.

[0042] Figure 20 The chemical structures of spacers (top) and various connectors are shown.

[0043] Figure 21 The chemical structures of various connectors are shown.

[0044] Figure 22 The chemical structures of various linkers attached to nucleotide moieties are shown.

[0045] Figure 23 The chemical structures of various linkers attached to nucleotide moieties are shown.

[0046] Figure 24 The chemical structures of various linkers attached to nucleotide moieties are shown.

[0047] Figure 25 The chemical structure of the biotinylated nucleotide arm is shown. In this example, the nucleotide moiety is attached to the linker via a propargylamine attachment at the 5-position of the pyrimidine base or the 7-position of the purine base.

[0048] Figure 26 A schematic diagram of guanine tetrads (e.g., G-tetrads) is shown.

[0049] Figure 27 A schematic diagram of the intramolecular G-quadruplex structure is shown.

[0050] Figure 28 A schematic diagram of the workflow for generating circularized lockable probes is shown, including hybridizing first and second lockable probes (respectively) with first and second target RNA molecules to generate first and second circularized lockable probes (respectively).

[0051] Figure 29 It was shown as Figure 28 The diagram shown illustrates the continuation of the workflow.

[0052] Figure 30 A schematic diagram of the workflow for generating circularized lockable probes is shown, including hybridizing first and second lockable probes (respectively) with first and second target RNA molecules to generate first and second circularized lockable probes (respectively).

[0053] Figure 31 It was shown as Figure 30 The diagram shows a continuation of the workflow.

[0054] Figure 32 A schematic diagram of the workflow for generating circularized lockable probes is shown, including hybridizing first and second lockable probes (respectively) with first and second target RNA molecules to generate first and second circularized lockable probes (respectively).

[0055] Figure 33 It was shown as Figure 32 The diagram shows a continuation of the workflow.

[0056] Figure 34 Three graphs of data from the sequencing run are shown, illustrating how the lock-in probe hybridizes with RNA within the biological sample, undergoes gap filling and ligation to generate circular nucleic acids, which then undergo rolling circle amplification to generate tandem molecules within the biological sample. These tandem molecules undergo a two-stage sequencing workflow of 100 sequencing cycles. The top graph shows intensity measurements. The middle graph shows background measurements. The bottom graph shows lag and forward measurements.

[0057] Figure 35A schematic diagram of the workflow for sequencing intracellular tandem structures is shown.

[0058] Figure 36 A schematic diagram of the workflow for sequencing intracellular tandem structures is shown.

[0059] Figure 37 A schematic diagram of the workflow for sequencing intracellular tandem structures is shown.

[0060] Figure 38 A schematic diagram of the workflow for sequencing intracellular tandem structures is shown.

[0061] Figure 39 A schematic diagram of the in-situ target detection workflow is shown, involving direct target detection and the formation of binding complexes.

[0062] Figure 40 A schematic diagram of the in-situ target detection workflow is shown, involving direct target detection, amplification reaction to form an amplicon, and formation of a binding complex with the amplicon.

[0063] Figure 41 The diagram illustrates the workflow for in situ RNA detection, involving direct RNA detection, amplification to form an amplicon, and the formation of a binding complex with the amplicon.

[0064] Figure 42 The diagram illustrates the workflow for in situ DNA detection, involving direct DNA detection, amplification reaction to form an amplicon, and the formation of a binding complex with the amplicon.

[0065] Figure 43 A schematic diagram of the workflow for in situ detection of peptides is shown, involving direct detection of the peptide, performing an amplification reaction to form an amplicon, and forming a binding complex with the amplicon. Detailed Implementation

[0066] This document provides kits, systems, and methods for in situ detection of target nucleic acids in biological samples. This disclosure provides novel solutions and methods to address many shortcomings of existing technologies. Multiple components can be used to facilitate the detection of target nucleic acids, including nucleic acid molecules, detectable nucleotide conjugates, and primer nucleic acid sequences. The nucleic acid molecule may contain an index sequence corresponding to the target nucleic acid. Detection may include binding the nucleic acid molecule to the target nucleic acid and contacting the nucleic acid molecule with the primer nucleic acid sequence to form a primer-bound index sequence. Nucleotide conjugates (e.g., detectable nucleotide conjugates) may be added to the sample, and binding complexes may form between at least two primer-bound index sequences and the detectable nucleotide conjugates. The binding complexes may be detected to reveal the identity of the terminal nucleotides of the primer-bound index sequences. This process can be repeated to reveal the identity of each nucleotide within the index sequence, thereby identifying the target nucleic acid.

[0067] The methods described herein may involve detecting one or more binding complexes as described above, and thereby identifying target nucleic acids. The systems described herein may include computing devices comprising computer programs having instructions to perform any of the methods described herein. The computing devices may include multiple components, including but not limited to one or more processors, operating systems, and memory. The kits described herein can be used to perform any of the methods described herein. The kits may include nucleic acid molecules, detectable nucleotide conjugates, and instructions for use.

[0068] definition

[0069] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and such definitions included herein should not be construed as indicating a material difference from the commonly understood meaning in the art.

[0070] Throughout this application, various embodiments may be presented in a range format. It should be understood that the range format description is for convenience and brevity only and should not be construed as a hard limitation on the scope of this disclosure. Therefore, a range description should be considered as having specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, a range description (such as 1 to 6) should be considered as having specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0071] As used in the specification and claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. For example, the term “sample” includes multiple samples, including mixtures thereof.

[0072] As used herein, the term “approximately” refers to a number plus or minus 10% of that number. The term “approximately” refers to a range minus 10% of its lowest value and plus 10% of its highest value.

[0073] As used herein, a "detectable nucleotide conjugate" refers to a composition comprising multiple nucleotides and a detectable label attached to a common nucleus. One or more nucleotides may include unlabeled and labeled nucleotides. Nucleotides may include blocking groups. The detectable label may be a fluorophore. The multiple nucleotides and the detectable label may be covalently or non-covalently attached to the common nucleus.

[0074] The terms “determine,” “measure,” “evaluate,” “assess,” “determine,” and “analyze” are generally used interchangeably herein to refer to various forms of measurement. These terms include determining the presence or absence of an element (e.g., detection). These terms may include quantitative, qualitative, or both quantitative and qualitative determinations. Assessments may be relative or absolute. “Detecting presence” may include determining the quantity of something present, in addition to determining whether something exists or does not exist based on the context.

[0075] As used herein, a “nucleic acid” (also known as a “polynucleotide” or “oligonucleotide”) is a polymer of two or more nucleotides, or a variant or functional segment thereof, bound together by covalent nucleoside bonds. In examples of naturally occurring nucleic acids, the nucleoside bonds are typically phosphodiester bonds. However, other examples may optionally include other nucleoside bonds, such as phosphate thioester bonds, and may or may not contain phosphate ester groups. Nucleic acids include double-stranded and single-stranded deoxyribonucleic acid (DNA), as well as double-stranded and single-stranded ribonucleic acid (RNA), DNA / RNA hybrids, peptide-nucleic acid (PNA), hybrids between PNA and DNA or RNA, and may also include other types of nucleic acid modifications.

[0076] As used herein, “nucleotide” means nucleotide, nucleoside, or analogue thereof. A nucleotide is a naturally occurring and chemically modified nucleotide, and may include, but is not limited to, nucleosides, ribonucleotides, deoxyribonucleotides, protein-nucleic acid residues, or derivatives thereof. Examples of nucleotides include adenine, thymine, uracil, cytosine, guanine, or residues thereof; deoxyadenine, deoxythymine, deoxyuracil, deoxycytosine, deoxyguanine, or residues thereof; adenine PNA, thymine PNA, uracil PNA, cytosine PNA, guanine PNA, or residues thereof or equivalents thereof; and N-glycosides or C-glycosides of purine or pyrimidine bases (e.g., deoxyribonucleosides containing 2-deoxy-D-ribose or ribonucleosides containing D-ribose).

[0077] As used herein, the term "complementarity" refers to the topological compatibility or mating of the interaction surfaces of a ligand molecule and its receptor. Therefore, receptors and their ligands can be described as complementary, and furthermore, the contact surface properties are complementary to each other. Regarding nucleic acid sequences, complementarity refers to the tendency of a nucleic acid sequence to hybridize with another nucleic acid sequence by virtue of the nucleotides constituting that sequence. Nucleic acids bind to each other through non-covalent interactions between nucleotides, based on base-pairing interactions between adenine (A) and thymine (T) and between guanine (G) and cytosine (C). When two nucleic acids are antiparallel to each other, the nucleotide bases at each position in the sequence will be complementary.

[0078] As used herein, a “branched polymer” refers to a polymer having multiple functional groups that facilitate the conjugation of bioactive molecules, such as nucleotides, and these functional groups may be attached to the side chains of the polymer or directly to the central core or main chain of the polymer. A branched polymer may have a linear main chain in which one or more functional groups detach from the main chain for conjugation. A branched polymer may also be a polymer having one or more side chains with sites suitable for conjugation. Examples of functional groups include, but are not limited to: hydroxyl, ester, amine, carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, hydrazine, thiol, alkyl acid, acyl halide, isocyanate, isothiocyanate, maleimide, vinyl sulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, diketone, methanesulfonate, toluenesulfonate, and trifluoroethylsulfonate.

[0079] As used herein, "polymerase" refers to an enzyme containing a nucleotide-binding moiety that facilitates the formation of a binding complex between a target nucleic acid and its complementary nucleotide. Polymerases may possess one or more activities, including but not limited to: base analog detection activity, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand displacement activity, and nucleotide binding and recognition. Polymerases may include catalytically inactivating polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes containing a nucleotide-binding moiety.

[0080] As used herein, “retention time” refers to the length of time a binding complex remains stable without any binding component dissociating from the binding complex. The binding complex may contain a target nucleic acid, a polymerase, and a detectable nucleotide conjugate. Retention time indicates the stability of the binding complex and the strength of the binding interaction. Retention time can be measured by observing the onset and / or duration of the binding complex (e.g., by observing a signal from a labeled component of the binding complex). For example, a labeled nucleotide or a labeled reagent containing one or more nucleotides may be present in the binding complex, thus allowing a signal from the label to be detected during the retention time of the binding complex. An example label is a fluorescent label.

[0081] It should be understood that the use of alternative terms (e.g., "or") is considered to refer to one or both of the alternatives or any combination thereof.

[0082] As used herein, the term “and / or” will be considered to mean that each of the specified features or components is explicitly disclosed with or without the other. For example, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include: “A and B”; “A or B”; “A” (A only); and “B” (B only). Similarly, the term “and / or” as used herein, such as “A, B and / or C”, is intended to cover each of the following aspects: “A, B and C”; “A, B or C”; “A or C”; “A or B”; “B or C”; “A and B”; “B and C”; “A and C”; “A” (A only); “B” (B only); and “C” (C only).

[0083] As used herein and in the appended claims, the terms “comprising,” “including,” “having,” and “containing,” and their grammatical variations, are intended to be non-limiting, such that one or more items in the list do not exclude other items that may be replaced or added to the listed items. It should be understood that whenever an aspect is described herein with the language “comprising,” other similar aspects described as “consisting of” and / or “substantially consisting of” are also provided.

[0084] As used herein, the terms “about” and “approximately” mean a value or composition within an acceptable error range for a particular value or composition, as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about” or “approximately” may mean within one or more standard deviations. Alternatively, depending on the limitations of the measurement system, “about” or “approximately” may mean a range of up to 10% (i.e., ±10%) or greater. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. Furthermore, particularly for biological systems or methods, the term may mean a value of up to one order of magnitude or up to 5 times. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition. Furthermore, in the case of providing ranges and / or subranges of values, the range and / or subranges may include the endpoints of the range and / or subranges.

[0085] As used herein, the term "polymerase" and its variants include enzymes comprising a domain that binds a nucleotide (or nucleoside), wherein the polymerase can form a complex having a template nucleic acid and a complementary nucleotide. A polymerase may have one or more activities, including but not limited to: base analog detection activity, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand substitution activity, and nucleotide binding and recognition. A polymerase can be any enzyme capable of catalyzing the polymerization of nucleotides (including their analogs) into a nucleic acid chain. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, a polymerase includes one or more active sites at which nucleotide binding and / or nucleotide polymerization catalysis can occur. In some embodiments, the polymerase includes other enzymatic activities, such as, for example, 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, the polymerase has strand substitution activity. Polymerases may include, but are not limited to: naturally occurring polymerases and any subunits and truncated forms thereof, mutant polymerases, variant polymerases, recombinant, fusion, or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives, or fragments thereof (e.g., catalytically active fragments) that retain the ability to catalyze nucleotide polymerization. Polymerases include catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes containing nucleotide-binding domains. In some embodiments, polymerases may be isolated from cells or produced using recombinant DNA technology or chemical synthesis methods. In some embodiments, polymerases may be expressed in prokaryotes, eukaryotes, viruses, or bacteriophages. In some embodiments, polymerases may be post-translational modified proteins or fragments thereof. Polymerases may be derived from prokaryotes, eukaryotes, viruses, or bacteriophages. Polymerases include DNA-guided DNA polymerases and RNA-guided DNA polymerases.

[0086] As used herein, the term "strand displacement" refers to the ability of a polymerase to locally separate double-stranded nucleic acid strands and synthesize a new strand in a template-based manner. Strand displacement polymerases displace the complementary strand from the template strand and catalyze the synthesis of the new strand. Strand displacement polymerases include mesophilic and thermophilic polymerases. Strand displacement polymerases include wild-type enzymes and variants (including exonuclease subtraction mutants, mutant versions, chimeric enzymes, and truncated enzymes). Examples of strand displacement polymerases include phi29 DNA polymerase, large fragment Bst DNA polymerase, large fragment Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-), the Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase can be a wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), a variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or a chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0087] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide,” as used herein, and other related terms, are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs generated using nucleotide analogs (e.g., peptide nucleic acids (PNAs) and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids comprise polymers of nucleotides, wherein the nucleotides comprise natural or non-natural bases and / or sugars. Nucleic acids contain naturally occurring nucleoside bonds, such as phosphodiester bonds. Nucleic acids may lack phosphate ester groups. Nucleic acids contain non-natural nucleoside bonds, including thiophosphate, sulfur-containing phosphate, or peptide nucleic acid (PNA) bonds. In some embodiments, nucleic acids comprise one type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0088] As used herein, the terms “operably linked” and “operably conjugated” or related terms refer to the juxtaposition of components. Juxtaposed components may be covalently linked together. For example, two nucleic acid components may be enzymatically linked together, wherein the bond conjugating the two components together comprises a phosphodiester bond. First and second nucleic acid components may be linked together, wherein the first nucleic acid component can confer function to the second nucleic acid component. For example, a bond between a primer-binding sequence and a sequence of interest forms a nucleic acid library molecule having a portion that can bind to a primer. In another instance, a transgene (e.g., a nucleic acid encoding a polypeptide or a nucleic acid sequence of interest) may be linked to a vector, wherein the bond allows the transgene sequence contained in the vector to be expressed or function. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects transgene expression. In some embodiments, the vector includes at least one host cell regulatory sequence, which includes a promoter sequence, an enhancer, a transcription and / or translation initiation sequence, a transcription and / or translation termination sequence, a polypeptide secretion signaling sequence, and so on. In some embodiments, the host cell regulatory sequence controls the level, timing, and / or location of transgene expression.

[0089] The terms “linked,” “connected,” “attached,” “additional,” and variations thereof encompass any type of fusion, binding, attachment, or association between any combination of compounds or molecules that possesses sufficient stability to withstand use in a particular procedure. This procedure may include, but is not limited to: nucleotide binding; nucleotide incorporation; deblocking (e.g., removal of chain terminations); washing; removal; flow; detection; imaging and / or identification. Such bonds may include, for example, covalent bonds, ionic bonds, hydrogen bonds, dipole-dipole bonds, hydrophilic bonds, hydrophobic bonds, or affinity bonds, bonds or associations involving van der Waals forces, mechanical bonds, and so on. In some embodiments, such bonds occur intramolecularly, for example, by linking the ends of single-stranded or double-stranded linear nucleic acid molecules together to form a cyclic molecule. In some embodiments, such bonds may occur between combinations of different molecules, or between molecules and non-molecules, including, but not limited to: bonds between nucleic acid molecules and solid surfaces; bonds between proteins and detectable reporter gene portions; bonds between nucleotides and detectable reporter gene portions; and so on. Some examples of bonding can be found, for example, Hermanson, G., “Bioconjugate Techniques”, 2nd edition (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998).

[0090] As used herein, the term "primer" and related terms refer to oligonucleotides capable of hybridizing with DNA and / or RNA polynucleotide templates to form double-stranded molecules. Primers include natural nucleotides and / or nucleotide analogs. Primers can be recombinant nucleic acid molecules. Primers can have any length, but typically range from 4 to 50 nucleotides. Typical primers include a 5' end and a 3' end. The 3' end of a primer may contain a 3'OH portion that acts as the initiation site for nucleotide polymerization in a polymerase-catalyzed primer extension reaction. Alternatively, the 3' end of a primer may lack a 3'OH portion or may contain a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any nucleotide or more along the length of the primer may be labeled with a detectable reporter gene portion. Primers may be in solution (e.g., soluble primers) or may be immobilized to a vector (e.g., capture primers).

[0091] The terms “template nucleic acid,” “template polynucleotide,” “target nucleic acid,” “target polynucleotide,” “template strand,” and other variations refer to the nucleic acid strand used as the base nucleic acid molecule in any iterative sequencing method described herein. The template nucleic acid can be single-stranded or double-stranded, or it can have single-stranded or double-stranded portions. The template nucleic acid can be obtained from naturally occurring sources, in recombinant forms, or chemically synthesized to include any type of nucleic acid analogue. The template nucleic acid can be linear, circular, or other forms. The template nucleic acid can include an insert portion having an insert sequence. The template nucleic acid can also include at least one adaptor sequence. The insert portion can be isolated in any form, including chromosomal, genomic, organelle (e.g., mitochondrial, chloroplast, or ribosome) recombinant molecules obtained from fresh-frozen paraffin-embedded tissue, puncture biopsy, circulating tumor cells, cell-free circulating DNA, or any type of nucleic acid library; cloned or amplified cDNA; RNA (such as precursor mRNA or mRNA); oligonucleotides; and whole-genome DNA. The insert can be isolated from any source, including organisms such as prokaryotes, eukaryotes (e.g., humans, plants, and animals), fungi, viral cells, tissues, normal or diseased cells or tissues, body fluids (including blood, urine, serum, lymph, tumors, saliva, anal and vaginal secretions, amniotic samples, sweat, and semen), environmental samples, culture samples, or synthetic nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. The insert can be isolated from any organ, including the head, neck, brain, mammary gland, ovary, cervix, colon, rectum, endometrium, gallbladder, intestine, bladder, prostate, testis, liver, lung, kidney, esophagus, pancreas, thyroid gland, pituitary gland, thymus, skin, heart, larynx, or other organs. The template nucleic acid can undergo nucleic acid analysis (including sequencing and compositional analysis).

[0092] The term "adaptor" and related terms refer to an oligonucleotide that can be operatively linked to a target polynucleotide, wherein the adaptor confers function on the co-linked adaptor-target molecule. Adaptors comprise DNA, RNA, chimeric DNA / RNA, or analogs thereof. Adaptors may include at least one ribonucleoside residue. Adaptors may be single-stranded, double-stranded, or have single-stranded and / or double-stranded portions. Adaptors may be configured in linear, stem-loop, hairpin, or Y-shaped forms. Adaptors can be of any length, including 4 to 100 nucleotides or longer. Adaptors may have blunt ends, overhanging ends, or a combination of both. Overhanging ends include 5' and 3' overhangs. The 5' end of a single-stranded adaptor or one strand of a double-stranded adaptor may have a 5' phosphate group or lack a 5' phosphate group. Adaptors may include a 5' tail that has not hybridized to the target polynucleotide (e.g., a tailed adaptor), or the adaptor may be tailless. The adaptor may include a sequence complementary to at least a portion of a primer (such as an amplification primer, sequencing primer, or capture primer (e.g., a soluble or immobilized capture primer)). The adaptor may contain a random or degenerate sequence. The adaptor may contain at least one inosine residue. The adaptor may contain at least one thiophosphate, thiophosphate, and / or phosphoramide bond. The adaptor may contain a barcode sequence that can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplex assays. In some embodiments, the barcode sequence uniquely identifies the target RNA. The adaptor may contain a unique identification sequence (e.g., a unique molecular index, UMI; or a unique molecular tag) that can be used to uniquely identify the nucleic acid molecule to which the adaptor is appended. In some embodiments, the unique identification sequence can be used to increase error correction and accuracy, reduce false-positive variant detection rates, and / or improve the sensitivity of variant detection. The adaptor may include at least one restriction enzyme recognition sequence, including any combination of any one or more of the following groups: type I, type II, type III, type IV, Hs, or type IIB.

[0093] In some embodiments, any of the amplification primer sequence, sequencing primer sequence, capture primer sequence, target capture sequence, circular anchoring sequence, sample barcode sequence, target barcode sequence, spatial barcode sequence, or anchoring region sequence may be about 3 to 50 nucleotides in length, about 5 to 40 nucleotides in length, or about 5 to 25 nucleotides in length.

[0094] The term "universal sequence" and related terms refer to a sequence common to two or more polynucleotide molecules in a nucleic acid molecule. For example, an adaptor with a universal sequence can be operatively bound to multiple polynucleotides, such that a co-bound population of molecules carries the same universal adaptor sequence. Examples of universal adaptor sequences include amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble or immobilized capture primers).

[0095] When used to refer to nucleic acid molecules, the terms "hybridize," "hybridizing," "hybridization," or other related terms refer to the hydrogen bonding between two different nucleic acids to form a double-stranded nucleic acid. Hybridization also includes the hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridized molecule with double-stranded regions. Hybridization can involve Watson-Crick or Hoogstein binding to form double-stranded nucleic acids or double-stranded regions within a nucleic acid molecule. The two different regions of a double-stranded nucleic acid or a single nucleic acid can be completely complementary or partially complementary. Complementary nucleic acid strands do not need to hybridize to each other across their entire length. Complementary base pairing can be standard AT or CG base pairing, or it can be other forms of base pairing interactions. Double-stranded nucleic acids can contain mismatched base-paired nucleotides.

[0096] When used to refer to nucleic acids, the terms "extend," "extending," "extension," and other variations refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation involves the polymerization of one or more nucleotides into the terminal 3' OH end of the nucleic acid chain, resulting in the extension of the nucleic acid chain. Nucleotide incorporation can be performed using native nucleotides and / or nucleotide analogs. Typically, but not always, nucleotide incorporation occurs in a template-dependent manner. Any suitable method for extending nucleic acid molecules can be used, which involves primer extension catalyzed by DNA polymerase or RNA polymerase.

[0097] The term "nucleotide" and related terms refer to a molecule comprising an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and at least one phosphate ester group. Canonical or non-canonical nucleotides are used in accordance with the terminology. In some embodiments, phosphate esters include monophosphate, diphosphate, or triphosphate, or corresponding phosphate ester analogs. The term "nucleoside" refers to a molecule comprising an aromatic base and a sugar. Nucleotides and nucleosides may be unlabeled or labeled with a detectable reporter gene motif.

[0098] Nucleotides (and nucleosides) typically contain heterocyclic bases, including substituted or unsubstituted nitrogen-containing parent heteroaromatic rings, commonly found in nucleic acids, including naturally occurring, substituted, modified, or engineered variants or analogues thereof. The bases of nucleotides (or nucleosides) are capable of forming Watson-Crick and / or Husstein hydrogen bonds with suitable complementary bases. Example bases include, but are not limited to, purines and pyrimidines, such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethylene adenine, N... 6 -Δ 2-Isopentenyladenine (6iA), N 6 -Δ 2 -Isopentenyl-2-methylthioadenine (2ms6iA), N 6 -Methyladenine, guanine (G), isoguanine, N 2 -Dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O 6 -Methylguanine; 7-deazo-purine, such as 7-deazoadenine (7-deazoaden-A) and 7-deazoguanine (7-deazo-G); pyrimidine, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4 -Methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydrouracil; D); indole, such as nitroindole and 4-methylindole; pyrrole, such as nitropyrrole; muscarin; inosine; hydroxymethylcytosine; 5-methylcytosine; base (Y); and methylated, glycosylated, and acylated base moieties; etc. Further examples of bases can be found in Fasman, 1989, “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press, Pocaraton, Florida.

[0099] Nucleotides (and nucleosides) typically contain a sugar moiety, such as a carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), an acyclic moiety (Martinez et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez et al., 1997 Bioorganic & Medicinal Chemistry Letters Vol. 7: 3013-3016), and other sugar moieties (Joeng et al., 1993 J. Med. Chem. 36: 2627-2638; Kim et al., 1993 J. Med. Chem. 36: 30-7; Eschenmosser 1999 Science 284: 2118-2124; and US Patent No. 5,558,991). The sugar moiety includes: ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoribosyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoribosyl; 3'-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[0100] In some embodiments, the nucleotide comprises a chain of one, two, or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, the nucleotide is an analogue having a phosphorus chain, wherein the phosphorus atoms are linked together by an intermediate O, S, NH, methylene, or ethylene group. In some embodiments, the phosphorus atom in the chain comprises a substituted side group (containing O, S, or BH3). In some embodiments, the chain comprises a phosphate ester group substituted with an analogue, including phosphoramide, thiophosphate, dithiophosphate, and O-methylphosphoramide groups.

