Direct in situ sequencing

Abstract

Binding complexes including a detectable nucleotide conjugate having a plurality of copies of a nucleotide attached to the particle are described. The binding complexes allow one to localize detectable signals to active regions of biochemical interaction, e.g., sites of protein-protein interaction, protein-nucleic acid interaction, nucleic acid hybridization, or enzymatic reaction, and can be used to identify sites of base incorporation in elongating nucleic acid chains during polymerase reactions and to provide improved base discrimination for sequencing and array-based applications.

Description

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 549,891, filedFebruary 5,2024, and U.S. Provisional Patent Application No. 63 / 652,540, filed May 28, 2024, which are herein incorporated by reference in their entireties for all purposes. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 52933-761.601-PCT SL.XML, created February 4, 2025, which is 6.16 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. BACKGROUND

[0003] Nucleic acid sequencing can be used to obtain information in a wide variety of biomedical contexts, including diagnostics, prognostics, biotechnology, and forensic biology. Various sequencing methods have been developed including Maxam-Gilbert sequencing and chain-termination methods, or de novo sequencing methods including shotgun sequencing and bridge PCR, or next-generation methods including polony sequencing, 454 pyrosequencing Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, HeliScope single molecule sequencing, SMRT® sequencing, and others. Despite advances in DNA sequencing, many challenges still remain unaddressed. SUMMARY

[0004] Aspects disclosed herein provide methods for identifyinga ribonucleic acid (RNA) in situ, the method comprising: (a) providing a cell or a tissue that has b een permeabilized, wherein the cell or the tissue comprises the RNA; (b) directing a probe to the cell or tissue, wherein the probe comprises (i) an RNA-binding sequence and (ii) an index sequence, under conditions sufficient to couple the RNA-binding sequence to at least a portion of the RNA; (c) amplifying the index sequence to generate one or more amplicons, wherein the one or more amplicons comprises multiple copies of the index sequence or derivative thereof; (d) directing a primer nucleic acid sequenceto the cell or the tissue under conditions sufficientto bind the primer nucleic acid sequence to a portion of the one or more amplicons to produce a primed index sequence; (e) directing the cell or the tissue with a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide in the primed index sequence or the derivative thereof, under conditions suitable to form a binding complex between the one or more nucleotide moieties and the nucleotide of the primed index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (f) detecting the binding complex within the cell or tissue, thereby identifying the RNA. In some embodiments, the method does not comprise performing a reverse transcription reaction of the RNA. In some embodiments, the method further comprises providing the cell in (a). In some embodiments, the cell is a cancer cell. In some embodiments, the method further comprises providing the tissue in (a). In some embodiments, the tissue is a formalin-fixed paraffin embedded tissue. In some embodiments, the tissue is a fresh-frozen tissue. In some embodiments, the cell or the tissue is extracted from a subject. In some embodiments, the subject is a human subject. In some embodiments, the index sequence comprises a nucleic acid. In some embodiments, the index sequence is 4-50 nucleotides in length. In some embodiments, at least a portion of the index sequence or derivative thereof recognizes the primer nucleic acid sequence. In some embodiments, the primer nucleic acid sequence comprises a blocking group. In some embodiments, the blocking group inhibits incorporation of another nucleotide into the primer nucleic acid sequence. In some embodiments, the blocking group comprises a 3’-0-azido group, a 3’-0-azidomethyl group, a 3’ -O-alkyl hydroxylamino group, a 3 ’-phosphorothioate group, a 3’-0-malonyl group, or a 3’-0-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 a reverse complement of the index sequence. In some embodiments, the primed index sequence comprises a double-stranded nucleic acid. In some embodiments, the primed index sequence comprises a single-stranded nucleic acid. In some embodiments, the cell or the tissue is immobilized on a 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 an interior surface of a flow cell. In some embodiments, the interior surface of the flow cell comprises one or more hydrophilic polymer layers. In some embodiments, the one or more hydrophilic polymer layers comprises abranchedpolymer. In some embodiments, the one or more hydrophilic polymer layers comprises a polymer comprising polyethylene glycol. In some embodiments, the nucleotide conjugate comprises a common core. In some embodiments, the one or more nucleotide moieties are coupled to the common core. In some embodiments, the common core comprises a polymer, a micelle, a liposome, a microparticle, a nanoparticle, or a quantum dot. In some embodiments, the polymer comprises a protein. In some embodiments, in (e) the nucleotide conjugate is included in a mixture of a plurality of nucleotide conjugates, wherein each of the plurality of nucleotide conjugates comprises different types of nucleotide moieties from each other. In some embodiments, the different types of the nucleotide moieties comprise at least three different types of the nucleotide moieties. In some embodiments, the one or more nucleotide moieties do not comprise a blocking group coupled thereto. In some embodiments, the detecting in (f) comprises imaging the cell or the tissue using one or more image sensors. In some embodiments, the one or more image sensors comprises a photodetector array. In some embodiments, the method further comprises washing the cell or the tissue following (f) to remove the nucleotide conjugate from the cell or the tissue. In some embodiments, the method further comprises 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 the primed index sequence or derivative thereof. In some embodiments, the detectable label comprises a fluorophore. In some embodiments, the detectable label comprises a quantum dot. In some embodiments, the conditions sufficient to couple the target-binding sequence to at least a portion of the target in (b) comprises an incubation temperature, wherein the incubation temperature is about 20-45°C. In some embodiments, the conditions sufficient to couple the target-binding sequence to at least a portion of the target in(b) comprises a salt. In some embodiments, the RNA is a messenger RNA. In some embodiments, the RNA is a micro RNA. In some embodiments, the RNA is a ribosomal RNA. In some embodiments, the RNA is a transfer RNA. In some embodiments, the RNA is a transfermessenger RNA. In some embodiments, the RNA comprises a modification. In some embodiments, the modification is a 5’ phosphorylation. In some embodiments, the modification is an inter-nucleotide linkage. In some embodiments, the probe comprises 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 nucleic acid. In some embodiments, the nucleic acid comprises deoxyribonucleic acid (DNA). In some embodiments, the nucleic acid comprises RNA. In some embodiments, the probe is a padlock probe. In some embodiments, the method further comprises, before (c), ligating a first end of the probe to a second end of the probe using a ligase. In some embodiments, the ligase is a T4 ligase. In some embodiments, the ligase is a SplintR ligase. In some embodiments, performing the amplification reaction in (c) comprises performinga rolling circle amplification reaction using a polymerizing enzyme. In some embodiments, the polymerizing enzyme is a phi29 polymerase. In some embodiments, the cell or the tissue is permeabilized using a solvent. In some embodiments, the solvent is methanol. In some embodiments, the solvent is acetone. In some embodiments, the cell or the tissue is permeabilized using a detergent. In some embodiments, the detergent is saponin. In some embodiments, the detergent is Triton X-100.

[0005] Aspects disclosed herein provide methods for identifying a polypeptide in situ, the method comprising: (a) providing a cell or a tissue that has been permeabilized, wherein the cell or the tissue comprises the polypeptide; (b) directing a polypeptide-binding probe to the cell or tissue, wherein the polypeptide-binding probe comprises a polypeptide binding moiety and an oligonucleotide coupled thereto, under conditions sufficient to couple the polypeptide-binding probe to the polypeptide; (c) directing a probe to the cell or the tissue, wherein the with a probe comprising (i) an oligonucleotide-binding probe and (ii) an index sequence under conditions sufficient to couple the oligonucleotide-binding probe to at least a portion of the oligonucleotide of the polypeptide-binding probe; (d) amplifying the index sequence to generate one or more amplicons, wherein the one or more amplicons comprises multiple copies of the index sequence or derivative thereof; (e) directing a primer nucleic acid sequence to 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 produce a primed index sequence; (f) directing the cell or the tissue with a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide in the primed index sequence or the one or more amplicons thereof, under conditions suitable to form a binding complex between the one or more nucleotide moieties and the nucleotide of the primed index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (g) detecting the binding complex within the cell or tissue, thereby identifying the polypeptide. In some embodiment, the method further comprises providing the cell in (a). In some embodiment, the cell is a cancer cell. In some embodiment, the method further comprises providing the tissue in (a). In some embodiment, the tissue is a formalin-fixed paraffin embedded tissue. In some embodiment, the tissue is a fresh-frozen tissue. In some embodiment, the cell or the tissue is extracted from a subject. In some embodiment, the subject is a human subject. In some embodiment, the index sequence comprises a nucleic acid. In some embodiment, the index sequence is 4-50 nucleotides in length. In some embodiment, at least a portion of the index sequence or derivative thereof recognizes the primer nucleic acid sequence. In some embodiment, the primer nucleic acid sequence comprises a blocking group. In some embodiment, the blocking group inhibits incorporation of another nucleotide into the primer nucleic acid sequence. In some embodiment, the blocking group comprises a 3’-0-azido group, a 3’-0-azidomethyl group, a 3’ -O-alkyl hydroxylamino group, a 3 ’-phosphorothioate group, a 3 ’-0-malonyl group, or a 3 ’-0-benzyl group. In some embodiment, the primer nucleic acid sequence does not include a 3’ hydroxyl group. In some embodiment, the primer nucleic acid sequence binds to at least a portion of the index sequence. In some embodiment, the primer nucleic acid sequence binds to at least a portion of a reverse complement of the index sequence. In some embodiment, the primed index sequence comprises a double stranded nucleic acid. In some embodiment, the primed index sequence comprises a singlestranded nucleic acid. In some embodiment, the cell or the tissue is immobilized on a surface of a substrate. In some embodiment, the surface of the substrate has a water contact angle of less than or equal to 45 degrees. In some embodiment, the surface of the substrate is an interior surface of a flow cell. In some embodiment, the interior surface of the flow cell comprises one or more hydrophilic polymer layers. In some embodiment, the one or more hydrophilic polymer layers comprises a branched polymer. In some embodiment, the one or more hydrophilic polymer layers comprises a polymer comprising polyethylene glycol. In some embodiment, the nucleotide conjugate comprises a common core. In some embodiment, the one or more nucleotide moieties are coupled to the common core. In some embodiment, the common core comprises a polymer, a micelle, a liposome, a microparticle, a nanoparticle, or a quantum dot. In some embodiment, the polymer comprises a protein. In some embodiment, in (e) the nucleotide conjugate is included in a mixture of a plurality of nucleotide conjugates, wherein each of the plurality of nucleotide conjugates comprises different types of nucleotide moieties from each other. In some embodiment, the different types of the nucleotide moieties comprise at least three different types of the nucleotide moieties. In some embodiment, the one or more nucleotide moieties do not comprise a blocking group coupled thereto. In some embodiment, the detecting in (f) comprises imaging the cell or the tissue using one or more image sensors. In some embodiment, the one or more image sensors comprises a photodetector array. In some embodiments, the method further comprises washing the cell or the tissue following (f) to remove the nucleotide conjugate from the cell or the tissue. In some embodiment, the method further comprises 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 the primed index sequence or derivative thereof. In some embodiment, the detectable label comprises a fluorophore. In some embodiment, the detectable label comprises a quantum dot. In some embodiment, the conditions sufficient to couplethetarget-bindingsequenceto at least a portion of the target in (b) comprises an incubation temperature, wherein the incubation temperature is about 20-45°C. In some embodiment, the conditions sufficient to couple the target-binding sequence to at least a portion of the target in (b) comprises a salt. In some embodiment, the polypeptide comprises a protein. In some embodiment, the protein is a cell-surface protein. In some embodiment, the protein is an intracellular protein. In some embodiment, the protein is a signaling protein. In some embodiment, the polypeptide comprises a protein complex. In some embodiment, the polypeptide binding moiety comprises an antibody or antibody fragment. In some embodiment, the polypeptide binding moiety comprises an aptamer. In some embodiment, the polypeptide bindingmoiety comprises a nanobody. In some embodiment, the oligonucleotide comprisesDNA. In some embodiment, the oligonucleotide is 4- 50 nucleotides in length. In some embodiment, the polypeptide binding moiety is covalently attached to the oligonucleotide. In some embodiment, the polypeptide binding moiety is non-covalently attached to the oligonucleotide. In some embodiment, the probe comprises nucleic acid. In some embodiment, the nucleic acid comprises DNA. In some embodiment, the nucleic acid comprises RNA. In some embodiment, the probe is a padlock probe. In some embodiment, before (d), ligating a first end of the probe to a second end of the probe using a ligase. In some embodiment, the ligase is a T4 ligase. In some embodiment, the ligase is a SplintR ligase. In some embodiment, performing the amplification reaction in (d) comprises performing a rolling circle amplification reaction using a polymerizing enzyme. In some embodiment, the polymerizing enzyme is a phi29 polymerase. In some embodiment, the cell or the tissue is permeabilized using a solvent. In some embodiment, the solvent is methanol. In some embodiment, the solvent is acetone. In some embodiment, the cell or the tissue is permeabilized using a detergent. In some embodiment, the detergent is saponin. In some embodiment, the detergent is Triton X-100.

[0006] Aspects disclosed herein provide methods for identifying a DNA in situ, the method comprising: (a) providing a cell or a tissue that has been permeabilized, wherein the cell or the tissue comprises the DNA, where the DNA is not cDNA; (b) directing a probe to the cell or the tissue, wherein the probe comprises a (i) DNA-binding sequence and (ii) an index sequence, under conditions sufficient to couple the DNA-binding sequence to at least a portion of the DNA; (c) amplifying the index sequence to generate one or more amplicons, wherein the one or more amplicons comprises multiple copies of the index sequence or derivative thereof; (d) directing a primer nucleic acid sequence to 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 produce a primed index sequence; (e) directingthe cell or the tissue with a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide in the primed index sequence or the derivatives thereof, under conditions suitable to form a binding complex between the one or more nucleotide moieties and the nucleotide of the primed index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (f) detecting the binding complex within the cell or tissue, thereby identifying the DNA. In some embodiments, the method further comprises providing the cell in (a). In some embodiments, the cell is a cancer cell. In some embodiments, the method further comprises providing the tissue in (a). In some embodiments, the tissue is a formalin-fixed paraffin embedded tissue. In some embodiments, the tissue is a fresh-frozen tissue. In some embodiments, the cell or the tissue is extracted from a subject. In some embodiments, the subject is a human subject. In some embodiments, the index sequence comprises a nucleic acid. In some embodiments, the index sequence is 4-50 nucleotides in length. In some embodiments, at least a portion of the index sequence or derivative thereof recognizes the primer nucleic acid sequence. In some embodiments, the primer nucleic acid sequence comprises a blocking group. In some embodiments, the blocking group inhibits incorporation of another nucleotide into the primer nucleic acid sequence. In some embodiments, the blocking group comprises a 3’-O-azido group, a 3’-0-azidomethyl group, a 3’ -O-alkyl hydroxylamino group, a 3 ’-phosphorothioate group, a 3’-0-malonyl group, or a 3’-0-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 a reverse complement of the index sequence. In some embodiments, the primed index sequence comprises a double-stranded nucleic acid. In some embodiments, the primed index sequence comprises a single-stranded nucleic acid. In some embodiments, the cell or the tissue is immobilized on a 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 an interior surface of a flow cell. In some embodiments, the interior surface of the flow cell comprises one or more hydrophilic polymer layers. In some embodiments, the one or more hydrophilic polymer layers comprises a branched polymer. In some embodiments, the one or more hydrophilic polymer layers comprises a polymer comprisingpoly ethylene glycol. In some embodiments, the nucleotide conjugate comprises a common core. In some embodiments, the one or more nucleotide moieties are coupled to the common core. In some embodiments, the common core comprises a polymer, a micelle, a liposome, a microparticle, a nanoparticle, ora quantum dot. In some embodiments, the polymer comprises a protein. In some embodiments, wherein in (e) the nucleotide conjugate is included in a mixture of a plurality of nucleotide conjugates, wherein each of the plurality of nucleotide conjugates comprises different types of nucleotide moieties from each other. In some embodiments, the different types of the nucleotide moieties comprise at least three different types of the nucleotide moieties. In some embodiments, the one or more nucleotide moieties do not comprise a blocking group coupled thereto. In some embodiments, the detecting in (f) comprises imaging the cell or the tissue using one or more image sensors. In some embodiments, the one or more image sensors comprises a photodetector array. In some embodiments, the method further comprises washing the cell or the tissue following (f) to remove the nucleotide conjugate from the cell or the tissue. In some embodiments, the method further comprises 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 the primed index sequence or derivative thereof. In some embodiments, the detectable label comprises a fluorophore. In some embodiments, the detectable label comprises a quantum dot. In some embodiments, the conditions sufficient to couple the target-binding sequence to at least a portion of the target in (b) comprises an incubation temperature, wherein the incubation temperature is about20-45°C. In some embodiments, the conditions sufficientto couple the targetbinding sequence to at least a portion of the target in (b) comprises a salt. In some embodiments, the DNA comprises a modification. In some embodiments, the modification is a methyl modification. In some embodiments, the modification is a phosphoryl modification. In some embodiments, the probe comprises 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 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 padlock probe. In some embodiments, the method further comprises before (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, performing the amplification reaction in (c) comprises performing a rolling circle amplification reaction using a polymerizing enzyme. In some embodiments, the polymerizing enzyme is a phi29 polymerase. In some embodiments, the cell or the tissue is permeabilized using a solvent. In some embodiments, the solventis methanol. In some embodiments, the solvent is acetone. In some embodiments, the cell or the tissue is permeabilized using a detergent. In some embodiments, the detergent is saponin. In some embodiments, the detergent is Triton X-100.

[0007] Aspects disclosed herein provide methods for identifying a ribonucleic acid in situ, the method comprising: (a) providing a cell or tissue that has been permeabilized, wherein the cell or the tissue comprises the ribonucleic acid; (b) directing a nucleic acid molecule to the cell or the tissue, wherein the nucleic acid molecule comprises a target-binding sequence, under conditions sufficientto couple the target-binding sequence to at least a portion of the ribonucleic acid, wherein the nucleic acid molecule further comprises an index sequence; (c) directing a primer nucleic acid sequenceto the cell or the tissue under conditions sufficientto bind the primer nucleic acid sequence to at least a portion of the one or more amplicons to produce a primed index sequence; (d) directing the cell or the tissue with a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide in the primed index sequence or derivative thereof, under conditions suitable to form a binding complex between the one or more nucleotide moieties and the nucleotide of the primed index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; (e) detecting the binding complex within the cell or tissue, thereby identifying the ribonucleic acid. In some embodiments, the method further comprises providing the cell in (a). In some embodiments, the cell is a cancer cell. In some embodiments, the method further comprises providingthe tissue in (a). In some embodiments, the tissue is a formalin-fixed paraffin embedded tissue. In some embodiments, the tissue is a fresh-frozen tissue. In some embodiments, the cell or the tissue is extracted from a subject. In some embodiments, the subject is a human subject. In some embodiments, the ribonucleic acid is a messenger ribonucleic acid. In some embodiments, the ribonucleic acid is a micro ribonucleic acid. In some embodiments, the ribonucleic acid is a ribosomal ribonucleic acid. In some embodiments, the ribonucleic acid is a transfer ribonucleic acid. In some embodiments, the ribonucleic acid is a transfer-messenger ribonucleic acid. In some embodiments, the ribonucleic acid comprises a modification. In some embodiments, the modification comprises a methyl group. In some embodiments, the nucleic acid molecule is a deoxyribonucleic acid. In some embodiments, the nucleic acid molecule is about 10-80 nucleotides in length. In some embodiments, the nucleic acid molecule is about 20-60 nucleotides in length. In some embodiments, the nucleic acid molecule is about 30-50 nucleotides in length. In some embodiments, the nucleic acid molecule comprises a modification. In some embodiments, the modification is a 5’ phosphorylation. In some embodiments, the modification is an internucleotide linkage. In some embodiments, the target-binding sequence is about 5-25 nucleotides in length. In some embodiments, the method further comprises introducing an 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 padlock probe. In some embodiments, the method further comprises 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 comprises amplifying the index sequence. In some embodiments, the amplifying comprises performing rolling circle amplification using a polymerizing enzyme. In some embodiments, the polymerizing enzyme is a phi29 polymerase. In some embodiments, the amplifying generates multiple copies of the index sequence or a reverse complement of the index sequence. In some embodiments, the conditions sufficient to couple the target-binding sequence to at least a portion of the ribonucleic acid in (b) comprises a salt. In some embodiments, the conditions sufficient to couple the target-binding sequence to at least a portion of the ribonucleic acid in (b) comprises an incubation temperature, wherein the incubation temperature is about 2045°C. In some embodiments, the conditions sufficient to couple the target-binding sequence to at least a portion of the ribonucleic acid in (b) comprises a chaotropic 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 a ribonucleic acid. In some embodiments, the nucleic acid is a 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 4-50 nucleotides in length. In some embodiments, atleastaportion of the index sequence recognizes the primer nucleic acid sequence. In some embodiments, the primer nucleic acid sequence comprises a blocking group. In some embodiments, the blocking group inhibits incorporation of another nucleotide into the primer nucleic acid sequence. In some embodiments, the blocking group comprises a 3’-0-azidogroup, a 3’-0-azidomethyl group, a 3’ -O-alkyl hydroxylamino group, a 3’-phosphorothioate group, a 3’-0-malonyl group, or a 3’-0-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 reverse complement of the index sequence. In some embodiments, the primed index sequence comprises a double-stranded nucleic acid. In some embodiments, the primed index sequence comprises a single-stranded nucleic acid. In some embodiments, the cell or the tissue is immobilized on 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 an interior surface of a flow cell. In some embodiments, the interior surface of the flow cell comprises one or more hydrophilic polymer layers. In some embodiments, the one or more hydrophilic polymerlayers comprises a branched polymer. In some embodiments, the one or more hydrophilic polymer layers comprises a polymer comprising polyethylene glycol. In some embodiments, the nucleotide conjugate comprises a common core. In some embodiments, the one or more nucleotide moieties are coupled to the common core. In some embodiments, the common core comprises a polymer, a micelle, a liposome, a microparticle, a nanoparticle, or a quantum dot. In some embodiments, the polymer comprises a protein. In some embodiments, in (d) the nucleotide conjugate is included in a mixture of a plurality of nucleotide conjugates, wherein each of the plurality of nucleotide conjugates comprises different types of nucleotide moieties from each other. In some embodiments, the different types of the nucleotide moieties comprise at least three different types of the nucleotide moieties. In some embodiments, the one or more nucleotide moieties do not comprise a blocking group coupled thereto. In some embodiments, the detecting in (e) comprises imagingthe cell orthe tissue using one or more image sensors. In some embodiments, the one or more image sensors comprises a photodetector array. In some embodiments, the method further comprises washing the cell or the tissue following (e) to remove the nucleotide conjugate from the cell or the tissue. In some embodiments, the method further comprises repeating steps (d) and (e) with a second nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a second nucleotide in the primed index sequence or derivative thereof. In some embodiments, the detectable label comprises a fluorophore. In some embodiments, the detectable label comprises a quantum dot.