[0101] The terms "reporter moiety," "reporter moieties," or related terms refer to compounds that generate or cause the generation of a detectable signal. A reporter moiety is sometimes referred to as a "tag." Any suitable reporter moiety can be used, including luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophores, radioisotopes, electrochemical, mass spectrometry, Raman spectroscopy, haptens, affinity tags, atoms, or enzymes. A reporter moiety produces a detectable signal caused by a chemical or physical change, such as heat, light, electricity, pH, salt concentration, enzymatic activity, or proximity events. Proximity events include two reporter moieties coming close to each other, associating with each other, or binding to each other. Those skilled in the art are well aware of selecting reporter moieties such that each reporter moiety absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from other reporter moieties, allowing for the monitoring of the presence of different reporter moieties in the same or different reactions. Two or more different reporter moieties with spectrally distinct emission profiles or with minimal overlap spectral emission profiles can be selected. The reporter gene portion can be linked (e.g., operatively linked) to nucleotides, nucleosides, nucleic acids, enzymes (e.g., polymerases or reverse transcriptases) or vectors (e.g., surfaces).

[0102] The reporter gene portion (or marker) includes a fluorescent marker or fluorophore. Examples of fluorescent moieties that can serve as fluorescent markers or fluorophores include, but are not limited to: fluorescein and fluorescein derivatives, such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthalenefluorescein, isothiocyanatefluorescein, NHS-fluorescein, iodoacetamidefluorescein, maleimidefluorescein, SAMSA-fluorescein, thioaminoureafluorescein, and carbazide methylthioacetylaminofluorescein; rhodamine and rhodamine derivatives, such as TRITC, TMR, rhodamine sulfonyl chloride, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, rhodamine B sulfonyl chloride, rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, and Texas Red hydrazide; coumarin and coumarin derivatives, such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, and AMCE-hydrazide; and BODIPY and its derivatives, such as BODIPY. FL C3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FLIA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503; Cascade Blue and its derivatives, such as Cascade Blue acetylazide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide; LuciferYellow and its derivatives, such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH; cyanides and their derivatives, such as indolonyl cyanide dyes, benzoindolonyl cyanide dyes, pyridiniumyl cyanide dyes, thiazolonyl cyanide dyes, quinolinelonyl cyanide dyes, imidazolonyl cyanide dyes, Cy3, Cy5; lanthanide chelates and their derivatives, such as BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates; Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler red dyes, CAL Flour dyes, JOE and its derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilichrome dyes, malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes, and others known in the art, such as Haugland, Molecular Probes Handbook, (Eugene, Oregon) 6th edition;Those described in Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed., Plenum Press, New York (1999) or Hermanson, Bioconjugate Techniques, 2nd ed., or their derivatives;Or any combination thereof. Anthocyanin dyes can exist in sulfonated or non-sulfonated forms and consist of two pseudoindole, benzoindole, pyridinium, thiazolylium, and / or quinolineonium groups separated by a polymethystylene bridge between two nitrogen atoms. Commercially available anthocyanin fluorophores include, for example, Cy3 (which may include 1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-yl}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium or 1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{ ...2-(3-{ [Oxypyrrolidine-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfonyl-1,3-dihydro-2H-indole-2-yl]prop-1-en-1-yl)-3,3-dimethyl-3H-indole-5-sulfonate], Cy5 (which may include 1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indole-2-yl]prop-1-en-1-yl)-3,3-dimethyl-3H-indole-2-yl]prop-1-en-1-yl)-3,3-dimethyl-3H-indole-5-sulfonate), Cy5 (which may include 1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indole-2-yl) (1-(6-((2,5-dioxopyrrolidone-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-(((2,5-dioxopyrrolidone-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfonylindoline-2-ylide)pent-1,3-diene-1-yl)-3,3-dimethyl-3H-indoline-1-on-5-sulfonate) and Cy7 (which may include 1-(5-carboxypentyl) Cy2 is an oxazole derivative, not a pseudoindole, and the benzo-derived Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule.

[0103] In some embodiments, the reporter gene portion may be a FRET pair, allowing for multiple classifications to be performed in a single excitation and imaging step. As used herein, FRET may include excitation exchange (Forster) transfer or electron exchange (Dexter) transfer.

[0104] When used to refer to nucleic acids, the terms "amplify," "amplifying," "amplification," and other related terms include generating multiple copies of the original polynucleotide template molecule, wherein the copies contain sequences complementary to the template sequence or sequences identical to the template sequence. In some embodiments, the copies contain sequences substantially identical to or substantially identical to sequences complementary to the template sequence.

[0105] As used herein, the term "support" refers to a substrate designed for the deposition of biological molecules or biological samples for assays and / or analyses. Examples of biological molecules to be deposited onto a support include nucleic acids (e.g., DNA, RNA), peptides, carbohydrates, lipids, single cells, or multiple cells. Examples of biological samples include, but are not limited to, saliva, sputum, mucus, blood, plasma, serum, urine, feces, sweat, tears, and fluids from tissues or organs.

[0106] In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porous. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example, including a capillary or the inner surface of a capillary.

[0107] In some embodiments, the surface of the support may be substantially smooth. In some embodiments, the support may have a regular or irregular texture, including bumps, etched surfaces, holes, three-dimensional supports, or any combination thereof.

[0108] In some embodiments, the support comprises beads of any shape, including spherical, hemispherical, cylindrical, barrel-shaped, ring-shaped, disc-shaped, rod-shaped, conical, triangular, cubic, polygonal, tubular, or linear.

[0109] The support can be made of any material, including but not limited to: glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)) or any combination thereof. Various combinations of both glass and plastic substrates are contemplated.

[0110] The support may have multiple (e.g., two or more) nucleic acid templates immobilized thereon. These multiple immobilized nucleic acid templates may have the same sequence or different sequences. In some embodiments, individual nucleic acid template molecules from the multiple nucleic acid templates are immobilized to different sites on the support. In some embodiments, two or more individual nucleic acid template molecules from the multiple nucleic acid templates are immobilized to sites on the support.

[0111] The term "array" refers to a carrier comprising an array of sites located at predetermined positions on the carrier. The sites can be discrete and separated by gaps. In some embodiments, the predetermined sites on the carrier can be arranged in rows or columns in one dimension, or in rows and columns in two dimensions. In some embodiments, the multiple predetermined sites are arranged in an organized manner on the carrier. In some embodiments, the multiple predetermined sites are arranged in any organized pattern, including straight lines, hexagonal patterns, grid patterns, patterns with reflective symmetry, patterns with rotational symmetry, etc. The spacing between different pairs of sites can be the same or different. In some embodiments, the support comprises at least 10 2 At least 10 sites 3 At least 10 sites 4 At least 10 sites 5 At least 10 sites 6 At least 10 sites 7 At least 10 sites 8 At least 10 sites 9 At least 10 sites 10 At least 10 sites 11 At least 10 sites 12 At least 10 sites 13 At least 10 sites 14 At least 10 sites 15 One or more sites, wherein the site is located at a predetermined position on the support. In some embodiments, a plurality of predetermined sites on the support (e.g., 10 sites) 2 Up to 10 15Nucleic acid templates are immobilized at multiple sites (one or more sites) to form a nucleic acid template array. In some embodiments, the nucleic acid templates are immobilized at multiple predetermined sites by hybridization with immobilized surface capture primers, or the nucleic acid templates are covalently attached to the surface capture primers. In some embodiments, the nucleic acid templates are immobilized at multiple predetermined sites, for example, at 10 sites. 2 Up to 10 15 At one or more predetermined sites. In some embodiments, a fixed nucleic acid template is amplified in a clonal manner to generate fixed nucleic acid clusters at multiple predetermined sites. In some embodiments, a single fixed nucleic acid cluster comprises a linear cluster, or comprises a single-stranded or double-stranded tandem.

[0112] In some embodiments, a carrier comprising a plurality of sites located at random positions on a carrier is referred herein to as a carrier having randomly positioned sites thereon. The positions of the randomly positioned sites on the carrier are not predetermined. The plurality of randomly positioned sites are arranged on the carrier in a disordered and / or unpredictable manner. In some embodiments, the carrier comprises at least 10 2 At least 10 sites 3 At least 10 sites 4 At least 10 sites 5 At least 10 sites 6 At least 10 sites 7 At least 10 sites 8 At least 10 sites 9 At least 10 sites 10 At least 10 sites 11 At least 10 sites 12 At least 10 sites 13 At least 10 sites 14 At least 10 sites 15 One or more sites, wherein the sites are randomly located on the carrier. In some embodiments, multiple randomly located sites on the carrier (e.g., 10 sites) 2 One to 10 15 Nucleic acid templates (one or more sites) are immobilized to form a nucleic acid template-immobilized vector. In some embodiments, the nucleic acid template is immobilized at multiple randomly located sites by hybridization with immobilized surface capture primers, or the nucleic acid template is covalently attached to the surface capture primers. In some embodiments, the nucleic acid template is immobilized at multiple randomly located sites, for example, at 10 sites. 2 One to 10 15 At one or more sites. In some embodiments, a fixed nucleic acid template is amplified in a clonal manner to generate fixed nucleic acid clusters at multiple randomly located sites. In some embodiments, a single fixed nucleic acid cluster comprises a linear cluster, or comprises a single-stranded or double-stranded tandem.

[0113] In some embodiments, a plurality of immobilized surface-capture primers on the vector are fluidly connected to each other to allow solutions of reagents (e.g., nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, etc.) to flow onto the vector, such that the plurality of immobilized surface-capture primers on the vector can react with the reagents substantially simultaneously in a large-scale parallel manner. In some embodiments, the fluid connectivity of the plurality of immobilized surface-capture primers can be used to perform nucleic acid amplification reactions (e.g., RCA, MDA, PCR, and bridge amplification) substantially simultaneously on the plurality of immobilized surface-capture primers.

[0114] In some embodiments, multiple immobilized nucleic acid clusters on a vector are fluidly connected to each other to allow solutions of reagents (e.g., enzymes, nucleotides, divalent cations, etc.) to flow onto the vector, such that the multiple immobilized nucleic acid clusters on the vector can react with the reagents substantially simultaneously in a massively parallel manner. In some embodiments, the fluid connectivity of the multiple immobilized nucleic acid clusters can be used to perform nucleotide binding assays and / or nucleotide polymerization reactions (e.g., primer extension or sequencing) substantially simultaneously on the multiple immobilized nucleic acid clusters, and optionally to perform detection and imaging of massively parallel sequencing.

[0115] When used to refer to immobilized enzymes, the term "immobilized" and related terms refer to an enzyme (e.g., polymerase) that is attached to a carrier, or to a coating on a carrier, or embedded in a matrix formed by a coating on a carrier via covalent bonds or non-covalent interactions.

[0116] When used to refer to immobilized nucleic acids, the term "immobilized" and related terms refer to nucleic acid molecules attached to a vector, or to a coating on a vector, or embedded in a matrix formed by a coating on a vector via covalent or non-covalent interactions, wherein the nucleic acid molecules include surface capture primers, nucleic acid template molecules, and extension products of the capture primers. Extension products of the capture primers include nucleic acid multimers (e.g., nucleic acid clusters).

[0117] In some embodiments, one or more nucleic acid templates are immobilized on a support, for example, at sites immobilized on the support. In some embodiments, the one or more nucleic acid templates are amplified clonally. In some embodiments, the one or more nucleic acid templates are amplified clonally from a support (e.g., in solution) and subsequently deposited onto and immobilized on the support. In some embodiments, the clonal amplification reaction of the one or more nucleic acid templates is performed on the support, resulting in immobilization on the support. In some embodiments, the one or more nucleic acid templates are amplified clonally using a nucleic acid amplification reaction (e.g., in solution or on a support), which includes any one or any combination of the following: polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridging amplification, isothermal bridging amplification, rolling circle amplification (RCA), loop-to-loop amplification, helicase-dependent amplification, recombinase-dependent amplification, and / or single-strand-binding (SSB) protein-dependent amplification.

[0118] The chapter titles used in this article are for layout purposes only and should not be construed as limiting the topics described.

[0119] method

[0120] This document provides methods for the in situ detection of one or more targets (e.g., RNA, DNA, peptides) in biological samples. The methods described herein do not include cDNA synthesis. Nucleic acid molecules containing target-binding sequences and index sequences can be introduced into biological samples, thereby conjugating them to targets (e.g., RNA, DNA, or peptides). Primer nucleic acid sequences can be added to the sample and bound to the index sequence to form a primer-binding index sequence. Nucleotide conjugates (e.g., detectable nucleotide conjugates) comprising a detectable label and one or more nucleotide moieties can be added to the sample to form binding complexes. The binding complexes, corresponding to the sequence associated with the index sequence, and thus corresponding to the identity of the target (e.g., RNA, DNA, or peptide), can be detected.

[0121] The binding complex involved in the methods disclosed herein may include at least one nucleotide conjugate (e.g., a detectable nucleotide conjugate) which may include multiple copies of the same nucleotide conjugated thereto. When the nucleotide is complementary to the index sequence, the nucleotide conjugate can form a binding complex with the index sequence to which the polymerase and primer bind, and this binding complex exhibits increased stability and longer retention time compared to binding complexes formed using a single unconjugated or unchained nucleotide.

[0122] The method described herein offers several advantages compared to other in situ workflows. For example, biological samples can be placed on solid supports, such as planar supports made of glass or plastic that can be fabricated in any shape and size. Assembling hybridization chambers on the supports may not be necessary. Preparing chemically washed glass beads for cell adhesion may not be required. The supports can be passivated with coatings that promote cell adhesion. The coatings do not need to be formulated into ligation-enhancing primers. The supports with cell samples attached can be readily adapted to readily available flow cell scaffolds / carriers that are fluidly connected to an automated fluid dispensing system and configured on a fluorescence microscope. Using the fluid dispensing system, any combination of steps in in situ sequencing, including cell seeding, cell fixation, cell permeabilization, probe hybridization, probe ligation reaction, amplification, and sequencing, can be performed automatically.

[0123] Another advantage of the method described herein is the formation of amplicones (e.g., tandem amplicons) within a biological sample. Amplicons can be single-stranded and collapse into compacted DNA nanospheres, each nanosphere carrying multiple tandem copies of a polynucleotide unit along its length, wherein the polynucleotide unit includes the target sequence of interest and at least one universal sequencing primer binding site. Each polynucleotide unit can bind a sequencing primer, a sequencing polymerase, and a nucleotide reagent (e.g., a detectable labeled multivalent molecule) to form a detectable sequencing complex (e.g., a detectable ternary complex). Each nanosphere can carry multiple detectable sequencing complexes. The compacted nature of the nanospheres increases the local concentration of the detectable labeled nucleotide reagent used during the sequencing workflow, which increases the signal intensity emitted from the nanospheres to give discrete detectable signals that can be imaged as fluorescent dots within the cellular sample. Each dot can correspond to an amplicon, and each amplicon corresponds to a target molecule in the biological sample. Multiple dots in a biological sample can be detected and imaged simultaneously. Alternatively, multiple dots in a single batch of a biological sample can be detected and imaged.

[0124] One aspect of this disclosure provides a method for in situ identification of a target (e.g., RNA, DNA, or peptide), the method using... Figure 39The components are shown. The method includes: (a) providing a permeated cell or tissue containing a target (3901); (b) guiding a nucleic acid molecule (3902) to the cell or tissue (3902) under conditions sufficient to couple a target-binding sequence to at least a portion of the target (e.g., RNA or DNA), wherein the nucleic acid molecule contains a target-binding sequence, and wherein the nucleic acid molecule contains an index sequence; (c) guiding a primer nucleic acid sequence (3905) to bind a primer nucleic acid sequence (3905) to at least a portion of one or more amplicones to generate a primer-binding index sequence. (3904) to cells or tissues; (d) to guide (3906) cells of tissues with a nucleotide conjugate (e.g., a detectable nucleotide conjugate) comprising: (i) a detectable marker and (ii) one or more nucleotide moieties complementary to nucleotides in an index sequence or a derivative thereof that binds to a primer; and (e) to detect the binding complex within cells or tissues, thereby identifying a target.

[0125] Another aspect of this disclosure provides a method for in situ identification of a target (e.g., RNA, DNA, or peptide) according to one aspect of this disclosure, the method using Figure 40The components are shown. The method includes: (a) providing a permeabilized cell or tissue containing a target (4001); (b) guiding a nucleic acid molecule (4003) to the cell or tissue (4002) under conditions sufficient to couple a target-binding sequence to at least a portion of the target (e.g., RNA, DNA, or polypeptide), wherein the nucleic acid molecule contains a target-binding sequence, and wherein the nucleic acid molecule contains an index sequence; (c) guiding a probe (e.g., a lock probe) (4005) (4004) to the cell or tissue under conditions sufficient to bind the probe to the nucleic acid molecule; (d) amplifying (4006) (e.g., performing a rolling circle amplification reaction) the index sequence to generate one or more amplicons, wherein the one or more amplicons contain multiple copies of the index sequence or a derivative thereof, thereby forming one or more amplicons (4001). 07); (e) guiding (4008) the primer nucleic acid sequence (4009) to a cell or tissue under conditions sufficient to bind the primer nucleic acid sequence (4007) to at least a portion of one or more amplicones to generate a primer-binding index sequence; (f) guiding cells of a tissue with a nucleotide conjugate (e.g., a detectable nucleotide conjugate) comprising: (i) a detectable marker and (ii) one or more nucleotide portions complementary to the nucleotides in the primer-binding index sequence or a derivative thereof; and (g) detecting the binding complex within the cell or tissue to identify the target.

[0126] Another aspect of this disclosure provides a method for in situ identification of RNA, the method using Figure 41The components are shown. The method includes: (a) providing a permeated cell or tissue containing RNA (4101); (b) guiding a probe (4103) to the cell or tissue (4102) under conditions sufficient to couple an RNA-binding sequence to at least a portion of the RNA, wherein the probe (4103) contains an RNA-binding sequence, and wherein the probe further contains an index sequence; (c) amplifying (4104) (e.g., performing a rolling circle amplification reaction) the index sequence to generate one or more amplicons, wherein the one or more amplicons contain multiple copies of the index sequence or a derivative thereof, thereby forming one or more amplicons (4105); (d) binding primer nucleic acid sequences to at least a portion of the one or more amplicons to generate (e) Guide (4106) primer nucleic acid sequence (4107) to cells or tissues under conditions of primer-binding index sequence; (e) Guide cells or tissues with nucleotide conjugates containing: (i) a detectable marker and (ii) one or more nucleotides complementary to nucleotides in the primer-binding index sequence or a derivative thereof; and (f) Detect the binding complex within cells or tissues to identify RNA.

[0127] Another aspect of this disclosure provides a method for in situ identification of DNA, the method using Figure 42The components are shown. The method includes: (a) providing a permeabilized cell or tissue containing DNA (4201), wherein the DNA is not cDNA; (b) guiding a DNA-binding sequence (4203) into the cell or tissue (4202) under conditions sufficient to couple a DNA-binding sequence to at least a portion of the DNA, wherein the probe contains an index sequence; (c) amplifying (4204) (e.g., performing a rolling circle amplification reaction) the index sequence to generate one or more amplicons, wherein the one or more amplicons contain multiple copies of the index sequence or a derivative thereof, thereby forming one or more amplicons (4205); (d) binding a primer nucleic acid sequence to at least a portion of the one or more amplicons to produce primer binding. (e) Guide (4206) primer nucleic acid sequence (4207) to cells or tissues under the condition of an index sequence; (e) guide cells or tissues with a nucleotide conjugate comprising: (i) a detectable marker and (ii) one or more nucleotides complementary to the nucleotides in the index sequence or a derivative thereof that bind to the primer; and (f) detect the binding complex within cells or tissues to identify DNA.

[0128] Another aspect of this disclosure provides a method for in situ identification of peptides (e.g., proteins), the method using... Figure 43The components are shown. The method includes: (a) providing permeabilized cells or tissues, wherein the cells or tissues contain a polypeptide (4301); (b) guiding a polypeptide-binding probe (4303) to a cell or tissue (4302) under conditions sufficient to couple a polypeptide-binding probe to at least a portion of the polypeptide, wherein the polypeptide-binding probe contains a polypeptide-binding moiety and an oligonucleotide (4304) coupled thereto; (c) guiding (4305) a cell or tissue containing an oligonucleotide-binding probe (4307) under conditions sufficient to couple an oligonucleotide-binding probe to at least a portion of an oligonucleotide or polypeptide-binding probe, wherein the probe further contains an index sequence; (d) amplifying (4307) the index sequence (e.g., performing a rolling circle amplification reaction) to generate one or more amplicons, wherein one or more amplicons contain the index sequence or a derivative thereof. (e) guiding (4309) the primer nucleic acid sequence (4310) to a cell or tissue under conditions sufficient to bind the primer nucleic acid sequence to at least a portion of the one or more amplicons to produce a primer-binding index sequence; (f) guiding the cell or tissue with a nucleotide conjugate comprising: (i) a detectable marker and (ii) one or more nucleotide portions complementary to the nucleotides in the primer-binding index sequence or a derivative thereof; and (f) detecting the binding complex within the cell or tissue to identify DNA.

[0129] i. biological samples

[0130] The methods described herein include detecting one or more targets (e.g., RNA, DNA, and / or peptides) in a biological sample. The biological sample may comprise tissue or a combination of tissue and aerosol. In some embodiments, the biological sample may comprise cells. In some embodiments, the biological sample may be cells. The biological sample may be derived from an organism. The biological sample may be derived from a cell culture. In some embodiments, previously harvested biological samples may be preserved. Cell samples may comprise whole cells, multiple whole cells, intact tissue, or intact tumor. In some embodiments, the biological sample comprises a fresh biological sample, a freshly frozen biological sample, a section of a biological sample, or an FFPE biological sample. In some embodiments, the biological sample may comprise one or more live or non-live cells. In some embodiments, the biological sample comprises intact cells or tissue. In some embodiments, the biological sample comprises undysed cells or tissue. In some embodiments, the biological sample comprises a single cell, multiple cells, tissue, organ, organism, or a section of these biological samples.

[0131] In some embodiments, the biological sample is derived from eukaryotes (such as animals, plants, fungi, protozoa), archaea, or eubacteria. The biological sample may be derived from prokaryotic or eukaryotic cells, such as adherent or non-adherent eukaryotic cells. The biological sample may be derived from primary or immortalized cell lines of rodents, pigs, cats, dogs, cattle, horses, primates, or humans. In some embodiments, the biological sample may be obtained from viruses, fungi, or prokaryotes. In some embodiments, the biological sample may be obtained from animals, insects, or plants. In some embodiments, the biological sample comprises cells infected with one or more viruses. In some embodiments, the biological sample may be obtained from any organism, including humans, apes, great apes, dogs, cats, cattle, horses, mice, pigs, goats, wolves, frogs, fish, plants, insects, or bacteria. In some embodiments, the biological sample may be obtained from organs, including but not limited to the brain, mammary glands, ovaries, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testes, liver, lungs, kidneys, esophagus, pancreas, thyroid gland, pituitary gland, thymus, skin, heart, larynx, or other organs.

[0132] Biological samples can be solid samples, such as tissue biopsies. Biological samples can be liquid samples, such as blood or blood components (e.g., serum or plasma). In some embodiments, biological samples are obtained from: skin, heart, lungs, kidneys, respiration, bone marrow, feces, semen, vaginal fluid, interstitial fluid derived from tumor tissue, breast, pancreas, cerebrospinal fluid, tissue, pharyngeal swabs, biopsies, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, body cavity fluid, sputum, pus, microbiota, meconium, breast milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric juice and digestive juices, tears, eye discharge, sweat, mucus, earwax, oil, glandular secretions, cerebrospinal fluid, hair, nails, skin cells, plasma, nasal swabs or nasopharyngeal lavage fluid, cerebrospinal fluid, umbilical cord blood, stressed fluids and / or other excretions or body tissues. Biological samples can be cell-free samples.

[0133] The biological sample may be tissue. The tissue may be extracted from a subject. In some embodiments, the subject may be a human subject. In some embodiments, the tissue may be formalin-fixed and paraffin-embedded. In some other embodiments, the tissue may be freshly frozen. The tissue can be sectioned, with section thicknesses ranging from approximately 1 to 30 µm, approximately 2 to 29 µm, approximately 3 to 28 µm, approximately 4 to 27 µm, approximately 5 to 26 µm, approximately 6 to 25 µm, approximately 7 to 24 µm, approximately 8 to 23 µm, approximately 9 to 22 µm, approximately 10 to 21 µm, approximately 11 to 20 µm, approximately 12 to 19 µm, approximately 13 to 18 µm, approximately 14 to 17 µm, approximately 15 to 16 µm, approximately 1 µm, approximately 2 µm, approximately 3 µm, approximately 4 µm, approximately 5 µm, approximately 6 µm, approximately 7 µm, approximately 8 µm, approximately 9 µm, approximately 10 µm, approximately 11 µm, approximately 12 µm, approximately 13 µm, approximately 14 µm, approximately 15 µm, approximately 16 µm, approximately 17 µm, approximately 18 µm, approximately 19 µm, approximately 20 µm, etc. Thicknesses of approximately 21 µm, 22 µm, 23 µm, 24 µm, 25 µm, 26 µm, 27 µm, 28 µm, 29 µm, or 30 µm.

[0134] In some embodiments, biological samples can be grown / cultured on a vector, and the cultured biological samples can be analyzed on the vector. Nucleic acids in the biological sample may not be released from cells and may not be captured on the vector. In some cases, nucleic acids in the biological sample may be retained in cells and sequenced in the cells (e.g., in situ sequencing). In some embodiments, nucleic acids in the biological sample may be included in an optional library preparation workflow by attaching at least one adaptor sequence (e.g., a universal adaptor sequence). Nucleic acids in the biological sample may be amplified in cells by rolling circle amplification.

[0135] Biological samples can be processed and / or analyzed in a flow cell. For example, a flow cell with cultured cells attached to it can be placed on a sequencing device configured with at least one fluid delivery device, at least one fluid device (e.g., a microfluidic device), at least one imaging device, and / or at least one sensor to detect signals from the sequencing reaction. The sequencing reaction can be performed using soluble sequencing primers. Sequencing primers can be designed to hybridize with a target sequence or a universal adaptor sequence. Sequencing primers may include or omit a sample index sequence (e.g., a sample barcode sequence). Nucleic acids in cultured cells can be sequenced using any nucleotide conjugate described herein (e.g., a detectable nucleotide conjugate). In some embodiments, the sequencing reaction can employ labeled or unlabeled nucleotide analogues. In some embodiments, the individual cycle time can be achieved in less than 30 minutes. In some embodiments, the field of view (FOV) can exceed 1 mm. 2 And it is used to scan large areas (> 10 mm) 2 The cycle time can be less than 5 minutes.

[0136] In some embodiments, biological samples can be placed on a carrier. This document provides solid carriers (e.g., low-nonspecificity binding carriers) that further comprise a low-nonspecificity binding surface.

[0137] Biological samples may contain cells. Cells described herein may be leukocytes, erythrocytes, platelets, epithelial cells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts, skeletal muscle cells, smooth muscle cells, gametes, or cells derived from the heart, lungs, brain, liver, kidneys, spleen, pancreas, thymus, bladder, stomach, colon, or small intestine. Cells may be normal or healthy. Alternatively or in combination, cells may be diseased cells (such as cancer cells) or pathogenic cells derived from an infected host. In some embodiments, cells belong to a subgroup of cells, such as immune cells (e.g., T cells, cytotoxic (killer) T cells, helper T cells, αβ T cells, γδ T cells, T cell progenitor cells, B cells, B cell progenitor cells, lymphoid stem cells, myeloid progenitor cells, lymphocytes, granulocytes, natural killer cells, plasma cells, memory cells, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells and / or macrophages, or any combination thereof), undifferentiated human stem cells, induced differentiated human stem cells, or rare cells (e.g., circulating tumor cells (CTCs), circulating epithelial cells, circulating endothelial cells, circulating endometrial cells, bone marrow cells, progenitor cells, foam cells, mesenchymal cells, or trophoblast cells). Other cells are also considered and consistent with the disclosure herein.

[0138] Biological samples may be embedded in wax, resin, epoxy resin, or agar. For example, biological samples may be immobilized in any one or any combination of two or more of the following: acetone, ethanol, methanol, formaldehyde, paraformaldehyde-Triton, or glutaraldehyde. Biological samples may be sectioned or unsectioned. Biological samples may be stained, destained, or unstained.

[0139] In some embodiments, the biological sample may be permeated in the presence of at least about 60%, 70%, 80%, 90% or more methanol (or ethanol) and incubated on ice for a period of time. The incubation period may be at least about 10, 15, 20, 25, 30, 35, 40, 50, 60 or more minutes.

[0140] In some embodiments, the method includes treating a biological sample with a permeation reagent that alters the cell membrane to allow experimental reagents to permeate into the cell. For example, the permeation reagent can remove membrane lipids from the cell membrane. In some embodiments, the biological sample may be treated with a permeation reagent comprising any combination of organic solvents, detergents, chemical compounds, cross-linking agents, and / or enzymes. In some embodiments, the organic solvent comprises acetone, ethanol, and methanol. In some embodiments, the detergent comprises saponins, Triton X-100, Tween-20, or sodium dodecyl sulfate (SDS), or a solution of sodium N-lauroyl sarcosinate. In some embodiments, the cross-linking agent comprises paraformaldehyde, a nonionic polyoxyethylene surfactant (e.g., NP40), or a combination thereof. In some embodiments, the enzyme comprises trypsin, pepsin, or a protease (e.g., proteinase K). In some embodiments, the protease may be used to permeate cells under alkaline or acidic conditions. In some embodiments, the permeation reagent comprises water and / or PBS. For example, cells fixed with 70% ethanol can be permeabilized for about 30 to 60 minutes, and the permeabilizing agent can be replaced with PBS-T (e.g., PBS with 0.05% Tween-20). In some embodiments, cells can be post-fixed with 3% paraformaldehyde and 0.1% glutaraldehyde for about 30 to 60 minutes, and washed multiple times with PBS-T.

[0141] Biological samples can be used to generate three-dimensional polymer matrices containing cellular and subcellular components (e.g., nucleic acid molecules) of the biological sample. The three-dimensional polymer matrix can be covalently or non-covalently coupled to the carrier described herein. In some embodiments, the three-dimensional polymer matrix is ​​porous and contains polymerized or cross-linked subcellular components, including target nucleic acid molecules. A polymer matrix can be formed within a biological sample (e.g., cells or tissues) by infusing one or more polymer precursors (e.g., monomers, such as, for example, ethylene oxide for polyethylene glycol) into the biological sample and allowing the one or more polymer precursors to polymerize or cross-link. Before, during, or after polymer matrix formation, the positions of portions of the biological sample (e.g., DNA, RNA, proteins) can be fixed using, for example, a fixative (e.g., formaldehyde). Porous matrices can be prepared according to various methods. For example, a polyacrylamide gel matrix can be copolymerized with biotinylated DNA molecules and an acrylamide-modified streptavidin monomer, using a suitable acrylamide:bisacrylamide ratio to control the cross-linking density. Further control over the molecular sieve size and density can be achieved by adding additional cross-linking agents (such as functionalized polyethylene glycol). Methods for immobilizing biological samples to a surface and generating a polymer matrix within a biological sample are provided in PCT / US2019 / 055434 (WO 2020 / 076976; Molecular Index in 3D Space), which are incorporated herein by reference in their entirety.

[0142] In any of the methods described herein, the biological sample may be infused with a swellable polyelectrolyte hydrogel (US Patent Nos. 10,309,879 and Chen 2015 Science 347:543, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the immobilized and permeabilized biological sample may be infused with sodium acrylate, acrylamide, and the crosslinking agent N-N'-methylenebisacrylamide. In some embodiments, ammonium persulfate (APS) initiator and tetramethylethylenediamine (TEMED) promoter are infused to achieve polymerization. In some embodiments, the biological sample may be infused with proteinase K for proteolysis and incubation in a digestion buffer. In some embodiments, the gel within the biological sample may be swollen by adding water.

[0143] In any of the methods described herein, the biological sample may be cultured on a vector. In some embodiments, the method includes culturing the biological sample on a vector under conditions suitable for amplifying the biological sample for 2 to 10 or more generations. The cultured biological sample may generate cell colonies. In some embodiments, the method includes culturing the biological sample to confluent or non-confluent stages. In some embodiments, the method includes culturing cell samples on a vector in a simple or complex cell culture medium. For example, cell culture media may include D-MEM high glucose (e.g., from Thermo Fisher Scientific, catalog number 11965118), fetal bovine serum (e.g., 10% FBS; e.g., from Thermo Fisher Scientific, catalog number A3160402), MEM non-essential amino acids (e.g., 0.1 mM MEM, e.g., from Thermo Fisher Scientific, catalog number 11140050), L-glutamine (e.g., 6 mM L-glutamine, e.g., from Thermo Fisher Scientific, catalog number A2916801), MEM sodium pyruvate (e.g., 1 mM sodium pyruvate, e.g., from Thermo Fisher Scientific, catalog number 11360070), and antibiotics (e.g., 1% penicillin-streptomycin-glutamine, e.g., from Thermo Fisher, catalog number 10378016). In some embodiments, the method includes culturing biological samples at humidity and temperature suitable for culturing cells on a carrier. Example suitable conditions include approximately 37°C and a humid atmosphere containing approximately 5-10% carbon dioxide in the air. Biological samples can be cultured using appropriate oxygen and / or nitrogen aeration.

[0144] In any of the methods described herein, the term "simple cell culture medium" or related terms refer to a cell culture medium that typically lacks components that support cell growth and / or proliferation in a culture. Simple cell culture media can be used, for example, to wash, suspend, or dilute biological samples. Simple cell culture media can be mixed with certain components to prepare a cell culture medium that can support cell growth and / or proliferation in a culture. Simple cell culture media contain any one or any combination of two or more of buffers, phosphate compounds, sodium compounds, potassium compounds, calcium compounds, magnesium compounds, and / or glucose. In some embodiments, simple cell culture media contain PBS (phosphate-buffered saline), DPBS (Dulbecco's phosphate-buffered saline), HBSS (Hank's balanced salt solution), DMEM (Dulbecco's Modified Eagle's Medium), EMEM (Eagle's Minimum Essential Medium), and / or EBSS. In some embodiments, biological samples may be placed in a simple cell culture medium before or during any of the nucleic acid methods described herein.

[0145] In any of the methods described herein, the term "composite cell culture medium" or related terms refer to a cell culture medium that can be used to support cell growth and / or proliferation in a culture without the need for supplementation or additives. A composite cell culture medium may include any combination of two or more of buffer systems (e.g., HEPES), inorganic salts, amino acids, proteins, peptides, carbohydrates, fatty acids, lipids, purines and their derivatives (e.g., hypoxanthine), pyrimidines and their derivatives, and / or trace elements. Composite cell culture media include fluids obtained from fluids or tissue extracts. Composite cell culture media include artificial cell culture media. In some embodiments, a composite cell culture medium may be a serum-containing medium, such as a composite cell culture medium comprising fluids such as fetal bovine serum, plasma, serum, lymph, human placental cord serum, and amniotic fluid. In some embodiments, a composite cell culture medium may be a serum-free medium, which is generally (but not necessarily) a defined cell culture medium. In some embodiments, a composite cell culture medium may be a chemically defined medium, which generally (but not necessarily) includes recombinant peptides and ultrapure inorganic and / or organic compounds. In some embodiments, the composite cell culture medium may be a protein-free medium, including, for example, MEM (Minimum Essential Medium) and RPMI-1640 (Roswell Park Memorial Institute). In some embodiments, the composite cell culture medium comprises IMDM (Iscove's Modified Dulbecco's Medium). In some embodiments, the composite cell culture medium comprises DMEM (Dulbecco's Modified Iscove's Medium). In some embodiments, the biological sample may be placed in the composite cell culture medium before or during any of the nucleic acid methods described herein.

[0146] In any of the methods described herein, the biological sample may comprise a fixed biological sample. In some embodiments, the biological sample may be treated with a fixation reagent (e.g., a fixing reagent) that preserves cells and their contents to inhibit degradation and may inhibit cell lysis. For example, the fixation reagent may preserve nucleic acids (e.g., RNA) carried by the biological sample. In some embodiments, the fixation reagent inhibits the loss of nucleic acids in the biological sample.

[0147] In some embodiments, the immobilization agent can crosslink the target to prevent the target from escaping from the biological sample. In some embodiments, the crosslinking immobilization agent comprises any combination of aldehydes, formaldehyde, paraformaldehyde, formalin, glutaraldehyde, imine esters, N-hydroxysuccinimide esters (NHS), and / or glyoxal (bifunctional aldehydes).

[0148] In some embodiments, the fixation reagent comprises at least one alcohol, including methanol or ethanol. In some embodiments, the fixation reagent comprises at least one ketone, including acetone. In some embodiments, the fixation reagent comprises acetic acid, glacial acetic acid, and / or picric acid. In some embodiments, the fixation reagent comprises mercuric chloride. In some embodiments, the fixation reagent comprises a zinc salt, said zinc salt comprising zinc sulfate or zinc chloride. In some embodiments, the fixation reagent can denature the peptide. In some embodiments, the fixation reagent comprises 4% w / v paraformaldehyde relative to water / PBS. In some embodiments, the fixation reagent comprises 10% formaldehyde at 35% neutral pH. In some embodiments, the fixation reagent comprises 2% v / v glutaraldehyde relative to water / PBS. In some embodiments, the fixation reagent comprises 25% 37% formaldehyde solution, 70% picric acid, and 5% acetic acid. In some embodiments, biological samples can be fixed on a carrier with 4% paraformaldehyde for about 30 to 60 minutes and washed with PBS. In some embodiments, the biological samples can be stained, destained, or unstained.

[0149] ii. Objective

[0150] The methods described herein relate to the detection of one or more targets. One or more targets may comprise nucleic acids (e.g., RNA and / or DNA), peptides, or combinations thereof. In some embodiments, one or more targets may comprise one or more naturally occurring nucleic acids, one or more recombinant nucleic acids, one or more synthetic nucleic acids, or combinations thereof. One or more targets comprising one or more nucleic acids may comprise linear nucleic acids, circular nucleic acids, branched nucleic acids, or combinations thereof. In some embodiments, one or more targets comprising one or more nucleic acids may comprise DNA. In some embodiments, one or more targets may comprise genomic DNA. In some embodiments, one or more targets may comprise viral DNA. In some embodiments, one or more targets may comprise cell-free DNA (cfDNA). In some embodiments, DNA may be genomic DNA, methylated DNA, unmethylated DNA, organelle DNA, or combinations thereof. In some cases, one or more targets may comprise PCR-amplified DNA. In some embodiments, one or more targets may comprise synthetic DNA. Synthetic DNA may comprise synthetic DNA probes. DNA may be fragmented, unfragmented, or combinations thereof. DNA may include one or more modifications. For example, DNA may include methylation modifications, phosphorylation modifications, locked nucleic acid modifications, or combinations thereof.

[0151] In some embodiments, one or more targets may comprise RNA. The RNA of one or more targets may comprise poly-A RNA and / or non-poly-A RNA. RNA may comprise coding RNA, non-coding RNA, or a combination thereof. RNA may comprise tRNA, rRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), antisense RNA, non-coding RNA, protein-coding RNA, or a combination thereof. One or more targets may comprise RNA that is a target RNA, a non-target RNA, or a combination thereof. In some cases, the RNA may have been transcribed and spliced. In some cases, the RNA may be unspliced. In some cases, the RNA may comprise wild-type RNA, mutant RNA, and / or spliced ​​variant RNA. In some cases, the RNA may comprise pre-spliced ​​RNA, partially spliced ​​RNA, and / or fully spliced ​​RNA. In some cases, the RNA may comprise coding RNA, non-coding RNA, housekeeping RNA, cell-specific RNA, tissue-specific RNA, disease-specific RNA, or a combination thereof. In some embodiments, RNA may comprise RNA expressed by one or more cells in response to stimuli such as heat, light, chemicals, or drugs. In some embodiments, RNA may include RNA found in healthy cells, diseased cells, or combinations thereof. In some embodiments, RNA may include RNA transcribed from a transgenic DNA sequence introduced into a biological sample using a recombinant DNA procedure. For example, RNA may be transcribed from a transgenic DNA sequence controlled by an inducible or constitutive promoter sequence. In some embodiments, RNA may include RNA transcribed from a non-transgenic DNA sequence. RNA may include one or more modifications. For example, RNA may include phosphorylation (e.g., 5' phosphorylation), internucleotide bonding, or combinations thereof.

[0152] Binding complexes associated with RNA, or amplicons generated by the binding of nucleic acid molecules to RNA, can be formed without reverse transcription of RNA. For example, RNA can be directly bound by a probe containing an RNA-binding sequence. The probe can be linked to form a circular nucleic acid and amplified using rolling circle amplification to generate one or more amplicons. One or more amplicons can then form a binding complex with the conjugated and detected nucleotide. In this case, RNA can be detected directly without reverse transcription.

[0153] One or more targets may comprise one or more polypeptides. One or more polypeptides of one or more targets may comprise one or more proteins, one or more peptides, or combinations thereof. One or more polypeptides may comprise one or more cell surface proteins, one or more intracellular proteins, one or more signaling proteins, one or more protein complexes, or combinations thereof. One or more polypeptides may comprise one or more antibodies or antibody fragments. In some cases, one or more polypeptides may include one or more modifications. One or more modifications of one or more polypeptides may include: formation of one or more phosphoramides (N-linked), one or more phosphorylations, one or more adenosylylations, one or more uridineylations, one or more propionylations, formation of one or more pyroglutamates, one or more S-glutathioneylations, one or more S-nitrosylations, one or more sulfonylations, one or more S-sulfinylations, one or more S-sulfonylations, one or more succinylations, one or more sulfation, one or more glycosylations, one or more carbamylations, one or more carbonylations, one or more spontaneous isopeptide bond formations, one or more biotinylations, one or more carbamylations, one or more oxidations, one or more myristoylations, one or more palmitoylations, one or more farnesylations, one or more geraniylgeraniylations, one or more glycosylation modifications, one or more glycosylphosphatidylinositol glycosylations, one or more glycosylphosphatidylinositol glycosylations, one or more esterylations, one or more flavin partial attachments, one or more heme C attachments, one or more... Multiple types of phosphoplastin thioethylamine, one or more retinaldehyde Schiff base formations, one or more translation factor modifications, one or more diphtheria amide formations, one or more phosphoglycerol ethanolamines, one or more hydroxyputrescine formations, one or more β-lysine additions to lysine, one or more acylation modifications (e.g., O-acylation, N-acylation, and S-acylation), one or more acetylations, one or more formylations, one or more alkylations, one or more amidations, one or more argininoylations, one or more polyglutamylations, one or more polyglycytylations, one or more butyrylations, one or more γ-carboxylations, one or more glycosylations, one or more polysialylations, one or more malonylations, one or more hydroxylations, one or more nucleotide additions, one or more phosphate esters (O-linked), one or more polyethylene glycolation, one or more ubiquitinations, one or more SUMOylations, one or more ubiquitin-like esters, one or more ISGylations, one or more citrullinations, one or more deamidations, one or more eliminations, or combinations thereof.

[0154] In some embodiments, one or more targets may comprise clonal amplified nucleic acids. In some embodiments, this disclosure provides methods including clonal amplification, which includes using polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridging amplification, isothermal bridging amplification, rolling circle amplification, circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-strand binding (SSB) protein-dependent amplification, or any combination thereof. In some embodiments, the methods described herein can be used to detect the clonal amplification products.

[0155] One or more targets disclosed herein, upon coupling with a surface to a biological sample, can have a fixed three-dimensional relationship within the biological sample. This fixed three-dimensional relationship at least partially enables the identification of spatial and cellular origins within the biological sample after nucleic acid identification using the systems and methods described herein. The description of nucleic acid and spatial analysis within biological samples is found in U.S. Patent Application No. 17 / 356,929, filed June 24, 2021, entitled "Multivalent Binding Compositions for Nucleic Acid Analysis," which is incorporated herein by reference in its entirety.

[0156] In some embodiments, one or more targets comprise one or more target nucleic acid sequences. In some embodiments, one or more targets may be a tandem of multiple repetitive sequences comprising one or more target nucleic acid sequences. Multiple copies of the target nucleic acid sequence within the tandem may be covalently linked using phosphodiester bonds. The tandem may be formed using amplification reactions, including rolling circle amplification.

[0157] In some embodiments, two or more targets can be detected. In some embodiments, the two or more targets may each contain the same target (e.g., the same nucleic acid sequence). In some embodiments, the two or more targets may contain different targets (e.g., different nucleic acid sequences). In some embodiments, one or more targets may contain at least about 1 target, at least about 2 targets, at least about 3 targets, at least about 4 targets, at least about 5 targets, at least about 6 targets, at least about 7 targets, at least about 8 targets, at least about 9 targets, at least about 10 targets, at least about 20 targets, at least about 30 targets, at least about 40 targets, at least about 50 targets, at least about 60 targets, at least about 70 targets, or at least about 80 targets. At least approximately 90 targets, at least approximately 100 targets, at least approximately 200 targets, at least approximately 500 targets, at least approximately 1000 targets, at least approximately 2000 targets, at least approximately 5000 targets, at least approximately 10000 targets, at least approximately 12000 targets, at least approximately 20000 targets, at least approximately 25000 targets, at least approximately 50000 targets, at least approximately 100000 targets, at least approximately 1000000 targets, or more. In some embodiments, one or more targets may comprise up to about 1 target, up to about 2 targets, up to about 3 targets, up to about 4 targets, up to about 5 targets, up to about 6 targets, up to about 7 targets, up to about 8 targets, up to about 9 targets, up to about 10 targets, up to about 20 targets, up to about 30 targets, up to about 40 targets, up to about 50 targets, up to about 60 targets, up to about 70 targets, or up to about 80 targets. The number of targets is approximately 90, 100, 200, 500, 1000, 2000, 5000, 10000, 12000, 20000, 25000, 50000, 100000, or 1,000000.

[0158] iii. Nucleic acid molecules

[0159] The methods described herein may involve conjugating one or more nucleic acid molecules to one or more targets for detection. The one or more nucleic acid molecules described herein may contain one or more probes. The one or more probes may contain one or more RNA-binding sequences, one or more DNA-binding sequences, one or more oligonucleotide-binding sequences, one or more index sequences, or combinations thereof. In some cases, the one or more nucleic acid molecules may contain one or more probes configured to bind to RNA. The one or more probes configured to bind to RNA may contain an RNA-binding sequence, an index sequence, or a combination thereof. The one or more probes configured to bind to RNA may contain a sequence at least partially complementary to a portion of an RNA sequence or its inverse complementary sequence. The sequence at least partially complementary to a portion of an RNA sequence or its inverse complementary sequence may be an RNA-binding sequence. In some cases, the one or more nucleic acid molecules may contain one or more probes configured to bind to DNA. The one or more probes configured to bind to DNA may contain a DNA-binding sequence, an index sequence, or a combination thereof. The one or more probes configured to bind to DNA may contain a sequence at least partially complementary to a portion of a DNA sequence or its inverse complementary sequence. The sequence at least partially complementary to a portion of a DNA sequence or its inverse complementary sequence may be a DNA-binding sequence. In some cases, the one or more nucleic acid molecules may contain one or more probes configured to bind to a peptide-binding probe. One or more probes configured to bind to a peptide-binding probe may comprise an oligonucleotide-binding probe that is at least partially complementary to an oligonucleotide of the peptide-binding probe. In some cases, one or more nucleic acid molecules may comprise a target-binding sequence, an index sequence, or a combination thereof. In some cases, one or more nucleic acid molecules may be configured to bind to at least a portion of RNA or its inverse complementary sequence.

[0160] In some embodiments, one or more nucleic acid template molecules may contain DNA. In some embodiments, one or more nucleic acid template molecules may contain RNA. In some embodiments, one or more nucleic acid molecules may contain a combination of DNA and RNA. In some embodiments, one or more nucleic acid molecules may include modifications. In some embodiments, modifications may include phosphorylation, internucleotide bonding, fluorescent dyes, or combinations thereof. In some embodiments, internucleotide bonding may include locked nucleic acid residues. In some embodiments, modifications may include 5' modifications, 3' modifications, internal modifications, or combinations thereof.

[0161] One or more nucleic acid molecules may have different lengths. In some embodiments, each of the one or more nucleic acid molecules may have the same length. In some embodiments, each of the one or more nucleic acid molecules may have different lengths. In some embodiments, at least one nucleic acid molecule or one or more nucleic acid molecules may have a second nucleic acid molecule with a length different from that of the one or more nucleic acid molecules.In some embodiments, the length of one or more nucleic acid molecules can be about 1 to 100 nucleotides, about 2 to 98 nucleotides, about 3 to 96 nucleotides, about 4 to 94 nucleotides, about 5 to 92 nucleotides, about 6 to 90 nucleotides, about 7 to 88 nucleotides, about 8 to 86 nucleotides, about 9 to 84 nucleotides, about 10 to 82 nucleotides, about 11 to 80 nucleotides, about 12 to 78 nucleotides, about 13 to 76 nucleotides, about 14 to 74 nucleotides, about 15 to 72 nucleotides, about 16 to 70 nucleotides, about 17 to 68 nucleotides, about 18 to 66 nucleotides, or about 19 to 64 nucleotides. Approximately 20 to 62 nucleotides, approximately 22 to 60 nucleotides, approximately 24 to 58 nucleotides, approximately 26 to 56 nucleotides, approximately 28 to 54 nucleotides, approximately 30 to 52 nucleotides, approximately 32 to 50 nucleotides, approximately 34 to 48 nucleotides, approximately 36 to 46 nucleotides, approximately 38 to 44 nucleotides, approximately 40 to 42 nucleotides, at least approximately 1 nucleotide, at least approximately 2 nucleotides, at least approximately 3 nucleotides, at least approximately 4 nucleotides, at least approximately 5 nucleotides, at least approximately 6 nucleotides, at least approximately 7 nucleotides, at least approximately 8 nucleotides, at least approximately 9 nucleotides, at least approximately 10 nucleotides, at least approximately 11 nucleotides, at least approximately 12 nucleosides. Acid, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, at least about 24 nucleotides, at least about 26 nucleotides, at least about 28 nucleotides, at least about 30 nucleotides, at least about 32 nucleotides, at least about 34 nucleotides, at least about 36 nucleotides, at least about 38 nucleotides, at least about 40 nucleotides, at least about 42 nucleotides, at least about 44 nucleotides, at least about 46 nucleotides, at least about 48 nucleotides, at least about 50 nucleotides, at least about 52 nucleotides. The nucleic acid molecule may contain at least about 54 nucleotides, at least about 56 nucleotides, at least about 58 nucleotides, at least about 60 nucleotides, at least about 62 nucleotides, at least about 64 nucleotides, at least about 66 nucleotides, at least about 68 nucleotides, at least about 70 nucleotides, at least about 72 nucleotides, at least about 74 nucleotides, at least about 76 nucleotides, at least about 78 nucleotides, at least about 80 nucleotides, at least about 82 nucleotides, at least about 84 nucleotides, at least about 86 nucleotides, at least about 88 nucleotides, at least about 90 nucleotides, at least about 92 nucleotides, at least about 94 nucleotides, at least about 96 nucleotides, at least about 98 nucleotides, or at least about 100 nucleotides. In some embodiments, the length of the nucleic acid molecule is about 10 to 80 nucleotides.In some embodiments, the length of the nucleic acid molecule is about 20 to 60 nucleotides. In some embodiments, the length of the nucleic acid molecule is about 30 to 50 nucleotides.