[0008] Aspects disclosed herein provide systems for identifying a RNA in situ, the system comprising: a computing device comprising one or more computer processors, an operating system configured to perform executable instructions, a memory, and a computer program including instructions comprising any one of the methods described herein. In some embodiments, the system further comprises the RNA.

[0009] Aspects disclosed herein provide systems for identifying a polypeptide in situ, the system comprising: a computing device comprising one or more computer processors, an operating system configured to perform executable instructions, a memory, and a computer program including instructions comprising any one of the methods described herein. In some embodiments, the system further comprises the polypeptide. In some embodiments, the system further comprises the polypeptide-binding probe.

[0010] Aspects disclosed herein provide systems for identifying a DNAm situ, the system comprising: a computing device comprising one or more computer processors, an operating system configured to perform executable instructions, a memory, and a computer program includinginstructions comprising any oneof the methods describedherein. In some embodiments, the system further comprises the DNA. In some embodiments, the system further comprises a cell. In some embodiments, the cell is a cancer cell. In some embodiments, the system further comprises a tissue. In some embodiments, the tissue is a formalin-fixed paraffin embedded tissue. In some embodiments, the tissue is a fresh-frozen tissue. In some embodiments, the cell or tissue is extracted from a subject. In some embodiments, the subject is a human subject. In some embodiments, the system further comprises the probe. In some embodiments, the probe is a padlock probe. In some embodiments, the padlock probe comprises a nucleic acid. In some embodiments, the system further comprises the 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 polymerizing enzyme. In some embodiments, the polymerizing enzyme is a phi29 polymerizing enzyme. In some embodiments, the system further comprises a salt. In some embodiments, the salt comprises NaCl, MgCh, orCaCh. In some embodiments, the systemfurther 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 comprises a detergent. In some embodiments, the detergent is saponin. In some embodiments, the detergent is Triton X-l 00. In some embodiments, the system further comprises a substrate. In some embodiments, the substrate is an interior surface of a flow cell. In some embodiments, the one or more computer processors comprises a central processing unit. In some embodiments, the one or more computer processors comprises a graphic processing unit. In some embodiments, the one or more computer processors comprises a general-purpose processing unit. In some embodiments, the one or more processors comprises a field-programmable gate array. In some embodiments, the system further comprises a fluidic system. In some embodiments, the fluidic system is configured to provide fluid flow control capability for delivering reagents to a sample in a flow cell.

[0011] Aspects disclosed herein provide systems for identifying a ribonucleic acid in situ, the system comprising: a computing device comprising one or more computer processors, an operating system configured to perform executable instructions, a memory, and a computer program including instructions comprising any one of the methods disclosed herein. In some embodiments, the system further comprises the cell. In some embodiments, the cell is a cancer cell. In some embodiments, the system further comprises the tissue. In some embodiments, the tissue is a formalin-fixed paraffin embedded tissue. In some embodiments, the tissue is a fresh-frozen tissue. In some embodiments, the cell or the tissue is extracted from a subject. In some embodiments, the subject is a human subject. In some embodiments, the system further comprises the ribonucleic acid. In some embodiments, the system further comprises the nucleic acid molecule. In some embodiments, the system further comprises a primer nucleic acid sequence. In some embodiments, the system further comprises an index-binding molecule. In some embodiments, the index-binding molecule comprises a nucleic acid. In some embodiments, Ihe index-binding molecule is a padlock probe. In some embodiments, the index-binding molecule is configured to bind to the index 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 polymerizing enzyme. In some embodiments, the polymerizing enzyme is a phi29 polymerase. In some embodiments, the system further comprises a salt. In some embodiments, the salt comprises NaCl, MgCh, or CaCh. In some embodiments, the system further comprises a solvent. In some embodiments, the solvent is a polar solvent. In some embodiments, the system further comprises a chaotropic reagent. In some embodiments, the system further comprises a substrate. In some embodiments, the substrate is an interior surface of a flow cell. In some embodiments, the one or more computer processors comprises a central processing unit. In some embodiments, the one or more computer processors comprises a graphic processing unit. In some embodiments, the one or more computer processors comprises a general-purpose processing unit. In some embodiments, the one or more processors comprises a field-programmable gate array. In some embodiments, the system further comprises a fluidic system. In some embodiments, the fluidic system is configured to provide fluid flow control capability for delivering reagents to a sample in a flow cell.

[0012] Aspects disclosed herein provide kits for identifying a RNA in situ, the kit comprising: (a) a probe comprising (i) an index sequence, and (ii) an RNA-binding sequence, wherein the RNA-binding sequence recognizes to at least a portion of the target; (b) a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide of a primed index sequence, wherein the nucleotide conjugate is configured to form a binding complex between the one or more nucleotide moieties and the nucleotide of the primed index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (b) instructions for identifying the RNA in situ, wherein the instructions comprise any one of the methods disclosed herein.

[0013] Aspects disclosed herein provide kits for identifying a polypeptide in situ, 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 to 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 nucleotide of a primed index sequence, wherein the nucleotide conjugate is configured to form a binding complex between the one or more nucleotide moieties and the nucleotide of the primed index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (d) instructions for identifying the polypeptide in situ, wherein the instructions comprise any one of the methods disclosed herein.

[0014] Aspects disclosed herein provide kits for identifying a DNA in situ, the kit comprising: (a) a probe comprising (i) an index sequence, and (ii) an RNA-binding sequence, wherein the RNA-binding sequence recognizes to at least a portion of the target; (b) a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide of a primed index sequence, wherein the nucleotide conjugate is configured to form a binding complex between the one or more nucleotide moieties and the nucleotide of the primed index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (c) instructions for identifying the RNA in situ, wherein the instructions comprise any one 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 derivative thereof to produce the primed index sequence. In some embodiments, the probe is a padlock probe. In some embodiments, the padlock 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 polymerizing enzyme. In some embodiments, the polymerizing enzyme is a phi29 polymerase. In some embodiments, the kit further comprises a buffer. In some embodiments, the buffer comprises a salt. In some embodiments, the salt comprises NaCl, MgCl2, or CaCl2. In some embodiments, the buffer comprises a solvent. In some embodiments, the solventis polar. In some embodiments, the buffer comprises a chaotropic agent. In some embodiments, the kit further comprises a solvent. In some embodiments, the solvent is methanol. In some embodiments, the solvent is acetone. In some embodiments, the kit further comprises a detergent. In some embodiments, the detergent is saponin. In some embodiments, the detergent is Triton X-100. In some embodiments, the kit further comprises a flow cell. In some embodiments, the flow cell comprises a sub strate configured to be coupled to the cell or the tissue.

[0015] Aspects disclosed herein provide kits for identifying a ribonucleic acid in situ, the kit comprising: (a) a nucleic acid molecule comprising (i) an index sequence, and (ii) a targetbinding 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 a nucleotide in the primed index sequence, wherein the nucleotide conjugate is configured to form a binding complex between the one or more nucleotide moieties and the nucleotide of the primed index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (c) instructions for identifying the ribonucleic acid in situ, wherein the instructions comprise any one 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 derivative thereof to produce a primed index sequence. In some embodiments, the kit further comprises an indexbindingmolecule. In some embodiments, the index-binding molecule comprises a nucleic acid. In some embodiments, the index-binding molecule is a padlock probe. In some embodiments, the index-binding molecule is configured to bind to the index sequence. 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 polymerizing enzyme. In some embodiments, the polymerizing enzyme is aphi29 polymerase. In some embodiments, the kitfurther comprises abuffer. In some embodiments, the buffer comprises a salt. In some embodiments, the salt comprises NaCl, MgCl2, or CaCl2. In some embodiments, the buffer comprises a solvent. In some embodiments, the solventis polar. In some embodiments, the buffer comprises a chaotropic agent. In some embodiments, the kit further comprises a flow cell. In some embodiments, the flow cell comprises a substrate configured to be coupled to the cell or the tissue.

[0016] Aspects disclosed herein provide methods for in situ sequencing, the methods comprising a) providing a biological sample deposited on a solid support, wherein the biological sample harbors a plurality of RNA including at least a first target RNA; b) providing a plurality of padlock probes wherein individual padlock probes in the plurality comprise a 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 and second binding arms, wherein the first and second binding arms of a padlock probe can hybridize to portions of a target RNA molecule to form an RNA-padlock probe complex, wherein individual padlock probes comprises any one or any combination of two or more sequences including: (i) a universal binding site for a sequencing primer; (ii) a universal binding site for an amplification primer; (iii) a universal binding site for a compaction oligonucleotide; and / or (iv) an optional target barcode sequence which uniquely identifies a target RNA; c) contacting the biological sample with the plurality of padlock probes wherein the contacting is conducted under a condition suitable for moving the plurality of the padlock probes into the biological sample and suitable for hybridizing individual padlock probes to at least a portion of their cognate target RNA inside the biological sample thereby forming a plurality of RNA-padlock probe complexes, wherein the first and second binding arms of individual padlock probes hybridize to proximal positions on a target RNA molecule to form a circularized padlock probe having a nick or gap between the hybridized first and second binding arms; d) contacting the biological sample with a padlock probe closing reagent, wherein the contacting is conducted under a condition suitable for moving the padlock probe closing reagent into the biological sample, and the condition is suitable for generating a plurality of covalently closed padlock probes; e) contacting the biological sample with a rolling circle amplification reagent, wherein the contacting is conducted under a condition suitable for moving the rolling circle amplification reagent into the biological sample; f) conducting rolling circle amplification inside the biological sample thereby generating a plurality of concatemers inside the biological sample, wherein the rolling circle amplification reaction is conducted in the presence of a plurality of compaction oligonucleotides, wherein individual compaction oligonucleotides comprise a 5' region that binds a first portion of a concatemer molecule and a 3' region that binds a second portion of the same concatemer molecule thereby pulling together distal portions of the concatemer molecule causing compaction of the concatemer molecule to form a DNA nanoball; and g)sequencing the plurality of concatemer molecules inside the biological sample by contacting the plurality of concatemers inside the biological sample with (i) a plurality of universal sequencing primers, (ii) a plurality of sequencing polymerases, and (iii) a plurality of detectably labeled multivalent molecules, under a condition suitable for hybridizing the plurality of universal sequencing primers to their respective universal sequencing primer binding sites on the concatemers and conducting a plurality of polymerase-catalyzed sequencing cycles thereby generatinga plurality of sequencing read products insidethe biological sample, wherein individual detectably labeled multivalent molecules comprise a core attached to a plurality of nucleotide- arms, wherein the nucleotide-arms are attached to a nucleotide moiety.

[0017] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCORPORATION BY REFERENCE

[0018] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The novel features of the inventive concepts are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present inventive concepts will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the inventive concepts are utilized, and the accompanying drawings of which:

[0020] FIGS. 1A-1H illustrate the steps utilizing a non-limiting examples of multivalent binding composition (e.g. nucleotide conjugate) for sequencing a target nucleic acid: FIG. 1A illustrates a non-limiting example 4-of attaching target nucleic acid to a surface; FIG. IB illustrates clonally the target nucleic acid to form clusters of amplified target nucleic acid molecules; FIG. IC illustrates a non-limiting example of priming the target nucleic acid to produce a primed target nucleic acid; FIG. ID illustrates a non-limiting example of contacting the primed target nucleic acid to the binding complex and polymerase to form a binding complex; FIG. IE illustrates a non-limiting example of the images of the binding complex captured on the surface; FIG. IF illustrates a non-limiting example of extending the primer strand by one nucleotide; FIG. 1G illustrates a non-limiting example of another cycle of contacting the primed target nucleic acid to the binding complex and polymerase to form a binding complex; and FIG. 1H illustrates non-limiting examples of the images of binding complex captured on the surface in subsequent sequencing cycles.

[0021] FIG. 2 A-2B illustrate a non-limiting example of detectingtargetnucleic acid using the nucleotide conjugates (e.g. detectable nucleotide conjugates). FIG. 2A shows the step of contacting the polymerase and nucleotide conjugates (e.g. detectable nucleotide conjugates) to some nucleic acid molecules; FIG 2B shows the binding complex formed between the polymerase, nucleotide conjugates (e.g. detectable nucleotide conjugates), and the target nucleic acid molecules.

[0022] FIG. 3 shows a flow chart outlining the steps for sequencing a target nucleic acid and extending the primer strand through a single base addition.

[0023] FIG. 4 shows a flow chart outlining the steps for sequencing a target nucleic acid and extending the primer strand through incorporating the nucleotide on the particle-nucleotide conjugate.

[0024] FIG. 5A-5C shows schematic representations of non-limiting examples of varying configurations of the nucleotide conjugates (e.g. detectable nucleotide conjugates): FIG. 5A shows nucleotide conjugates (e.g. detectable nucleotide conjugates) having various multi-arm configurations; FIG. 5B shows a polymer-nucleotide conjugate having the polymer branch radiating from the center; and FIG. 5C shows nucleotide conjugates (e.g. detectable nucleotide conjugates) having the binding moiety biotin.

[0025] FIG. 6 shows a generalized graphical depiction of the increase in signal intensity that has been observed during binding, persistence, and washing and removal of multivalent substrates.

[0026] FIGS. 7A-7G show fluorescence images of multivalentpolyethyleneglycol(PEG) polymer-nucleotide (base-labeled) conjugates, having an effective nucleotide concentration of 500 nanomolar (nM) and varying PEG branch length, after contacting to a support surface comprising DNA templates (comprising G or A as the first base; prepared using rolling circle amplification (RCA)) in an exposure buffer comprising 20 nM klenow polymerase and 2.5 mM Sr+2. Images were acquired after washing with an imaging buffer having the same composition as the exposure buffer but lacking nucleotides and polymerase. Panels show images obtained using multivalent PEG-nucleotide ligands with arm lengths as follows. FIG. 7A: IK PEG. FIG. 7B: 2K PEG. FIG. 7C: 3K PEG. FIG. 7D: 5K PEG. FIG. 7E: 10KPEG. FIG. 7F: 20KPEG. FIG. 7G shows images obtained using 10K PEG and an inactive klenow polymerase comprising the mutation D882H. FIG. 7H shows images obtained using 10K PEG and an inactive klenow polymerase comprising the mutation D882E. FIG. 71 shows images obtained using 10K PEG and WO 2025 / 170937                                   PCT / US2025 / 014502 an inactive klenow polymerase comprising the mutation D882A. FIG. 7G shows images obtained using 10K PEG and an active wild type klenow polymerase.

[0027] FIG. 8 shows a quantitative representation of the fluorescence intensities in the images shown in FIGS. 7A-7F, separated by color value, with orange trace corresponding to the red label (Cy3 label; A bases) and blue trace correspondingto the green label (Cy 5 lab el; G bases).

[0028] FIGS. 9A-9J show fluorescence images ofthe steps in a sequencing reaction using multivalent PEG-substrate compositions. FIG. 9A. Red and green fluorescent images post exposure of DNARCA templates (G and A first base) to 500 nM base labeled nucleotides (A-Cy3 and G-Cy5) in exposure buffer containing 20 nM Klenow polymerase and 2.5 mM Sr+2. Images were collected after washing with imaging buffer with the same composition as the exposure buffer but containing no nucleotides or polymerase. Contrast was scaled to maximize visualization of the dimmest signals, but no signals persisted following washing with imaging buffer (a. inset). FIGS. 9B-9E: fluorescence images showing multivalent PEG-nucleotide (baselabeled) ligands PB1 (FIG. 9B), PB2 (FIG. 9C), PB3 (FIG. 9D), and PB5 (FIG. 9E) having an effective nucleotide concentration of 500 nM after mixing in the exposure buffer and imaging in the imaging buffer as described above. FIG. 9F: fluorescence image showing multivalent PEG-nucleotide (base-labeled) ligand PB5 at 2.5uM after mixing in the exposure buffer and imaging in the imaging buffer as above. FIGS. 9G-9I. Fluorescence images showing further base discrimination by exposure of the binding complex to inactive mutants of klenow polymerase (FIG. 9G. D882H; FIG. 9H. D882E; FIG. 91. D882A) vs. the wild type Klenow (control) enzyme (FIG. 9J)

[0029] FIGS. 10A-10B show the efficacy of the multivalent reporter compositions in determining the base sequence of aDNA sequence over 5 sequencing cycles: FIG. 10A shows images and expected sequences for templates taken after each sequencing cycle; and FIG. 10B shows aligned sequencing results utilizing the images taken in FIG. 10A.

[0030] FIG. 11 shows normalized fluorescence from multivalent substrates bound to DNA clusters as in FIG. 9, with the substrate complexes formed in the presence (condition B) and absence (condition A) of Triton-XlOO (0.016%).

[0031] FIG. 12 shows normalized fluorescence of multivalent substrates and free nucleotides. (Top) Two replicates of a multivalent substrate bound to DNA clusters (Conditions A and B) vs. binding complexes formed using labeled free nucleotides (Condition C) after 1 minute; (Bottom) Time course of fluorescence from multivalent substrate complexes over the course of 60 min.

[0032] FIG. 13 shows a non-limiting example of a computing device; in this case, a device with one or more processors, memory, storage, and a network interface. WO 2025 / 170937                                   PCT / US2025 / 014502

[0033] FIG. 14 shows a non-limiting example of a web / mobile application provision system; in this case, a system providing browser-based and / or native mobile user interfaces.

[0034] FIG. 15 shows a non-limiting example of a cloud-based web / mobile application provision system; in this case, a system comprising an elastically load balanced, auto-scaling web server and application server resources as well synchronously replicated databases.

[0035] FIG. 16 shows a schematic of a multivalent molecule comprising a core attached to a plurality of nucleotide-arms

[0036] FIG. 17 shows a schematic of a multivalent molecule comprising a dendrimer core attached to a plurality of nucleotide arms.

[0037] FIG. 18 shows a schematic of a multivalent molecule comprising a core attached to a plurality of nucleotide arms, where the nucleotide arms comprise a biotin, a spacer, a linker, and a nucleotide moiety.

[0038] FIG. 19 shows a schematic of a nucleotide-arm comprising a core attachment moiety, a spacer, a linker, and a nucleotide moiety.

[0039] FIG. 20 shows the chemical structure for a spacer (top), and various linkers.

[0040] FIG. 21 shows chemical structures of various linkers.

[0041] FIG. 22 shows chemical structures of various linker attached to nucleotide moieties.

[0042] FIG. 23 shows chemical structures of various linker attached to nucleotide moieties.

[0043] FIG. 24 shows chemical structures of various linker attached to nucleotide moieties.

[0044] FIG. 25 shows the chemical structure of a biotinylated nucleotide arm. In this example, the nucleotide moiety is connected to the linker via a propargyl amine attachment at the 5 position of a pyrimidine base or the 7 position of a purine base.

[0045] FIG. 26 shows a schematic of a guanine tetrad (e.g. G-tetrad).

[0046] FIG. 27 shows a schematic of an intramolecular G-quadruplex structure.

[0047] FIG. 28 shows a schematic of a workflow for generating circularized padlock probes, comprisinghybridizingfirstand second padlock probes to the first and second targetRNA molecules (respectively) to generate first and second circularized padlock probes (respectively).

[0048] FIG. 29 shows schematic of a workflow that is a continuation of the schematic shown in FIG. 28.

[0049] FIG. 30 shows a schematic of a workflow for generating circularized padlock probes, comprisinghybridizingfirstand second padlock probes to the first and second targetRNA molecules (respectively) to generate first and second circularized padlock probes (respectively).

[0050] FIG. 31 shows a schematic of a workflow that is a continuation of the workflow shown in FIG. 30.

[0051] FIG. 32 shows a schematic of a workflow for generating circularized padlock probes, comprisinghybridizingfirstand second padlock probes to the first and second targetRNA molecules (respectively) to generate first and second circularized padlock probes (respectively).

[0052] FIG. 33 shows a schematic of a workflow that is a continuation of the workflow shown in FIG. 32.

[0053] FIG. 34 shows three graphs of data from a sequencing run wherein padlock probes were hybridized to RNA inside a biological sample, subj ected to gap fill-in and ligation to generate circular nucleic acids which were subjected to rolling circle amplification to generate concatemer molecules inside the biological sample. The concatemers were subjected to a two-stage sequencing workflow for 100 sequencing cycles. The top graph shows intensity measurements. The middle graph shows background measurements. The bottom graph shows phasing and prephasing measurements.

[0054] FIG. 35 shows generated inside a cell.

[0055] FIG. 36 shows generated inside the cell.

[0056] FIG. 37 shows generated inside the cell.

[0057] FIG. 38 shows a schematic of a workflow for sequencing a concatemer that is a schematic of a workflow for sequencing a concatemer that is a schematic of a workflow for sequencing a concatemer that is a schematic of a workflow for sequencing a concatemer that is generated inside the cell.

[0058] FIG. 39 shows a schematic of a workflow for detecting a target in situ involving direct detection of the target and formation of a binding complex.

[0059] FIG. 40 shows a schematic of a workflow for detecting a target in situ involving direct detection of the target, performing an amplification reaction to form an amplicon, and formation of a binding complex with the amplicon.

[0060] FIG. 41 shows a schematic of a workflow for detecting an RNA in situ involving direct detection of the RNA, performing an amplification reaction to form an amplicon, and formation of a binding complex with the amplicon.

[0061] FIG. 42 shows a schematic of a workflow for detecting a DNA in situ involving direct detection of the DNA, performing an amplification reaction to form an amplicon, and formation of a binding complex with the amplicon. WO 2025 / 170937                                   PCT / US2025 / 014502

[0062] FIG. 43 shows a schematic of a workflow for detecting a polypeptide in situ involving direct detection of the polypeptide, performing an amplification reaction to form an amplicon, and formation of a binding complex with the amplicon. DETAILED DESCRIPTION

[0063] Provided herein are kits, systems, and methods related to detecting a target nucleic acid in situ in a biological sample. The present disclosure provides novel solutions and approaches to addressing many of the shortcomings of existing technologies. Multiple components may be used to facilitate the detection of the target nucleic acid, including a nucleic acid molecule, a detectable nucleotide conjugate, and a primer nucleic acid sequence. The nucleic acid molecule may comprise an index sequence that corresponds with the target nucleic acid. The detecting may comprise binding the nucleic acid molecule to the target nucleic acid and contacting the nucleic acid molecule with a primer nucleic acid sequence to form a primed index sequence. The nucleotide conjugate (e.g. detectable nucleotide conjugatejmay be added to the sample and a binding complex may be formed between at least two copies of the primed index sequence and the detectable nucleotide conjugate. The binding complex may be detected to reveal the identity of the terminal nucleotide of the primed index sequence. This process may be repeated to reveal the nucleotide identities of each of the nucleotides within the index sequence, thereby identifying the target nucleic acid.

[0064] The methods described herein may relate to detecting one or more binding complexes as described above and thereby identifying the target nucleic acid. The systems described herein may comprise a computing device comprising a computer program with instructions to perform any one of the methods described herein. The computing device may comprise a number of components, including but not limited to one or more processors, an operating system, and a memory. The kits described herein may be used for performing any one of the methods described herein. The kits may include a nucleic acid molecule, a detectable nucleotide conjugated, and instructions for use. DEFINITIONS

[0065] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intendedto have the same meaning as is 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 ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0066] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0067] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0068] As used herein, the term “about” a numb er refers to th at numb er plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.

[0069] As used herein, a “detectable nucleotide conjugate” refers to a composition comprising a plurality of nucleotides and a detectable label attached to a common core. The one or more nucleotides can include unlabeled and labeled nucleotides. The nucleotides can include a blocking group. The detectable label can be a fluorophore. The plurality of nucleotides and the detectable label can be attached to the common core covalently or non-covalently.

[0070] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0071] As used herein, “nucleic acid” (also referred to as a “polynucleotide” or “oligonucleotide”) is a polymer of two or more nucleotides joined by covalent intemucleosidic linkages, or variants or functional fragments thereof. In naturally occurring examples of nucleic acids, the intemucleoside linkage is typically a phosphodiester bond. However, other examples optionally comprise other internucleoside linkages, such as phosphorothiolate linkages and may or may not comprise a phosphate group. Nucleic acids include double- and single-stranded deoxyribonucleic acid (DNA), as well as double- and single-stranded ribonucleic acid (RNA), DNA / RNA hybrids, peptide-nucleic acids (PNAs), hybrids between PNAs and DNA or RNA, and may also include other types of nucleic acid modifications.

[0072] As used herein, a “nucleotide” refers to a nucleotide, nucleoside, oranalogthereof. The nucleotide refers to both naturally occurring and chemically modified nucleotides and can include but are not limited to a nucleoside, a ribonucleotide, a deoxyribonucleotide, a protein -nucleic acid residue, or derivatives. Examples of the nucleotide includes an adenine, a thymine, a uracil, a cytosine, a guanine, or residue thereof; a deoxyadenine, a deoxythymine, a deoxyuracil, a deoxy cytosine, a deoxyguanine, or residue thereof; a adenine PNA, a thymine PNA, a uracil PNA, a cytosine PNA, a guanine PNA, or residue or equivalents thereof, an N- or C-glycoside of a purine or pyrimidine base (e.g., a deoxyribonucleoside containing 2-deoxy-D-ribose or ribonucleoside containing D-ribose).

[0073] “Complementary” as used herein, refers to the topological compatibility or matching together of interacting surfaces of a ligand molecule and its receptor. Thus, the receptor and its ligand can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other. With respect to nucleic acid sequences, complementary refers the sequence of a nucleic acid having the propensity to hybridize to another nucleic acid sequence by virtue of the nucleotides that makeup the nucleic acid sequence. Nucleic acids bind to each other through the formation of 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 aligned antiparallel to each other, the nucleotide bases at each position in the sequences will be complementary.

[0074] “Branched polymer”, as used herein, refers to a polymer having a plurality of functional groups that help conjugate a biologically active molecule such as a nucleotide, and the functional group can be either on the side chain of the polymer or directly attaches to a central core or central backbone of the polymer. The branched polymer can have linear backbone with one or more functional groups coming off the backbone for conjugation. The branched polymer can also be a polymer having one or more sidechains, wherein the side chain has a site suitable for conjugation. Examples of the functional group include but are limited to hydroxyl, ester, amine, carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, hydrazide, thiol, alkanoic acid, acid halide, isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate.

[0075] “Polymerizing enzyme” as used herein, refers to an enzyme that contains a nucleotide bindingmoiety and helps formation of abindingcomplexbetweenatargetnucleicacid and a complementary nucleotide. The polymerase can have one or more activities including, but notlimited to, base analog detection activities, DNA polymerization activity, reversetranscriptase activity, DNA binding, strand displacement activity, and nucleotide binding and recognition. The WO 2025 / 170937                                   PCT / US2025 / 014502 polymerase can include catalytically inactive polymerase, catalytically active polymerase, reverse transcriptase, and other enzymes containing a nucleotide binding moiety.

[0076] “Persistence time,” as used herein, refers to the length of time that a binding complex remains stable without any binding component dissociates from the binding complex. The binding complex may comprise a target nucleic acid, a polymerase, and a detectable nucleotide conjugate. The binding complex may comprise the target nucleic acid, a polymerase and nucleotide (e.g., a detectable nucleotide). The persistence time is indicative of the stability of the binding complex and strength of the binding interactions. Persistence time can be measured by observing the onset and / or duration of a binding complex, such as by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides may be present in a binding complex, thus allowing the signal from the label to be detected during the persistence time of the binding complex. One example label is a fluorescent label.

[0077] It is understood the use of the alternative term (e.g., “or”) is taken to mean either one or both or any combination thereof of the alternatives.

[0078] The term “and / or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other. For example, the term “and / oi” as used in a phrase such as “A and / or B” herein is intended to include: “A andB”; “A orB”; “A” (A alone); and “B” (B alone). In a similar manner, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass 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 alone); “B” (B alone); and “C” (C alone).

[0079] As used herein and in the appended claims, terms “comprising”, “including”, “having” and “containing”, and their grammatical variants, as used herein are intended to be nonlimiting so that one item or multiple items in a list do not exclude other items that can be substituted or added to the listed items. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0080] As used herein, the terms “about” and “approximately” refer to a value or composition that is within an acceptable error range for the 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, “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art. Alternatively, “about” or “approximately” can mean a range of up to 10% (i.e., ±10%) or more depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-foldof a value. When particular values or compositions are provided in the instant 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. Also, where ranges and / or subranges of values are provided, the ranges and / or subranges can include the endpoints of the ranges and / or subranges.

[0081] The term “polymerase” and its variants, as used herein, comprises an enzyme comprising a domain that binds a nucleotide (or nucleoside) where the polymerase can form a complex having a template nucleic acid and a complementary nucleotide. The polymerase can have one or more activities including, but not limited to, base analog detection activities, DNA polymerization activity, reverse transcriptase activity, DNAbinding, strand displacement activity, and nucleotide binding and recognition. A polymerase can be any enzyme that can catalyze polymerization of nucleotides (including analogs thereof) into a nucleic acid strand. Typically but not necessarily such nucleotide polymerization can occur in a template-dependent fashion. Typically, a polymerase comprises one or more active sites at which nucleotide binding and / or catalysis of nucleotide polymerization can occur. In some embodiments, a polymerase includes other enzymatic activities, such as for example, 3 'to 5' exonuclease activity or 5 'to 3' exonuclease activity. In some embodiments, a polymerase has strand displacing activity. A polymerase can include without limitation naturally occurring polymerases and any subunits and truncations 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 that retain the ability to catalyze nucleotide polymerization (e.g., catalytically active fragment). The polymerase includes catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes comprising a nucleotide binding domain. In some embodiments, a polymerase can be isolated from a cell, or generated using recombinant DNA technology or chemical synthesis methods. In some embodiments, a polymerase can be expressed in prokaryote, eukaryote, viral, or phage organisms. In some embodiments, a polymerase can be post-translationally modified proteins or fragments thereof. A polymerase can be derived from a prokaryote, eukaryote, virus or phage. A polymerase comprises DNA-directed DNA polymerase and RNA-directed DNA polymerase.

[0082] As used herein, the term “strand displacing” refers to the ability of a polymerase to locally separate strands of double-stranded nucleic acids and synthesize a new strand in a template-based manner. Strand displacing polymerases displace a complementary strand from a template strand and catalyze new strand synthesis. Strand displacing polymerases include mesophilic and thermophilic polymerases. Strand displacing polymerases include wild type enzymes, and variants including exonuclease minus mutants, mutant versions, chimeric enzymes and truncated enzymes. Examples of strand displacing polymerases include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bea DNA polymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase. The phi29DNA polymerase canbe wild type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0083] Theterms “nucleic acid”, "polynucleotide" and "oligonucleotide" and other related terms used herein are used interchangeably andreferto polymers of nucleotides and are notlimited 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), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids (PNA) and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or doublestranded. Nucleic acids comprise polymers of nucleotides, where the nucleotides include natural or non-natural bases and / or sugars. Nucleic acids comprise naturally-occurring intemucleosidic linkages, for example phospho di ester linkages. Nucleic acids can lack a phosphate group. Nucleic acids comprise non-natural intemucleoside linkages, including phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, nucleic acids comprise a one type of polynucleotides or a mixture of two or more different types of polynucleotides.

[0084] The term “operably linked” and “operably joined” or related terms as used herein refers to juxtaposition of components. The juxtaposition components can be linked together covalently. For example, two nucleic acid components can be enzymatically ligated together where the linkage that joins together the two components comprises phosphodiester linkage. A first and second nucleic acid component can be linked together, where the first nucleic acid component can confer a function on a second nucleic acid component. For example, linkage 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 example, a transgene (e.g., a nucleic acid encoding a polypeptide ora nucleic acid sequence of interest) can be ligated to a vector where the linkage permits expression or functioning of the transgene sequence contained in the vector. In some embodiments, a transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects expression of the transgene. In some embodiments, the vector comprises at least one host cell regulatory sequence, including a promoter sequence, enhancer, transcription and / or translation initiation sequence, transcription and / or translation termination sequence, polypeptide secretion signal sequences, and the like. In some embodiments, the host cell regulatory sequence controls expression of the level, timing and / or location of the transgene.

[0085] The terms “linked”, “joined”, “attached”, “appended” and variants thereof comprise any type of fusion, bond, adherence or association between any combination of compounds or molecules that is of sufficient stability to withstand use in the particular procedure. The procedure can include but are not limited to: nucleotide binding; nucleotide incorporation; de-blocking(e.g., removal of chain-terminating moiety); washing; removing; flowing; detecting imaging and / or identifying. Such linkage can comprise, for example, covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonding, bonds or associations involving van der Waals forces, mechanical bonding, and the like. In some embodiments, such linkage occurs intramolecularly, for example linking together the ends of a single-stranded or double-stranded linear nucleic acid molecule to form a circular molecule. In some embodiments,, such linkage can occur between a combination of different molecules, or between a molecule and a non-molecule, including but not limited to: linkage between a nucleic acid molecule and a solid surface; linkage between a protein and a detectable reporter moiety; linkage between a nucleotide and detectable reporter moiety; and the like. Some examples of linkages can be found, for example, in Hermanson, G., “Bioconjugate Techniques”, Second Edition (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques forthe Biomedical Sciences”, London: Macmillan (1998); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998).

[0086] The term “primer” and related terms used herein refers to an oligonucleotide that is capable of hybridizing with a DNA and / or RNA polynucleotide template to form a duplex molecule. Primers comprise natural nucleotides and / or nucleotide analogs. Primers can be recombinant nucleic acid molecules. Primers may have any length, but typically range from 4-50 nucleotides. A typical primer comprises a 5 ’ end and 3 ’ end. The 3 ’ end of the primer can include a 3’ OH moiety which serves as a nucleotide polymerization initiation site in a polymerase-catalyzed primer extension reaction. Alternatively, the 3’ end of the primer can lack a 3’ OH moiety, or can include a terminal 3’ blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any one nucleotide, or more than one nucleotide, along the length of the primer can be labeled with a detectable reporter moiety. A primer can be in solution (e.g., a soluble primer) or can be immobilized to a support (e.g., a capture primer).

[0087] The term “template nucleic acid”, “template polynucleotide”, “target nucleic acid” “target polynucleotide”, “template strand” and other variations refer to a nucleic acid strand that serves as the basis nucleic acid molecule for any of the reiterative sequencing methods describe herein. The template nucleic acid can be single-stranded or double-stranded, or the template nucleic acid can have single-stranded or double-stranded portions. The template nucleic acid can be obtained from a naturally-occurring source, recombinant form, or chemically synthesized to include any type of nucleic acid analog. The template nucleic acid can be linear, circular, or other forms. The template nucleic acids can include an insert portion having an insert sequence. The template nucleic acids can also include at least one adaptor sequence. The insert portion can be isolated in any form, including chromosomal, genomic, organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned, amplified, cDNA, RNA such as precursor mRNA or mRNA, oligonucleotides, whole genomic DNA, obtained from fresh frozen paraffin embedded tissue, needle biopsies, circulating tumor cells, cell free circulating DNA, or any type of nucleic acid library. The insert portion can be isolated from any source including from organisms such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, viruses cells, tissues, normal or diseased cells ortissues, body fluids includingblood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, semen, environmental samples, culture samples, or synthesized nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. The insert portion can be isolated from any organ, includinghead, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testicles, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs. Thetemplate nucleicacid canbe subjected to nucleic acid analysis, including sequencing and composition analysis.

[0088] The term “adaptor” and related terms refers to oligonucleotides that can be operably linked to a target polynucleotide, where the adaptor confers a function to the co-joined adaptor-target molecule. Adaptors compriseDNA, RNA, chimericDNA / RNA, or analogs thereof. Adaptors can include at least one ribonucleoside residue. Adaptors can be single -stranded, doublestranded, or have single-stranded and / or double-stranded portions. Adaptors canbe configured to be linear, stem-looped, hairpin, or Y-shaped forms. Adaptors canbe any length, including 4-100 nucleotides or longer. Adaptors can have blunt ends, overhang ends, or a combination of both. Overhangends include 5’ overhangand 3’ overhangends. The 5’ end of a single-stranded adaptor, or one strand of a double-stranded adaptor, can have a 5 ’ phosphate group or lack a 5 ’ phosphate group. Adaptors can include a 5 ’ tail that does not hybridize to a target polynucleotide (e.g., tailed adaptor), or adaptors can be non-tailed. An adaptor can include a sequence that is complementary to at least a portion of a primer, such as an amplification primer, a sequencing primer, or a capture primer (e.g., soluble or immobilized capture primers). Adaptors can include a random sequence or degenerate sequence. Adaptors can include at least one inosine residue. Adaptors can include at least one phosphorothioate, phosphorothiolate and / or phosphoramidate linkage. Adaptors can include a barcode sequence which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. In some embodiments, a barcode sequence uniquely identifies a target RNA. Adaptors can include a unique identification sequence (e.g., unique molecular index, UMI; or a unique molecular tag) that can be used to uniquely identify a nucleic acid molecule to which the adaptor is appended. In some embodiments, a unique identification sequence can be used to increase error correction and accuracy, reduce the rate of false-positive variant calls and / or increase sensitivity of variant detection. Adaptors can include at least one restriction enzyme recognition sequence, including any one or any combination of two or more selected from a group consisting of type I, type II, type III, type IV, type Hs or type IIB.

[0089] In some embodiments, any of the amplification primer sequences, sequencing primer sequences, capture primer sequences, target capture sequences, circularization anchor sequences, sample barcode sequences, target barcode sequences, spatial barcode sequences, or anchor region sequences can be about 3-50 nucleotides in length, or about 5-40 nucleotides in length, or about 5-25 nucleotides in length.

[0090] The term “universal sequence” and related terms refers to a sequence in a nucleic acid molecule that is common among two or more polynucleotide molecules. For example, an adaptor having a universal sequence can be operably joined to a plurality of polynucleotides so that the population of co-joined molecules carry the same universal adaptor sequence. Examples of universal adaptor sequences include an amplification primer sequence, a sequencing primer sequence or a capture primer sequence (e.g., soluble or immobilized capture primers).

[0091] When used in reference to nucleic acid molecules, the terms “hybridize” or “hybridizing” or “hybridization” or other related terms refers to hydrogen bonding between two different nucleic acids to form a duplex nucleic acid. Hybridization also includes hydrogen bondingbetween two differentregions of a single nucleic acid molecule to form a self -hybridizing molecule havinga duplex region. Hybridization can comprise Watson-Crick or Hoogstein binding to form a duplex double-stranded nucleic acid, or a double-stranded region within a nucleic acid molecule. The double-stranded nucleic acid, or the two differentregions of a single nucleic acid, may be wholly complementary, or partially complementary. Complementary nucleic acid strands need not hybridize with each other across their entire length. The complementary base pairing can be the standard A-T or C-G base pairing, or can be other forms of base-pairing interactions. Duplex nucleic acids can include mismatched base-paired nucleotides.

[0092] When used in reference to nucleic acids, the terms “extend”, “extending”, “extension” and other variants, refers to incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation comprises polymerization of one or more nucleotides into the terminal 3’ OH end of a nucleic acid strand, resulting in extension of the nucleic acid strand. Nucleotide incorporation can be conducted with natural nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent fashion. Any suitable method of extending a nucleic acid molecule may be used, including primer extension catalyzed by a DNA polymerase or RNA polymerase.

[0093] The term “nucleotides” and related terms refers to a molecule comprising an aromatic base, a five carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Canonical or non-canonical nucleotides are consistent with use of the term. The phosphate in some embodiments comprises a monophosphate, diphosphate, or triphosphate, or corresponding phosphate analog. The term “nucleoside” refers to a molecule comprising an aromatic base and a sugar. Nucleotides and nucleosides can be non-labeled or labeled with a detectable reporter moiety.

[0094] Nucleotides (and nucleosides) typically comprise a hetero cyclic base including substituted or unsubstituted nitrogen-containing parent heteroaromatic ring which are commonly found in nucleic acids, including naturally-occurring, substituted, modified, or engineered variants, or analogs of the same. The base of a nucleotide (or nucleoside) is capable of forming Watson-Crick and / or Hoogstein hydrogen bonds with an appropriate complementary base. Example bases include, but are not limited to, purines and pyrimidines such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N6-A2-isopentenyladenine (6iA), N6-A2-isopentenyl-2-methylthioadenine (2ms6iA), N6-methyladenine, guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O6-methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O4-methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; nebularine; inosines; hydroxymethylcytosines; 5 -methycytosines; base (Y); as well as methylated, glycosylated, and acylated base moieties; and the like. Additional example bases can be found in Fasman, 1989, in “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press, Boca Raton, Fla.