[0162] In some embodiments, a nucleic acid molecule may be conjugated to a target (e.g., a nucleic acid). In some embodiments, more than one nucleic acid molecule may be conjugated to a target (e.g., a nucleic acid). In some embodiments, a nucleic acid molecule (e.g., a probe containing an oligonucleotide binding probe) may be conjugated to a probe that binds to a target (e.g., a peptide binding probe that binds to a peptide). In some embodiments, more than one nucleic acid molecule (e.g., more than one probe containing an oligonucleotide binding probe) may be conjugated to a probe that binds to a target (e.g., a peptide binding probe that binds to a peptide). In some embodiments, a first group of nucleic acid molecules may be conjugated to a first target, and a second group of nucleic acid molecules may be conjugated to a second target. In some embodiments, the number of nucleic acid molecules in the first group and the second group are the same. In some embodiments, the number of nucleic acid molecules in the first group and the second group are different. In some embodiments, the number of nucleic acid molecules in the first group is greater than the number of nucleic acid molecules in the second group. In some embodiments, the number of nucleic acid molecules in the first group is less than the number of nucleic acid molecules in the second group. In some embodiments, a first group of nucleic acid molecules (e.g., probes containing oligonucleotide binding probes) may be coupled to a first probe binding to a first target (a first peptide binding probe binding to a first polypeptide), and a second group of nucleic acid molecules (e.g., probes containing oligonucleotide binding probes) may be coupled to a second target (e.g., a second peptide binding probe binding to a second polypeptide). In some embodiments, the number of nucleic acid molecules in the first and second groups is the same. In some embodiments, the number of nucleic acid molecules in the first and second groups is different. In some embodiments, the number of nucleic acid molecules in the first group is greater than the number of nucleic acid molecules in the second group. In some embodiments, the number of nucleic acid molecules in the first group is less than the number of nucleic acid molecules in the second group.

[0163] In some embodiments, one or more nucleic acid molecules contain at least one binding site (e.g., an RNA binding sequence, a DNA binding sequence, an oligonucleotide binding probe, or a target binding sequence). The binding site may contain a nucleic acid sequence that hybridizes to one or more targets for detection. Binding sites may have multiple lengths. In some embodiments, one binding site may have a different length than a second binding site.In some embodiments, the length of one or more nucleic acid molecules can be about 1 to 100 nucleotides, about 2 to 98 nucleotides, about 3 to 96 nucleotides, about 4 to 94 nucleotides, about 5 to 92 nucleotides, about 6 to 90 nucleotides, about 7 to 88 nucleotides, about 8 to 86 nucleotides, about 9 to 84 nucleotides, about 10 to 82 nucleotides, about 11 to 80 nucleotides, about 12 to 78 nucleotides, about 13 to 76 nucleotides, about 14 to 74 nucleotides, about 15 to 72 nucleotides, about 16 to 70 nucleotides, about 17 to 68 nucleotides, about 18 to 66 nucleotides, or about 19 to 64 nucleotides. Approximately 20 to 62 nucleotides, approximately 22 to 60 nucleotides, approximately 24 to 58 nucleotides, approximately 26 to 56 nucleotides, approximately 28 to 54 nucleotides, approximately 30 to 52 nucleotides, approximately 32 to 50 nucleotides, approximately 34 to 48 nucleotides, approximately 36 to 46 nucleotides, approximately 38 to 44 nucleotides, approximately 40 to 42 nucleotides, at least approximately 1 nucleotide, at least approximately 2 nucleotides, at least approximately 3 nucleotides, at least approximately 4 nucleotides, at least approximately 5 nucleotides, at least approximately 6 nucleotides, at least approximately 7 nucleotides, at least approximately 8 nucleotides, at least approximately 9 nucleotides, at least approximately 10 nucleotides, at least approximately 11 nucleotides, at least approximately 12 nucleotides At least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, at least about 24 nucleotides, at least about 26 nucleotides, at least about 28 nucleotides, at least about 30 nucleotides, at least about 32 nucleotides, at least about 34 nucleotides, at least about 36 nucleotides, at least about 38 nucleotides, at least about 40 nucleotides, at least about 42 nucleotides, at least about 44 nucleotides, at least about 46 nucleotides, at least about 48 nucleotides, at least about 50 nucleotides, at least about 52 nucleotides, up to The length can be approximately 54 nucleotides, at least 56 nucleotides, at least 58 nucleotides, at least 60 nucleotides, at least 62 nucleotides, at least 64 nucleotides, at least 66 nucleotides, at least 68 nucleotides, at least 70 nucleotides, at least 72 nucleotides, at least 74 nucleotides, at least 76 nucleotides, at least 78 nucleotides, at least 80 nucleotides, at least 82 nucleotides, at least 84 nucleotides, at least 86 nucleotides, at least 88 nucleotides, at least 90 nucleotides, at least 92 nucleotides, at least 94 nucleotides, at least 96 nucleotides, at least 98 nucleotides, or at least 100 nucleotides. In some embodiments, the target binding sequence is approximately 5 to 25 nucleotides in length.

[0164] In some embodiments, one or more nucleic acid molecules contain at least one index sequence. This at least one index sequence may indicate the identity of one or more targets. In some embodiments, a single index sequence corresponds to one or more targets. In some embodiments, more than one index sequence corresponds to one or more targets. In some embodiments, one or more nucleic acid molecules contain at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 index sequences. The at least one index sequence may be a sequence orthogonal to the sequences of one or more targets. In some embodiments, the at least one index sequence includes at least a portion of the sequences of one or more targets.

[0165] In some cases, a nucleic acid molecule in one or more nucleic acid molecules may contain a binding site (e.g., a target binding site) adjacent to the index sequence. For example, the binding site (e.g., a target binding site) may be directly adjacent to the index sequence. In another instance, the binding site may be separated from the index sequence by at least one nucleotide. In some cases, the nucleic acid molecule may bind to a target (e.g., RNA), and the index sequence may not be bound. For example, the nucleic acid molecule may contain a target binding sequence that hybridizes with RNA, and the nucleic acid molecule may contain an index sequence that does not hybridize with RNA (e.g., unhybridized). The index sequence may be single-stranded and may hybridize with another nucleic acid sequence (e.g., an index binding sequence).

[0166] Each of the at least one index sequence can have multiple lengths. The length of the index sequence can be about 1 to 25 nucleotides, about 2 to 24 nucleotides, about 3 to 23 nucleotides, about 4 to 22 nucleotides, about 5 to 21 nucleotides, about 6 to 20 nucleotides, about 7 to 19 nucleotides, about 8 to 18 nucleotides, about 9 to 17 nucleotides, about 10 to 16 nucleotides, about 11 to 15 nucleotides, about 12 to 14 nucleotides, at least about 1 nucleotide, at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, at least about 6 nucleotides, or at least about 6 nucleotides. The index sequence may contain 1 nucleotide, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, or at least about 25 nucleotides. In some embodiments, the index sequence is 4 to 50 nucleotides in length.

[0167] In some embodiments, the index sequence includes a primer binding site. In some embodiments, when the nucleic acid molecule includes more than one index sequence, each index sequence includes a primer binding site. The primer binding site can be configured to hybridize with the primer nucleic acid sequence described herein. The primer binding site can hybridize with the primer nucleic acid sequence. The primer binding site of different index sequences can have various lengths. The length of the primer binding sequence can be about 1 to 25 nucleotides, about 2 to 24 nucleotides, about 3 to 23 nucleotides, about 4 to 22 nucleotides, about 5 to 21 nucleotides, about 6 to 20 nucleotides, about 7 to 19 nucleotides, about 8 to 18 nucleotides, about 9 to 17 nucleotides, about 10 to 16 nucleotides, about 11 to 15 nucleotides, about 12 to 14 nucleotides, at least about 1 nucleotide, at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, or at least about 25 nucleotides.

[0168] An index sequence can be uniquely associated with one or more targets used for detection. One or more targets can comprise RNA, DNA, peptides, or combinations thereof. In some embodiments, the index sequence can be associated with one or more targets and at least one other analyte. In some embodiments, the other analyte can be a nucleic acid or a peptide. In some embodiments, the other analyte can be RNA. In some embodiments, the other analyte can comprise naturally occurring nucleic acids, recombinant nucleic acids, synthetic nucleic acids, or combinations thereof. In some embodiments, the other analyte can be DNA. In some embodiments, the other analyte can be genomic DNA. In some embodiments, the other analyte can be viral DNA. In some embodiments, the other analyte can be cfDNA. In some embodiments, the other analyte can be poly(A) RNA and / or non-poly(A) RNA. RNA can include coding RNA, non-coding RNA, or combinations thereof. RNA can comprise tRNA, rRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), antisense RNA, non-coding RNA, protein-coding RNA, or combinations thereof.

[0169] In some embodiments, other analytes may be peptides. In some embodiments, peptides may be proteins, peptides, or combinations thereof. In some embodiments, proteins may be antibodies, antibody fragments, ribosomal proteins, transcription factors, histones, enzymes, structural proteins, chemokines, cytokines, or combinations thereof.

[0170] The nucleic acid molecules described herein may contain one or more lock probes. One or more lock probes may contain nucleic acids. The nucleic acids of one or more lock probes may contain DNA, RNA, or a combination thereof. One or more lock probes may contain one or more modifications, including but not limited to phosphorylation (e.g., 5' phosphorylation), lock nucleic acids, or a combination thereof. In some embodiments, the lock probe contains canonical nucleotides and / or nucleotide analogs. In some embodiments, the lock probe is modified to confer resistance to nuclease degradation (e.g., ribonuclease degradation). For example, the lock probe includes at least one phosphothioester bond at its 5' end, which can make the lock probe resistant to nuclease degradation. In some embodiments, the lock probe contains 2-5 or more consecutive phosphothioester bonds at its 5' end. In some embodiments, the lock probe contains at least one ribonucleotide and / or at least one 2'-O-methyl, 2'-O-methoxyethyl (MOE), or 2'-fluoronucleotide. In some embodiments, the lock probe contains a phosphorylated 3' end. In some embodiments, the lock probe contains at least one lock nucleic acid (LNA) base. In some embodiments, the lock probe contains a phosphorylated 5' end (e.g., using a polynucleotide kinase).

[0171] One or more locking probes may include a first end, a second end, an index sequence, additional sequences, or combinations thereof. The first end of the locking probe may contain a sequence at least partially complementary to a target sequence (e.g., RNA or DNA) or its inverse complementary sequence. The first end of the locking probe may contain a sequence at least partially complementary to an oligonucleotide of a peptide-binding probe or a portion of its inverse complementary sequence. The second end of the locking probe may contain a sequence at least partially complementary to a target sequence (e.g., RNA or DNA) or its inverse complementary sequence. The second end of the locking probe may contain a sequence at least partially complementary to an oligonucleotide of a peptide-binding probe or a portion of its inverse complementary sequence. In some cases, when both the first and second ends bind to a target (e.g., RNA or DNA) or both bind to an oligonucleotide of a peptide-binding probe, the first end and the second end of the locking probe are directly adjacent. For example, the first end of the locking probe can hybridize with a first sequence of RNA, and the second end of the locking probe can hybridize with a second sequence of RNA, wherein the first and second sequences are consecutive sequences of RNA, such that when bound to the locking probe, there are no unhybridized nucleotides on the RNA at the junction of the first and second ends of the locking probe. In another example, the first end of the locking probe can hybridize with a first portion of an oligonucleotide of a peptide-binding probe, and the second end of the locking probe can hybridize with a second portion of an oligonucleotide of a peptide-binding probe, wherein the first and second portions are consecutive sequences of oligonucleotides, such that when bound to the locking probe, there are no unhybridized nucleotides on the oligonucleotide at the junction of the first and second ends of the locking probe.

[0172] In some embodiments, a single locking probe has a first random sequence at its first end that hybridizes to a first region of a target (e.g., an RNA molecule), and a second random sequence at its second end that hybridizes to a second region of the same target, wherein a slit or gap is formed between the first and second hybridization arms. In some embodiments, a single locking probe in a set of locking probes (e.g., multiple locking probes) comprises a first end and a second end that hybridize to the same target region of a target (e.g., an RNA molecule) to form multiple target-locking probe complexes having the same target sequence.

[0173] In some cases, when both the first and second ends of the lock probe bind to a target (e.g., RNA or DNA) or both bind to an oligonucleotide of the peptide-binding probe, the first and second ends of the lock probe are adjacent to each other and separated by a gap (e.g., by at least one nucleotide). For example, the first end of the lock probe may hybridize to a first sequence of RNA, and the second end of the lock probe may hybridize to a second sequence of RNA, and the first and second sequences are discontinuous sequences of RNA, such that when bound to the lock probe, at the junction of the first and second ends of the lock probe, at least one unhybridized nucleotide is present on the RNA. In another example, the first end of the lock probe may hybridize to a first portion of an oligonucleotide of the peptide-binding probe, and the second end of the lock probe may hybridize to a second portion of an oligonucleotide of the peptide-binding probe, and the first and second portions are discontinuous sequences of the oligonucleotide, such that when bound to the lock probe, at the junction of the first and second ends of the lock probe, at least one unhybridized nucleotide is present on the oligonucleotide. When a gap separates the first and second ends of a lock probe, a gap-filling reaction can be performed to fill unhybridized nucleotides in the oligonucleotide of the target or peptide-binding probe. The gap-filling reaction can be carried out by a polymerase (e.g., DNA polymerase or RNA polymerase). In some embodiments, the gap between the first and second binding arms of the hybridization is 1 to 30 bases in size. The 3'OH end of the hybridized lock probe can serve as the initiation site for a reverse transcriptase-catalyzed filling reaction (e.g., a gap-filling reaction) using the target RNA molecule as a template. After the filling reaction, the remaining nick can be enzymatically ligated to generate a covalently closed circular nucleic acid.

[0174] In some embodiments, the gap-filling reaction comprises contacting a circular nucleic acid with a reverse transcriptase and multiple nucleotides. In some embodiments, the reverse transcriptase includes, in some examples, a reverse transcriptase derived from AMV (avian myeloblastoma virus), M-MuLV (Moloni mouse leukemia virus), or HIV (human immunodeficiency virus). In some embodiments, the reverse transcriptase comprises a recombinant enzyme exhibiting reduced RNase H activity, such as REVERTAID (e.g., from Thermo Fisher Scientific, catalog number EP0441). In some embodiments, the reverse transcriptase may be a commercially available enzyme, including MULTISCRIBE (e.g., from Thermo Fisher Scientific, catalog number 4311235), THERMOSCRIPT (e.g., from Thermo Fisher Scientific, catalog number 12236-014), or ARRAYSCRIPT (e.g., from Ambion, catalog number AM2048). In some embodiments, the reverse transcriptase includes SUPERSCRIPT II (e.g., catalog number 18064014), SUPERSCRIPT III (e.g., catalog number 18080044), or SUPERSCRIPT IV (e.g., catalog number 18090010) (all SUPERSCRIPT enzymes from Invitrogen). In some embodiments, the reverse transcription reaction may include an RNase inhibitor.

[0175] One or more lock probes can be circularized to generate one or more circular nucleic acids. For example, a first end of a lock probe can be ligated to a second end of a lock probe to form a circular nucleic acid. Lock probes can be ligated by ligases, chemical reactions, or a combination thereof. In some cases, one end of the lock probe (e.g., the first or second end) may include a phosphorylation modification (e.g., 5' phosphorylation). A ligase can ligate the phosphorylated end of the lock probe to the other ends of the lock probe to form a circular nucleic acid. In some embodiments, the ligase can be a DNA ligase. In some embodiments, the ligase can be an RNA ligase. In some embodiments, the ligase can be a T4 ligase, SplintR ligase, T3 ligase, T4 ligase, T7 ligase, TtcB ligase, Taq ligase, or a combination thereof. In some embodiments, the ligase can distinguish between matched and mismatched hybridization ends to ensure target-specific hybridization. In some cases, one end of the lock probe may contain a bioorthogonal modification (e.g., an azide or alkyne). In some cases, the bioorthogonal modifications at one end of a lock probe can chemically react with the other end of the lock probe (which may include a second bioorthogonal modification) to form a covalent bond between the two ends, thereby forming a circular nucleic acid molecule.

[0176] In some embodiments, the lock probe includes at least one universal adaptor sequence, which includes a sample barcode sequence, an amplification primer binding site, a sequencing primer binding site, and / or a compacted oligonucleotide binding site. In some embodiments, the lock probe includes at least one unique identifier sequence (e.g., a unique molecular index (UMI)). In some embodiments, the lock probe includes at least one restriction enzyme recognition sequence.

[0177] In some embodiments, a set of locking probes (e.g., multiple locking probes) comprises at least two subgroups (e.g., two subfamilies) of locking probes. Figure 28 , 30 (and 32). In some embodiments, individual lock probes in the first subgroup of lock probes include a first binding arm and a second binding arm that hybridize with the same target region (e.g., the first target region) of a first target RNA molecule to form a first plurality of RNA-lock probe complexes having the same RNA sequence. In some embodiments, individual lock probes in the second subgroup of lock probes include a first binding arm and a second binding arm that hybridize with the same target region (e.g., the second target region) of a second target RNA molecule to form a second plurality of RNA-lock probe complexes having the same RNA sequence. In some embodiments, the first and second subgroups of lock probes hybridize with different target regions of the same target RNA molecule. In some embodiments, the first and second subgroups of lock probes hybridize with different target regions of different target RNA molecules. In some embodiments, the group of lock probes includes 2 to 10 lock probe subgroups, or 10 to 25 lock probe subgroups, or 25 to 50 lock probe subgroups, or up to 100 lock probe subgroups. In some embodiments, the lock probe set includes at least 100 lock probe subsets, at least 500 lock probe subsets, at least 1000 lock probe subsets, at least 10,000 lock probe subsets, or more lock probe subsets.

[0178] iv. Peptide-binding probe

[0179] This document discloses peptide-binding probes that can be used in the in-situ detection methods for targets (e.g., peptides) described herein. The peptide-binding probe can be configured to bind one or more peptides. The peptide-binding probe may comprise a peptide-binding moiety and an oligonucleotide. The peptide-binding moiety can be directly or indirectly coupled to the oligonucleotide using one or more covalent interactions, one or more non-covalent interactions, or a combination thereof. For example, the peptide-binding moiety may comprise a peptide, and the peptide of the peptide-binding moiety may be covalently linked to the oligonucleotide. Furthermore, the peptide-binding moiety may comprise a peptide, and the peptide of the peptide-binding moiety may be non-covalently linked to the oligonucleotide via a biotin-streptavidin interaction. The peptide-binding moiety can be linked to the oligonucleotide using one or more adapters. In some embodiments, one or more adapters may comprise streptavidin (or an avidin-like moiety), biotin, an amine group, a disulfide group, or a combination thereof. In some embodiments, one or more adapters may be non-cleavable or non-removable. In some embodiments, one or more adapters may be cleavable or removable. For example, one or more adapters may be light-cleavable (e.g., ultraviolet light), chemically cleavable (e.g., dithiothreitol), thermally cleavable, enzymatically cleavable, or a combination thereof.

[0180] The oligonucleotide of the peptide-binding probe can contain DNA, RNA, or a combination thereof. In some cases, the oligonucleotide can contain one or more non-natural nucleotides. One or more non-natural nucleotides can contain one or more of the following: 2-methoxyethoxy A, 2-methoxyethoxy MeC, 2-methoxyethoxy G, 2-methoxyethoxy T, 8-oxo dG, deoxyadenosine, N6-methylrA, 5-bromo dU, deoxyuridine, 2,6-diaminopurine, dideoxy-C, deoxyinosine, hydroxymethyl dC, isodeoxy-dG, isodeoxy-dC, 5-methyl dC, or 5-nitroindole, or a combination thereof. The oligonucleotide can contain one or more locked nucleic acids (LNAs). The oligonucleotide can contain single-stranded nucleic acids, double-stranded nucleic acids, or a combination thereof. Oligonucleotides can have a length of at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, or more. Oligonucleotides can have a length of up to about 2 nucleotides, up to about 3 nucleotides, up to about 4 nucleotides, up to about 5 nucleotides, up to about 6 nucleotides, up to about 7 nucleotides, up to about 8 nucleotides, up to about 9 nucleotides, up to about 10 nucleotides, up to about 11 nucleotides, up to about 12 nucleotides, up to about 13 nucleotides, up to about 14 nucleotides, up to about 15 nucleotides, up to about 16 nucleotides, up to about 17 nucleotides, up to about 18 nucleotides, up to about 19 nucleotides, up to about 20 nucleotides, up to about 25 nucleotides, up to about 30 nucleotides, up to about 40 nucleotides, up to about 45 nucleotides, up to about 50 nucleotides, up to about 55 nucleotides, up to about 60 nucleotides, up to about 70 nucleotides, up to about 80 nucleotides, up to about 90 nucleotides, up to about 100 nucleotides, or fewer.Oligonucleotides can have a length of about 2 to 100 nucleotides, about 3 to 90 nucleotides, about 4 to 80 nucleotides, about 5 to 70 nucleotides, about 6 to 60 nucleotides, about 7 to 55 nucleotides, about 8 to 50 nucleotides, about 9 to 45 nucleotides, about 10 to 40 nucleotides, about 11 to 30 nucleotides, about 12 to 25 nucleotides, about 13 to 20 nucleotides, about 14 to 19 nucleotides, about 15 to 18 nucleotides, or about 16 to 17 nucleotides.

[0181] Peptide-binding probes may comprise peptides, nucleic acids, linkers, or combinations thereof. In some cases, peptide-binding probes may comprise peptides. Peptides may comprise antibodies, antibody fragments, nanobodies, affinity molecules, or combinations thereof. Antibodies or antibody fragments may comprise multiple isotypes, including but not limited to IgG, IgM, IgA, IgD, IgE, or combinations thereof. In some cases, peptide-binding probes may comprise antibodies or antibody fragments, and the peptide-binding moiety may contain a paratope. In some cases, peptide-binding probes may comprise antibodies or antibody fragments, and the peptide-binding moiety may contain an antigen-binding site. In some cases, peptide-binding probes may comprise nucleic acids. The nucleic acid of a peptide-binding probe may contain an aptamer. The aptamer may be configured to bind to a target detected in the methods described herein.

[0182] v. index-combined sequence

[0183] This document discloses nucleic acid molecules containing index-binding sequences that can be used in the methods described herein for in situ detection of targets (e.g., RNA, DNA, or peptides). For example, the index-binding sequence of a nucleic acid molecule can cause the nucleic acid molecule to bind to an index sequence to trigger amplification or detection of the index sequence. In some embodiments, the nucleic acid molecule containing the index-binding sequence is a lock probe. In some embodiments, the nucleic acid molecule containing the index-binding sequence is a molecular inversion probe. In some embodiments, the nucleic acid molecule containing the index-binding sequence may contain a second index sequence. The index-binding sequence can be configured to be coupled to an index sequence. In some embodiments, the index-binding sequence can hybridize with an index sequence. In some embodiments, the index-binding sequence may contain at least one binding region that recognizes and binds at least a portion of the index sequence. In some embodiments, the index-binding sequence may contain more than one binding region that recognizes and binds to an index sequence. For example, a lock probe may have two index-binding sequences at its 5' and 3' ends, which, when hybridized to the respective 5' and 3' ends of the index sequence, trigger a ligation reaction to ligate the 5' and 3' ends of the lock probe.

[0184] In some embodiments, the index-binding sequence may bind to the index sequence to form a double-stranded region. In some cases, the double-stranded region is continuous. In some embodiments, the double-stranded region includes at least one nick. In some embodiments, the double-stranded region may be separated by a single-stranded region. In some cases, the length of the single-stranded region may be one or more nucleotides. For example, the two ends (5' and 3' ends) of a lock probe may bind to the index sequence to form a double-stranded region, wherein the two ends of the lock probe are separated by a nick. In some embodiments, the index-binding sequence may be ligated after hybridization with the index sequence. In the example of a lock probe, after ligation at the 5' and 3' ends, the lock probe forms a circular nucleic acid. In some embodiments, the ligation reaction may be performed by a ligase. In some embodiments, the ligase may be a DNA ligase. In some embodiments, the ligase may be an RNA ligase. In some embodiments, the ligase may be a T4 ligase, SplintR ligase, T3 ligase, T7 ligase, TtcB ligase, or a combination thereof. In some embodiments, the ligation reaction may form a circular nucleic acid.

[0185] vi. Amplification reaction

[0186] This document discloses amplification reactions that can be used for in situ detection of targets (e.g., RNA, DNA, or peptides). Amplification reactions can be used to enhance target-associated signals. In some cases, amplification reactions can generate multiple copies of the target, its inverse complementary sequence, or the target (e.g., a nucleic acid), one or more target-associated index sequences, or combinations thereof. Amplification reactions can generate multiple copies of the amplified sequence. In some cases, amplification reactions can generate multiple copies of the inverse complementary sequence of the amplified sequence. Amplification reactions may include replication of the target or its inverse complementary sequence or target-associated index sequence, or may include additional nucleic acid hybridization to generate additional binding sites for the detection portions described herein.