[0095] Nucleotides (and nucleosides) typically comprise a sugar moiety, such as carbocyclic moiety (Ferraro and Gotor 2000 Chern. Rev. 100: 4319-48), acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez, etal., 1997 Bioorganic & Medicinal Chemistry Letters vol. 7: 3013-3016), and other sugar moieties (Joeng, etal., 1993 J. Med. Chem. 36: 2627-2638; Kim, et al., 1993 J. Med. Chern. 36: 30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No. 5,558,991). The sugar moiety comprises: ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoriboxyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoriboxyl; 3'-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[0096] In some embodiments, nucleotides comprise a chain of one, two or three phosphorus atoms where the chain is typically attached to the 5’ carbon of the sugar moiety via an ester or phosphoramide linkage. In some embodiments, the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene or ethylene. In some embodiments, the phosphorus atoms in the chain include substituted side groups includingO, S orBH3. In some embodiments, the chain includesphosphate groups substituted with analogs includingphosphoramidate,phosphorothioate, phosphordithioate, and O-methylphosphoroamidite groups.

[0097] The term “reporter moiety”, “reporter moieties” or related terms refers to a compound that generates, or causes to generate, a detectable signal. A reporter moiety is sometimes called a “label”. Any suitable reporter moiety may be used, including luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent, phosphorescent, chromophore, radioisotope, electrochemical, mass spectrometry, Raman, hapten, affinity tag, atom, or an enzyme. A reporter moiety generates a detectable signal resulting from a chemical or physical change (e.g., heat, light, electrical, pH, salt concentration, enzymatic activity, or proximity events). A proximity event includes two reporter moieties approaching each other, or associating with each other, or binding each other. It is well known to one skilled in the art to select reporter moieties so that each absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from the other reporter moieties to permit monitoring the presence of different reporter moieties in the same reaction or in different reactions. Two or more different reporter moieties can be selected having spectrally distinct emission profiles, or having minimal overlapping spectral emission profiles. Reporter moieties can be linked (e.g., operably linked) to nucleotides, nucleosides, nucleic acids, enzymes (e.g., polymerases or reverse transcriptases), or support (e.g., surfaces).

[0098] A reporter moiety (or label) comprises a fluorescent label or a fluorophore. Example fluorescent moieties which may serve as fluorescent labels or fluorophores include, but are not limited to fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA -fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives such as BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanine and derivatives such as indolium based cyanine dyes, benzo-indoliumbased cyanine dyes, pyridium based cyanine dyes, thiozolium based cyanine dyes, quinolinium based cyanine dyes, imidazolium based cyanine dyes, Cy 3, Cy5, lanthanide chelates and 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 derivatives thereof, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin 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, Oreg.) 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or any combination thereof. Cyanine dyes may exist in either sulfonated or non-sulfonated forms, and consist of two indolenin, benzo-indolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3, (which may comprise 1-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-2-(3-{l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-l-en-l-yl)-3,3-dimethyl-3H-indolium or l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-2-(3-{l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-l,3-dihydro-2H-indol-2-ylidene}prop-l-en-l-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which may comprise 1-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-2-((lE,3E)-5-((E)-l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-l,3 -dien-1-yl)-3,3-dimethyl-3H-indol-l-ium or 1-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-ox oh exyl)-2-((1E, 3E)-5-((E)-l -(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-ox oh exyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-l,3-dien-l-yl)-3,3-dimethyl-3H-indol-l-ium-5-sulfonate), and Cy7 (which may WO 2025 / 170937                                   PCT / US2025 / 014502 comprise            l-(5-carboxypentyl)-2-[(lE,3E,5E,7Z)-7-(l-ethyl-l,3-dihydro-2H-indol-2- ylidene)hepta-l,3,5-trien-l-yl]-3H-indolium or 1 -(5-carb oxypentyl)-2-[(lE,3E,5E, 7Z)-7-(l-ethyl-5 -sulf o-1,3-dihy dro-2H-indol-2-ylidene)hepta-1,3,5-trien-1 -y l]-3H-indolium-5 -sulf onate), where “Cy” stands for 'cyanine', and the first digit identifies the number of carbon atoms between two indolenine groups. Cy2 which is an oxazole derivative rather than indolenin, and the benzo-derivatized Cy3.5, Cy5.5 and Cy7.5 are exceptions to this rule.

[0099] In some embodiments, the reporter moiety can be a FRET pair, such that multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET may comprise excitation exchange (Forster) transfers, or electron-exchange (Dexter) transfers.

[00100] When used in referenceto nucleic acids, the terms “amplify”, “amplifying”, “amplification”, and other related terms include producing multiple copies of an original polynucleotide template molecule, where the copies comprise a sequence that is complementary to the template sequence, or the copies comprise a sequence that is the same as the template sequence. In some embodiments, the copies comprise a sequence that is substantially identical to a template sequence, or is substantially identical to a sequence that is complementary to the template sequence.

[00101] The term “support” as used herein refers to a substrate that is designed for 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), polypeptides, saccharides, lipids, a single cell or multiple cells. Examples of biological samples include but are not limited to saliva, phlegm, mucus, blood, plasma, serum, urine, stool, sweat, tears and fluids from tissues or organs.

[00102] 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 porosity. 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 comprising a capillary or interior surface of a capillary.

[00103] In some embodiments, the surface of the support can be substantially smooth. In some embodiments, the support can be regularly or irregularly textured, including bumps, etched, pores, three-dimensional scaffolds, or any combination thereof.

[00104] In some embodiments, the support comprises a bead having any shape, including spherical, hemi-spherical, cylindrical, barrel-shaped, toroidal, disc-shaped, rod-like, conical, triangular, cubical, polygonal, tubular or wire-like.

[00105] The support can be fabricated from any material, including but not limited to glass, fused-silica, silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), WO 2025 / 170937                                   PCT / US2025 / 014502 polymethylmethacrylate (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 compositions of both glass and plastic substrates are contemplated.

[00106] The support can have a plurality (e.g., two or more) of nucleic acid templates immobilized thereon. The plurality of immobilized nucleic acid template s havethe same sequence or have different sequences. In some embodiments, individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized to a different site on the support. In some embodiments, two or more individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized to a site on the support.

[00107] The term “array” refers to a support comprising a plurality of sites located at pre-determined locations on the support to form an array of sites. The sites can be discrete and separated by interstitial regions. In some embodiments, the pre-determined sites on the support can be arranged in one dimension in a row or a column, or arranged in two dimensions in rows and columns. In some embodiments, the plurality of pre-determined sites is arranged on the support in an organized fashion. In some embodiments, the plurality of pre-determined sites is arranged in any organized pattern, including rectilinear, hexagonal patterns, grid patterns, patterns having reflective symmetry, patterns having rotational symmetry, or the like. The pitch between different pairs of sites can be that same or can vary. In some embodiments, the support comprises at least 102 sites, at least 103 sites, at least 104 sites, at least 105 sites, at least 106 sites, at least 107 sites, at least 108 sites, atleast 109 sites, at least 1010 sites, atleast 1011 sites, at least 1012 sites, at least 1013 sites, at least 1014 sites, at least 1015 sites, or more, where the sites are located at predetermined locations on the support. In some embodiments, a plurality of pre-determined sites on the support (e.g., 102- 1015 sites or more) are immobilized with nucleic acid templates to form a nucleic acid template array. In some embodiments, the nucleic acid templates that are immobilized at a plurality of pre-determined sites by hybridization to immobilized surface capture primers, or the nucleic acid templates are covalently attached to the surface capture primer. In some embodiments, the nucleic acid templates that are immobilized at a plurality of pre-determined sites, for example immobilized at 102 - 1015 sites or more. In some embodiments, the immobilized nucleic acid templates are clonally-amplified to generate immobilized nucleic acid clusters at the plurality of pre-determined sites. In some embodiments, individual immobilized nucleic acid clusters comprise linearclusters, or comprise single-stranded or double-stranded concatemers.

[00108] In some embodiments, a support comprising a plurality of sites located at random locations on the support is referred to herein as a support having randomly located sites thereon. The location of the randomly located sites on the support are not pre-determined. The plurality of randomly-located sites is arranged on the support in a disordered and / or unpredictable fashion. In some embodiments, the support comprises at least 102 sites, at least 103 sites, at least 104 sites, at least 105 sites, at least 106 sites, at least 107 sites, at least 108 sites, at least 109 sites, atleast 1010 sites, atleast 1011 sites, atleast 1012 sites, atleast 1013 sites, atleast 1014 sites, atleast 1015 sites, or more, where the sites are randomly located on the support. In some embodiments, a plurality of randomly located sites on the support (e.g., 102- 1015 sites or more) are immobilized with nucleic acid templates to form a support immobilized with nucleic acid templates. In some embodiments, the nucleic acid templates that are immobilized at a plurality of randomly located sites by hybridization to immobilized surface capture primers, or the nucleic acid templates are covalently attached to the surface capture primer. In some embodiments, the nucleic acid templates that are immobilized at a plurality of randomly located sites, for example immobilized at 102 - 1015 sites or more. In some embodiments, the immobilized nucleic acid templates are clonally-amplified to generate immobilized nucleic acid clusters at the plurality of randomly located sites. In some embodiments, individual immobilized nucleic acid clusters comprise linear clusters, or comprise single-stranded or double-stranded concatemers.

[00109] In some embodiment, the plurality of immobilized surface capture primers on the support are in fluid communication with each other to permit flowing a solution of reagents (e.g., nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, and the like) onto the support so that the plurality of immobilized surface capture primers on the support can be essentially simultaneously reacted with the reagents in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized surface capture primers can be used to conduct nucleic acid amplification reactions (e.g., RCA, MD A, PCR and bridge amplification) essentially simultaneously on the plurality of immobilized surface capture primers.

[00110] In some embodiment, the plurality of immobilized nucleic acid clusters on the support are in fluid communication with each other to permit flowing a solution of reagents (e.g., enzymes, nucleotides, divalent cations, and the like) onto the support so that the plurality of immobilized nucleic acid clusters on the support can be essentially simultaneously reacted with the reagents in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized nucleic acid clusters can be usedto conduct nucleotide binding assays and / or conduct nucleotide polymerization reactions (e.g., primer extension or sequencing) essentially simultaneously on the plurality of immobilized nucleic acid clusters, and optionally to conduct detection and imaging for massively parallel sequencing.

[00111] When used in reference to immobilized enzymes, the term “immobilized” and related terms refer to enzymes (e.g., polymerases) that are attached to a support through WO 2025 / 170937                                   PCT / US2025 / 014502 covalentbond ornon-covalentinteraction, or attached to a coating on the support, or buried within a matrix formed by a coating on the support.

[00112] When used in reference to immobilized nucleic acids, the term “immobilized” and related terms refer to nucleic acid molecules that are attached to a support through covalent bond or non-covalent interaction, or attached to a coating on the support, or buried within a matrix formed by a coating on the support, where the nucleic acid molecules include surface capture primers, nucleic acid template molecules and extension products of capture primers. Extension products of capture primers includes nucleic acid concatemers (e.g, nucleic acid clusters).

[00113] In some embodiments, one or more nucleic acid templates are immobilized on the support, for example immobilized at the sites on the support. In some embodiments, the one or more nucleic acid templates are clonally-amplified. In some embodiments, the one or more nucleic acid templates are clonally-amplified off the support (e.g., in-solution) and then deposited onto the support and immobilized on the support. In some embodiments, the clonal amplification reaction of the one or more nucleic acid templates is conducted on the support resulting in immobilization on the support. In some embodiments, the one or more nucleic acid templates are clonally-amplified (e.g., in solution or on the support) using a nucleic acid amplification reaction, including any one or any combination of: polymerase chain reaction (PCR), multiple displacement amplification (MD A), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, and / or single-stranded binding (SSB) protein-dependent amplification.

[00114] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. METHODS

[00115] Provided herein are methods related to detecting one or more targets (e.g. RNAs, DNAs, polypeptides) in situ in a biological sample. The methods described herein do not comprise cDNA synthesis. A nucleic acid molecule containing a target-binding sequence and an index sequence may be introduced to the biological sample such that it couples to the target (e.g. the RNA, the DNA, or the polypeptide). A primer nucleic acid sequence may be added to the sample and binds to the index sequence to form a primed index sequence. A nucleotide conjugate (e.g. a detectable nucleotide conjugate), which includes a detectable label and one or more nucleotide moieties may be added to the sample to form a binding complex. The binding complex WO 2025 / 170937                                   PCT / US2025 / 014502 may be detected, which corresponds to the sequence associated with the index sequence and thereby the identity of the target (e.g. the RNA, the DNA, or the polypeptide).

[00116] The binding complex involved in the methods disclosed herein can include at least one nucleotide conjugate (e.g. detectable nucleotide conjugate), which may include a plurality of copies of the same nucleotide conjugated thereto. When the nucleotide is complementary to the index sequence, the nucleotide conjugate may form abindingcomplex with a polymerase and the primed index sequence, and the binding complex exhibits increased stability and longer persistence time than the binding complex formed using a single unconjugated or untethered nucleotide.

[00117] The methods described herein offer several advantages over other in situ workflows. For example, the biological sample may be placed on a solid support, for example a planar support comprising glass or plastic which can be fabricated into any shape and size. Assembly of a hybridization chamber on the support may not be needed. Preparation of chemically-washed glass beads for cell adherence may also not be needed. The support can be passivated with a coating that promotes cell adhesion to the support. The coating does not need to be formulated to include tethered capture primers. The support, having a cell sample adhered thereon, can be easily adapted to fit into an existing flow cell holder / cradle which is fluidically connected to an automated fluid dispensing system and configured on a fluorescent microscope. Any combination of the steps for conducting in situ sequencing can be performed in an automated mode usingthe fluid dispensing system, including cell seeding, cell fixation, cell permeabilization, probe hybridization, probe ligation reaction, amplification, and sequencing.

[00118] Another advantage of the methods described herein, is the formation of amplicons (e.g. concatemers) inside the biological sample. The amplicons may be single-stranded and collapse into compact DNA nanoballs, where each nanoball carries numerous tandem copies of a polynucleotide unit along their lengths, where the polynucleotide unit includes a target sequence-of-interest and at least a universal sequencing primer binding site. Each polynucleotide unit can bind a sequencing primer, a sequencing polymerase and a nucleotide reagent (e.g., detectably labeled multivalent molecules), to form a detectable sequencing complex (e.g., a detectable ternary complex). Each nanoball may carry numerous detectable sequencing complexes. The compact nature of the nanoballs increases the local concentration of detectably -labeled nucleotide reagents that are used during the sequencing workflow which increases the signal intensity emitted from a nanoball to give a discrete detectable signal which can be imaged as a fluorescent spot inside the biological sample. Each spot may correspond to an amplicon and each amplicon corresponds to a target molecule in the biological sample. Multiple spots can be WO 2025 / 170937                                   PCT / US2025 / 014502 detected and imaged simultaneously in the biological sample. Alternatively, multiple spots can be detected and imaged in separate batches in the biological sample.

[00119] An aspect of the disclosure provides a method for identifying a target (e.g an RNA, a DNA, or a polypeptide) in situ using the components shown in FIG. 39. The method comprises (a) providing a cell or tissue that has been permeabilized, wherein the cell or tissue comprises the target (3901); (b) directing a nucleic acid molecule (3902) to the cell or tissue (3902), wherein the nucleic acid molecule comprises a target-binding sequence under conditions sufficient to couple the target-binding sequence to at least a portion of the target (e.g. an RNA, or a DNA), wherein the nucleic acid molecule comprises an index sequence; (c) directing (3904) a primer nucleic acid sequence (3905) to the cell or tissue under conditions sufficient to bind the primer nucleic acid sequence (3905) to at least a portion of the one or more amplicons to produce a primed index sequence; (d) directing (3906) the cell of tissue with a nucleotide conjugate (e.g a detectable nucleotide conjugate) comprising(i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide in the primed index sequence or derivative thereof, under conditions suitable to form abinding complex (3907)betweentheoneormorenucleotidemoieties and the nucleotide of the primed index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (e) detecting the binding complex within the cell or tissue, thereby identifying the target.

[00120] Another aspect of the disclosure provides a method for An aspect of the disclosure provides a method for identifying a target (e.g. an RNA, a DNA, or a polypeptide) in situ using the components shown in FIG. 40. The method comprises (a) providing a cell or tissue that has been permeabilized, wherein the cell or tissue comprises the target (4001); (b) directing a nucleic acid molecule (4003) to the cell or tissue (4002), wherein the nucleic acid molecule comprises a target-binding sequence under conditions sufficient to couple the target-binding sequence to at least a portion of the target (e.g. an RNA, a DNA or a polypeptide), wherein the nucleic acid molecule comprises an index sequence; (c) directing (4004) a probe (e.g. a padlock probe) (4005) to the cell or tissue under conditions sufficient to binding the probe to the nucleic acid molecule; (d) amplifying (4006) (e.g. performing a rolling circle amplification reaction) of the index sequence to generate one or more amplicons, wherein the one or more amplicons comprises multiple copies of the index sequence or derivative thereof to form one or more amplicons (4007); (e) directing (4008) a primer nucleic acid sequence (4009) to the cell or tissue under conditions sufficient to bind the primer nucleic acid sequence (4007) to at least a portion of the one or more amplicons to produce a primed index sequence; (f) directing the cell of tissue with a nucleotide conjugate (e.g. a detectable nucleotide conjugate) comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide in the primed index WO 2025 / 170937                                   PCT / US2025 / 014502 sequence or derivative thereof, under conditions suitable to form a binding complex (4010) between the one or more nucleotide moieties and the nucleotide of the primed index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (g) detecting the binding complex within the cell or tissue, thereby identifying the target.

[00121] Another aspect of the disclosure provides a method for identifying a, RNA in situ using the components shown in FIG. 41. The method comprises (a) providing a cell or tissue that has been permeabilized, wherein the cell or tissue comprises the RNA (4101); (b) directing a probe (4103) to the cell or tissue (4102), wherein the probe (4103) comprises an RNA-binding sequence under conditions sufficient to couple the RNA-binding sequence to at least a portion of the RNA, wherein the probe further comprises an index sequence; (c) amplifying (4104) (e.g. performing a rolling circle amplification reaction) of the index sequence to generate one or more amplicons, wherein the one or more amplicons comprises multiple copies of the index sequence or derivativethereof to form one or more amplicons (4105); (d) directing (4106) a primer nucleic acid sequence (4107) to the 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 primed index sequence; (e) directing the cell or tissue with a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide in the primed index sequence or the derivative thereof, under conditions suitable to form a binding complex (4108) between the one or more nucleotide moieties and the nucleotide of the primed index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (f) detecting the binding complex within the cell or tissue, thereby identifying the RNA.

[00122] Another aspect of the disclosure provides a method for identifying a, DNA in situ using the components shown in FIG. 42. The method comprises (a) providing a cell or tissue that has been permeabilized, wherein the cell or tissue comprises the DNA (4201), wherein the DNA is not cDNA; (b) directing a DNA-binding sequence (4203) to the cell or tissue (4202), wherein the probe comprises an index sequence, under conditions sufficient to couple the DNA-binding sequence to at least a portion of the DNA; (c) amplifying (4204) (e.g. performing a rolling circle amplification reaction) of the index sequence to generate one or more amplicons, wherein the one or more amplicons comprises multiple copies of the index sequence or derivative thereof to form one or more amplicons (4205); (d) directing (4206) a primer nucleic acid sequence (4207) to the 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 primed index sequence; (e) directing the cell or tissue with a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide in the primed index sequence or the derivative thereof, under conditions suitable to form a binding complex (4208) between the one or more WO 2025 / 170937                                   PCT / US2025 / 014502 nucleotide moieties and the nucleotide of the primed index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (f) detecting the binding complex within the cell or tissue, thereby identifying the DNA.

[00123] Another aspect of the disclosure provides a method for identifying a polypeptide (e.g. a protein) in situ usingthe components shown in FIG. 43. The method comprises (a) providing a cell or tissue that has been permeabilized, wherein the cell or tissue comprises the polypeptide (4301); (b) directing a polypeptide-binding probe (43 03) to the cell or tissue (4302), wherein the polypeptide-binding probe comprises an polypeptide-binding moiety and an oligonucleotide (4304) coupled thereto, under conditions sufficient to couple the polypeptide-binding probe to at least a portion of the polypeptide; (c) directing (4305) the cell or tissue wilh a probe (4307) comprising an oligonucleotide binding probe under conditions sufficient to couple the oligonucleotide-binding probe to at least a portion of the oligonucleotide or the polypeptide-binding probe, wherein the probe further comprises an index sequence; (c) amplifying (4307) (e.g. performing a rolling circle amplification reaction) of the index sequence to generate one or more amplicons, wherein the one or more amplicons comprises multiple copies of the index sequence or derivative thereof to form one or more amplicons (4308); (e) directing (4309) a primer nucleic acid sequence (4310) to the 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 primed index sequence; (f) directing the cell or tissue with a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide in the primed index sequence or the derivative thereof, under conditions suitable to form a binding complex (4311) between the one or more nucleotide moieties and the nucleotide of the primed index sequence without incorporating the one or more nucleotide moieties into the primer nucleic acid sequence; and (f) detecting the binding complex within the cell or tissue, thereby identifying the DNA. i. Biological samples

[00124] The methods described herein comprise detecting one or more targets (e.g RNAs, DNAs, and / or polypeptides) in a biological sample. The biological sample may comprise a tissue or be a tissue. In some embodiments, the biological sample may comprise a cell. In some embodiments, the biological sample may be a cell. The biological sample may be from an organism. The biological sample may be from a cell culture. In some embodiments, the biological sample may be preserved, havingbeen harvested previously. The biological sample may comprise a whole cell, a plurality of whole cells, an intact tissue or an intact tumor. In some embodiments, the biological sample may comprise a fresh biological sample, a freshly-frozen biological sample, a sectioned biological sample, or an FFPE biological sample. In some embodiments, the biological sample may comprise one or more living cells or non-living cells. In some embodiments, the biological sample comprises an intact cell or tissue. In some embodiments, the biological sample comprises a cell or tissue that is / are not lysed. In some embodiments, the biological sample comprises a single cell, a plurality of cells, a tissue, an organ, an organism, or section of these biological samples.

[00125] In some embodiments, the biological sample is derived from eukaryotes (such as animals, plants, fungi, protista), archaebacteria, 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 a primary or immortalized cell line from a rodent, porcine, feline, canine, bovine, equine, primate, or human cell lines. In some embodiments, the biological sample may be obtained from a virus, fungus, or prokaryote. In some embodiments, the biological sample can be obtained from an animal, insect or plant. In some embodiments, the biological sample comprises one or more virally-infected cells. In some embodiments, the biological sample can be obtained from any organism including human, simian, ape, canine, feline, bovine, equine, murine, porcine, caprine, lupine, ranine, piscine, plant, insect or bacteria. In some embodiments, the biological sample can be obtained from an organ including but not limited to brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testicles, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs.

[00126] The biological sample may be a solid sample, such as a tissue biopsy. The biological sample may be a fluid sample, such as blood ora component of blood (e.g., serum or plasma). In some embodiments, the biological sample is obtained from skin, heart, lung, kidney, breath, bone marrow, stool, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, breast, pancreas, cerebral spinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gall bladder, colon, intestine, brain, cavity fluids, sputum, pus, micropiota, meconium, breastmilk, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluid, tears, ocular fluids, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, fingernails, skin cells, plasma, nasal swab or nasopharyngeal wash, spinal fluid, cord blood, emphatic fluids, and / or other excretions or body tissues. The biological sample may be a cell-free sample.

[00127] The biological sample may be a 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 paraffin embedded. In some other embodiments, the tissue may be fresh-frozen. The tissue maybe sliced into slices, wherein the thickness of the slice maybe about 1-30 pm, about 2-29 pm, about 3-28 pm, about 4-27 pm, about 5-26 pm, about 6-25 pm, about 7-24 pm, about 8-23 pm, about 9-22 pm, about 10-21 pm, about 11-20 pm, about 12-19 pm, about 13-18 pm, about 14-17 pm, about 15-16 pm,about 1 pm, about2 pm, about3 pm, about4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 21 pm, about 22 pm, about 23 pm, about 24 pm, about 25 pm, about 26 pm, about 27 pm, about 28 pm, about 29 pm, or about 30 pm thick.

[00128] In some embodiments, the biological sample can be grown / cultured on a support and analysis of the cultured biological sample can be conducted on the support. Nucleic acids in the biological sample may not be released from the cells and are not captured on the support. In some cases, the nucleic acids in the biological sample may be retained in the cells and sequencedin the cells (e.g., in situ sequencing). In some embodiments, the nucleic acids in the biological sample may be subjected to an optional library prep workflow by appending at least one adaptor sequence, for example a universal adaptor sequence. The nucleic acids in the biological sample may be amplified in the cells by conducting rolling circle amplification.

[00129] The biological sample may be processed and / or analyzed in a flow cells. For example, flow cell, having cultured cells attached thereon, can be placed on a sequencing apparatus configured with at least one fluidic delivery device, at least one fluidics device (e.g., microfluidics device), at least one imaging device and / or at least one sensor to detect signals from the sequencing reactions. The sequencing reactions can be conducted with soluble sequencing primers. The sequencing primers can be designed to hybridize to a target sequence or a universal adaptor sequence. The sequencing primers can include or lack a sample index sequence (e.g, sample barcode sequence). The nucleic acids in the cultured cells can be sequenced by employing any of the nucleotide conjugates (e.g. detectable nucleotide conjugates) described herein. In some embodiments, the sequencing reactions can employ labeled or non-labeled nucleotide analogs. In some embodiments, individual cycle times can be achieved in less than 30 minutes. In some embodiments, the field of view (FOV) can exceed 1 mm2 and the cycle time for scanning large area (> 10 mm2) can be less than 5 minutes.

[00130] In some embodiments, the biological sample can be placed on a support. Provided herein are solid supports (e.g., low non-specific binding supports) further comprising a low non-specific binding surface

[00131] The biological sample may comprise cells. The cells described herein may be white blood cells, red blood cells, platelets, epithelial cells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts, skeletal muscle cells, smooth muscle cells, gametes, or cells from the heart, lungs, brain, liver, kidney, spleen, pancreas, thymus, bladder, stomach, colon, or small intestine. The cells may be normal or healthy cells. Alternately or in combination, the cells may be diseased cells, such as cancerous cells, or from pathogenic cells that are infecting a host. In some embodiments, the cell belongs to a subset of cells, such as immune cell (e.g., T cells, cytotoxic (killer) T cells, helper T cells, alphabeta T cells, gamma delta T cells, T cell progenitors, B cells, B-cell progenitors, 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, human stem cells that have been induced to differentiate, 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 trophoblasts). Other cells are contemplated and consistent with the disclosure herein.

[00132] The biological sample can be embedded in a wax, resin, epoxy or agar. The biological sample can be fixed, for example in any one or any combination of two or more of acetone, ethanol, methanol, formaldehyde, paraformaldehyde-Triton or glutaraldehyde. The biological sample can be sectioned or non-sectioned. The biological sample can be stained, destained or non-stained.

[00133] In some embodiments, the biological sample may be permeabilized 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 period of time for incubation can be at least about 10,15, 20,25, 30, 35, 40, 50, 60 or more minutes.

[00134] In some embodiments, the methods comprisetreatingthe biological sample with a permeabilization reagent that alters the cell membrane to permit penetration of experimental reagents into the cells. For example, the permeabilization reagent may remove membrane lipids from the cell membrane. In some embodiments, the biological sample can be treated with a permeabilization reagent which comprises any combination of an organic solvent, detergent, chemical compound, cross-linking agent and / or enzyme. In some embodiments, the organic solvents comprise acetone, ethanol, and methanol. In some embodiments, the detergents comprise saponin, Triton X-100, Tween-20, or sodium dodecyl sulfate (SDS), or N-lauroylsarcosine sodium salt solution. 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 protease (e.g. proteinase K). In some embodiments, the cells can be permeabilized using an alkaline condition, or an acidic condition with a protease enzyme. In some embodiments, the permeabilization reagent comprises water and / or PBS. For example, the fixed cells can be permeabilized with 70% ethanol for about 30-60 minutes, and the permeabilizing reagent can be exchanged with PBS-T (e.g., PBS with WO 2025 / 170937                                   PCT / US2025 / 014502 0.05% Tween-20). In some embodiments, the cells can be post-fixed with 3% paraformaldehyde and 0.1% glutaraldehyde for about 30-60 minutes, and washed with PBS-T multiple times.

[00135] The biological sample can be used to generate a three-dimensional polymer matrix comprising the cellular and sub-cellular components (e.g., nucleic acid molecules) of the biological sample. The three-dimensional polymer matrix can be coupled to the support described herein, covalently or non-covalently. In some embodiments, the three-dimensional polymer matrix is porous and comprises polymerized or cross-linked sub-cellular components, including the target nucleic acid molecules. A polymer matrix may be formed within a biological sample (e.g., a cell or tissue) by flowing one or more polymer precursors (e.g., monomers, such as, for example, ethylene oxide for polyethene glycol) into the biological sample and subjecting the one or more polymer precursors to polymerization or cross-linking. Priorto, during, or subsequent to formation of the polymer matrix, positions of moieties (e.g., DNA, RNA, protein) within the biological sample may be fixed, usingfor example, a fixation agent (e.g., formaldehyde). Aporous matrix may be made according to various methods. For example, a polyacrylamide gel matrix can be polymerized with biotinylated DNA molecules and acrydite-modified streptavidin monomers, using a suitable acrylamide:bis-acrylamide ratio to control the cross-linking density. Additional control over the molecular sieve size and density can be achieved by adding additional crosslinkers such as functionalized polyethylene glycols. Enablement for fixing the biological sample to a surface, as well as generating a polymer matrix within a biological sample, is provided in PCT / US2019 / 05543 4 (WO 2020 / 076976; Three Dimensional Spatial Molecular Indexing), which is hereby incorporated by reference in its entirety.

[00136] In any of the methods described herein, the biological sample may be infused with a swellable polyelectrolyte hydrogel (U.S. patent No. 10,309,879 and Chen 2015 Science 347:543, the contents ofthese documents are incorporated by referencein their entireties). In some embodiments, a fixed and permeabilized biological sample can be infused with sodium acrylate, acrylamide and a cross-linker N-N’-methylenebisacrylamide. In some embodiments, ammonium persulfate (APS) initiator and tetramethylethylenediamine (TEMED) accelerator were infused to achieve polymerization. In some embodiments, the biological sample can be infused with proteinase K for proteolysis and incubated in a digestion buffer. In some embodiments, the gel inside the biological sample can be swelled by addition of water.

[00137] In any of the methods described herein, the biological sample can be cultured on the support. In some embodiments, the methods comprise culturing the biological sample on the support under a condition suitable for expanding the biological sample for 2-10 generations or more. The cultured biological sample can generate a colony of cells. In some embodiments, the methods comprise culturing the biological sample to confluence or non- confluence. In some embodiments, the methods comprise culturing the biological sample on the support in a simple or complex cell culture media. For example, the cell culture media comprises D-MEM high glucose (e.g., from Thermo Fisher Scientific, catalog No. 11965118), fetal bovine serum (e.g., 10%FBS; for example from Thermo Fisher Scientific, catalogNo. A3160402), MEM non-essential amino acids (e.g., 0.1 mM MEM, for example from Thermo Fisher Scientific, catalogNo. 11140050), L-glutamine (e.g., 6 mM L-glutamine, for example from Thermo Fisher Scientific, catalog No. A2916801), MEM sodium pyruvate (e.g., 1 mM sodium pyruvate, for example from Thermo Fisher Scientific, catalog No. 11360070), and an antibiotic (e.g., 1% penicillin-streptomycin-glutamine, for example from Thermo Fisher, catalogNo. 10378016). In some embodiments, the methods comprise culturing the biological sample at a humidity and temperature that is suitable for culturing the cell(s) on the support. Example suitable conditions comprise approximately 37 °C with a humidified atmosphere of approximately 5-10% carbon dioxide in air. The biological sample can be cultured with suitable aeration with oxygen and / or nitrogen.

[00138] In any of the methods described herein, the term “simple cell media” or related terms refers to a cell media that typically lacks ingredients to support cell growth and / or proliferation in culture. Simple cell media can be used for example to wash, suspend, or dilute the biological sample. Simple cell media can be mixed with certain ingredients to prepare a cell media that can support cell growth and / or proliferation in culture. A simple cell media comprises any one or any combination of two or more of a buffer, a phosphate compound, a sodium compound, a potassium compound, a calcium compound, a magnesium compound and / or glucose. In some embodiments, the simple cell media comprises 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, the biological sample can be placed in a simple cell media prior to or during the step of conducting any of the nucleic acid methods described herein.

[00139] In any of the methods described herein, the term “complex cell media” or related terms refers to a cell media that can be used to support cell growth and / or proliferation in culture without supplementation or additives. Complex cell media can include any combination of two or more of a buffering system (e.g., HEPES), inorganic salt(s), amino acid(s), protein(s), polypeptide(s), carbohydrate(s), fatty acid(s), lipid(s), purine(s) and their derivatives (e.g., hypoxanthine), pyrimidine(s) and their derivatives, and / or trace element(s). Complex cell media includes fluids obtained from a fluid or tissue extract. Complex cell media includes artificial cell media. In some embodiments, complex cell media can be a serum-containing media, for example complex cell media includes fluids such as fetal bovine serum, blood plasma, blood serum, lymph fluid, human placental cord serum and amniotic fluid. In some embodiments, complex cell media can be a serum-free media, which are typically (but not necessarily) defined cell culture media. In some embodiments, complex cell media can be a chemically-defined media which typically (but not necessarily) include recombinant polypeptides, and ultra-pure inorganic and / or organic compounds. In some embodiments, complex cell media can be a protein-freemedia which include for example MEM (minimal essential media) and RPMI-1640 (Roswell Park Memorial Institute). In some embodiments, the complex cell media comprises IMDM (Iscove’s Modified Dulbecco’s Medium. In some embodiments, the complex cell media comprisesDMEM(Dulbecco’sModified Eagle’s Medium). In some embodiments, the biological sample can be placed in a complex cell media prior to or during the step of conducting any of the nucleic acid methods described herein.

[00140] In any of the methods described herein, the biological sample may comprise a fixed biological sample. In some embodiments, the biological sample can be treated with a fixation reagent (e .g., a fixing reagent) that preserves the cell and its contents to inhibit degradation and can inhibit cell lysis. For example, the fixation reagent can preserve nucleic acids (e.g. RNA) harbored by the biological sample. In some embodiments, the fixation reagent inhibits loss of nucleic acids from the biological sample.

[00141] In some embodiments, the fixation reagent can cross-link the target to prevent the target from escaping the biological sample. In some embodiments, a cross-linking fixation reagent comprises any combination of an aldehyde, formaldehyde, paraformaldehyde, formalin, glutaraldehyde, imidoesters, N-hydroxysuccinimide esters (NHS) and / or glyoxal (a bifunctional aldehyde).

[00142] 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 comprising zinc sulphate or zinc chloride. In some embodiments, the fixation reagent can denature polypeptides. In some embodiments, the fixation reagent comprises 4% w / v of paraformaldehyde to water / PBS. In some embodiments, the fixation reagent comprises 10% of 35% formaldehyde at a neutral pH. In some embodiments, the fixation reagent comprises 2% v / v of glutaraldehyde to water / PBS. In some embodiments, the fixation reagent comprises 25% of 37% formaldehyde solution, 70% picric acid and 5% acetic acid. In some embodiments, the biological sample can be fixed on the support with 4% paraformaldehyde for about 30-60 minutes and washed with PBS. In some embodiments, the biological sample can be stained, de-stained or un-stained. iL Targets

[00143] The methods described herein relate to detecting one or more targets. The one of more targets may comprise a nucleic acid (e.g. an RNA, and / or a DNA) a polypeptide, or a combination thereof. In some embodiments, the one or more targets may comprise one or more naturally-occurringnucleic acids, oneormore recombinantnucleic acids, one or more synthesized nucleic acids, or a combination thereof. The one or more targets comprising one or more nucleic acids may comprise linear forms of nucleic acids, circular forms of nucleic acids, branched forms of nucleic acids, or a combination thereof. In some embodiments, the one or more targets comprising one or more nucleic acids may comprise DNA. In some embodiments, the one or more targets may comprise genomic DNA. In some embodiments, the one ormore targets may comprise viral DNA. In some embodiments, the one or more targets may comprise cell free DNA (cfDNA). In some embodiments, the DNA may be genomic DNA, methylated DNA, un-methylated DNA, organellar DNA, or a combination thereof. In some cases, the one or more targets may comprise PCR amplified DNA. In some embodiments, the one or more targets may comprise synthetic DNA. The synthetic DNA may comprise synthetic DNA probes. The DNA may be fragmented, unfragmented, or a combination thereof. The DNA may comprise one or more modifications. For example, the DNA may comprise a methyl modification, a phosphorylationmodification, a locked nucleic acid modification, or a combination thereof.

[00144] In some embodiments, the one or more targets may comprise RNA. The RNA of the one ormore targets may comprise poly-A RNA and / or non-poly-a RNA. The RNA may comprise coding RNA, non-coding RNA, or a combination thereof. The RNA may comprise tRNA, rRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), piwi-interactingRNA (piRNA), antisense RNA, noncoding RNA, protein-encoding RNA, or a combination thereof. The one or more targets may comprise RNA that is target RNA, non-target RNA, or a combination thereof. In some cases, the RNA may have been transcribed and have undergone splicing. In some cases, the RNA may not have been spliced. In some cases, the RNA may comprise wild type RNA, mutant RNA or splice variant RNA. In some cases, the RNA may comprise pre-spliced RNA, partially spliced RNA, 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, the RNA may comprise RNA expressed by one or more cells in response to a stimulus such as heat, light, a chemical or a drug. In some embodiments, the RNA may comprise RNA found in healthy cells, diseased cells, or a combination thereof. In some embodiments, the RNA may comprise RNA transcribed from transgenic DNA sequences that are introduced into the biological sample usingrecombinantDNA 47 procedures. For example, the RNA can be transcribed from a transgenic DNA sequence that is controlled by an inducible or constitutive promoter sequence. In some embodiments, the RNA may comprise RNA that is transcribed from DNA sequences that are not transgenic. The RNA may comprise one or more modifications. For example, the RNA may comprise a phosphorylation (e.g. a 5’ phosphorylation), an inter-nucleotide linkage, or a combination thereof.

[00145] A binding complex associated with the RNA or an amplicon generated from binding a nucleic acid molecule to the RNA may be formed without performing reverse transcription of the RNA. For example, the RNA may be bound directly by a probe comprising an RNA-binding sequence. The probe may be ligated to form a circular nucleic acid and amplified using rolling circle amplification to generate one or more amplicons. The one or more amplicons may form a binding complex with a nucleotide conjugated and detected. In this case, the RNA is detected directly without performing reverse transcription.

[00146] The one or more targets may comprise one or more polypeptides. The one of more polypeptides of the one or more targets may comprise one or more proteins, one or more peptides, or a combination thereof. The one or more polypeptides may comprise one or more cellsurface proteins, one or more intracellular proteins, one or more signaling proteins, one or more protein complexes, or a combination thereof. The one or more polypeptides may comprise one or more antibodies or antibody fragments. In some cases, the one or more polypeptides may comprise one or more modifications. The one or more modifications of the one or more polypeptides may comprise one or more phosphoramidate (N-linked) formations, one or more phosphorylations, one or more adenylylations, one or more uridylylations, one or more propionylations, one or more pyroglutamate formations, one or more S-glutathionylations, one or more S-nitrosylations, one or more S-sulfenylations, one or more S-sulfinylations, one or more S-sulfonylations, one or more succinylations, one or more sulfations, one or more glycations, 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 famesylations, one or more geranylgeranylations, one or more glypiations, one or more glycosylphosphatidylinositols, one or more lipoylations, one or more flavin moiety attachments, one or more heme C attachments, one or more phosphopantetheinylations, one or more retinylidene Schiff base formations, one or more modifications of translation factors, one or more diphthamide formations, one or more ethanolamine phosphoglycerols, one or more hypusine formations, one or more beta-Lysine addition on a lysines, one or more acylations (e.g. O-acylation, N-acylation, and S-acylation), one or more acetylations, one or moreformylations, one or more alkylations, one or more amidations, one or more arginylations, one or more polyglutamylations, one or more polyglycylations, one or more butyrylations, one or more gamma-carboxylations, one or more glycosylation, one or more poly sialylations, one or more malonylations, one or more hydroxylations, one or more nucleotide additions, one or more phosphate esters (O-linked), one or more pegylations, one or more ubiquitinations, one or more SUMOylations, one or more neddylations, one or more ISGylations, one or more citrullinations, one or more deamidations, one or more eliminylations, or a combination thereof.

[00147] In some embodiments, the one or more targets may comprise nucleic acids that are clonally-amplified. In some embodiments, the present disclosure provides a method comprising clonal amplification comprising the use of a polymerase chain reaction (PCR), multiple displacement amplification (MD A), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification, circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-stranded binding (SSB) protein-dependent amplification, or any combination thereof. In some embodiments, the product of clonal amplification may be detected using the methods described herein.

[00148] The one or more targets of the instant disclosure may have a fixed threedimensional relationship within the biological sample after the biological sample is coupled to the surface. This fixed three-dimensional relationship, at least partially, enables the identification of spatial and cellular origin within the biological sample following nucleic acid identification using the systems and methods described herein. Nucleic acid analysis inside a biological sample and spatial analysis is described in U.S. Patent Application No. 17 / 356,929, filed June 24, 2021, entitled “Multivalent Binding Composition for Nucleic Acid Analysis”, which is hereby incorporated by reference in its entirety.

[00149] In some embodiments, the one or more targets comprises one or more target nucleic acid sequences. In some embodiments, the one or more targets may be a concatemer comprising multiple repeats of the one or more target nucleic acid sequences. The multiple copies of the target nucleic acid sequence within the concatemer may be covalently linked using phosphodiester bonds. The concatemer may be formed using an amplification reaction, including a rolling circle amplification reaction.