[0187] In some cases, the amplification reaction can generate one or more amplicons. One or more amplicons may include one or more copies of a target-associated index sequence or its inverse complementary sequence. One or more amplicons may contain one or more derivatives of a target, an index sequence, or a combination thereof. For the methods described herein, a nucleic acid derivative (e.g., a derivative of an index sequence) refers to the inverse complementary sequence of a nucleic acid sequence, such as the inverse complementary sequence of an index sequence. One or more amplicons may contain one or more copies of a portion of a target (e.g., RNA or DNA) or an inverse complementary sequence of a target (e.g., RNA or DNA). One or more amplicons may contain one or more copies of an oligonucleotide of a polypeptide-binding sequence or a portion of its inverse complementary sequence. One or more amplicons may contain one or more copies of an index-binding sequence or a portion of its inverse complementary sequence.

[0188] In some cases, amplification reactions may include rolling circle amplification (RoBLA), polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), helicase-dependent amplification (HAD), multiple displacement amplification (MDA), multiple cross-displacement amplification (MCDA), transcription-mediated amplification, ligase chain reaction, or combinations thereof. Amplification reactions may include one or more temperature incubation steps. In some cases, amplification reactions may occur at the same temperature (e.g., isothermally). In some cases, amplification reactions may occur at multiple temperatures. For example, RoBLA may be performed at 42°C for the duration of the amplification reaction. In some cases, the temperature during the amplification reaction may vary over a wide range. In some cases, the incubation temperature for the amplification reaction may be at least about 15°C, at least about 18°C, at least about 20°C, at least about 25°C, at least about 30°C, at least about 32°C, at least about 34°C, at least about 36°C, at least about 38°C, at least about 40°C, at least about 42°C, at least about 44°C, at least about 46°C, at least about 48°C, at least about 50°C, at least about 52°C, at least about 54°C, at least about 56°C, at least about 58°C, at least about 60°C, at least about 62°C, at least about 64°C, at least about 66°C, at least about 68°C, at least about 70°C, at least about 72°C, at least about 74°C, at least about 76°C, at least about 78°C, at least about 80°C, at least about 82°C, at least about 84°C, at least about 86°C, at least about 88°C, at least about 90°C, at least about 95°C, or higher. In some cases, the incubation temperature for the amplification reaction can be up to approximately 15°C, up to approximately 18°C, up to approximately 20°C, up to approximately 25°C, up to approximately 30°C, up to approximately 32°C, up to approximately 34°C, up to approximately 36°C, up to approximately 38°C, up to approximately 40°C, up to approximately 42°C, up to approximately 44°C, up to approximately 46°C, up to approximately 48°C, up to approximately 50°C, up to approximately 52°C, or up to approximately 54°C. Up to approximately 56°C, up to approximately 58°C, up to approximately 60°C, up to approximately 62°C, up to approximately 64°C, up to approximately 66°C, up to approximately 68°C, up to approximately 70°C, up to approximately 72°C, up to approximately 74°C, up to approximately 76°C, up to approximately 78°C, up to approximately 80°C, up to approximately 82°C, up to approximately 84°C, up to approximately 86°C, up to approximately 88°C, up to approximately 90°C, up to approximately 95°C or higher. In some cases, the incubation temperature for the amplification reaction can be up to approximately 18 to 90°C, approximately 20 to 88°C, approximately 25 to 86°C, approximately 30 to 84°C, approximately 32 to 82°C, approximately 34 to 80°C, approximately 36 to 78°C, approximately 38 to 76°C, approximately 40 to 74°C, approximately 42 to 72°C, approximately 44 to 70°C, approximately 46 to 68°C, approximately 48 to 66°C, approximately 50 to 64°C, approximately 52 to 62°C, approximately 54 to 60°C, or approximately 56 to 58°C. The amplification reaction can proceed for a specified duration.In some cases, the duration of the amplification reaction can be related to the amount of signal generated during the detection step of the method described herein. For example, in some cases, a longer amplification reaction can generate a stronger signal associated with the target. The amplification reaction can be for at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 1 hour, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 15 hours, at least about 20 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, or longer. The amplification reaction can last for up to approximately 5 minutes, up to approximately 10 minutes, up to approximately 15 minutes, up to approximately 30 minutes, up to approximately 45 minutes, up to approximately 1 hour, up to approximately 1 hour, up to approximately 3 hours, up to approximately 4 hours, up to approximately 5 hours, up to approximately 6 hours, up to approximately 7 hours, up to approximately 8 hours, up to approximately 9 hours, up to approximately 10 hours, up to approximately 12 hours, up to approximately 15 hours, up to approximately 20 hours, up to approximately 24 hours, up to approximately 36 hours, up to approximately 48 hours, or less. The amplification reaction time can range from approximately 5 minutes to 48 hours, from approximately 10 minutes to 36 hours, from approximately 15 minutes to 24 hours, from approximately 30 minutes to 20 hours, from approximately 45 minutes to 15 hours, from approximately 1 hour to 12 hours, from approximately 2 hours to 10 hours, from approximately 3 hours to 9 hours, from approximately 4 hours to 8 hours, or from approximately 5 hours to 7 hours.

[0189] In some cases, rolling circle amplification (RBA) reactions can be performed. RBA can include amplifying circular nucleic acids. Circular nucleic acids can contain one or more nucleic acid molecules as described herein. For example, lock probes can be ligated to form circular nucleic acids used during RBA reactions. Lock probes forming circular nucleic acids can be probes containing a DNA-binding sequence and an index sequence, probes containing an RNA-binding sequence and an index sequence, oligonucleotide-binding probes and an index sequence, or an index-binding sequence. RBA reactions can generate one or more amplicons. The amplicons in one or more amplicons can contain one or more copies of components of the circular nucleic acid. For example, circular nucleic acids can be generated by ligating a lock probe containing a DNA-binding sequence index sequence. This circular nucleic acid can be used to perform RBA reactions to generate one or more amplicons containing one or more copies of a DNA-binding sequence or its reverse complementary sequence, and one or more copies of an index sequence or its reverse complementary sequence. In another example, circular nucleic acids can be generated by ligating a lock probe containing an RNA-binding sequence index sequence. The circular nucleic acid can be used for rolling circle amplification to generate one or more amplicons containing one or more copies of an RNA-binding sequence or its inverse complementary sequence, and one or more copies of an index sequence or its inverse complementary sequence. In another example, the circular nucleic acid can be generated by attaching a lock probe containing an oligonucleotide binding probe index sequence. The circular nucleic acid can be used for rolling circle amplification to generate one or more amplicons containing one or more copies of an oligonucleotide binding probe or its inverse complementary sequence, and one or more copies of an index sequence or its inverse complementary sequence. In another example, the circular nucleic acid can be generated by attaching a lock probe containing an index binding sequence (optionally an index sequence). The circular nucleic acid can be used for rolling circle amplification to generate one or more amplicons containing one or more copies of an index binding sequence or its inverse complementary sequence, and optionally one or more copies of an index sequence or its inverse complementary sequence.

[0190] In some embodiments, the amplification reaction is carried out by a polymerase. In some embodiments, the polymerase may be a polymerase. In some embodiments, the polymerase may be a DNA polymerase. In some embodiments, the polymerase may be an RNA polymerase. In some embodiments, the polymerase may have strand substitution capability. In some embodiments, the polymerase may be a PHI29 polymerase, a Taq polymerase, a T4 polymerase, a T7 polymerase, or a combination thereof. For example, the amplification reaction may include rolling circle amplification, and one or more amplicons may be generated using a PHI29 polymerase.

[0191] In some embodiments, rolling circle amplification (RoBA) includes contacting a covalently circumscribed nucleic acid with a soluble amplification primer (e.g., a universal RoBA primer), a strand displacement DNA polymerase, a plurality of nucleotides, or a combination thereof, under conditions suitable for hybridizing individual amplification primers with a circular nucleic acid and for primer extension using the circular nucleic acid as a template molecule to generate one or more amplicons. In some embodiments, the plurality of nucleotides in the RoBA reaction may comprise any mixture of two or more of dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, any of the RoBA reactions described herein may be performed with or without a plurality of compacted oligonucleotides.

[0192] In some embodiments, when the rolling circle amplification reaction comprises multiple nucleotides including dUTPs, the resulting amplicon can be crosslinked with a crosslinking reactive group by treating the biological sample with succinimide ester (NHS), maleimide (sulfonyl-SMCC), imine ester (DMP), carbodiimide (DCC, EDC), or phenyl azide. In some embodiments, the polymerization of the crosslinking reactive group can be initiated by light or UV light. In some embodiments, the resulting amplicon can be crosslinked with a matrix by treating the biological sample with crosslinked agarose, crosslinked dextran, or crosslinked polyethylene glycol (PEG), polyacrylamide, cellulose alginate, or polyamide. In some embodiments, the PEG contains a sulfonyl-NHS ester moiety at one or both ends, such as PEGylated bis(sulfosuccinimide) octanoate (e.g., BS(PEG)9 from Thermo Fisher Scientific, catalog number 21582).

[0193] In some embodiments, the rolling circle amplification reaction can be carried out at a constant temperature (e.g., isothermal), wherein the constant temperature is from room temperature to about 30°C, or about 30 to 40°C, or about 40 to 50°C, or about 50 to 65°C.

[0194] In some embodiments, the DNA polymerase having strand substitution activity may be selected from the group consisting of: phi29 DNA polymerase, a large fragment of Bst DNA polymerase, a large fragment of Bsu DNA polymerase, and Bca (exonuclease) DNA polymerase, the Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, or Deep Vent DNA polymerase. In some embodiments, the phi29 DNA polymerase may be a wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon) or a variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or a chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0195] In some embodiments, rolling circle amplification primers may be modified to increase resistance to nuclease degradation. In some embodiments, the rolling circle amplification primers contain at least one phosphate-thioester diester bond at their 5' end, which can make the amplification primers resistant to exonuclease degradation. In some embodiments, the rolling circle amplification primers contain 2-5 or more consecutive phosphate-thioester diester bonds at their 5' end. In some embodiments, the rolling circle amplification primers contain at least one ribonucleotide and / or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide.

[0196] In some embodiments, rolling circle amplification (RoBA) can be performed in the presence of a plurality of compacting oligonucleotides that, when hybridized with an amplicon, compact the size and / or shape of the amplicon to form compacted nanospheres. In some embodiments, the compacting oligonucleotides comprise single-stranded oligonucleotides having a first region at one end that hybridizes with a portion of the amplicon and a second region at the other end that hybridizes with another portion of the same amplicon, wherein hybridization of the compacting oligonucleotide with a given amplicon compacts the size and / or shape of the amplicon.

[0197] The compacting oligonucleotide comprises a 5' region, an optional internal region (intermediate region), and a 3' region. The 5' and 3' regions of the compacting oligonucleotide can hybridize with different parts of the amplicon to pull the distal portions of the amplicon together, thereby compacting the amplicon to form DNA nanospheres. For example, the 5' region of the compacting oligonucleotide is designed to hybridize with the first part of the amplicon (e.g., a universal compacting oligonucleotide binding site), and the 3' region of the compacting oligonucleotide is designed to hybridize with the second part of the amplicon (e.g., a universal compacting oligonucleotide binding site). Including the compacting oligonucleotide during RCA promotes the formation of DNA nanospheres with a more compact size and shape compared to amplicons generated in the absence of the compacting oligonucleotide. The compaction and stabilization properties of the DNA nanospheres improve the accuracy of in situ sequencing by increasing signal intensity, and the nanospheres maintain their shape and size during multiple sequencing cycles.

[0198] In some embodiments, the compacted oligonucleotide comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The compacted oligonucleotide can be of any length, including 20-150 nucleotides, 30-100 nucleotides, or 40-80 nucleotides.

[0199] In some embodiments, the compacted oligonucleotide comprises a 5' region and a 3' region, and optionally an intermediate region between the 5' and 3' regions. The intermediate region can be of any length, for example, about 2-20 nucleotides in length. The intermediate region comprises a homopolymer having consecutive identical bases (e.g., AAA, GGG, CCC, TTT, or UUU). The intermediate region may also comprise a non-homogeneous sequence.

[0200] The 5' region of the compacted oligonucleotide can be completely or partially complementary to the first part of the amplicon along its length. The 3' region of the compacted oligonucleotide can be completely or partially complementary to the second part of the amplicon along its length. The 5' region of the compacted oligonucleotide can hybridize with the first universal sequence portion of the amplicon. The 3' region of the compacted oligonucleotide can hybridize with the second universal sequence portion of the amplicon.

[0201] In some embodiments, the 5' region of the compacted oligonucleotide may have the same sequence as the 3' region. The 5' region of the compacted oligonucleotide may have a different sequence than the 3' region. In some embodiments, the 3' region of the compacted oligonucleotide may have a sequence opposite to the 5' region. In some embodiments, the 5' region of the compacted oligonucleotide may have a sequence opposite to the 3' region.

[0202] In some embodiments, the 3' region of any compacted oligonucleotide may include three additional bases at the terminal 3' end, which contain 2'-O-methyl RNA bases (e.g., designated as mUmUmU), or the terminal 3' end may lack the additional 2'-O-methyl RNA bases.

[0203] In some embodiments, the compacted oligonucleotide includes one or more modified bases or bonds at its 5' or 3' end to confer certain functions. In some embodiments, the compacted oligonucleotide includes at least one phosphate thioester bond at its 5' and / or 3' end to confer exonuclease resistance. In some embodiments, at least one nucleotide at or near the 3' end includes a 2' fluorobase conferring exonuclease resistance. In some embodiments, the 3' end of the compacted oligonucleotide includes at least one 2'-O-methyl RNA base that blocks polymerase-catalyzed elongation. For example, the 3' end of the compacted oligonucleotide includes three bases, said three bases being 2'-O-methyl RNA bases (e.g., designated mUmUmU). In some embodiments, the compacted oligonucleotide includes a 3' reverse dT at its 3' end that blocks polymerase-catalyzed elongation. In some embodiments, the compacted oligonucleotide includes 3' phosphorylation that blocks polymerase-catalyzed elongation. In some embodiments, the internal region of the compacted oligonucleotide includes at least one locked nucleic acid (LNA), which increases the thermal stability of the duplex formed by hybridizing the compacted oligonucleotide with an amplicon. In some embodiments, the compacted oligonucleotide includes a phosphorylated 5' end (e.g., using a polynucleotide kinase).

[0204] In some embodiments, the compacted oligonucleotide includes three additional bases at the 3' end, which comprises a 2'-O-methyl RNA base (e.g., designated mUmUmU), or the 3' end lacks an additional 2'-O-methyl RNA base.

[0205] In some embodiments, the compacted oligonucleotide may include at least one region having consecutive guanines. For example, the compacted oligonucleotide may include at least one region having 2, 3, 4, 5, 6 or more consecutive guanines. In some embodiments, the compacted oligonucleotide comprises four consecutive guanines capable of forming a guanine tetrad structure (see [link to previous embodiment]). Figure 18 The tetrad structure of guanine can be stabilized via Hoogsteen hydrogen bonding. The tetrad structure of guanine can also be stabilized by a central cation (including potassium, sodium, lithium, rubidium, or cesium).

[0206] At least one compacted oligonucleotide can form a guanine tetrad ( Figure 12 ) and hybridize with the universal binding sequence in the amplicon, which can lead to the amplicon folding to form an intramolecular G-quadruplex structure ( Figure 13 The amplicon can self-collapse to form compacted nanospheres. The formation of guanine tetrads and G-tetramers within the nanospheres increases their stability, allowing them to maintain their compacted size and shape, thus withstanding variations in pH, temperature, and / or repeated reagent flows during sequencing within biological samples.

[0207] In some embodiments, the plurality of compacted oligonucleotides in the rolling circle amplification reaction have the same sequence. Alternatively, the plurality of compacted oligonucleotides in the rolling circle amplification reaction comprises a mixture of two or more different populations of compacted oligonucleotides with different sequences.

[0208] In some embodiments, the immobilized amplicon can self-collapse into compacted nucleic acid nanospheres. The nanospheres can be imaged and FWHM measurements can be obtained to determine the shape / size of the nanospheres.

[0209] In some embodiments, including a compacting oligonucleotide in the rolling circle amplification reaction can promote the collapse of tandem aggregates into DNA nanospheres. Using compacting oligonucleotides for RCA helps maintain the compacted size and shape of the DNA nanospheres during multiple sequencing cycles, which can improve the FWHM (half-maximum full width) of the dot image of the DNA nanospheres within the biological sample. In some embodiments, the DNA nanospheres do not disperse during multiple sequencing cycles. In some embodiments, the dot image of the DNA nanospheres is not magnified during multiple sequencing cycles. In some embodiments, the dot image of the DNA nanospheres remains discrete dots during multiple sequencing cycles. The dot image can be represented as Gaussian dots, and the size can be measured in FWHM. Smaller dot sizes, as indicated by a smaller FWHM, are generally associated with an improved image of the dots. In some embodiments, the FWHM of the nanosphere dots can be about 10 μm or smaller.

[0210] vii. Reaction conditions

[0211] This document discloses methods for in situ detection of one or more targets (e.g., nucleic acids or peptides) under certain reaction conditions. In some cases, one or more nucleic acid molecules (e.g., probes) can be introduced into a biological sample containing one or more targets, provided that conditions are sufficient to conjugate a binding sequence of a nucleic acid molecule (e.g., an RNA-binding sequence, a DNA-binding sequence, or a target-binding sequence) to one or more targets (e.g., RNA or DNA). In some cases, one or more nucleic acid molecules (e.g., probes or index-binding molecules) can be introduced into a biological sample containing one or more targets, provided that conditions are sufficient to conjugate a binding sequence of a nucleic acid molecule (e.g., an oligonucleotide-binding probe or an index-binding sequence) to a target (e.g., a peptide or RNA) to one or more probes or nucleic acid molecules (e.g., one or more peptide-binding probes or one or more target-binding sequences). In some cases, one or more primer nucleic acid sequences can be introduced into a biological sample containing one or more targets, provided that conditions are sufficient to conjugate one or more primer nucleic acid sequences to one or more index sequences or their inverse complementary sequences. Conditions sufficient to couple 1) the binding sequence described herein with one or more targets, 2) nucleic acid molecules with peptide-binding probes or target-binding sequences, and / or 3) one or more primer nucleic acid sequences with one or more index sequences or their reverse complementary sequences may include reagents, temperature, incubation time, concentration, or combinations thereof.

[0212] In some embodiments, conditions sufficient to couple 1) the binding sequence described herein with one or more targets, 2) a nucleic acid molecule with a peptide-binding probe or target-binding sequence, and / or 3) one or more primer nucleic acid sequences with one or more index sequences or their reverse complementary sequences may include reagents. In some embodiments, the reagents may comprise salts. In some embodiments, the salts may include NaCl, MgCl2, CaCl2, etc. 2、 KCl, Na₂SO₄, or combinations thereof. In some further embodiments, the coupling conditions include the presence of strontium ions, magnesium ions, and / or calcium ions. In some embodiments, the reagent may comprise a buffer solution. In some embodiments, the buffer solution may comprise phosphate-buffered saline, Tris, ethylenediaminetetraacetic acid, or combinations thereof.

[0213] In some embodiments, the reagent may comprise a solvent. In some embodiments, the solvent may be a polar solvent. In some embodiments, the solvent may be a nonpolar solvent. In some embodiments, the solvent may be an aprotic solvent. In some embodiments, the solvent may be a protic solvent.

[0214] In some embodiments, the reagent may comprise a liquid-dissociating reagent. In some embodiments, the liquid-dissociating reagent may comprise glycine, arginine, histidine hydrochloride, formamide, dimethyl sulfoxide, dimethylformamide, urea, thiourea, guanidine hydrochloride, or combinations thereof.

[0215] In some embodiments, conditions sufficient to couple 1) the binding sequence described herein with one or more targets, 2) a nucleic acid molecule with a peptide-binding probe or target-binding sequence, and / or 3) one or more primer nucleic acid sequences with one or more index sequences or their reverse complementary sequences may include temperature. Temperature may be an incubation temperature maintained for a period sufficient to couple the target binding sequence with the target nucleic acid. Temperatures can be approximately 10 to 74°C, approximately ℃, approximately 12 to 72°C, approximately 14 to 70°C, approximately 16 to 68°C, approximately 18 to 66°C, approximately 20 to 64°C, approximately 22 to 62°C, approximately 24 to 60°C, approximately 26 to 58°C, approximately 28 to 56°C, approximately 30 to 54°C, approximately 32 to 52°C, approximately 34 to 50°C, approximately 36 to 48°C, approximately 38 to 46°C, approximately 40 to 44°C, at least approximately 10°C, at least approximately 12°C, at least approximately 14°C, at least approximately 16°C, at least approximately 18°C, at least approximately 20°C, at least approximately 22°C At least about 24°C, at least about 26°C, at least about 28°C, at least about 30°C, at least about 32°C, at least about 34°C, at least about 36°C, at least about 38°C, at least about 40°C, at least about 42°C, at least about 44°C, at least about 46°C, at least about 48°C, at least about 50°C, at least about 52°C, at least about 54°C, at least about 56°C, at least about 58°C, at least about 60°C, at least about 62°C, at least about 64°C, at least about 66°C, at least about 68°C, at least about 70°C, at least about 72°C, or at least about 74°C.

[0216] In some embodiments, conditions sufficient to conjugate 1) the binding sequence described herein with one or more targets, 2) a nucleic acid molecule with a peptide-binding probe or target-binding sequence, and / or 3) one or more primer nucleic acid sequences with one or more index sequences or their reverse complementary sequences may include incubation time. Incubation time may be sufficient to conjugate the target binding sequence with the target nucleic acid. The incubation time can be at least about 10 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 55 minutes, at least about 1 hour, at least about 1.5 hours, at least about 2 hours, at least about 2.5 hours, at least about 3 hours, at least about 3.5 hours, at least about 4 hours, at least about 4.5 hours, at least about 5 hours, at least about 5.5 hours, at least about 6 hours, at least about 6.5 hours, at least about 7 hours, at least about 7.5 hours, at least about 8 hours, at least about 8.5 hours, at least about 9 hours, at least about 9.5 hours, at least about 10 hours, at least about 10.5 hours, at least about 11 hours, at least about 11.5 hours, or at least about 12 hours.

[0217] In some embodiments, conditions sufficient to couple 1) the binding sequence described herein with one or more targets, 2) nucleic acid molecules with peptide-binding probes or target-binding sequences, and / or 3) one or more primer nucleic acid sequences with one or more index sequences or their reverse complementary sequences may include concentration. Concentration may be related to the concentration of one or more nucleic acid molecules. In some embodiments, the concentration may be about 0.1 to 10000 nM, about 0.2 to 5000 nM, about 0.3 to 1000 nM, about 0.4 to 900 nM, about 0.5 to 800 nM, about 0.6 to 700 nM, about 0.7 to 600 nM, about 0.8 to 500 nM, about 0.9 to 400 nM, about 1 to 300 nM, about 2 to 250 nM, about 3 to 200 nM, about 4 to 150 nM, about 5 to 100 nM, about 6 to 50 nM, about 7 to 25 nM, about 8 to 20 nM, about 9 to 15 nM, at least about 0.1 nM, at least about 0.2 nM, at least about 0.3 nM, at least about 0.4 nM, at least about 0.5 nM, at least about 0.6 nM, at least about 0.7 nM, at least about 0.8 nM. nM, at least about 0.9 nM, at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 50 nM, at least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 250 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1000 nM, at least about 5000 nM, or at least about 10000 nM.In some embodiments, the concentration of one or more nucleic acid molecules may be up to about 0.1 nM, up to about 0.2 nM, up to about 0.3 nM, up to about 0.4 nM, up to about 0.5 nM, up to about 0.6 nM, up to about 0.7 nM, up to about 0.8 nM, up to about 0.9 nM, up to about 1 nM, up to about 2 nM, up to about 3 nM, up to about 4 nM, up to about 5 nM, up to about 6 nM, up to about 7 nM, up to about 8 nM, up to about 9 nM, up to about 10 nM, up to about 15 nM, up to about 20 nM, up to about 25 nM, up to about 50 nM, up to about 100 nM, up to about 150 nM, up to about 200 nM, up to about 250 nM, up to about 300 nM, up to about 400 nM. nM, up to about 500 nM, up to about 600 nM, up to about 700 nM, up to about 800 nM, up to about 900 nM, up to about 1000 nM, up to about 5000 nM, or up to about 10000 nM.

[0218] viii. Primer nucleic acid sequence

[0219] This document discloses primer nucleic acid sequences that can be used in the methods described herein for in situ detection of targets (e.g., RNA). The methods disclosed herein may include one or more primer nucleic acid sequences. One or more primer nucleic acid sequences may be configured to bind to an index sequence or its inverse complementary sequence. One or more primer nucleic acid sequences may comprise DNA, RNA, or a combination thereof. Primer nucleic acid sequences may be configured to be coupled to an index sequence. In some embodiments, primer nucleic acid sequences may hybridize with an index sequence. In some embodiments, primer nucleic acid sequences may comprise at least one binding region that recognizes and binds to at least a portion of the index sequence. In some embodiments, primer nucleic acid sequences may comprise more than one binding region that recognizes and binds to an index sequence. One or more primer nucleic acid sequences may form a double-stranded region upon binding to an index sequence or its inverse complementary sequence, as described herein. One or more primer nucleic acid sequences may bind directly to the nucleic acid molecules described herein. For example, a single-stranded oligonucleotide may comprise a sequence complementary to a portion of an index sequence and form a double-stranded nucleic acid region after hybridization with the index sequence. In some embodiments, one or more primer nucleic acid sequences may bind to the index-binding sequence described herein. In some embodiments, one or more primer nucleic acid sequences may bind to the amplification product generated by the amplification reaction described herein. In some embodiments, the primer nucleic acid sequence binds to at least a portion of the index sequence. In some embodiments, the primer nucleic acid sequence binds to at least a portion of the reverse complementary sequence of the index sequence. In some embodiments, one or more primer nucleic acid sequences bind directly to the target nucleic acid described herein. In some embodiments, the primer nucleic acid sequence is a sequencing primer. In some embodiments, the primer nucleic acid sequence is an amplification primer.

[0220] One or more primer nucleic acid sequences may comprise a nucleic acid molecule of a given length. Each of the one or more primer nucleic acid sequences may be of the same length or different lengths. In some embodiments, at least two of the one or more primer nucleic acid sequences have the same length. In some embodiments, at least two of the one or more primer nucleic acid sequences have different lengths. One or more primer nucleic acid sequences may have multiple lengths.In some embodiments, one or more primer nucleic acid sequences may have the following lengths: about 1 to 100 nucleotides, about 2 to 98 nucleotides, about 3 to 96 nucleotides, about 4 to 94 nucleotides, about 5 to 92 nucleotides, about 6 to 90 nucleotides, about 7 to 88 nucleotides, about 8 to 86 nucleotides, about 9 to 84 nucleotides, about 10 to 82 nucleotides, about 11 to 80 nucleotides, about 12 to 78 nucleotides, about 13 to 76 nucleotides, about 14 to 74 nucleotides, about 15 to 72 nucleotides, about 16 to 70 nucleotides, about 17 to 68 nucleotides, about 18 to 66 nucleotides, about 19 to 64 nucleotides. 1 nucleotide, about 20 to 62 nucleotides, about 22 to 60 nucleotides, about 24 to 58 nucleotides, about 26 to 56 nucleotides, about 28 to 54 nucleotides, about 30 to 52 nucleotides, about 32 to 50 nucleotides, about 34 to 48 nucleotides, about 36 to 46 nucleotides, about 38 to 44 nucleotides, about 40 to 42 nucleotides, at least about 1 nucleotide, at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides Nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, at least about 24 nucleotides, at least about 26 nucleotides, at least about 28 nucleotides, at least about 30 nucleotides, at least about 32 nucleotides, at least about 34 nucleotides, at least about 36 nucleotides, at least about 38 nucleotides, at least about 40 nucleotides, at least about 42 nucleotides, at least about 44 nucleotides, at least about 46 nucleotides, at least about 48 nucleotides, at least about 50 nucleotides, at least about 52 nucleotides. At least about 54 nucleotides, at least about 56 nucleotides, at least about 58 nucleotides, at least about 60 nucleotides, at least about 62 nucleotides, at least about 64 nucleotides, at least about 66 nucleotides, at least about 68 nucleotides, at least about 70 nucleotides, at least about 72 nucleotides, at least about 74 nucleotides, at least about 76 nucleotides, at least about 78 nucleotides, at least about 80 nucleotides, at least about 82 nucleotides, at least about 84 nucleotides, at least about 86 nucleotides, at least about 88 nucleotides, at least about 90 nucleotides, at least about 92 nucleotides, at least about 94 nucleotides, at least about 96 nucleotides, at least about 98 nucleotides, or at least about 100 nucleotides in length.

[0221] In some embodiments, one or more primer nucleic acid sequences inhibit the incorporation of another nucleotide into the primer nucleic acid sequence. In some embodiments, one or more primer nucleic acid sequences do not include a 3' hydroxyl group. In some embodiments, one or more primer nucleic acid sequences may include a 3' hydroxyl group substituted by a chain termination portion. In some embodiments, one or more primer nucleic acid sequences include a blocking group. In some embodiments, the blocking group may include an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group, a silyl group, or a combination thereof.

[0222] In some embodiments, a primer nucleic acid sequence is coupled to an index or an inverse complementary sequence of the index to generate a primer-binding index sequence. The primer-binding index sequence may comprise a single-stranded nucleic acid, a double-stranded nucleic acid, or a combination thereof. In some embodiments, the primer-binding index sequence is formed between a nucleic acid molecule and a primer nucleic acid sequence, as described herein. In some embodiments, the primer-binding index sequence is formed between a target molecule and a primer nucleic acid sequence, as described herein. In some cases, the primer-binding index sequence is formed between an amplicon and a primer nucleic acid sequence, as described herein.

[0223] In some embodiments, the primer nucleic acid sequence may bind to at least a portion of one or more amplicons described herein. For example, the primer nucleic acid sequence may bind to a portion of one or more index sequences within one or more amplicons to generate a primer-binding index sequence. In some cases, the primer nucleic acid sequence may bind to a portion of one or more index sequences adjacent to one or more amplicons to form a primer-binding index sequence. For example, a portion of one or more applicants adjacent to one or more index sequences may be upstream or downstream of one or more index sequences, and when the primer nucleic acid sequence binds to a portion of one or more amplicons adjacent to one or more tag sequences, it can form a double-stranded nucleic acid sequence of one or more index sequences adjacent to one or more amplicons.

[0224] In some embodiments, the primer nucleic acid sequence can bind directly to the target to form a primer-binding index sequence. Figure 1C Non-limiting steps are shown for annealing primer 103 to target 102 to form primer-binding index sequence 104. Figure 1BOnly one primer used in the annealing step is shown, but more than one primer can be used depending on the type of target nucleic acid. In some cases, the adaptor used to attach the target to the surface may have the same sequence as the primer used to prepare the target nucleic acid for primer binding. Primers may include forward amplification primers, reverse amplification primers, sequencing primers and / or molecular barcode sequences or any combination thereof. In some cases, a single primer sequence can be used in the hybridization step. In some cases, multiple different primer sequences can be used in the hybridization step. Figure 1D As shown, the primer-binding sequence 104 can be combined with a nucleotide conjugate (e.g., a detectable nucleotide conjugate) and polymerase 106 to form a binding complex.

[0225] The methods described herein relate to the detection of a target using a detectable nucleotide conjugate. In some embodiments, the target is ribonucleic acid (RNA). In some embodiments, a nucleotide conjugate (e.g., a detectable nucleotide conjugate) is introduced into a biological sample containing the target under conditions sufficient to form a binding complex.

[0226] In some embodiments, the nucleotide conjugate (e.g., a detectable nucleotide conjugate) comprises a detectable tag. In some embodiments, the detectable tag may be covalently conjugated to the nucleotide conjugate (e.g., the detectable nucleotide conjugate). In some embodiments, the detectable tag may be non-covalently conjugated to the nucleotide conjugate (e.g., the detectable nucleotide conjugate). In some embodiments, the detectable tag may comprise a quantum dot. In some embodiments, the detectable tag may comprise a fluorophore. Examples of fluorescent components include, but are not limited to: fluorescein and fluorescein derivatives, such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthalenefluorescein, isothiocyanatefluorescein, NHS-fluorescein, iodoacetamidefluorescein, maleimidefluorescein, SAMSA-fluorescein, thioaminoureafluorescein, and carbazide methylthioacetylaminofluorescein; rhodamine and rhodamine derivatives, such as TRITC, TMR, rhodamine sulfonyl chloride, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, rhodamine B sulfonyl chloride, rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, and Texas Red hydrazide; coumarin and coumarin derivatives, such as AMCA, AMCA-NHS, AMCA-sulfonyl-NHS, AMCA-HPDP, DCIA, and AMCE-hydrazide; and BODIPY and its derivatives, such as BODIPY. FLC3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 acylhydrazine, BODIPY493 / 503 C3 acylhydrazine, BODIPY FL C3 acylhydrazine, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503; Cascade Blue and its derivatives, such as Cascade Blue acetylazide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue acylhydrazine; Lucifer Yellow and its derivatives, such as Lucifer Yellow iodoacetamide, Lucifer Yellow... CH; anthocyanins and their derivatives, such as indolonyl anthocyanin dyes, benzoindolonyl anthocyanin dyes, pyridiniumyl anthocyanin dyes, thiazolonyl anthocyanin dyes, quinolinelonyl anthocyanin dyes, imidazolonyl anthocyanin dyes, Cy3, Cy5; lanthanide chelates and their derivatives, such as BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates;Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE and its derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilichrome dyes, malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes, and others known in the art, such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oregon) 6th edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd edition, Plenum Press, New York (1999) or Hermanson, Bioconjugate Techniques, 2nd edition, or their derivatives;Or any combination thereof. Anthocyanin dyes can exist in sulfonated or non-sulfonated forms and consist of two pseudoindole, benzoindole, pyridinium, thiazolylium, and / or quinolineonium groups separated by a polymethystylene bridge between two nitrogen atoms. Commercially available anthocyanin fluorophores include, for example, Cy3 (which may include 1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-yl}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium or 1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{ ...2-(3-{ [Oxypyrrolidine-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfonyl-1,3-dihydro-2H-indole-2-yl]prop-1-en-1-yl)-3,3-dimethyl-3H-indole-5-sulfonate], Cy5 (which may include 1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indole-2-yl]prop-1-en-1-yl)-3,3-dimethyl-3H-indole-2-yl]prop-1-en-1-yl)-3,3-dimethyl-3H-indole-5-sulfonate), Cy5 (which may include 1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indole-2-yl) (1-(6-((2,5-dioxopyrrolidone-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-(((2,5-dioxopyrrolidone-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfonylindoline-2-ylide)pent-1,3-diene-1-yl)-3,3-dimethyl-3H-indoline-1-on-5-sulfonate) and Cy7 (which may include 1-(5-carboxypentyl) Cy2 is an oxazole derivative, not a pseudoindole, and the benzo-derived Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule.

[0227] In some embodiments, the detection markers can be FRET pairs, allowing for multiple classifications to be performed in a single excitation and imaging step. As used herein, FRET can include excitation exchange (Forster) transfer or electron exchange (Dexter) transfer.

[0228] In some embodiments, one or more nucleotide conjugates (e.g., detectable nucleotide conjugates) are labeled with fluorophores, and the detection step includes fluorescence imaging; and in particular, the fluorescence imaging includes dual-wavelength excitation / four-wavelength emission fluorescence imaging. In some embodiments, four different nucleotide conjugates (e.g., detectable nucleotide conjugates), each containing a different nucleotide or nucleotide analogue, are used to determine the identity of the terminal nucleotide, wherein the four different nucleotide conjugates (e.g., detectable nucleotide conjugates) are each labeled with their respective fluorophores, and the detection step includes simultaneous excitation at wavelengths sufficient to excite all four fluorophores, and imaging the fluorescence emission at wavelengths sufficient to detect each respective fluorophore. In some embodiments, four different nucleotide conjugates (e.g., detectable nucleotide conjugates), each containing a different nucleotide or nucleotide analogue, are used to determine the identity of the terminal nucleotide, wherein the four different nucleotide conjugates (e.g., detectable nucleotide conjugates) are labeled Cy3, Cy3.5, Cy5, and Cy5.5, respectively, and wherein the detection step includes simultaneous excitation at any two of 532 nm, 568 nm, and 633 nm, and imaging of fluorescence emission at approximately 570 nm, 592 nm, 670 nm, and 702 nm, respectively; and / or wherein fluorescence imaging includes dual-wavelength excitation / dual-wavelength emission fluorescence imaging. In some embodiments, four different nucleotide conjugates (e.g., detectable nucleotide conjugates), each comprising a different nucleotide or nucleotide analogue, are used to identify the terminal nucleotide. One, two, three, or four different nucleotide conjugates (e.g., detectable nucleotide conjugates) are each labeled with a different fluorophore or a set of fluorophores. The detection step includes simultaneous excitation at wavelengths sufficient to excite one, two, three, or four fluorophores or a set of fluorophores, and imaging the fluorescence emission at wavelengths sufficient to detect each respective fluorophore. In some embodiments, three different nucleotide conjugates (e.g., detectable nucleotide conjugates), each comprising a different nucleotide or nucleotide analogue, are used to identify the terminal nucleotide. One, two, or three different nucleotide conjugates (e.g., detectable nucleotide conjugates) are each labeled with a different fluorophore or a set of fluorophores. The detection step includes simultaneous excitation at wavelengths sufficient to excite one, two, or three fluorophores or a set of fluorophores, and imaging the fluorescence emission at wavelengths sufficient to detect each respective fluorophore. The detection of a fourth nucleotide is determined or determinable by reference to a “dark” or unlabeled spot or the location of the target nucleotide.In some embodiments, the binding complex consists of three types of nucleotide conjugates (e.g., detectable nucleotide conjugates), wherein each of the three types of nucleotide conjugates (e.g., detectable nucleotide conjugates) contains a different type of nucleotide. In some embodiments, the detection of the binding complex is performed in the absence of unbound or present polymeric nucleotide conjugates in solution.

[0229] Figure 1DNon-limiting examples of nucleotide conjugates (e.g., detectable nucleotide conjugates) include four nucleotide conjugates (e.g., detectable nucleotide conjugates) 105a, 105b, 105c, and 105d. Each nucleotide conjugate (e.g., detectable nucleotide conjugate) has multiple copies of the nucleotide attached to the particle, and the four nucleotide conjugates (e.g., detectable nucleotide conjugates) respectively cover four types of nucleotides. Nucleotide conjugates (e.g., detectable nucleotide conjugates) having a nucleotide complementary to the next base on the target can form a binding complex with a polymerase and a target nucleic acid. In some cases, the binding complex may include one, two, or three nucleotide conjugates (e.g., detectable nucleotide conjugates). In some embodiments, each different type of nucleotide conjugate (e.g., detectable nucleotide conjugate) may be labeled with a separate tag. In some embodiments, three of the four nucleotide conjugates may be labeled, and the fourth may be unlabeled or conjugated with an undetectable tag. In some embodiments, one, two, three, or four nucleotide conjugates (e.g., detectable nucleotide conjugates) may be labeled. These conjugates may be labeled with the same label or individually with a label corresponding to the identity of the nucleotide they are conjugated with. Three, two, one, or no nucleotide conjugates (e.g., detectable nucleotide conjugates) may be unlabeled or conjugated with an undetectable label. In some embodiments, a four-color detection method may be used to detect polymerase complexes incorporating nucleotide conjugates (e.g., detectable nucleotide conjugates), such that the sample contains conjugates corresponding to all four nucleotides, each with a separate label corresponding to its conjugated nucleotide. In some embodiments, the four nucleotide conjugates (e.g., detectable nucleotide conjugates) may be simultaneously exposed to or in contact with the target; in some other embodiments, the four nucleotide conjugates (e.g., detectable nucleotide conjugates) may be individually or sequentially exposed to or in contact with the target in groups of two or three. In some embodiments, a three-color detection method can be used to detect polymerase complexes incorporating nucleotide conjugates (e.g., detectable nucleotide conjugates) such that the sample contains conjugates corresponding to three of four nucleotides, wherein the three conjugates have individual labels corresponding to their conjugated nucleotides, and one conjugate is unlabeled or conjugated to an undetectable label. In some embodiments, only three types of conjugates are provided such that the sample contains conjugates corresponding to three of four nucleotides, wherein the three conjugates have individual labels corresponding to their conjugated nucleotides, and one conjugate is absent. In some embodiments, the identity of the nucleotide corresponding to the unlabeled or absent nucleotide conjugate can be determined relative to the position of a “dark” spot and / or the position of a known target that does not show a fluorescent signal. In some embodiments, the detection of the binding complex is performed in the absence of unbound or present polymeric nucleotide conjugates in solution.

[0230] In some embodiments, a nucleotide conjugate (e.g., a detectable nucleotide conjugate) comprises one or more nucleotides or nucleotide analogs. In some embodiments, a nucleotide conjugate (e.g., a detectable nucleotide conjugate) may comprise a single type of nucleotide. In some embodiments, a nucleotide conjugate (e.g., a detectable nucleotide conjugate) comprises more than one type of nucleotide. In some embodiments, the nucleotide comprises ATP, ADP, AMP, dATP, dADP, dAMP, TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, or dGMP.

[0231] In some embodiments, this disclosure provides a reagent comprising one or more nucleotide conjugates (e.g., detectable nucleotide conjugates) disclosed herein and a buffer. For example, in some embodiments, this disclosure provides a reagent comprising one, two, three, four or more nucleotide conjugates (e.g., detectable nucleotide conjugates), wherein each nucleotide conjugate (e.g., detectable nucleotide conjugate) comprises a single type of nucleotide. In some embodiments, the reagents of this disclosure comprise one, two, three, four or more nucleotide conjugates (e.g., detectable nucleotide conjugates), wherein each nucleotide conjugate (e.g., detectable nucleotide conjugate) comprises a single type of nucleotide or nucleotide analogue, and wherein the nucleotide or nucleotide moiety may correspond to one or more of the following groups: ATP, ADP, AMP, dATP, dADP and dAMP; one or more of the following groups: TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP and dUMP; one or more of the following groups: CTP, CDP, CMP, dCTP, dCDP and dCMP; and one or more of the following groups: GTP, GDP, GMP, dGTP, dGDP and dGMP. In some other instances or further instances, this disclosure provides a reagent comprising or further comprising one, two, three, four or more nucleotide conjugates (e.g., detectable nucleotide conjugates), wherein each nucleotide conjugate (e.g., detectable nucleotide conjugate) comprises a single type of nucleotide or nucleotide moiety, and wherein said nucleotide or nucleotide moiety may correspond to one or more of the group consisting of ATP, ADP, AMP, dATP, dADP, dAMP, TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP and dGMP.

[0232] In some embodiments, nucleotide conjugates (e.g., detectable nucleotide conjugates) are included in a mixture of multiple nucleotide conjugates (e.g., detectable nucleotide conjugates), each of which contains nucleotide moieties of a different type from each other. The types of nucleotides may be ATP, ADP, AMP, dATP, dADP, dAMP, TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, dGMP, or combinations thereof.

[0233] In some embodiments, the nucleotide conjugate (e.g., a detectable nucleotide conjugate) comprises a polymer having multiple branches. In some embodiments, the multiple branches comprise copies of nucleotides coupled thereto. In some embodiments, the polymer has a star, comb, cross-linked, bottle-brush, or dendritic configuration. In some embodiments, the nucleotide conjugate (e.g., a detectable nucleotide conjugate) comprises one or more binding groups comprising avidin, biotin, affinity tags, or any combination thereof. In some embodiments, the binding group is positioned between the polymer branches and the nucleotides.

[0234] In some embodiments, the nucleotide conjugate (e.g., a detectable nucleotide conjugate) may comprise a common core. The common core may comprise multiple copies of the same nucleotide covalently or non-covalently bound to the common core. Examples of a common core may include polymer particles; metal particles; quantum dots; liposomes; emulsion particles, or any other particles (e.g., nanoparticles, microparticles, etc.). In some embodiments, the polymer particles are or comprise branched polymers; dendrites; cross-linked polymer particles, such as agarose, polyacrylamide, acrylates, methacrylates, cyanoacrylates, methyl methacrylate particles; glass particles; ceramic particles; or protein particles. In some embodiments, the common core is a branched polymer. In some embodiments, the common core is or comprises a protein. In some embodiments, the protein is streptavidin, avidin, or a combination thereof. In some embodiments, the nucleotide conjugate (e.g., a detectable nucleotide conjugate) comprises multiple polymer branches radiating from the common core, some of which are attached to nucleotides and / or some of which are attached to oligonucleotides.

[0235] In some embodiments, this disclosure provides a nucleotide conjugate (e.g., a detectable nucleotide conjugate) comprising a branched polymer having two or more branches and two or more copies of a nucleotide, wherein the nucleotide is attached to a first plurality of said branches or arms, and optionally, one or more interacting portions are attached to a second plurality of said branches or arms. In some embodiments, the nucleotide conjugate (e.g., a detectable nucleotide conjugate) may further comprise one or more tags on the polymer. In some embodiments, this disclosure provides the nucleotide conjugate (e.g., a detectable nucleotide conjugate) wherein the nucleoside has a surface density of at least four nucleotides per polymer. In some embodiments, this disclosure provides the nucleotide conjugate (e.g., a detectable nucleotide conjugate) comprising or incorporating a nucleotide or nucleotide analogue modified to prevent incorporation into an extended nucleic acid chain during a polymerase reaction. In some embodiments, the nucleotide conjugate (e.g., a detectable nucleotide conjugate) may comprise or incorporate a nucleotide or nucleotide analogue reversibly modified to prevent incorporation into an extended nucleic acid chain during a polymerase reaction. In some embodiments, this disclosure provides the nucleotide conjugate (e.g., a detectable nucleotide conjugate) wherein one or more tags comprise a fluorescent label, a FRET donor, and / or a FRET acceptor. In some embodiments, the nucleotide conjugate (e.g., a detectable nucleotide conjugate) may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more branches or arms, or 2, 4, 8, 16, 32, 64, or more branches or arms. In some embodiments, the branches or arms may radiate from a central portion. In some embodiments, the nucleotide conjugate (e.g., a detectable nucleotide conjugate) may comprise one or more interacting portions, which may comprise avidin or streptavidin; a biotin moiety; an affinity tag; an enzyme, antibody, microantibody, receptor, or other protein; a non-protein tag; a metal affinity tag; or any combination thereof. In some embodiments, this disclosure provides the nucleotide conjugates (e.g., detectable nucleotide conjugates) wherein the polymer includes polyethylene glycol, polypropylene glycol, polyvinyl acetate, polylactic acid, or polyglycolic acid. In some embodiments, this disclosure provides the nucleotide conjugates (e.g., detectable nucleotide conjugates) wherein a nucleotide or nucleotide analog is attached to a branch or arm via a linker; and particularly wherein the linker comprises PEG, and wherein the PEG portion has an average molecular weight of about 1K, about 2K, about 3K, about 4K, about 5K, about 10K, about 15K, about 20K, about 50K, about 100K, about 150K, or about 200K, or greater than about 200K.In some embodiments, this disclosure provides the nucleotide conjugate (e.g., a detectable nucleotide conjugate) wherein the linker comprises PEG, and wherein the PEG portion has an average molecular weight between about 5K and about 20K. In some embodiments, this disclosure provides the nucleotide conjugate (e.g., a detectable nucleotide conjugate) wherein at least one nucleotide or nucleotide analog comprises deoxyribonucleotide, ribonucleotide, deoxyribonucleoside, or ribonucleoside; and / or wherein the nucleotide or nucleotide analog is conjugated to the linker via the 5' end of the nucleotide or nucleotide analog. In some embodiments, this disclosure provides the nucleotide conjugate (e.g., a detectable nucleotide conjugate) wherein one of the nucleotides or nucleotide analogs comprises deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine, adenosine, guanosine, 5-methyluridine, and / or cytidine; and wherein the length of the linker is between 1 and 1,000 nm.

[0236] In any of the methods described herein, any sequencing method described herein may employ at least one nucleotide. A nucleotide comprises a base, a sugar, and at least one phosphate ester group. In some embodiments, at least one nucleotide of a plurality of nucleotides comprises an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and one or more phosphate ester groups (e.g., 1 to 10 phosphate ester groups). The plurality of nucleotides may comprise at least one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. The plurality of nucleotides may comprise a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, at least one nucleotide of a plurality of nucleotides is not a nucleotide analog. In some embodiments, at least one nucleotide of a plurality of nucleotides comprises a nucleotide analog.

[0237] In some embodiments, in any of the sequencing methods described herein, at least one nucleotide of the plurality of nucleotides comprises a chain of one, two, or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, at least one nucleotide of the plurality of nucleotides is an analogue having a phosphorus chain, wherein the phosphorus atoms are linked together by an intermediate O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atom in the chain comprises a substituted side group (containing O, S, or BH3). In some embodiments, the chain comprises a phosphate ester group substituted with an analogue, including phosphoramide, thiophosphate, dithiophosphate, and O-methylphosphoramide groups.

[0238] In some embodiments, in any of the sequencing methods described herein, at least one nucleotide of a plurality of nucleotides comprises a terminator nucleotide analog having a chain-terminating portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain-terminating portion may inhibit polymerase-catalyzed incorporation of subsequent nucleotide portions or free nucleotides into the nascent chain during primer extension reactions. In some embodiments, the chain-terminating portion is attached to the 3' sugar hydroxyl position, wherein the sugar comprises a ribose or deoxyribose portion. In some embodiments, the chain-terminating portion may remove / cleave from the 3' sugar hydroxyl position to produce a nucleotide having a 3'OH sugar group, which may be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain-terminating portion comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the chain termination portion may be cleaved / removed from the nucleotide, for example by reacting the chain termination portion with a chemical agent, pH change, light, or heat. In some embodiments, the alkyl, alkenyl, alkynyl, and allyl chain termination portions may be cleaved with tetrakis(triphenylphosphine)palladium(O) (Pd(PPh3)4), with piperidine, or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the aryl and benzyl chain termination portions may be cleaved with H2 Pd / C. In some embodiments, the amine, amide, ketone, isocyanate, phosphate, thio, and disulfide chain termination portions may be cleaved with phosphine or with a thiol group (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the carbonate chain termination portion may be cleaved with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). In some embodiments, the urea and silyl chain termination portions may be cleaved with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride.

[0239] In some embodiments, in any of the sequencing methods described herein, at least one nucleotide of a plurality of nucleotides comprises a terminator nucleotide analog having a chain-terminating portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain-terminating portion comprises an azide, an azide group, or an azidemethyl group. In some embodiments, the chain-terminating portion comprises a 3'-O-azido group or a 3'-O-azidomethyl group. In some embodiments, the chain-terminating portion azide, azide group, and an azidemethyl group can be cleaved / removed with a phosphine compound. In some embodiments, the phosphine compound comprises a derived trialkylphosphine portion or a derived triarylphosphine portion. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), bis(sulfotriphenyl)phosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[0240] In some embodiments, in any of the sequencing methods described herein, the nucleotide includes a chain termination portion selected from the group consisting of: 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-mercapto, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tert-butyl, 3'-fluorenylmethoxycarbonyl, 3'-tert-butoxycarbonyl, 3'-O-alkylhydroxyamino, 3'-thiophosphate, and 3'-O-benzyl, or derivatives thereof.