[00150] In some embodiments, two or more targets may be detected. In some embodiments, the two or more targets can each comprise the same targets (e.g. the same nucleic acid sequence). In some embodiments, the two or more targets may comprise different targets (e.g. different nucleic acid sequences). In some embodiments the one or more targets may comprise 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, at least about 80 targets, at least about 90 targets, at least about 100 targets, at least about200 targets, at least about 500 targets, at least about 1000 targets, at least about2000 targets, at least about 5000 targets, at least about 10000 targets, at least about 12000 targets, at least about 20000 targets, at least about 25000 targets, at least about 50000 targets, at least about 100000 targets, at least about 1000000 targets, or more. In some embodiments the one or more targets may comprise atmostaboutl target, at mo st about 2 targets, atmostabout3 targets, at most about 4 targets, at most about 5 targets, at most about 6 targets, at most about 7 targets, at most about 8 targets, at most about 9 targets, at most about 10 targets, at most about 20 targets, at most about 30 targets, at most about 40 targets, at most about 50 targets, at most about 60 targets, at most about 70 targets, at most about 80 targets, at most about 90 targets, at most about 100 targets, at mostabout200 targets, at most about 500 targets, atmost about 1000 targets, at mostabout2000 targets, at most about 5000 targets, at most about 10000 targets, at most about 12000 targets, at most about 20000 targets, at most about 25000 targets, at most about 50000 targets, at most about 100000 targets, or at most about 1000000 targets. m.   Nucleic a cid molecules

[00151] The methods described herein may involve coupling one or more nucleic acid molecules to the one or more targets for detection. The one or more nucleic acid molecules as described herein may comprise one or more probes. The one or more probes may comprise one or more RNA-binding sequences, one or more DNA-binding sequences, one or more oligonucleotide-binding sequences, one or more index sequences, or a combination thereof. In some cases, the one or more nucleic acid molecules may comprise one or more probes configured to bind to an RNA. The one or more probes configuredto bind to the RNA may comprise an RNA-binding sequence, an index sequence, or a combination thereof. The one or more probes configured to bind to the RNA my comprise a sequence that is at least partially complementary to a portion of the RNA sequence or reverse complement thereof. The sequence that is at least partially complementary to a portion of the RNA sequence or reverse complement thereof may be an RNA-binding sequence. In some cases, the one or more nucleic acid molecules may comprise one or more probes configured to bind to a DNA. The one or more probes configured to bind to the DNA may comprise an DNA-binding sequence, an index sequence, or a combination thereof. The one or more probes configured to bind to the DNA my comprise a sequence that is at least partially complementary to a portion of the DNA sequence or reverse complement thereof. The sequence that is at least partially complementary to a portion of the DNA sequence or reverse complement thereof may be an DNA-binding sequence. In some cases, the one or more nucleic acid molecules may comprise one or more probes configured to bind to a polypeptide-binding probe. The one or more probes configured to bind to the polypeptide-binding probe may comprise an oligonucleotide-binding probe that is at least partially complementary to an oligonucleotide of the polypeptide-binding probe. In some cases, the one or more nucleic acid molecules may comprise a target-binding sequence, an index sequence, or a combination thereof. In some cases, the one or more nucleic acid molecules may be configured to bind to at least a portion of an RNA or a reverse complement thereof.

[00152] In some embodiments, the one or more nucleic acid molecules may comprise DNA. In some embodiments, the one or more nucleic acid molecules may comprise RNA. In some embodiments, the one or more nucleic acid moleculesmay comprisea combination of DNA and RNA. In some embodiments, the one or more nucleic acid molecule may comprise a modification. In some embodiments, the modification may include a phosphorylation, an internucleotide linkage, a fluorescent dye, or a combination thereof. In some embodiments, the internucleotide linkage may comprise a locked nucleic acid residue. In some embodiments, the modification may comprise a 5 ’ modification, a 3 ’ modification, an internal modification, or a combination thereof.

[00153] The one or more nucleic acid molecules may have a variety of 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 a different length. In some embodiments, atleast one nucleic acid molecule orthe one or more nucleic acid molecules has a different length than a second nucleic acid molecule of the one or more nucleic acid molecules. In some embodiments, the length of the one or more nucleic acid molecules may be about 1-100 nucleotides, about 2-98 nucleotides, about 3-96 nucleotides, about 4-94 nucleotides, about 5-92 nucleotides, about 6-90 nucleotides, about 7-88 nucleotides, about 8-86 nucleotides, about 9-84 nucleotides, about 10-82 nucleotides, about 11-80 nucleotides, about 12-78 nucleotides, about 13-76 nucleotides, about 14-74 nucleotides, about 15-72 nucleotides, about 1670 nucleotides, about 17-68 nucleotides, about 18-66 nucleotides, about 19-64 nucleotides, about 20-62 nucleotides, about 22-60 nucleotides, about 24-58 nucleotides, about 26-56 nucleotides, about 28-54 nucleotides, about 30-52 nucleotides, about 32-50 nucleotides, about 3448 nucleotides, about36-46 nucleotides, about38-44 nucleotides, about40-42 nucleotides, atleast 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 18 nucleotides, at least about 22 nucleotides, at least about 28 nucleotides, at least about 34 nucleotides, at least about 40 nucleotides, at least about 46 nucleotides, at least about 52 nucleotides, at least about 58 nucleotides, at least about 64 nucleotides, at least about 70 nucleotides, at least about 76 nucleotides, at least about 82 nucleotides, at least about 88 nucleotides, at least about 94 nucleotides, at least about 16 nucleotides, at least about 19 nucleotides, at least about 24 nucleotides, at least about 30 nucleotides, at least about 36 nucleotides, at least about 42 nucleotides, at least about 48 nucleotides, at least about 54 nucleotides, at least about 60 nucleotides, at least about 66 nucleotides, at least about 72 nucleotides, at least about 78 nucleotides, at least about 84 nucleotides, at least about 90 nucleotides, at least about 96 nucleotides, at least about 17 nucleotides, at least about 20 nucleotides, at least about 26 nucleotides, at least about 32 nucleotides, at least about 38 nucleotides, at least about 44 nucleotides, at least about 50 nucleotides, at least about 56 nucleotides, at least about 62 nucleotides, at least about 68 nucleotides, at least about 74 nucleotides, at least about 80 nucleotides, at least about 86 nucleotides, at least about 92 nucleotides, at least about 98 nucleotides, or at least about 100 nucleotides. In some embodiments, the nucleic acid molecule is about 10-80 nucleotides in length. In some embodiments, the nucleic acid molecule is about 2060 nucleotides in length. In some embodiments, the nucleic acid molecule is about 30-50 nucleotides in length.

[00154] In some embodiments, one nucleic acid molecule may couple to a target (e.g. a nucleic acid). In some embodiments, more than one nucleic acid molecule may couple to a target (e.g. a nucleic acid). In some embodiments, one nucleic acid molecule (e.g. a probe comprising an oligonucleotide-binding probe) may couple to a probe that is bound to a target (e.g a polypeptide-bindingprobethatis bound to a polypeptide). In some embodiments, more than one nucleic acid molecule (e.g. more than one probe comprising an oligonucleotide-binding probe) may couple to a probe that is bound to a target((e.g. a polypeptide-binding probe that is bound to a polypeptide). In some embodiments, a first set of nucleic acid molecules may couple to a first target and a second set of nucleic acid molecules may couple to a second target. In some embodiments, the number of nucleic acid molecules in the first set and second set are the same. In some embodiments, the number of nucleic acid molecules in the first set and second set are different. In some embodiments, the number of nucleic acid molecules in the first set is greater than the number of nucleic acid molecules in the second set. In some embodiments, the number of nucleic acid molecules in the first set is less than the number of nucleic acid molecules in the second set. In some embodiments, a first set of nucleic acid molecules (e.g. probes comprising oligonucleotide-binding probes) may couple to a first probe that is bound to a first target (a first polypeptide-binding probe that is bound to a first polypeptide) and a second set of nucleic acid molecules (e.g. probes comprising oligonucleotide-binding probes) may couple to a second target (e.g. a second polypeptide-binding probe that is bound to a second polypeptide). In some embodiments, the number of nucleic acid molecules in the first set and second set are the same. In some embodiments, the number of nucleic acid molecules in the first set and second set are different. In some embodiments, the number of nucleic acid molecules in the first set is greater than the number of nucleic acid molecules in the second set. In some embodiments, the number of nucleic acid molecules in the first set is less than the number of nucleic acid molecules in the second set.

[00155] In some embodiments, a nucleic acid molecule of the one or more nucleic acid molecule comprises 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 comprise a nucleic acid sequence that hybridizes to the one or more targets for detection. The binding site may have a variety of lengths. In some embodiments, one binding site may have a different length than a second binding site. In some embodiments, the length of the one or more nucleic acid molecules may be about 1-100 nucleotides, about 2-98 nucleotides, about 3-96 nucleotides, about 4-94 nucleotides, about 5-92 nucleotides, about 6-90 nucleotides, about 7-88 nucleotides, about 8-86 nucleotides, about 9-84 nucleotides, about 10-82 nucleotides, about 11- 80 nucleotides, about 12-78nucleotides, about 13-76 nucleotides, about 14-74nucleotides, about 15-72 nucleotides, about 16-70 nucleotides, about 17-68 nucleotides, about 18-66 nucleotides, about 19-64 nucleotides, about 20-62 nucleotides, about 22-60 nucleotides, about 24-58 nucleotides, about26-56 nucleotides, about28-54 nucleotides, about 3 0-5 2 nucleotides, about32-50 nucleotides, about 34-48 nucleotides, about 36-46 nucleotides, about 38-44 nucleotides, about 40-42 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, at least about 13 nucleotides, at least about 14 nucleotides, at least about 17 nucleotides, at least about 20 nucleotides, at least about 26 nucleotides, at least about 32 nucleotides, at least about 38 nucleotides, at least about 44 nucleotides, at least about 15 nucleotides, at least about 18 nucleotides, at least about 22 nucleotides, at least about 28 nucleotides, at least about 34 nucleotides, at least about 40 nucleotides, at least about 46 nucleotides, at least about 16 nucleotides, at least about 19 nucleotides, at least about 24 nucleotides, at least about 30 nucleotides, at least about 36 nucleotides, at least about 42 nucleotides, at least about 48 nucleotides, at nucleotides, at nucleotides, at nucleotides, at nucleotides, at nucleotides, at nucleotides, at nucleotides, at least about 50 least about 56 least about 62 least about 68 least about 74 least about 80 least about 86 least about 92 nucleotides, at nucleotides, at nucleotides, at nucleotides, at nucleotides, at nucleotides, at nucleotides, at nucleotides, at least about 52 least about 58 least about 64 least about 70 least about 76 least about 82 least about 88 least about 94 nucleotides, at nucleotides, at nucleotides, at nucleotides, at nucleotides, at nucleotides, at nucleotides, at nucleotides, at least about 54 least about 60 least about 66 least about 72 least about 78 least about 84 least about 90 least about 96 nucleotides, at least about 98 nucleotides, or at least about 100 nucleotides in length. In some embodiments, the target-binding sequence is about 5-25 nucleotides in length.

[00156] In some embodiments, the one or more nucleic acid molecules comprise at least one index sequence. The at least one index sequence may denote the identity of the one or more targets. In some embodiments, a single index sequence corresponds one or more targets. In some embodiments, more than one index sequence corresponds to one or more targets. In some embodiments, the one or more nucleic acid molecules comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 index sequences. The at least one index sequence may be sequence orthogonal to the sequence of the one or more targets. In some embodiments, the at least one index sequence includes at least a portion of a sequence of the one or more targets.

[00157] In some cases, a nucleic acid molecule of the one or more nucleic acid molecules may comprise a binding site (e.g. a target binding site) that is adjacent to an index sequence. For example, the binding site (e.g. a target binding site) may be directly proximal to the index sequence. In another example, the binding site may be separated by at least one nucleotide from the index sequence. In some cases, the nucleic acid molecule may be bound to a target (e.g an RNA) and the index sequence may be unbound. For example, a nucleic acid molecule may comprise a target-binding sequence that is hybridized to an RNA and the nucleic acid molecule may comprise an index sequence that is not hybridized (e.g. unhybridized) to the RNA. The index sequence maybe single-stranded and amenable to hybridization by another nucleic acid sequence (e.g. an index-binding sequence).

[00158] Each of the at least one index sequence may have a variety of lengths. The length of an index sequence may be about 1-25 nucleotides, about 2-24 nucleotides, about 3-23 nucleotides, about 4-22 nucleotides, about 5-21 nucleotides, about 6-20 nucleotides, about 7-19 nucleotides, about8-18 nucleotides, about9-17 nucleotides,about 10-16 nucleotides, about Ills nucleotides, about 12-14 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, atleastabout7 nucleotides, atleastabout8 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 4-50 nucleotides in length.

[00159] In some embodiments, the index sequence comprises a primer binding site. In some embodiments, where a nucleic acid molecule comprises more than one index sequence, each index sequence comprises a primer binding site. The primer binding site may be configured to hybridize to a primer nucleic acid sequence as described herein. The primer binding site may hybridize to a primer nucleic acid sequence. The primer binding sites of different index sequences may have a variety of lengths. The length of an primer binding site may be about 1-25 nucleotides, about 2-24 nucleotides, about 3-23 nucleotides, about 4-22 nucleotides, about 5-21 nucleotides, about 6-20 nucleotides, about 7-19 nucleotides, about 8-18 nucleotides, about 9-17 nucleotides, about 10-16 nucleotides, about 11-15 nucleotides, about 12-14 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 nucleotides, at least about 15 nucleotides, at nucleotides, at least about 18 nucleotides, at nucleotides, at least about 21 nucleotides, at least about 13 nucleotides, at least about 14 least about 16 nucleotides, at least about 17 least about 19 nucleotides, at least about 20 least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, or at least about 25 nucleotides.

[00160] The index sequence may be uniquely associated with the one or more targets for detection. The one or more targets may comprise an RNA, a DNA, a polypeptide, or a combination thereof. In some embodiments, an index sequence maybe associated with the one or more targets and at least one other analyte. In some embodiments, the other analyte may be a nucleic acid or a polypeptide. In some embodiment, the other analyte may be an RNA. In some embodiments, the other analyte may comprise naturally-occurring nucleic acid, recombinant nucleic acid, synthesized nucleic acid, or a combination thereof. In some embodiments, the other analyte may be a DNA. In some embodiments, the other analyte may be genomic DNA. In some embodiments, the other analyte may be viral DNA. In some embodiments, the other analyte may be cfDNA. In some embodiments, the other analyte may be poly-ARNA and / or non-poly-a RNA. The RNAmay comprise codingRNA, non-codingRNA, ora combination thereof. TheRNAmay comprise tRNA, rRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), WO 2025 / 170937                                   PCT / US2025 / 014502 microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), antisense RNA, non-coding RNA, protein-encoding RNA, or a combination thereof.

[00161] In some embodiments the other analyte may be a polypeptide. In some embodiments, the polypeptide may be a protein, a peptide, or a combination thereof. In some embodiments, the protein may be an antibody, antibody fragment, ribosomal protein, transcription factor, histone, enzyme, structural protein, chemokine, cytokine, or a combination thereof.

[00162] The nucleic acid molecules described herein may comprise one or more padlock probes. The one or more padlock probes may comprise nucleic acid. The nucleic acid of the one or more padlock probes may comprise DNA, RNA, or a combination thereof. The one or more padlock probes may comprise one or more modifications, including but not limited to a phosphorylation (e.g. a 5 ’ phosphorylation), a locked nucleic acid, or a combination thereof. In some embodiments, the padlock probes comprise canonical nucleotides and / or nucleotide analogs. In some embodiments, the padlock probes are modified to confer resistance to nuclease degradation (e.g., ribonuclease degradation). For example, the padlock probes comprise at least one phosphorothioate diester bond at their 5’ ends which can render the padlock probes resistant to nuclease degradation. In some embodiments, the padlock probes comprise 2-5 or more consecutive phosphorothioate diester bonds at their 5’ ends. In some embodiments, the padlock probes comprise at least one ribonucleotide and / or at least one 2’ -O-methyl, 2’-O-methoxyelhyl (MOE), 2’ fluoro-base nucleotide. In some embodiments, the padlock probes comprise phosphorylated 3 ’ ends. In some embodiments, the padlock probes comprise at least one locked nucleic acid (LNA) base. In some embodiments, the padlock probes comprise a phosphorylated 5’ end (e.g., using a polynucleotide kinase).

[00163] A padlock probe of the one or more padlock probes may comprise a first end, a second end, an index sequence, additional sequence, or a combination thereof. The first end of the padlock probe may comprise a sequence that is at least partially complementary to a sequence ofa target (e.g. an RNA or a DNA) or reverse complement thereof. The first end of the padlock probe may comprise a sequence that is at least partially complementary to a portion of an oligonucleotide of a polypeptide-binding probe or reverse complement thereof. The second end of the padlock probe may comprise a sequence that is at least partially complementary to a sequence of a target (e.g. an RNA or a DNA) or reverse complement thereof. The second end of the padlock probe may comprise a sequence that is at least partially complementary to a portion of an oligonucleotide of a polypeptide-binding probe or reverse complement thereof. In some cases, the first end of the padlock probe is directly adjacent to the second end of the padlock probe when both the first end and the second end are bound to a target (e.g. an RNA or a DNA) or bound to an oligonucleotide of a polypeptide-binding probe. For example, the first end of the padlock probe may hybridize to a first sequence of the RNA and the second end of the padlock probe may hybridize to a second sequence of an RNA and the first sequence and the second sequence are contiguous sequences of the RNA such that there are no unhybridized nucleotides on the RNA when bound to the padlock probe atthe junction of the first end and the second end of thepadlock probe. In another example, the first end of the padlock probe may hybridize to a first portion of an oligonucleotide of a polypeptide-binding probe and the second end of the padlock probe may hybridize to a second portion of the oligonucleotide of the polypeptide-binding probe and the first portion and the second portion are contiguous sequences of the oligonucleotides such that there are no unhybridized nucleotides on the oligonucleotide when bound to the padlock at the junction of the first end and the second end of the padlock probe.

[00164] In some embodiments, the first end of an individual padlock probe has a first random sequence that hybridizes to a first region of a target (e.g. an RNA molecule), and the second end of the individual padlock probe has a second random sequence that hybridizes to a second region of the same target, where a nick or gap is formed between the hybridized first and second binding arms. In some embodiments, individual padlock probes in a set of padlock probes (e.g., a plurality of padlock probes) comprise first and second ends that hybridize to the same target regions of the targets (e.g. RNA molecules) to form a plurality of target-padlock probe complexes having the same target sequence.

[00165] In some cases, the first end of the padlock probe is adjacent to the second end of the padlock probe and separated by a gap (e, g. separated by at least one nucleotide) when both the first end and the second end are bound to a target (e.g. an RNA or a DNA) or bound to an oligonucleotide of a polypeptide-binding probe. For example, the first end of the padlock probe may hybridize to a first sequence of the RNA and the second end of the padlock probe may hybridize to a second sequence of an RNA and the first sequence and the second sequence are noncontiguous sequences of the RNA such that there is at least one unhybridized nucleotide on the RNA when bound to the padlock probe atthe junction of the first end and the second end of the padlock probe. In another example, the first end of the padlock probe may hybridize to a first portion of an oligonucleotide of a polypeptide-binding probe and the second end of the padlock probe may hybridize to a second portion of the oligonucleotide of the polypeptide-binding probe and the first portion and the second portion are noncontiguous sequences of the oligonucleotides such that there is at least one unhybridized nucleotide on the oligonucleotide when bound to the padlock atthe junction of the first end and the second end of the padlock probe. In cases where the first end of the padlock probe and the second end of the padlock probe are separated by a gap, a gap-filling reaction can be performed to fill in the unhybridized nucleotides of the target or oligonucleotide of the polypeptide-binding probe. The gap-filling reaction canbe performed by a polymerizing enzyme, e.g. a DNA polymerase or anRNA polymerase. In some embodiments, the size of the gap between the hybridized firstand second binding arms is 1-30 bases. The 3’OH end of hybridized padlock probe can serve as an initiation site for a reverse transcriptase-catalyzed fill-in reaction (e.g., gap fill-in reaction) using the target RNA molecule as a template. After the fill-in reaction, the remaining nick can be enzymatically ligated to generate covalently closed circular nucleic acid.

[00166] In some embodiments, the gap-filling reaction comprises contacting the circular nucleic acid with a reverse transcriptase enzyme and a plurality of nucleotides. In some embodiments, the reverse transcriptase comprises In some embodiments, the reverse transcriptase enzyme comprises a reverse transcriptase enzyme from AMV (avian myeloblastosis virus), M-MuLV (moloney murine leukemia virus), or HIV (human immunodeficiency virus). In some embodiment, the reverse transcriptase enzyme comprises a recombinant enzyme that exhibits reduced RNase H activity, for example REVERTAID (e.g., from Thermo Fisher Scientific, catalog No. EP0441). In some embodiments, the reverse transcriptase can be a commercially -available enzyme, including MULTISCRIBE (e.g., from Thermo Fisher Scientific, catalog # 4311235), THERMOSCRIPT (e.g., from Thermo Fisher Scientific, catalog # 12236-014), or ARRAYSCRIPT (e.g., from Ambion, catalogNo. AM2048). In some embodiments, the reverse transcriptase enzyme comprises SUPERSCRIPT II (e.g., catalogNo. 18064014), SUPERSCRIPT III (e.g., catalogNo. 18080044), or SUPERSCRIPT IV enzymes (e.g., catalogNo. 18090010) (all SUPERSCRIPT enzymes from Invitrogen). In some embodiments, the reverse transcription reaction can include an RNase inhibitor.

[00167] The one or more padlock probes may be circularized to generate one or more circular nucleic acids. For example, a first end of a padlock probe may be ligated to a second end of the padlock probe to form a circular nucleic acid. The padlock probe may be ligated by a ligase, a chemical reaction, or a combination thereof. In some cases, one end of a padlock probe (e.g. a first end or a second end) may comprise a phosphorylation modification (e.g. a 5’ phosphorylation). A ligase may ligate the end of the padlock probe comprising the phosphorylation modification to the other end of the padlock probe to form a circular nucleic acid. 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 maybe a T4 ligase, a SplintRligase, a T3 ligase, a T4 ligase, a T7 ligase, an TtcB ligase, a Taq ligase or a combination thereof. In some embodiments, the ligase can discriminate between matched and mis-matched hybridized ends to ensure target-specific hybridization. In some cases, one end of a padlock probe may comprise a biorthogonal modification (e.g. an azide or an alkyne). In some cases, a chemical re action between WO 2025 / 170937                                   PCT / US2025 / 014502 the biorthogonal modification of the one end of the padlock probe may be reacted to another end of the padlock probe, which may comprise a second biorthogonal modification to form a covalent bond between the two ends in order to form a circular nucleic acid molecule.

[00168] In some embodiments, the padlock probes comprise at least one universal adaptor sequence including a sample barcode sequence, an amplification primer binding site, a sequencing primer binding site and / or a compaction oligonucleotide binding site. In some embodiments, the padlock probes comprise at least one unique identification sequence (e.g., unique molecular index (UMI). In some embodiments, the padlock probes comprise at least one restriction enzyme recognition sequence.

[00169] In some embodiments, a set of padlock probes (e.g., a plurality of padlock probes) comprise atleasttwo sub-sets (e.g., two sub-populations) of padlockprobes (e.g., FIGs. 28, 30 and 32). In some embodiments, individual padlock probes in a first sub-set of padlock probes comprise first and second binding arms that hybridize to the same target regions (e.g., a first target region) of the first target RNA molecules to form a first plurality of RNA-padlock probe complexes having the same RNA sequence. In some embodiments, individual padlock probes in a second sub-setofpadlock probes comprisefirstand secondbindingarmsthathybridize to the same target regions (e.g., a second target region) of the second target RNA molecules to forma second plurality of RNA-padlock probe complexes having the same RNA sequence. In some embodiments, the first and second sub-sets of padlock probes hybridize to different target regions of the same target RNA molecules. In some embodiments, the first and second sub-sets of padlock probes hybridize to different target regions of different target RNA molecules. In some embodiments, the set of padlock probes comprise 2-10 sub-sets of padlock probes, or 10-25 subsets of padlock probes, or 25-50 sub-sets of padlock probes, or up to 100 sub-sets of padlock probes. In some embodiments, the set of padlock probes comprise at least 100 sub-sets of padlock probes, at least 500 sub-sets of padlock probes, at least 1000 sub-sets of padlock probes, at least 10,000 sub-sets of padlock probes, or more sub-sets of padlock probes. iv.   Polypeptide-binding probes

[00170] Disclosed herein are polypeptide-binding probesthat are useful in the methods described herein for detecting a target (e.g. a polypeptide) in situ. The polypeptide-binding probes may be configured to bind one or more polypeptides. The polypeptide-binding probes may comprise a polypeptide binding moiety and an oligonucleotide. The polypeptide binding moiety may be coupled to the oligonucleotide either directly or indirectly using one or more covalent interactions, one or more non-covalent interactions, or a combination thereof. For example, the polypeptide binding moiety may comprise a polypeptide and the polypeptide of the polypeptide binding moiety may be covalently linked to the oligonucleotide. Additionally, the polypeptide binding moiety may comprise a polypeptide and the polypeptide of the polypeptide binding moiety may be non-covalently linked to the oligonucleotide via a biotin-streptavidin interaction. The polypeptide binding moiety may be linked to the oligonucleotide using one or more linkers. In some embodiments, the one or more linkers may comprise streptavidin (or an avidin -like moiety), biotin, an amine group, a disulfide group, or a combination thereof. In some embodiments, the one or more linkers may not be cleavable or not removable. In some embodiments, the one or more linkers may be cleavable or removable. For example, the one or more linkers can be cleavable with light (e.g., UV light), chemically-cleavable (e.g., dithiothreitol), heat- cleavable, enzymatically cleavable, or a combination thereof.

[00171] The oligonucleotide of the polypeptide-binding probe may comprise DNA, RNA, or a combination thereof. In some cases, the oligonucleotide may comprise one or more non-natural nucleotides. The one or more non-natural nucleotides may comprise one or more of 2-MethoxyEthoxy A, 2-MethoxyEthoxy MeC, 2-MethoxyEthoxy G, 2-MethoxyEthoxy T, 8-oxo dG, deoxyadenosine, N6-Methyl rA, 5-Bromo dU, deoxyUridine, 2,6-Diaminopurine, Dideoxy-C, deoxyinosine, Hydroxymethyl dC, Iso-dG, Iso-dC, 5-Methyl dC, or 5-Nitroindole or a combination thereof. The oligonucleotide may comprise one or more locked nucleic acids (LNA). The oligonucleotide may comprise single-stranded nucleic acids, double-stranded nucleic acids, or a combination thereof. The oligonucleotide may 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, atleastabout7 nucleotides, atleastabout8 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. The oligonucleotide may have a length of at most about 2 nucleotides, at most about 3 nucleotides, at most about 4 nucleotides, at most about 5 nucleotides, at most about 6 nucleotides, at most about 7 nucleotides, at most about 8 nucleotides, at most about 9 nucleotides, at most about 10 nucleotides, at most about 11 nucleotides, at most about 12 nucleotides, at most about 13 nucleotides, at most about 14 nucleotides, at most about 15 nucleotides, at most about 16 nucleotides, at most about 19 nucleotides, at most about 30 nucleotides, at most about 50 nucleotides, at most about 70 nucleotides, at most about 17 nucleotides, at most about 20 nucleotides, at most about 40 nucleotides, at most about 55 nucleotides, at most about 80 nucleotides, at most about 18 nucleotides, at most about 25 nucleotides, at most about 45 nucleotides, at most about 60 nucleotides, at most about 90 nucleotides, at most about 100 nucleotides, or less. The oligonucleotide may have a length of about2-100 nucleotides, about 3-90 nucleotides, about4-80 nucleotides, about 5-70nucleotides, about 6-60 nucleotides, about 7-55 nucleotides, about 8-50 nucleotides, about 9-45 nucleotides, about 10-40 nucleotides, about 11-30 nucleotides, about 12-25 nucleotides, about 13-20 nucleotides, about 14-19 nucleotides, about 15-18 nucleotides, or about 16-17 nucleotides.

[00172] The polypeptide-bindingprobe may comprise a polypeptide, a nucleic acid, a linker, or a combination thereof. In some cases, the polypeptide-binding probe may comprise a polypeptide. The polypeptide may comprise an antibody, an antibody fragment, a nanobody, an affimer, or a combination thereof. The antibody or antibody fragment may comprise a variety of isotypes including but not limited to IgG, IgM, IgA, IgD, IgE, ora combination thereof. In some cases, the polypeptide-binding probe may comprise an antibody or antibody fragment and the polypeptide binding moiety may comprise an a paratope. In some cases, the polypeptide-binding probe may comprise an antibody or antibody fragment and the polypeptide binding moiety may comprise an antigen binding site. In some cases, the polypeptide-bindingprobe may comprise a nucleic acid. The nucleic acid of the polypeptide-bindingprobe may comprise an aptamer. The aptamer may be configured to bind to the target that is detected in the methods described herein. v. Index-binding sequences

[00173] Disclosed herein are nucleic acid molecules comprising index-binding sequences that are useful in the methods described herein for detecting a target (e.g., an RNA, a DNA, or a polypeptide) in situ. For example, the index-binding sequences of the nucleic acid molecules may enable the nucleic acid molecule to bind the index sequence to initiate an amplification or detection of the index sequence. In some embodiments, the nucleic acid molecule comprisingthe index-binding sequence is a padlock probe. In some embodiments, the nucleic acid molecule comprising the index-binding sequence is a molecular inversion probe. In some embodiments, the nucleic acid molecule comprisingthe index-binding sequence may comprise a second index sequence. The index-binding sequence may be configured to couple to an index sequence. In some embodiments, the index-binding sequencemay hybridize to an index sequence. In some embodiments, the index-binding sequence may comprise at least one binding region that recognizes and binds at least a portion of an index sequence. In some embodiments, the index- WO 2025 / 170937                                   PCT / US2025 / 014502 binding sequence may comprise more than one binding region that recognizes and binds an index sequence. For example, a padlock probe may have two index-binding sequences at a 5’ end and 3’ end of the padlock probe that, when each hybridized to the 5’ end and 3’ end of the index sequence, initiates a ligation reaction thereby adjoining the 5’ and 3’ ends of the padlock probe.

[00174] In some embodiments, the index-binding sequence may bind to the index sequence to form a double-stranded region. In some instances, the double-stranded region is continuous. In some embodiments, the double-stranded region comprises at least one nick. In some embodiments, the double-stranded region may be separated by a single-stranded region. In some cases, the single-stranded region may be one or more nucleotides in length. For example, both ends (the 5’ end and the 3 ’ end) of a padlock probe may bind to the index sequence to form a double-stranded region with a nick separating the two ends of the padlock probe. In some embodiments, the index-binding sequence may be ligated after hybridization to the index sequence. In the example of the padlock probe, upon ligation of the 5’ end to the 3’ end, the padlock probe forms a circular nucleic acid. In some embodiments, a 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, a SplintR ligase, a T3 ligase, a T7 ligase, an TtcB ligase, or a combination thereof. In some embodiments, the ligation reaction may form a circular nucleic acid. vi. Amplification reactions

[00175] Disclosed herein are amplification reactions useful for the detection of targets (e.g. RNA, DNA, or polypeptides) in situ. The amplification reactions may be used to increase a signal associated with a target. In some cases, the amplification reactions may generate multiple copies of a target, reverse complement or a target (e.g. a nucleic acid), one or more index sequences associated with a target, or a combination thereof. The amplification reaction may generate multiple copies of the sequence that is amplified. In some instances, the amplification reaction may generate multiple copies of the reverse complement of the sequencebeing amplified. The amplification reactions may comprise replication of a target or reverse complement thereof or an index sequence associated with the target or may comprise hybridization of additional nucleic acid molecules to generate additional binding sites for a detection moiety as described herein.

[00176] In some cases, one or more amplicons may be generated by the amplification reactions. The one or more amplicons may comprise one or more copies of an index sequence or reverse complement thereof associated with a target. The one or more amplicons may comprise one or more derivatives of the target, the index sequence, or a combination thereof. A derivative of a nucleic acid for the methods described herein, e.g. the derivative of an index sequence, refers to the reverse complement of the nucleic acid sequence, e.g. the reverse complement of the index sequence. The one or more amplicons may comprise one or more copies of a portion of a target (e.g. an RNA or a DNA) or a reverse complement of a portion of a target (e.g. an RNA or a DNA). The one or more amplicons may comprise one or more copies of a portion of an oligonucleotides of a polypeptide-binding sequence or reverse complement thereof. The one or more amplicons may comprise one or more copies of a portion of an index-binding sequence or reverse complement thereof.

[00177] In some cases, the amplification reactions may comprise a rolling circle amplification reaction, a polymerase chain reaction (PCR), a 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), a transcription mediated amplification, a ligase chain reaction, or a combination thereof. The amplification reactions may include one or more temperature incubation steps. In some cases, the amplification reaction may take place at the same temperature (e.g. isothermally). In some cases, the amplification reaction may take place at multiple temperatures. For example, a rolling circle amplification reaction may be performed at 42°C for the duration of the amplification reaction. In some cases, the temperature may vary throughout a range during the amplification reaction. In some cases, an incubation temperature of 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 about30°C, at least about 32°C, at least about 34°C, at least about 36°C, at least about 3 8°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 more. In some cases, an incubation temperature of the amplification reaction may be at most about 15°C, at most about 18°C, at most about 20°C, at most about 25°C, at most about 30°C, at most about 32°C, at most about 34°C, at most about 36°C, at most about 3 8°C, at most about 40°C, at most about 42°C, at most about 44°C, at most about 46°C, at most about 48°C, at most about 50°C, at most about 52°C, at most about 54°C, at most about 56°C, at most about 58°C, at most about 60°C, at most about 62°C, at most about 64°C, at most about 66°C, at most about 68°C, at most about 70°C, at most about 72°C, at most about 74°C, at most about 76°C, at most about 78°C, at most about 80°C, at most about 82°C, at most about 84°C, at most about 86°C, at most about 88°C, at most about 90°C, at most about 95°C, or more. In some cases, an incubation temperature of the amplification reaction may be at most about 18-90°C, about 20-88°C, about 25-86°C, about30-84°C, about 32-82°C, about 3480°C, about36-78°C, about38-76°C, about40-74°C, about 42-72°C, about44-70°C, about46-68°C, about 48-66°C, about 50-64°C, about 52-62°C, about 54-60°C, or about 56-58 °C. The amplification reactions may be performed for a duration of time. In some cases, the duration of time of the amplification may be correlated to the amount of signal that is generated during the detection step the methods described herein. For example, in some cases, a longer amplification reaction may generate a larger signal associated with a target. The amplification reaction may be at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 30 minutes, atleast about45 minutes, atleastabout 1 hour, atleastabouthours, atleastabout3 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, atleast about 24 hours, atleast about 3 6 hours, at least about 48 hours, or more. The amplification reaction may be at most about 5 minutes, at most about 10 minutes, at most about 15 minutes, at most about 30 minutes, at most about 45 minutes, at most about 1 hour, at most about hours, at most about 3 hours, at most about 4 hours, at most about 5 hours, at most about 6 hours, at most about 7 hours, at most about 8 hours, at most about 9 hours, at most about 10 hours, at most about 12 hours, at most about 15 hours, at most about 20 hours, at most about 24 hours, at most about 36 hours, at most about 48 hours, or less. The amplification reaction may be about 5 minutes-48 hours, about 10 minutes-36 hours, about 15 minutes-24 hours, about30 minutes-20hours, about45 minutes-15 hours, about 1 hour -12 hours, about2 hours-10 hours, about 3 hours-9 hours, about 4 hours-8 hours, or about 5 hours-7 hours.

[00178] In some cases, a rolling circle amplification reaction may be performed. The rolling circle amplification may comprise amplifying a circular nucleic acid. The circular nucleic acid may comprise one or more nucleic acid molecules as described herein. For example, a padlock probe may be ligated to form a circular nucleic acid that is used during a rolling circle amplification reaction. The padlock probe that forms a circular nucleic acid may be a probe comprising a DNA-binding sequence and an index sequence, a prob e comprising an RNA-binding sequence and an index sequence, an oligonucleotide-binding probe and an index sequence, or an index-binding sequence. The rolling circle amplification rection may generate one or more amplicons. An amplicon of the one or more amplicons may comprise one or more copies of the component of the circular nucleic acid. For example, the circular nucleic acid may be generated by ligating a padlock probe that comprises a DNA-binding sequence an index sequence. A rolling circle amplification reaction may be performed using this circular nucleic acid to generate one or more amplicons that comprise one or more copies of the DNA-binding sequence or reverse complementthereof and one or more copies of the index sequence or reverse complementthereof. In another example, the circular nucleic acid may be generated by ligating a padlock probe that comprises an RNA-binding sequence an index sequence. A rolling circle amplification reaction may be performedusingthis circular nucleic acid to generate one or more amplicons that comprise one or more copies of the RNA-binding sequence or reverse complement thereof and one or more copies of the index sequence or reverse complement thereof. In another example, the circular nucleic acid may be generated by ligating a padlock probe that comprises an oligonucleotide-binding probe an index sequence. A rolling circle amplification reaction may be performed using this circular nucleic acid to generate one or more amplicons that comprise one or more copies of the oligonucleotide-binding probe or reverse complementthereof and one or more copies of the index sequence or reverse complementthereof. In another example, the circular nucleic acid may be generated by ligating a padlock probethat comprises an index-binding sequence an optionally an index sequence. A rolling circle amplification reaction may be performedusingthis circular nucleic acid to generate one or more amplicons that comprise one or more copies of the indexbinding sequence or reverse complementthereof and optionally one or more copies of the index sequence or reverse complement thereof.

[00179] In some embodiments, the amplification reaction is performed by a polymerizing enzyme. In some embodiments, the polymerizing enzyme 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 stranddisplacement capabilities. In some embodiments, the polymerase may be phi29 polymerase, taq polymerase, T4 polymerase, T7 polymerase, or a combination thereof. For example, the amplification reaction may comprise a rolling circle amplification reaction and a phi29 polymerase may be used to generate one or more amplicons.

[00180] In some embodiments, the rolling circle amplification reaction comprises contacting the covalently circular nucleic acids with a soluble amplification primer (e.g., a universal rolling circle amplification primer), a strand-displacing DNA polymerase, a plurality of nucleotides, or a combination thereof, under a condition suitable for hybridizing individual amplification primers to a circular nucleic acids, and under a condition suitable for conducting primer extension using the circular nucleic acids as a template molecule to generate one or more amplicons. In some embodiments, the plurality of nucleotides in the rolling circle amplification reaction may comprise any mixture of two or more of dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, any of the rolling circle amplification reactions described herein can be conducted in the presence or in the absence of a plurality of compaction oligonucleotides.

[00181] In some embodiments, when the rolling circle amplification reaction includes a plurality of nucleotide which includes dUTP, the resulting amplicon can be cross-linked to a cross-linking reactive group by treatingthe biological sample with a succinimide ester (N H S), makdmide (Sulfo-SMCC). imidoester (DMP), carbodiimide (DCC, EDC) or phenyl azide. In some embodiments, polymerization of the cross-linking reactive group can be initiatedwith light or UV light. In some embodiments, the resulting amplicon can be cross-linked to a matrix by treating the biological sample with a cross-linked agarose, cross-linked dextran or cross-linked polyethylene glycol (PEG), polyacrylamide, cellulose alginate or polyamide. In some embodiments, the PEG comprises a sulfo-NHS ester moiety at one or both ends, for example a PEGylated bis(sulfosuccinimidyl)suberate) (e.g., BS(PEG)9 from Thermo Fisher Scientific, catalog No. 21582).

[00182] In some embodiments, the rolling circle amplification reaction can be conducted ata constant temperature (e.g., isothermal) wherein the constant temperature is atroom temperature to about 30 °C, or about 30 - 40 °C, or about 40 - 50 °C, or about 50 - 65 °C.

[00183] In some embodiments, the DNA polymerase having a strand displacing activity can be selected from a group consisting of phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase, and Bea (exo-) DNA polymerase, Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, or Deep Vent DNA polymerase. In some embodiments, the phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), and chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[00184] In some embodiments, the rolling circle amplification primers can be modified to increase resistance to nuclease degradation. In some embodiments, the rolling circle amplification primers comprise at least one phosphorothioate diester bond at their 5 ’ ends which can render the amplification primers resistant to exonuclease degradation. In some embodiments, the rolling circle amplification primers comprise 2-5 or more consecutivephosphorothioate diester bonds at their 5’ ends. In some embodiments, the rolling circle amplification primers comprise at least one ribonucleotide and / or at least one 2 ’-O-methyl or 2 ’-O-meth oxyethyl (MOE) nucleotide.

[00185] In some embodiments, the rolling circle amplification reaction can be conducted in the presence of a plurality of compaction oligonucleotides which, when hybridized to an amplicon, compacts the size and / or shape of the amplicon to form a compact nanoball. In some embodiments, the compaction oligonucleotides comprise single stranded oligonucleotides having a first region at one end that hybridizes to a portion of an amplicon and a second region at WO 2025 / 170937                                   PCT / US2025 / 014502 the other end that hybridizes to another portion of the same amplicon, where hybridization of the compaction oligonucleotide to a given amplicon compacts the size and / or shape of the amplicon.

[00186] The compaction oligonucleotides include a 5’ region, an optional internal region (intervening region), and a 3’ region. The 5’ and 3’ regions of the compaction oligonucleotide can hybridize to different portions of the amplicon to pull together distal portions of the amplicon causing compaction of the amplicon to form a DNA nanoball. For example, the 5’ region of the compaction oligonucleotide is designed to hybridize to a first portion of the amplicon (e.g., a universal compaction oligonucleotide binding site), and the 3’ region of the compaction oligonucleotide is designed to hybridized to a second portion of the amplicon (e.g, a universal compaction oligonucleotide binding site). Inclusion of compaction oligonucleotides during RCA can promote formation of DNA nanoballs having tighter size and shape compared to amplicons generated in the absence of the compaction oligonucleotides. The compact and stable characteristics of the DNA nanoballs improves in situ sequencing accuracy by increasing signal intensity and the nanoballs retain their shape and size during multiple sequencing cycles.

[00187] In some embodiments, the compaction oligonucleotides comprise single stranded oligonucleotides comprising DNA, RNA, or a combination of DNA and RNA. The compaction oligonucleotides can be any length, including 20-150 nucleotides, or 30-100 nucleotides, or 40-80 nucleotides in length.

[00188] In some embodiments, the compaction oligonucleotides comprises a 5’ region and a 3’ region, and optionally an intervening region between the 5’ and 3’ regions. The intervening region can be any length, for example about 2-20 nucleotides in length. The intervening region comprises a homopolymer having consecutive identical bases (e.g., AAA, GGG, CCC, TTT or UUU). The intervening region comprises a non-homopolymer sequence.