[0241] In some embodiments, in any of the methods for sequencing described herein, the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter gene portion. The detectable reporter gene portion comprises a fluorophore. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to the nucleotide base with a cleavable / removable adapter. In some embodiments, at least one nucleotide of the plurality of nucleotides is not labeled with a detectable reporter gene portion. In some embodiments, a specific detectable reporter gene portion (e.g., a fluorophore) attached to a nucleotide may correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow detection and identification of the nucleotide base.

[0242] In some embodiments, in any of the methods described herein for sequencing nucleic acid molecules, the cleavable linker on the nucleotide base comprises a cleavable moiety comprising an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the cleavable linker on the base can be cleaved / removed from the base by reacting the cleavable moiety with a chemical agent, a pH change, light, or heat. In some embodiments, the cleavable alkyl, alkenyl, alkynyl, and allyl moiety can be cleaved with tetrakis(triphenylphosphine)palladium(O) (Pd(PPh3)4), with piperidine, or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the cleavable aryl and benzyl moiety can be cleaved with H2 Pd / C. In some embodiments, cleavable amines, amides, ketones, isocyanates, phosphates, thiocyanates, and disulfides can be cleaved using phosphine or with a thiol group, including β-mercaptoethanol or dithiothreitol (DTT). In some embodiments, cleavable carbonates can be cleaved using potassium carbonate (K₂CO₃) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, cleavable ureas and silyl groups can be cleaved using tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0243] In some embodiments, in any of the sequencing methods described herein, the cleavable linker on the nucleotide base comprises a cleavable portion comprising an azide, an azide group, or an azide methyl group. In some embodiments, the cleavable portion of the azide, azide group, and azide methyl group can be cleaved / removed using a phosphine compound. In some embodiments, the phosphine compound comprises a derived trialkylphosphine moiety or a derived triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), bis(sulfotriphenyl)phosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[0244] In some embodiments, in any of the sequencing methods described herein, the chain termination portion (e.g., at the 2' and / or 3' positions of the sugar) and the cleavable linker on the nucleotide base have the same or different cleavable portions. In some embodiments, the chain termination portion linked to the base (e.g., at the 2' and / or 3' positions of the sugar) and the detectable reporter gene portion can be chemically cleaved / removed with the same chemical agent. In some embodiments, the chain termination portion linked to the base (e.g., at the 2' and / or 3' positions of the sugar) and the detectable reporter gene portion can be chemically cleaved / removed with different chemical agents.

[0245] Nucleotides can be linked to a common nucleus via linkers, and nucleotides can be attached to one end or site of the polymer. Nucleotides can be conjugated to the common nucleus via their 5' ends. In some common nucleus-nucleotide conjugates, one nucleotide is attached to one end or site of the polymer. In some common nucleus-nucleotide conjugates, multiple nucleotides are attached to one end or site of the polymer. The conjugated nucleotides can be spatially accessible to one or more proteins, one or more enzymes, and nucleotide-binding moieties. In some embodiments, the nucleotides can be provided separately from the nucleotide-binding moieties (such as polymerases). In some embodiments, the linker does not contain luminescent or light-absorbing groups.

[0246] The common nucleus may also have a binding portion. In some embodiments, the common nucleus may self-associate without using separate interacting portions. In some embodiments, the common nucleus may self-associate due to buffering or salting conditions, such as in the case of calcium-mediated interactions of hydroxyapatite particles, lipid or polymer-mediated interactions of micelles or liposomes, or salt-mediated aggregation of metal (such as iron or gold) nanoparticles.

[0247] Nucleotide conjugates (e.g., detectable nucleotide conjugates) may have one or more tags. Examples of tags include, but are not limited to: fluorophores, spin tags, metals or metal ions, colorimetric tags, nanoparticles, PET tags, radioactive tags, or other such tags that can make the nucleotide conjugates (e.g., detectable nucleotide conjugates) detectable by methods known in the art for detecting macromolecules or molecular interactions. Tags may be attached to nucleotides (e.g., by attachment to the 5' phosphate moiety of the nucleotide), to the common core itself (e.g., to a PEG subunit), to the ends of the polymer, to the central portion, or to any other location within the polymer-nucleotide conjugate that a person skilled in the art would recognize as sufficient to make the detectable nucleotide conjugates (such as the common core) detectable by methods known in the art or described elsewhere herein. In some embodiments, one or more tags are provided to correspond to or distinguish a particular detectable nucleotide conjugate.

[0248] An example of a nucleotide conjugate (e.g., a detectable nucleotide conjugate) is a polymer-nucleotide conjugate. Some non-limiting examples of nucleotide conjugates (e.g., detectable nucleotide conjugates) are shown in Figures 5A through 5C. For example, Figure 5A shows nucleotide conjugates (e.g., detectable nucleotide conjugates) having various configurations; Figure 5B shows nucleotide conjugates (e.g., detectable nucleotide conjugates) having polymer branches radiating from the center; and Figure 5C shows nucleotide conjugates (e.g., detectable nucleotide conjugates) having a binding moiety (such as biotin).

[0249] Examples of branched polymers include polyethylene glycol (PEG), polypropylene glycol, polyvinyl alcohol, polylactic acid, polyglycolic acid, polyglycine, polyvinyl acetate, dextran, or other such polymers, or copolymers incorporating any two or more of the foregoing or other polymers known in the art. In some embodiments, the polymer is PEG. In another embodiment, the polymer may have PEG branches.

[0250] Suitable polymers may be characterized by the incorporation of repeating units of functional groups suitable for derivatization (such as amine, hydroxyl, carbonyl, or allyl groups). The polymer may also have one or more pre-derived substituents, such that one or more specific subunits will be incorporated into the derivatization site or branching site, regardless of whether other subunits are incorporated into the same site, substituent, or portion. Pre-derived substituents may include, or may further include, for example: nucleotides, nucleosides, nucleotide analogs, labels (such as fluorescent labels, radioactive labels, or spin labels), interacting moieties, additional polymeric moieties, or any combination thereof.

[0251] In some embodiments, the nucleotide conjugate (e.g., a detectable nucleotide conjugate) may comprise a polymer. The polymer may have multiple branches. The branched polymer may have various configurations, including but not limited to star-shaped (“starburst”), aggregated star-shaped (“out-of-order”), bottle brush, or dendritic forms. The branched polymer may radiate from a central attachment point or central portion, or may incorporate multiple branches, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more branches. In some embodiments, each subunit of the polymer may optionally constitute an independent branch.

[0252] The length and size of the branches can vary based on the type of polymer. In some branched polymers, the branches may have lengths between 1 nm and 1,000 nm, 1 nm and 100 nm, 1 nm and 200 nm, 1 nm and 300 nm, 1 nm and 400 nm, 1 nm and 500 nm, 1 nm and 600 nm, 1 nm and 700 nm, 1 nm and 800 nm, or 1 nm and 900 nm, or greater, or lengths falling within or between any of the values ​​disclosed herein. In some branched polymers, the branches may have dimensions corresponding to any value within the range defined by 1K, 2K, 3K, 4K, 5K, 10K, 15K, 20K, 30K, 50K, 80K, 100K, or any value within the range defined by any two of the foregoing. The apparent molecular weight of a polymer can be calculated from the known molecular weights of a representative number of subunits (e.g., determined by size exclusion chromatography, mass spectrometry, or any other method known in the art). The polymer may have multiple branches. The number of branches in a polymer can be 2, 3, 4, 5, 6, 7, 8, 12, 16, 24, 32, 64, 128, or more, or a number falling within the range defined by any two of these values.

[0253] For detectable nucleotide conjugates, branched polymers with 4, 8, 16, 32, or 64 branches may have nucleotides attached to the ends of the PEG branches, such that each end has 0, 1, 2, 3, 4, 5, 6, or more nucleotides attached thereto. In a non-limiting example, a branched polymer having between 3 and 128 PEG arms has one or more nucleotides attached to the ends of the polymer branches, such that each end has 0, 1, 2, 3, 4, 5, 6, or more nucleotides or nucleotide analogs attached thereto. In some embodiments, the branched polymer or dendrite has an even number of arms. In some embodiments, the branched polymer or dendrite has an odd number of arms.

[0254] In some embodiments, the nucleotide conjugate (e.g., a detectable nucleotide conjugate) may comprise a polymer, wherein each branch or subgroup of the polymer may have a portion thereto attached comprising a nucleotide (e.g., adenine, thymine, uracil, cytosine, or guanine residues, or derivatives or mimics thereof), and this portion is capable of binding to a polymerase, reverse transcriptase, or other nucleotide-binding domain. Optionally, the portion may be incorporated into the elongated nucleic acid chain during a polymerase reaction. In some cases, the portion may be blocked, preventing its incorporation into the elongated nucleic acid chain during a polymerase reaction. In some other instances, the portion may be reversibly blocked, preventing its incorporation into the elongated nucleic acid chain during a polymerase reaction until such blocking is removed, after which the portion may then be incorporated into the elongated nucleic acid chain during a polymerase reaction.

[0255] Nucleotides can be conjugated to polymer side chains via their 5' ends. In some cases, nucleotides can be modified to inhibit or prevent their incorporation into the elongating nucleic acid chain during polymerase reactions. For example, nucleotides may include 3' deoxynucleotides, 3' azide nucleotides, 3'-methyl azide nucleotides, or other such nucleotides known or possibly known in the art, making them incompatible with the elongating nucleic acid chain during polymerase reactions. In some embodiments, nucleotides may include a 3'-O-azido group, a 3'-O-azidomethyl group, a 3'-thiophosphate group, a 3'-O-malonyl group, a 3'-O-alkylhydroxyamino group, or a 3'-O-benzyl group. In some embodiments, the nucleotide lacks a 3'-hydroxyl group.

[0256] The polymer may further have a binding moiety in each branch or subgroup of branches. Some examples of binding moieties include, but are not limited to: biotin, avidin, streptavidin, etc., multihistidine domains, complementary pairing nucleic acid domains, G-quartet forming nucleic acid domains, calmodulin, maltose-binding proteins, cellulases, maltose, sucrose, glutathione-S-transferase, glutathione, O-6-methylguanine-DNA methyltransferase, benzylguanine and its derivatives, benzylcysteine ​​and its derivatives, antibodies, epitopes, protein A, and protein G. The binding moieties can be any interacting molecule or fragment thereof known in the art that binds or facilitates interactions between proteins, between proteins and ligands, between proteins and nucleic acids, between nucleic acids, or between small molecule interacting domains or portions.

[0257] In some embodiments, the nucleotide conjugates provided herein (e.g., detectable nucleotide conjugates) may comprise one or more elements of complementary interacting moieties. Examples of complementary interacting moieties include, for example, biotin and avidin; SNAP-benzylguanosine; antibodies or FABs and epitopes; IgG FC and protein A, protein G, protein A / G, or protein L; maltose-binding proteins and maltose; lectins and homopolysaccharides; ion-chelating moieties; complementary nucleic acids; nucleic acids capable of forming triple-stranded or triple-helical interactions; nucleic acids capable of forming G-tetramers, etc. Those skilled in the art will readily recognize that numerous pairs of moieties exist and are widely used due to their strong and specific interactions with each other; and therefore, any such complementary pair or group is considered suitable for the construction or conjecture of the nucleotide conjugates (e.g., detectable nucleotide conjugates) of this disclosure. In some embodiments, nucleotide conjugates disclosed herein (e.g., detectable nucleotide conjugates) may include nucleotide conjugates in which one element of the complementary interacting portion is attached to a molecule or multivalent ligand, and other elements of the complementary interacting portion are attached to individual molecules or multivalent ligands (e.g., detectable nucleotide conjugates). In some embodiments, nucleotide conjugates disclosed herein (e.g., detectable nucleotide conjugates) may include nucleotide conjugates in which two or more elements of the complementary interacting portion are attached to a single molecule or multivalent ligand (e.g., detectable nucleotide conjugates). In some embodiments, nucleotide conjugates disclosed herein (e.g., detectable nucleotide conjugates) may include nucleotide conjugates in which two or more elements of the complementary interacting portion are attached to a single arm of a single molecule or multivalent ligand or to a single position on the single molecule or multivalent ligand (e.g., detectable nucleotide conjugates). In some embodiments, nucleotide conjugates disclosed herein (e.g., detectable nucleotide conjugates) may include nucleotide conjugates in which two or more elements of the complementary interacting portion are attached to the same arm of a single molecule or multivalent ligand or to the same position on the single molecule or multivalent ligand (e.g., detectable nucleotide conjugates). In some embodiments, a nucleotide conjugate (e.g., a detectable nucleotide conjugate) containing one element of a complementary interaction portion and a nucleotide conjugate (e.g., a detectable nucleotide conjugate) containing another element of a complementary interaction portion can be mixed simultaneously or sequentially. In some embodiments, interactions between molecules or particles as disclosed herein allow association or aggregation of multiple molecules or particles, resulting in, for example, an increase in detectable signal. In some embodiments, fluorescence, colorimetric, or radiometric signals are enhanced. In other embodiments, other interaction portions as disclosed herein or known in the art are contemplated.In some embodiments, nucleotide conjugates (e.g., detectable nucleotide conjugates) as provided herein can be provided such that one or more molecules comprising a first interacting moiety (such as, for example, one or more imidazole or pyridine moieties) and one or more additional molecules comprising a second interacting moiety (such as, for example, histidine residues) are simultaneously or sequentially mixed. In some embodiments, the nucleotide conjugate (e.g., detectable nucleotide conjugate) comprises 1, 2, 3, 4, 5, 6 or more imidazole or pyridine moieties. In some embodiments, the nucleotide conjugate (e.g., detectable nucleotide conjugate) comprises 1, 2, 3, 4, 5, 6 or more histidine residues. In such embodiments, the presence of divalent cations (such as nickel, manganese, magnesium, calcium, strontium, etc.) can facilitate interactions between molecules or particles as provided. In some embodiments, for example, a (His)3 group can interact with a (His)3 group on another molecule or particle via coordination with a nickel or manganese ion.

[0258] ix. Combining complex

[0259] The methods described herein can involve detecting a target by forming a binding complex between an index sequence bound to a primer described herein and a nucleotide conjugate described herein (e.g., a detectable nucleotide conjugate). The target can be ribonucleic acid (RNA). The binding complex can include one or more non-covalent interactions.

[0260] In any of the methods described herein, sequencing employs at least one multivalent molecule comprising multiple nucleotide arms attached to a nucleus and having any conformation, including starburst, out-of-order, or bottle-brush conformations (e.g., Figures 5A to 5C). In some embodiments, the multivalent molecule comprises: (1) a nucleus; and (2) multiple nucleotide arms comprising (i) a nucleus attachment portion, (ii) a spacer comprising a PEG portion, (iii) a linker, and (iv) a nucleotide portion, wherein the nucleus is attached to the multiple nucleotide arms, the spacer is attached to the linker, and the linker is attached to the nucleotide portion (e.g., Figures 16 to 18 In some embodiments, the nucleotide moiety comprises a base, a sugar, and at least one phosphate ester group, and the linker is attached to the nucleotide moiety via a base. In some embodiments, the linker comprises an aliphatic chain or an oligomeric glycol chain, wherein the two linker chains have 2 to 6 subunits. In some embodiments, the linker further comprises an aromatic moiety. Example nucleotide arms are shown in... Figure 19 Example multivalent molecules are shown in Figures 5A to 5C. Figures 16 to 18 The example spacer is shown in [the image / image]. Figure 6 The example connector is shown in (the image above). Figure 20 (See image below) and Figure 21 The example nucleotides attached to the linker are shown in [the diagram]. Figures 22 to 24 The example biotinylated nucleotide arm is shown in [the image]. Figure 25 As shown in the image.

[0261] Forming binding complexes within the context of the methods and systems described herein can have certain advantages. Increased binding of nucleotides to enzymes (e.g., polymerases) or enzyme complexes can be achieved by increasing the effective concentration of nucleotides. This increase can be achieved by increasing the concentration of nucleotides in free solution or by increasing the amount of nucleotides near the relevant binding site. This increase can also be achieved by physically confining multiple nucleotides to a limited volume, thus resulting in a localized increase in concentration, and such structures can therefore bind to the binding site with a higher apparent affinity than observed in the case of unconjugated, unchained, or otherwise unconfined individual nucleotides. An example way to achieve such confinement is by providing a binding complex in which multiple nucleotides bind to particles (such as polymers, branched polymers, dendrites, micelles, liposomes, microparticles, nanoparticles, quantum dots, or other suitable particles known in the art).

[0262] Binding complexes can be used to localize detectable signals to active sites of biochemical interactions, such as protein-nucleic acid interactions, nucleic acid hybridization reactions, or enzymatic reactions (such as polymerase reactions). For example, the binding complexes described herein can be used to identify sites of base incorporation in elongated nucleic acid chains during polymerase reactions and provide base differentiation for sequencing and array-based applications. The increased binding between the target and the nucleotide in the binding complex when the nucleotide is complementary to the target nucleic acid provides an enhanced signal that significantly improves base detection accuracy and reduces imaging time.

[0263] Furthermore, the formation of the binding complex described herein allows sequencing signals from a given sequence to originate from a cluster region containing multiple copies of the target sequence. Sequencing methods incorporating multiple copies of the target sequence offer the following advantages: due to the presence of multiple simultaneous sequencing reactions within the defined region, each simultaneous sequencing reaction provides its own signal, which can therefore be amplified. The presence of multiple signals within the defined region also reduces the impact of any single skipped cycle due to the fact that signals from a large number of correctly detected bases can overwhelm signals from a small number of skipped or incorrectly detected bases, thus providing a method for reducing phase modulation errors and / or improving read lengths in sequencing reactions.

[0264] As described herein, the formation of binding complexes and their associated components can result in one or more of the following: (i) stronger signals and higher base interpretation accuracy compared to conventional nucleic acid amplification and sequencing methods; (ii) better differentiation of sequence-specific signals from background signals; (iii) reduced amount of starting material required; (iv) increased sequencing rate and shorter sequencing time; (v) reduced phase modulation error; and (vi) increased read length in sequencing reactions.

[0265] In some embodiments, the nucleotide moiety comprises a chain of one, two, or three phosphorus atoms, wherein the chain is typically linked to the 5' carbon of the sugar moiety via ester or phosphoramide bonding. In some embodiments, at least one nucleotide moiety is a nucleotide analog having a phosphorus chain, wherein the phosphorus atoms are linked together by intermediate O, S, NH, methylene, or ethylene groups. In some embodiments, the phosphorus atoms in the chain include substituted side groups (containing O, S, or BH3). In some embodiments, the chain includes phosphate ester groups substituted with analogs, including phosphoramide, thiophosphate, dithiophosphate, and O-methylphosphoramide groups.

[0266] In some embodiments, the multivalent molecule comprises a core attached to a plurality of nucleotide arms, wherein each nucleotide arm comprises a nucleotide moiety, the nucleotide unit being a nucleotide analog having a chain-terminating portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at both the 2' and 3' positions of the sugar. In some embodiments, the nucleotide moiety comprises a chain-terminating portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain-terminating portion may inhibit polymerase-catalyzed incorporation of subsequent nucleotide moieties or free nucleotides into the nascent chain during primer extension reactions. In some embodiments, the chain-terminating portion is attached to the 3' sugar hydroxyl position, wherein the sugar comprises a ribose or deoxyribose moiety. In some embodiments, the chain-terminating portion may be removed / cleaved from the 3' sugar hydroxyl position to produce a nucleotide having a 3'OH sugar group, which may be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain-terminating portion includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the chain-terminating portion may be cleaved / removed from the nucleotide moiety, for example by reacting the chain-terminating portion with a chemical agent, a pH change, light, or heat. In some embodiments, the alkyl, alkenyl, alkynyl, and allyl groups of the chain-terminating portion may be cleaved with tetrakis(triphenylphosphine)palladium(O) (Pd(PPh3)4), with piperidine, or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the aryl and benzyl groups of the chain-terminating portion may be cleaved with H2 Pd / C. In some embodiments, the amine, amide, ketone, isocyanate, phosphate, thio, and disulfide groups of the chain-terminating portion may be cleaved with phosphine or with a thiol group (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the chain-terminating carbonate portion can be cleaved with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the chain-terminating urea and silyl groups can be cleaved with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0267] In some embodiments, the nucleotide moiety comprises a chain-terminating portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain-terminating portion comprises an azide, an azide group, or an azidemethyl group. In some embodiments, the chain-terminating portion comprises a 3'-O-azido group or a 3'-O-azidomethyl group. In some embodiments, the chain-terminating portion azide, azide group, and an azidemethyl group can be cleaved / removed by a phosphine compound. In some embodiments, the phosphine compound comprises a derived trialkylphosphine moiety or a derived triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), bis(sulfotriphenyl)phosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[0268] In some embodiments, the nucleotide portion comprises a chain termination portion selected from the group consisting of: 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-mercapto, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tert-butyl, 3'-fluorenylmethoxycarbonyl, 3'-tert-butoxycarbonyl, 3'-O-alkylhydroxyamino, 3'-thiophosphate, and 3-O-benzyl or derivatives thereof.

[0269] In some embodiments, the multivalent molecule includes a core attached to a plurality of nucleotide arms, wherein the nucleotide arms include spacers, linkers, and nucleotide moieties, and wherein the core, linkers, and / or nucleotide moieties are labeled with a detectable reporter gene moiety. In some embodiments, the detectable reporter gene moiety includes a fluorophore. In some embodiments, a specific detectable reporter gene moiety (e.g., a fluorophore) attached to the multivalent molecule may correspond to a base of the nucleotide moiety (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow for the detection and identification of nucleotide bases.

[0270] In some embodiments, at least one nucleotide arm of the multivalent molecule has a nucleotide portion attached to a detectable reporter gene portion. In some embodiments, the detectable reporter gene portion is attached to a nucleotide base. In some embodiments, the detectable reporter gene portion includes a fluorophore. In some embodiments, a specific detectable reporter gene portion (e.g., a fluorophore) attached to the multivalent molecule may correspond to a base of the nucleotide portion (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow for the detection and identification of nucleotide bases.

[0271] In some embodiments, the core of the multivalent molecule comprises an avidin-like or streptavidin-like moiety, and the core-attached portion comprises biotin. In some embodiments, the core comprises a streptavidin-type or avidin-type moiety (containing avidin protein), and any derivative, analog, or other non-natural form of avidin that can bind to at least one biotin moiety. Other forms of the avidin moiety include natural and recombinant avidin and streptavidin, as well as derived molecules such as non-glycosylated avidin and truncated streptavidin. For example, the avidin moiety includes deglycosylated forms of avidin, bacterial streptavidin produced by Streptomyces (e.g., Streptomyces averdin), and derived forms such as N-acylavidin, such as N-acetyl, N-phthaloyl, and N-succinylavidin, and commercially available products EXTRAVIDIN, CAPTAVIDIN, NEUTRAVIDIN, and NEUTRAALITE AVIDIN.

[0272] In some embodiments, the binding complex may be formed between one or more nucleotides of a detectable nucleotide conjugate described herein and a nucleotide of a primer-binding index sequence described herein. In some embodiments, the binding complex may be formed between one or more nucleotides of a detectable nucleotide conjugate, a nucleotide of a primer-binding index sequence, and a polymerase (e.g., DNA polymerase). In some embodiments, the binding complex comprises one type of detectable nucleotide conjugate. In some embodiments, the binding complex comprises two or more types of nucleotide conjugates (e.g., detectable nucleotide conjugates). In some embodiments, each of the two or more types of nucleotide conjugates (e.g., detectable nucleotide conjugates) comprises a different type of nucleotide. In some embodiments, the binding complex comprises a blocked nucleotide (e.g., 3'-O-azidomethyl, 3'-O-alkylhydroxyamino, or 3'-O-methylnucleotide). In some embodiments, the blocked nucleotide is located at the 3' position of the primer nucleic acid sequence of the primer-binding index sequence. In some embodiments, the nucleotide of the nucleotide conjugate (e.g., the detectable nucleotide conjugate) is a blocked nucleotide.

[0273] In some embodiments, the binding complex may be multivalent. In some embodiments, the binding complex may be formed between two or more nucleotides of a detectable nucleotide conjugate described herein and a nucleotide of a primer-binding index sequence described herein. In some embodiments, the binding complex may be formed between two or more nucleotides of a detectable nucleotide conjugate, a nucleotide of a primer-binding index sequence, and a polymerase (e.g., DNA polymerase). In some embodiments, the binding complex comprises one type of detectable nucleotide conjugate. In some embodiments, the binding complex comprises two or more types of nucleotide conjugates (e.g., detectable nucleotide conjugates). In some embodiments, each of the two or more types of nucleotide conjugates (e.g., detectable nucleotide conjugates) comprises a different type of nucleotide. In some embodiments, the binding complex comprises a blocked nucleotide (e.g., 3'-O-azidomethyl, 3'-O-alkylhydroxyamino, or 3'-O-methylnucleotide). In some embodiments, the blocked nucleotide is located at the 3' position of the primer nucleic acid sequence of the primer-binding index sequence. In some embodiments, the nucleotide of the nucleotide conjugate (e.g., the detectable nucleotide conjugate) is a blocked nucleotide.

[0274] In some embodiments, the nucleotide moiety involved in the formation of the binding complex is not incorporated. In some embodiments, the binding complex further comprises a polymerase. In some embodiments, this disclosure provides a method in which the polymerase molecule is catalytically inactivated, such as in which the polymerase molecule is catalytically inactivated due to mutation, chemical modification, or lack of necessary ions or cofactors. In some embodiments, this disclosure also provides a method in which the polymerase molecule is catalytically active, and / or in which the binding complex does not contain blocked nucleotides.

[0275] In some embodiments, any method described herein for sequencing nucleic acid molecules may include forming a binding complex, wherein the binding complex comprises (i) a polymerase, a nucleic acid tandem molecule that forms a double strand with a primer, and nucleotides, or the binding complex comprises (ii) a polymerase, a nucleic acid tandem molecule that forms a double strand with a primer, and nucleotide units of a multivalent molecule. In some embodiments, the binding complex has a residence time greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 second. The binding complex has a residence time of greater than about 0.1 to 0.25 seconds, or about 0.25 to 0.5 seconds, or about 0.5 to 0.75 seconds, or about 0.75 to 1 second, or about 1 to 2 seconds, or about 2 to 3 seconds, or about 3 to 4 seconds, or about 4 to 5 seconds, and / or the method is carried out at or above 15°C, at or above 20°C, at or above 25°C, at or above 35°C, at or above 37°C, at or above 42°C, at or above 55°C, at or above 60°C, or at or above 72°C, or at or above 80°C, or within the range defined by any of the foregoing. The binding complex (e.g., a ternary complex) remains stable before being subjected to conditions that cause dissociation between the polymerase, template molecule, primer and / or nucleotide moieties or any of the nucleotides. For example, dissociation conditions include contacting the binding complex with any or any combination of a detergent, EDTA, and / or water. In some embodiments, this disclosure provides a method wherein the binding complex is deposited onto, attached to, or hybridized to a surface that exhibits a contrast-to-noise ratio greater than 20 in a detection step. In some embodiments, this disclosure provides a method wherein contact is performed under conditions that stabilize the binding complex when the nucleotide or nucleotide moiety is complementary to the next base of the template nucleic acid, and destabilize the binding complex when the nucleotide or nucleotide moiety is not complementary to the next base of the template nucleic acid.

[0276] In some embodiments, in any sequencing method employing multivalent molecules, a plurality of first complex polymerases are bound to a plurality of multivalent molecules to form at least one affinity complex. The method includes the steps of: (a) binding a first nucleic acid primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of a tandem template molecule, thereby forming a first binding complex, wherein a first nucleotide portion of the first multivalent molecule binds to the first sequencing polymerase; and (b) binding a second nucleic acid primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same tandem template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the second sequencing polymerase. This includes the first and second binding complexes of the same multivalent molecule forming an affinity complex. In some embodiments, the first sequencing polymerase comprises any wild-type or mutant polymerase described herein. In some embodiments, the second sequencing polymerase comprises any wild-type or mutant polymerase described herein. The tandem template molecule comprises a tandem repeat sequence of the sequence of interest and at least one universal sequencing primer binding site. The first and second nucleic acid primers may bind to the sequencing primer binding site along the tandem template molecule. Example multivalent molecules in Figures 21 to 24 As shown in the image.

[0277] In some embodiments, in any sequencing method employing multivalent molecules, the method includes binding a plurality of first complex polymerases to a plurality of multivalent molecules to form at least one affinity complex, the method comprising the steps of: (a) contacting a plurality of sequencing polymerases and a plurality of nucleic acid primers to different portions of a tandem nucleic acid tandem molecule to form at least first and second complex polymerases on the same tandem template molecule; (b) contacting the plurality of multivalent molecules to at least the first and second complex polymerases on the same tandem template molecule under conditions suitable for binding individual multivalent molecules from a plurality of multivalent molecules to the first and second complex polymerases, wherein at least a first nucleotide portion of the individual multivalent molecule binds to the first complex polymerase, the first complex polymerase including a first primer that hybridizes to a first portion of the tandem template molecule, thereby forming a first binding complex (e.g., a first ternary complex). The process involves (c) detecting the first and second binding complexes on the same tandem template molecule, and (d) identifying the first nucleotide portion in the first binding complex to determine the sequence of the first portion of the tandem template molecule, and identifying the second nucleotide portion in the second binding complex to determine the sequence of the second portion of the tandem template molecule. In some embodiments, multiple sequencing polymerases comprise any wild-type or mutant sequencing polymerase described herein. The tandem template molecule comprises a tandem repeat sequence of the sequence of interest and at least one universal sequencing primer binding site. Multiple nucleic acid primers can bind to the sequencing primer binding site along the tandem template molecule. Example multivalent molecules in... Figures 21 to 24 As shown in the image.

[0278] In some embodiments, the binding complex is deposited onto, attached to, or hybridized to a surface that exhibits a contrast-to-noise ratio greater than 20 in the detection step. In some embodiments, this disclosure provides a method in which the composition is deposited under buffered conditions incorporating a polar aprotic solvent. In some embodiments, contact is performed under conditions that stabilize the binding complex when the nucleotide is complementary to the next base of the target and destabilize the binding complex when the nucleotide is not complementary to the next base of the target nucleic acid.

[0279] In some embodiments, the binding complex can be used to replace a single unconjugated or unchained nucleotide to form a binding complex with the polymerase and the target, and the local concentration of the nucleotide can be increased several times, thereby enhancing the signal intensity. The formed binding complex can also have a longer residence time, which in turn helps to shorten the imaging step. The high signal intensity resulting from the use of nucleotide conjugates (e.g., detectable nucleotide conjugates) can be maintained throughout the binding and imaging steps. The strong binding between the polymerase, the primer-binding index sequence, and the nucleotide or nucleotide analogue also means that the formed binding complex will remain stable during the washing step, and the signal will remain at a high intensity when other reaction mixtures and mismatched nucleotide analogues are washed away. After the imaging step, the binding complex can be unstable and the primer-binding index sequence can be extended by one base. In some embodiments, the one nucleotide used to extend the primer-binding index sequence by one base can be unmodified. In some embodiments, the one nucleotide used to extend the primer-binding index sequence by one base may not contain a label.

[0280] In some embodiments, the binding complexes described herein may comprise one or more buffers, salts, ions, or additives. Representative additives in some embodiments may include, but are not limited to: betaine, spermidine, detergents (such as polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether (Triton X-100), ethyl 2-[2-[3,4-bis(2-hydroxyethoxy)oxacyclopentan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl lactone (Tween 20), sodium dodecyl sulfate (SDS) or nonylphenol polyoxyethylene ether (NP-40)), ethylene glycol, polyethylene glycol, dextran, polyvinyl alcohol, vinyl alcohol, methylcellulose, heparin, heparan sulfate, glycerol, sucrose, 1,2-propanediol, dimethyl sulfoxide (DMSO), N,N,N-trimethylglycine, ethanol, ethoxyethanol, propylene glycol, polypropylene glycol, block copolymers (such as Pluronic) (r) series polymers), arginine, histidine, imidazole or any combination thereof, or any substance known in the art as a DNA "relaxant" (a compound that has the effect of altering the length of DNA, altering the number of intrapolymer linkages or crossovers, or altering the conformational dynamics of DNA molecules to increase the accessibility of intrastrand sites to DNA binding sites).

[0281] The binding complex may include zwitterionic compounds as additives. Other representative additives can be found in Lorenz, TC, *Journal of Visual Experimentation* (63), e3998, doi:10.3791 / 3998 (2012), whose disclosure regarding additives for use in facilitating nucleic acid binding or kinetics, or in facilitating processes involving the manipulation, use, or storage of nucleic acids, is hereby incorporated by reference. In some embodiments, representative cations may include, but are not limited to, sodium, magnesium, strontium, potassium, manganese, calcium, lithium, nickel, cobalt, or other such cations known in the art to promote nucleic acid interactions, such as self-association, secondary or tertiary structure formation, base pairing, surface association, peptide association, protein binding, etc.

[0282] This disclosure relates to binding complexes having multiple nucleotides conjugated to particles (e.g., polymers, branched polymers, dendrites, or equivalent structures). Contacting the binding complex with a target bound by a polymerase and primers can result in the formation of a detectable binding complex, thereby enabling more accurate determination of the bases of the target nucleic acid.

[0283] In some embodiments, the binding complex can be used to replace a single unconjugated or unchained nucleotide to form a complex with the polymerase and the target nucleic acid. In some cases, by forming the binding complex, the local concentration of the nucleotide can be increased several times, thereby enhancing the signal strength, particularly the correct signal relative to the mismatch. The binding complexes described herein may include at least one nucleotide conjugate (e.g., a detectable nucleotide conjugate) for interacting with the target nucleic acid. Multivalent compositions may also include two, three, or four different nucleotide conjugates (e.g., detectable nucleotide conjugates), each having a different nucleotide conjugated to a common nucleus.

[0284] The binding complex can contain one, two, three, four, or more nucleotide conjugates (e.g., detectable nucleotide conjugates), each of which contains a different type of nucleotide. The first type of nucleotide conjugate (e.g., detectable nucleotide conjugates) can contain nucleotides selected from the group consisting of ATP, ADP, AMP, dATP, dADP, and dAMP. The second type of nucleotide conjugate (e.g., detectable nucleotide conjugates) can contain nucleotides selected from the group consisting of TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, and dUMP. The third type of nucleotide conjugate (e.g., detectable nucleotide conjugates) can contain nucleotides selected from the group consisting of CTP, CDP, CMP, dCTP, dCDP, and dCMP. The fourth type of nucleotide conjugate (e.g., detectable nucleotide conjugates) can contain nucleotides selected from the group consisting of GTP, GDP, GMP, dGTP, dGDP, and dGMP. In some embodiments, each nucleotide conjugate (e.g., a detectable nucleotide conjugate) comprises a single type of nucleotide, each corresponding to one or more nucleotides selected from the group consisting of: ATP, ADP, AMP, dATP, dADP, dAMP, TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, and dGMP. Each binding complex may further be associated with one or more tags containing a specific nucleotide conjugated to each conjugate. Example tags include fluorescent tags, colorimetric tags, electrochemical tags (such as, for example, glucose or other reducing sugars, or thiols or other redox active moieties), luminescent tags, spin tags, radioactive tags, steric hindrance tags, affinity tags, etc.

[0285] In some embodiments, the multivalent molecule includes a nucleus attached to a plurality of nucleotide arms, wherein the plurality of nucleotide arms have the same type of nucleotide moiety selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0286] In some embodiments, the multivalent molecule comprises a nucleus attached to a plurality of nucleotide arms, wherein each arm comprises a nucleotide moiety. The nucleotide moiety comprises an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and one or more phosphate groups (e.g., 1 to 10 phosphate groups). The plurality of multivalent molecules may comprise one type of multivalent molecule having a nucleotide moiety of one type selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. The plurality of multivalent molecules may comprise a mixture of any combination of two or more types of multivalent molecules, wherein the individual multivalent molecules in the mixture comprise a nucleotide moiety of one type selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0287] Figure 6 This illustrates how the use of nucleotide conjugates (e.g., detectable nucleotide conjugates) can improve signal intensity during the binding, retention, and washing / removal steps of the methods described herein. Due to the increased local concentration of nucleotides on the polymer-nucleotide conjugate, binding between the polymerase, the primer-bound target strand, and the nucleotide becomes more favorable when the nucleotide is complementary to the next base of the target nucleic acid. The resulting binding complex has a longer retention time, which in turn helps to shorten the imaging step. The high signal intensity resulting from the use of nucleotide conjugates...

Claims

1. A method for in situ identification of ribonucleic acid (RNA), the method comprising: (a) Providing permeable cells or tissue, wherein the cells or tissue contain the RNA; (b) Guide the probe to the cell or tissue under conditions sufficient to couple the RNA binding sequence to at least a portion of the RNA, wherein the probe comprises (i) the RNA binding sequence and (ii) an index sequence; (c) Amplify the index sequence to generate one or more amplicons, wherein the one or more amplicons contain multiple copies of the index sequence or a derivative thereof; (d) Guide the primer nucleic acid sequence into the cell or tissue under conditions sufficient to bind the primer nucleic acid sequence to a portion of the one or more amplicones to generate a primer-binding index sequence; (e) Guiding the cell or tissue with a nucleotide conjugate under conditions suitable for forming a binding complex between nucleotides of an index sequence that binds to one or more nucleotide moieties of the primer without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; the nucleotide conjugate comprising: (i) a detectable marker and (ii) the one or more nucleotide moieties of the index sequence or its derivative that bind to the primer that are complementary to the nucleotides of the primer. as well as (f) Detect the binding complex within the cell or tissue to identify the RNA.

2. The method according to claim 1, wherein the method does not include performing a reverse transcription reaction of the RNA.

3. The method according to any one of claims 1 or 2, further comprising providing the cell in (a).

4. The method according to claim 1, wherein the cell is a cancer cell.

5. The method according to any one of claims 1 or 2, further comprising providing the tissue in (a).

6. The method according to claim 5, wherein the tissue is formalin-fixed paraffin-embedded tissue.

7. The method of claim 5, wherein the tissue is freshly frozen tissue.

8. The method according to any one of claims 1 or 2, wherein the cells or the tissue are extracted from the subject.

9. The method of claim 8, wherein the subject is a human subject.

10. The method according to any one of claims 1 to 9, wherein the index sequence comprises nucleic acid.

11. The method of claim 10, wherein the length of the index sequence is 4 to 50 nucleotides.

12. The method according to any one of claims 1 to 11, wherein at least a portion of the index sequence or a derivative thereof identifies the primer nucleic acid sequence.

13. The method according to any one of claims 1 to 11, wherein the primer nucleic acid sequence comprises a blocking group.

14. The method of claim 13, wherein the blocking group inhibits the incorporation of another nucleotide into the primer nucleic acid sequence.

15. The method according to claim 13, wherein the blocking group comprises a 3'-O-azido group, a 3'-O-azidomethyl group, a 3'-O-alkylhydroxyamino group, a 3'-thiophosphate group, a 3'-O-malonyl group, or a 3'-O-benzyl group.

16. The method according to any one of claims 1 to 15, wherein the primer nucleic acid sequence does not include a 3' hydroxyl group.

17. The method according to any one of claims 1 to 16, wherein the primer nucleic acid sequence binds to at least a portion of the index sequence.

18. The method according to any one of claims 1 to 17, wherein the primer nucleic acid sequence binds to at least a portion of the inverse complementary sequence of the index sequence.

19. The method according to any one of claims 1 to 18, wherein the primer-bound index sequence comprises a double-stranded nucleic acid.

20. The method according to any one of claims 1 to 19, wherein the primer-bound index sequence comprises a single-stranded nucleic acid.

21. The method according to any one of claims 1 to 20, wherein the cells or the tissue are fixed to the surface of the substrate.

22. The method of claim 21, wherein the surface of the substrate has a water contact angle of less than or equal to 45 degrees.

23. The method of claim 21, wherein the surface of the substrate is the inner surface of the flow cell.

24. The method of claim 23, wherein the inner surface of the flow cell comprises one or more hydrophilic polymer layers.

25. The method of claim 24, wherein the one or more hydrophilic polymer layers comprise a branched polymer.

26. The method of claim 24, wherein the one or more hydrophilic polymer layers comprise a polymer containing polyethylene glycol.

27. The method according to any one of claims 1 to 26, wherein the nucleotide conjugate comprises a common core.

28. The method of claim 27, wherein one or more nucleotide moieties are coupled to the common nucleus.

29. The method of claim 27 or 28, wherein the common core comprises a polymer, micelles, liposomes, microparticles, nanoparticles, or quantum dots.

30. The method of claim 29, wherein the polymer comprises a protein.

31. The method according to any one of claims 1 to 30, wherein the nucleotide conjugate in (e) is included in a mixture of a plurality of nucleotide conjugates, wherein each of the plurality of nucleotide conjugates comprises nucleotide moieties of different types from each other.

32. The method of claim 31, wherein the different types of nucleotide moieties comprise at least three different types of nucleotide moieties.

33. The method according to any one of claims 1 to 32, wherein the one or more nucleotide moieties do not contain a blocking group coupled thereto.

34. The method according to any one of claims 1 to 33, wherein the detection in (f) comprises imaging the cell or the tissue using one or more image sensors.

35. The method of claim 34, wherein the one or more image sensors comprise a photodetector array.

36. The method according to any one of claims 1 to 35, further comprising washing the cells or tissue after (f) to remove the nucleotide conjugate from the cells or tissue.

37. The method of claim 36, further comprising repeating (e) and (f) with a second nucleotide conjugate comprising (i) a detectable marker and (ii) one or more nucleotide portions complementary to a second nucleotide in an index sequence or a derivative thereof that binds to the primer.

38. The method according to any one of claims 1 to 37, wherein the detectable marker comprises a fluorophore.

39. The method according to any one of claims 1 to 38, wherein the detectable marker comprises a quantum dot.

40. The method according to any one of claims 1 to 39, wherein the conditions sufficient to couple the target binding sequence to at least a portion of the target in (b) include an incubation temperature, wherein the incubation temperature is about 20°C to 45°C.

41. The method according to any one of claims 1 to 40, wherein the condition in (b) sufficient to couple the target binding sequence to at least a portion of the target includes a salt.

42. The method according to any one of claims 1 to 41, wherein the RNA is messenger RNA.

43. The method according to any one of claims 1 to 41, wherein the RNA is microRNA.

44. The method according to any one of claims 1 to 41, wherein the RNA is ribosomal RNA.

45. The method according to any one of claims 1 to 41, wherein the RNA is transfer RNA.

46. ​​The method according to any one of claims 1 to 41, wherein the RNA is a transfer messenger RNA.

47. The method according to any one of claims 1 to 46, wherein the RNA comprises a modification.

48. The method of claim 47, wherein the modification is 5' phosphorylation.

49. The method of claim 47, wherein the modification is an internucleotide bond.

50. The method according to any one of claims 1 to 49, wherein the probe comprises nucleic acid.

51. The method of claim 50, wherein the nucleic acid comprises deoxyribonucleic acid (DNA).

52. The method of claim 50, wherein the nucleic acid comprises RNA.

53. The method according to any one of claims 1 to 49, wherein the RNA binding sequence comprises nucleic acid.

54. The method of claim 53, wherein the nucleic acid comprises deoxyribonucleic acid (DNA).

55. The method of claim 53, wherein the nucleic acid comprises RNA.

56. The method according to any one of claims 1 to 55, wherein the probe is a locking probe.

57. The method of claim 56, further comprising, prior to (c), using a ligase to connect the first end of the probe to the second end of the probe.

58. The method of claim 57, wherein the ligase is a T4 ligase.

59. The method of claim 57, wherein the ligase is SplintR ligase.

60. The method according to any one of claims 1 to 59, wherein the amplification reaction in (c) comprises rolling circle amplification using a polymerase.

61. The method according to claim 60, wherein the polymerase is phi29 polymerase.

62. The method according to any one of claims 1 to 61, wherein the cells or the tissue are permeabilized using a solvent.

63. The method of claim 62, wherein the solvent is methanol.

64. The method of claim 62, wherein the solvent is acetone.

65. The method according to any one of claims 1 to 61, wherein the cells or the tissue are permeated using a detergent.

66. The method of claim 65, wherein the detergent is a saponin.

67. The method of claim 65, wherein the detergent is Triton X-100.

68. A method for in situ identification of polypeptides, the method comprising: (a) Providing permeated cells or tissue, wherein the cells or tissue contain the polypeptide; (b) Guide the polypeptide-binding probe to the cell or tissue under conditions sufficient to conjugate the polypeptide-binding probe to the polypeptide, wherein the polypeptide-binding probe comprises a polypeptide-binding moiety and an oligonucleotide conjugated thereto. (c) Guide the probe to the cell or tissue under conditions sufficient to couple the oligonucleotide-binding probe to at least a portion of the oligonucleotide of the polypeptide-binding probe, wherein the probe comprises (i) the oligonucleotide-binding probe and (ii) an index sequence. (d) Amplify the index sequence to generate one or more amplicones, wherein the one or more amplicones contain multiple copies of the index sequence or a derivative thereof; (e) Guide the primer nucleic acid sequence into the cell or the tissue under conditions sufficient to bind the primer nucleic acid sequence to at least a portion of the one or more amplicons to generate a primer-binding index sequence; (f) Guide the cell or tissue with a nucleotide conjugate under conditions suitable for forming a binding complex between nucleotides of an index sequence that binds to one or more nucleotide moieties of the primer without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence. The nucleotide conjugate comprises: (i) a detectable marker and (ii) the one or more nucleotide moieties of the index sequence that binds to the primer or the one or more amplicon thereof that are complementary to the nucleotides of the primer. as well as (g) Detect the binding complex within the cells or tissues to identify the polypeptide.

69. A method for in situ identification of DNA, the method comprising: (a) Providing permeated cells or tissue, wherein the cells or tissue contain the DNA, wherein the DNA is not cDNA; (b) Guide the probe to the cell or tissue under conditions sufficient to couple the DNA binding sequence to at least a portion of the DNA, wherein the probe comprises (i) the DNA binding sequence and (ii) an index sequence; (c) Amplify the index sequence to generate one or more amplicons, wherein the one or more amplicons contain multiple copies of the index sequence or a derivative thereof; (d) Guide the primer nucleic acid sequence into the cell or the tissue under conditions sufficient to bind the primer nucleic acid sequence to at least a portion of the one or more amplicons to generate a primer-binding index sequence; (e) Guiding the cell or tissue with a nucleotide conjugate under conditions suitable for forming a binding complex between nucleotides of an index sequence that binds to one or more nucleotide moieties of the primer without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; the nucleotide conjugate comprising: (i) a detectable marker and (ii) the one or more nucleotide moieties of the index sequence or its derivative that bind to the primer that are complementary to the nucleotides of the primer. as well as (f) Detect the binding complex within the cell or tissue to identify the DNA.

70. A method for in-situ identification of ribonucleic acid, the method comprising: (a) Providing permeated cells or tissue, wherein the cells or tissue contain the ribonucleic acid; (b) Guide a nucleic acid molecule to the cell or tissue under conditions sufficient to couple a target-binding sequence to at least a portion of the ribonucleic acid, wherein the nucleic acid molecule contains the target-binding sequence, and wherein the nucleic acid molecule further contains an index sequence; (c) Guide the primer nucleic acid sequence into the cell or the tissue under conditions sufficient to bind the primer nucleic acid sequence to at least a portion of the one or more amplicons to generate a primer-binding index sequence; (d) Guiding the cell or tissue with a nucleotide conjugate under conditions suitable for forming a binding complex between nucleotides of an index sequence that binds to one or more nucleotide moieties of the primer without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; the nucleotide conjugate comprising: (i) a detectable label and (ii) the one or more nucleotide moieties complementary to the nucleotides in the index sequence or a derivative thereof that binds to the primer. (e) Detect the binding complex within the cells or tissues to identify the ribonucleic acid.

71. A system for in situ identification of RNA, the system comprising: A computing device comprising one or more computer processors, an operating system configured to execute executable instructions, a memory, and a computer program comprising instructions containing the method according to any one of claims 1 to 67.

72. A system for in-situ identification of polypeptides, the system comprising: A computing device comprising one or more computer processors, an operating system configured to execute executable instructions, a memory, and a computer program comprising instructions containing the methods of claim 68.

73. A system for in situ identification of DNA, the system comprising: A computing device comprising one or more computer processors, an operating system configured to execute executable instructions, a memory, and a computer program comprising instructions containing the methods of claim 69.

74. A system for in-situ identification of ribonucleic acid, the system comprising: A computing device comprising one or more computer processors, an operating system configured to execute executable instructions, a memory, and a computer program comprising instructions containing the method of claim 70.

75. A kit for in situ identification of RNA, the kit comprising: (a) A probe comprising (i) an index sequence and (ii) an RNA binding sequence, wherein the RNA binding sequence recognizes at least a portion of the target; (b) A nucleotide conjugate comprising (i) a detectable marker and (ii) one or more nucleotide moieties complementary to a primer-binding index sequence, wherein the nucleotide conjugate is configured to form a binding complex between the one or more nucleotide moieties and the nucleotides of the primer-binding index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence. as well as (c) Instructions for in situ identification of the RNA, wherein the instructions comprise the method according to any one of claims 1 to 67.

76. A kit for in situ identification of peptides, the kit comprising: (a) A polypeptide-binding probe comprising (i) a polypeptide-binding moiety and (ii) an oligonucleotide coupled thereto; (b) A probe comprising (i) an index sequence and (ii) an oligonucleotide binding probe, wherein the oligonucleotide binding probe recognizes at least a portion of the oligonucleotide; (c) A nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a primer-binding index sequence, wherein the nucleotide conjugate is configured to form a binding complex between the one or more nucleotide moieties and the nucleotides of the primer-binding index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence. as well as (d) Instructions for in-situ identification of the polypeptide, wherein the instructions comprise the method according to claim 68.

77. A kit for in situ identification of DNA, the kit comprising: (a) A probe comprising (i) an index sequence and (ii) an RNA binding sequence, wherein the RNA binding sequence recognizes at least a portion of the target; (b) A nucleotide conjugate comprising (i) a detectable marker and (ii) one or more nucleotide moieties complementary to a primer-binding index sequence, wherein the nucleotide conjugate is configured to form a binding complex between the one or more nucleotide moieties and the nucleotides of the primer-binding index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence. as well as (c) Instructions for in situ identification of the RNA, wherein the instructions comprise the method according to claim 69.

78. A kit for in situ identification of ribonucleic acid, the kit comprising: (a) A nucleic acid molecule comprising (i) an index sequence and (ii) a target binding sequence, wherein the target binding sequence is complementary to at least a portion of the ribonucleic acid sequence; (b) A nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to nucleotides in an index sequence that binds to the primer, wherein the nucleotide conjugate is configured to form a binding complex between the one or more nucleotide moieties and the nucleotides in the index sequence that binds to the primer without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence. as well as (c) Instructions for in-situ identification of the ribonucleic acid, wherein the instructions comprise the method according to claim 70.

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