[00189] The 5’ region of the compaction oligonucleotides can be wholly complementary or partially complementary along its length to a first portion of an amplicon. The 3’ region of the compaction oligonucleotides can be wholly complementary or partially complementary along its length to a second portion of an amplicon. The 5’ region of the compaction oligonucleotides can hybridize to a first universal sequence portion of an amplicon. The 3’ region of the compaction oligonucleotides can hybridize to a second universal sequence portion of an amplicon.

[00190] In some embodiments, the 5 ’ region of the compaction oligonucleotide can have the same sequence as the 3’ region. The 5’ region of the compaction oligonucleotide can have a sequence that is different from the 3’ region. In some embodiments, the 3’ region of the compaction oligonucleotide can have a sequence that is a reverse sequence of the 5’ region. In WO 2025 / 170937                                   PCT / US2025 / 014502 some embodiments, the 5’ region of the compaction oligonucleotide can have a sequence that is a reverse sequence of the 3’ region.

[00191] In some embodiments, the 3’ region of any of the compaction oligonucleotides can include an additional three bases at the terminal 3 ’ end which comprises 2’-O-methyl RNA bases (e.g., designated mUmUmU) or the terminal 3 ’ end lacks additional 2’ -O-methyl RNA bases.

[00192] In some embodiments, the compaction oligonucleotides comprise one or more modified bases or linkages at their 5’ or 3’ ends to confer certain functionalities. In some embodiments, the compaction oligonucleotides comprise at least one phosphorothioate linkages at their 5’ and / or 3’ ends to confer exonuclease resistance. In some embodiments, at least one nucleotide at or near the 3 ’ end comprises a 2’ fluoro base which confers exonuclease resistance. In some embodiments, the 3’ end of the compaction oligonucleotides comprise at least one 2’-O-methyl RNA base which blocks polymerase-catalyzed extension. For example, the 3 ’ end of the compaction oligonucleotide comprises three bases comprising 2’-O-methyl RNA base (e.g., designated mUmUmU). In some embodiments, the compaction oligonucleotides comprise a 3’ inverted dT at their 3’ ends which blocks polymerase-catalyzed extension. In some embodiments, the compaction oligonucleotides comprise 3’ phosphorylation whichblockspolymerase-catalyzed extension. In some embodiments, the internal region of the compaction oligonucleotides comprise at least one locked nucleic acid (LNA) which increases the thermal stability of duplexes formed by hybridizing a compaction oligonucleotide to an amplicon. In some embodiments, the compaction oligonucleotides comprise a phosphorylated 5’ end (e.g., using a polynucleotide kinase).

[00193] In some embodiments, the compaction oligonucleotides includes an additional three bases at the terminal 3’ end which comprises 2’-O-methyl RNA bases (e.g., designated mUmUmU) or the terminal 3’ end lacks additional 2’-O-methyl RNA bases.

[00194] In some embodiments, the compaction oligonucleotides can include atleast one region having consecutive guanines. For example, the compaction oligonucleotides can include at least one region having 2, 3, 4, 5, 6 or more consecutive guanines. In some embodiments, the compaction oligonucleotides comprise four consecutive guanines which can form a guanine tetrad structure (see FIG. 18). The guanine tetrad structure can be stabilized via Hoogsteen hydrogen bonding. The guanine tetrad structure can be stabilized by a central cation including potassium, sodium, lithium, rubidium or cesium.

[00195] At least one compaction oligonucleotide can form a guanine tetrad (Figure 12) and hybridize to the universal binding sequences in an amplicon which can cause the amplicon to fold to form an intramolecular G-quadruplex structure (Figure 13). The amplicons can self- WO 2025 / 170937                                   PCT / US2025 / 014502 collapse to form compact nanoballs. Formation of the guanine tetrads and G-quadruplexes in the nanoballs may increase the stability of the nanoballs to retain their compact size and shape which can withstand changes in pH, temperature and / or repeated flows of reagents during sequencing inside the biological sample.

[00196] In some embodiments, the plurality of compaction oligonucleotides in the rolling circle amplification reaction have the same sequence. Alternatively, the plurality of compaction oligonucleotides in the rolling circle amplification reaction comprise a mixtu re of two or more different populations of compaction oligonucleotides having different sequences.

[00197] In some embodiment, the immobilized amplicon can self-collapse into a compact nucleic acid nanoball. The nanoballs can be imaged and a FWHM measurement can be obtained to give the shape / size of the nanoballs.

[00198] In some embodiments, inclusion of compaction oligonucleotides in the rolling circle amplification reaction can promote collapsing of a concatemer into a DNA nanob all. Conducting RCA with compaction oligonucleotides helps retain the compact size and shape of a DNA nanoball during multiple sequencing cycles which can improve FWHM (full width half maximum) of a spotimage of the DNAnanoball inside a biological sample. In some embodiments, the DNA nanoball does notunravel during multiple sequencing cycles. In some embodiments, the spot image of the DNA nanoball does not enlarge during multiple sequencing cycles. In some embodiments, the spot image of the DNA nanoball remains a discrete spot during multiple sequencing cycles. The spot image can be represented as a Gaussian spot and the size can be measured as a FWHM. A smaller spot size as indicated by a smaller FWHM typically correlates with an improved image of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 um or smaller. vii. Reaction conditions

[00199] Disclosed herein, are methods that relate to detection of one or more targets (e.g. nucleic acids or polypeptides) in situ under certain reaction conditions. In some cases, one or more nucleic acid molecules (e.g. probes) may be introduced to the biological sample comprising the one or more targets under conditions sufficient to couple a binding sequence of a nucleic 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. a probe or an index-bindingmolecule) may be introduced to the biological sample comprising one or more targets under sufficient conditions to couple a binding sequence of the nucleic acid molecule (e.g. an oligonucleotide-binding probe or an index-binding sequence) to one or more probes or nucleic acid molecules (e.g. one or more polypeptide-binding probes or one or more target-binding sequences) that are bound to a target (e.g. a polypeptide or an RNA). In some cases, one or more primer nucleic acid sequences may be introduced to the biological sample comprising one or more targets under conditions sufficient to couple the one or more primer nucleic acid sequences to one or more index sequences or the reverse complement thereof. Conditions sufficient to couple 1) the binding sequences as described herein to the one or more targets 2) nucleic acid molecules to polypeptide-binding probes or target-binding sequences, and / or 3) one or more primer nucleic acid sequences to one or more index sequences or the reverse complement thereof may include a reagent, a temperature, an incubation time, a concentration, or a combination thereof.

[00200] In some embodiments, Conditions sufficient to couple 1) the binding sequences as described herein to the one or more targets 2) nucleic acid molecules to polypeptide-binding probes or target-binding sequences, and / or 3) one or more primer nucleic acid sequences to one or more index sequences or the reverse complement thereof may include a regent. In some embodiments, the reagent may comprise a salt. In some embodiments, the salt may comprise NaCl, MgCl2, CaCl2, KC1, Na2SO4, or a combination thereof. In some further embodiments, the coupling conditions comprise the presence of strontium ions, magnesium ions, and / or calcium ions. In some embodiments, the reagent may comprise a buffer. In some embodiments, the buffer may comprise a phosphate buffered saline, tris, ethylenediaminetetraacetic acid, or a combination thereof.

[00201] 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.

[00202] In some embodiments, the reagent may comprise a chaotropic reagent. In some embodiments, the chaotropic reagent may comprise glycine, arginine, histidine hydrochloride, formamide, dimethyl sulfoxide, dimethylformamide, urea, thiourea, guanidine hydrochloride, or a combination thereof.

[00203] In some embodiments Conditions sufficient to couple 1) the binding sequences as described herein to the one or more targets 2) nucleic acid molecules to polypeptide-binding probes or target-binding sequences, and / or 3) one or more primer nucleic acid sequences to one or more index sequences or the reverse complement thereof may include a temperature. The temperature may be an incubation temperature maintained during the time sufficientto couple the target-binding sequence to the target nucleic acid. The temperature may be about 10-74°C, about °C, about 12-72°C, about 14-70°C, about 16-68°C, about 18-66°C, about20-64°C, about 22-62°C, about24-60°C, about26-58°C, about28-56°C, about 30-54°C, about 32-52°C, about 34-50°C, about 36-48°C, about 38-46°C, about 40-44°C, at least about 10°C, at least aboutl2°C, at least aboutl4°C, at least aboutl6°C, at least aboutl8°C, at least about20°C, at least about22°C, at least about24°C, at least about26°C, at least about28°C, at least about30°C, at least about32°C, at least about34°C, at least about36°C, at least about38°C, at least about40°C, at least about42°C, at least about44°C, at least about46°C, at least about48°C, at least about50°C, at least about52°C, at least about54°C, at least about56°C, at least about58°C, at least about60°C, at least about62°C, at least about64°C, at least about66°C, at least about68°C, at least about70°C, at least about72°C, or at least about74°C.

[00204] In some embodiments Conditions sufficient to couple 1) the binding sequences as described herein to the one or more targets 2) nucleic acid molecules to polypeptide-binding probes or target-binding sequences, and / or 3) one or more primer nucleic acid sequences to one or more index sequences or the reverse complement thereof may include an incubation time. The incubation time may be a time sufficient to couple the target-binding sequence to the target nucleic acid. The incubation time may 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 hours, at least about 1.5 hours, at least about2 hours, at least about2.5 hours, at least about3 hours, at least about3.5 hours, at least about4 hours, at least about4.5 hours, at least about5 hours, at least about5.5 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, atleast about 10.5 hours, atleast about 11 hours, atleast about 11.5 hours, or at least about 12 hours.

[00205] In some embodiments, Conditions sufficient to couple 1) the binding sequences as described herein to the one or more targets 2) nucleic acid molecules to polypeptide-binding probes or target-binding sequences, and / or 3) one or more primer nucleic acid sequences to one or more index sequences or the reverse complement thereof may include a concentration. The concentration may relate to the concentration of the one or more nucleic acid molecules. In someembodimentstheconcentrationmay be about0.1-10000 nM, about0.2-5000 nM, about 0.31000 nM, about 0.4-900 nM, about 0.5-800 nM, about 0.6-700 nM, about 0.7-600 nM, about 0.8500 nM, about0.9-400 nM, about 1-300 nM, about2-250 nM, about3-200 nM, about4-150 nM, about 5-100 nM, about 6-50 nM, about7-25nM, about 8-20 nM, about9-15nM, atleast about 0.1 nM, atleast 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, 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, atleast about 150 nM, at least about 200 nM, at least about 250 nM, atleast 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 the one or more nucleic acid molecules may be at most about at most about 0.1 nM, at most about 0.2 nM, at most about 0.3 nM, at most about 0.4 nM, at most about 0.5 nM, at most about 0.6 nM, at most about 0.7 nM, at most about 0.8 nM, at most about 0.9 nM, at most about 1 nM, at most about 2 nM, at most about 3 nM, at most about 4 nM, at most about 5 nM, at most about 6 nM, at most about 7 nM, at most about 8 nM, at most about 9 nM, at most about 10 nM, at most about 15 nM, at most about 20 nM, at most about 25 nM, at most about 50 nM, at most about 100 nM, at most about 150 nM, at most about200 nM, at most about250 nM, at most about300 nM, at mostabout400 nM, at most about 500 nM, at most about 600 nM, at most about 700 nM, at most about 800 nM, at most about 900 nM, at most about 1000 nM, at most about 5000 nM, or at most about 10000 nM viii.   Primer nucleic acid sequences

[00206] Disclosed herein are primer nucleic acid sequences which are useful in the methods described herein for detecting a target (e.g., RNA) in situ. The methods disclosed herein may include one or more primer nucleic acid sequences. The one or more primer nucleic acid sequence may be configured to bind to an index sequence a reverse complement of an index sequence. The one or more primer nucleic acid sequences may comprise DNA, RNA, or a combination thereof. The primer nucleic acid sequence may be configured to couple to an index sequence. In some embodiments, the primer nucleic acid sequence may hybridize to an index sequence. In some embodiments, the primernucleic acid may comprise at least one binding region that recognizes and binds at least a portion of an index sequence. In some embodiments, the primer nucleic acid may comprise more than one binding region that recognizes and binds an index sequence. The one or more primer nucleic acid sequences may form double-stranded region when bound to an index sequence or reverse complement thereof, as described herein. The one or more primernucleic acid sequences may bind directly to a nucleic acid molecule as described herein. For example, a single-stranded oligonucleotide may comprisea sequence that is complementary to a portion of an index sequence and form a double-stranded nucleic acid region upon hybridization of the single-stranded oligonucleotide to the index sequence. In some embodiments, the one or more primernucleic acid sequence may bind to an index-binding sequence as described herein. In some embodiments, the one or more primer nucleic acid sequences may bind to an amplification product generated from an amplification reaction as described herein. 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 reverse complement of an index sequence. In some embodiments, the one or more primer nucleic acid sequences binds directly to the target nucleic acid, as 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.

[00207] The one or more primer nucleic acid sequences may comprise nucleic acid molecules with a given length. Each of the one or more primer nucleic acid sequences may be 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. The one or more primer nucleic acid sequences may have a variety of lengths. In some embodiments, the one or more primer nucleic acid sequenceshas a length of 1-100nucleotides, about2-98 nucleotides, about 3-96nucleotides, about 4-94 nucleotides, about 5-92 nucleotides, about 6-90 nucleotides, about 7-88 nucleotides, about 8-86 nucleotides, about 9-84 nucleotides, about 10-82 nucleotides, about 11 -80 nucleotides, about 12-78 nucleotides, about 13-76 nucleotides, about 14-74 nucleotides, about 15-72 nucleotides, about 16-70 nucleotides, about 17-68 nucleotides, about 18-66 nucleotides, about 1964 nucleotides, about 20-62 nucleotides, about 22-60 nucleotides, about 24-58 nucleotides, about 26-56 nucleotides, about 28-54 nucleotides, about 30-52 nucleotides, about 32-50 nucleotides, about 34-48 nucleotides, about 36-46 nucleotides, about 38-44 nucleotides, about 4042 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 1 nucleotides, at least about 11 nucleotides, at least about 14 nucleotides, at least about 17 nucleotides, at least about 20 nucleotides, at least about 26 nucleotides, at least about 32 nucleotides, at least about 38 nucleotides, at least about 44 nucleotides, at least about 50 nucleotides, at least about 56 nucleotides, at least about 62 nucleotides, at least about 68 nucleotides, at least about 9 nucleotides, at least about 12 nucleotides, at least about 15 nucleotides, at least about 18 nucleotides, at least about 22 nucleotides, at least about 28 nucleotides, at least about 34 nucleotides, at least about 40 nucleotides, at least about 46 nucleotides, at least about 52 nucleotides, at least about 58 nucleotides, at least about 64 nucleotides, at least about 70 nucleotides, at least about 10 nucleotides, at least about 13 nucleotides, at least about 16 nucleotides, at least about 19 nucleotides, at least about 24 nucleotides, at least about 30 nucleotides, at least about 36 nucleotides, at least about 42 nucleotides, at least about 48 nucleotides, at least about 54 nucleotides, at least about 60 nucleotides, at least about 66 nucleotides, at least about 72 nucleotides, at nucleotides, at nucleotides, at nucleotides, at least about 74 least about 80 least about 86 least about 92 nucleotides, at nucleotides, at nucleotides, at nucleotides, at least about 76 least about 82 least about 88 least about 94 nucleotides, at nucleotides, at nucleotides, at nucleotides, at least about 78 least about 84 least about 90 least about 96 nucleotides, at least about 98 nucleotides, or at least about 100 nucleotides in length.

[00208] In some embodiments, the one or more primer nucleic acid sequences inhibits incorporation of another nucleotide into the primer nucleic acid sequence. In some embodiments, the one or more primer nucleic acid sequences does not include a 3 ’ hydroxyl group. In some embodiments, the one or more primer nucleic acid sequencesmay comprise a 3 ’ hydroxyl group substituted with a chain terminating moiety. In some embodiments, the one or more primer nucleic acid sequences comprises a blocking group. In some embodiments, the blocking group may comprise alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thiol group, disulfide group, carbonate group, urea group, silyl group, or a combination thereof.

[00209] In some embodiments, coupling of a primer nucleic acid sequence to an index or a reverse complement of an index generates a primed index sequence. The primed index sequence may comprise a single-stranded nucleic acid, a double-stranded nucleic acid, or a combination thereof. In some embodiments, the primed index sequence is formed between a nucleic acid molecule and a primer nucleic acid sequence, as described herein. In some embodiments, the primed index sequence is formed between a target molecule and a primer nucleic acid sequence, as described herein. In some cases, the primed index sequence is formed between an amplicon and a primer nucleic acid sequence, as described herein.

[00210] In some embodiments, the primer nucleic acid sequence may bind to at least a portion of the 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 the one or more amplicons to generate a primed index sequence. In some cases, the primer nucleic acid sequence may bind to a portion of the one more amplicons that is proximal to one or more index sequences to form a primed index sequences. For example, the portion of the one or more applicants that is proximal to the one or more index sequences may be upstream or downstream from the one or more index sequences and when the primer nucleic acid sequence binds to the portion of the one or more amplicons that is proximal to the one or more index sequences it may form a double-stranded nucleic acid sequence proximal to the one or more index sequences of the one or more amplicons.

[00211] In some embodiments, a primer nucleic acid sequence may bind directly to a target to form a primed index sequence. FIG. IC illustrates a non-limiting step of annealing a primer 103 to the target 102 to form a primed index sequence 104. FIG. IB only shows one primer being used in the annealing step, but more than one primer may be used depending on the types of target nucleic acid. In some instances, an adapter that is used to attach the target to the surface may have the same sequence as the primer used to prepare the primed target nucleic acid. The primer may comprise forward amplification primers, reverse amplification primers, sequencing primers, and / or molecular barcoding sequences, or any combination thereof. In some instances, one primer sequence may be used in the hybridization step. In some instances, a plurality of differentprimer sequences may be used in the hybridization step. As shown in Fig. ID, the primed sequence 104 may be combined with a nucleotide conjugate (e.g. detectable nucleotide conjugatejand a polymerase 106 to form a binding complex.

[00212] The methods described herein involve detection of a target using a detectable nucleotide conjugate. In some embodiments, the target is a ribonucleic acid. In some embodiments, the nucleotide conjugate (e.g. detectable nucleotide conjugatejis introduced to the biological sample comprising the target under conditions sufficient to form binding complex.

[00213] In some embodiments, the nucleotide conjugate (e.g. detectable nucleotide conjugatejcomprises a detectable label. In some embodiments, the detectable label may be conjugated to the nucleotide conjugate (e.g. detectable nucleotide conjugatejvia covalent bond. In some embodiments, the detectable label may be conjugated to the nucleotide conjugate (e.g. detectable nucleotide conjugatejvianon-covalentbond. In some embodiments,the detectable label may comprise a quantum dot. In some embodiments, the detectable label may comprise a fluorophore. Example fluorescent moieties include, but are not limited to, fluorescein and fluorescein derivatives suchas carboxyfluorescein, tetrachlorofluorescein, hexachloro fluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives suchas AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives such as BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPYFL C3 hydrazide,BODIPYFL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanine and derivatives such as indolium based cyanine dyes, benzo-indolium based cyanine dyes, pyridium based cyanine dyes, thiozolium based cyaninedyes,quinolinium based cyanine dyes, imidazolium based cyanine dyes, Cy 3, Cy5, lanthanide chelates and 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 derivatives thereof, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, Malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyesand others knownin the art such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum PressNew York (1999), or Hermanson,Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or any combination thereof. Cyanine dyes may exist in either sulfonated or non-sulfonated forms, and consist of two indolenin, benzo-indolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethinebridge between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3, (which may comprise l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-2-(3-{l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-3,3-dimethyl-l,3-dihydro-2H-indol-2-ylidene}prop-l-en-l-yl)-3,3- dimethyl-3H-indolium or l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-2-(3-{l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-l,3-dihydro-2H-indol-2- ylidene}prop-l-en-l-yl)-3,3-dimethyl-3H-indolium-5-sulfonate),Cy5 (which may comprise 1-(6((2,5 -dioxopy rrolidin-1 -yl)oxy)-6-oxohexy 1)-2-(( 1 E,3E)-5 -((E)-1 -(6-((2,5 -dioxopy rrolidin-1 -yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-l,3-dien-l-yl)-3,3-dimethyl-3H-indol-l-ium or 1-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-2-((IE,3E)-5-((E)-l-(6-((2,5-dioxopyrrolidin-1 -yl)oxy)-6-oxohexy 1)-3,3 -dimethyl-5 -sulfoindolin-2-ylidene)penta-1,3-dien-1 -yl)-3,3-dimethyl-3H-indol-l-ium-5-sulfonate), and Cy7 (which may comprise 1-(5-carboxypentyl)-2-[(lE,3E,5E,7Z)-7-(l-ethyl-l,3-dihydro-2H-indol-2-ylidene)hepta-l,3,5-trien-l-yl]-3H-indolium or l-(5-carboxypentyl)-2-[(lE,3E,5E,7Z)-7-(l-ethyl-5-sulfo-l,3-dihydro-2H-indol-2-ylidene)hepta-l,3,5-trien-l-yl]-3H-indolium-5-sulfonate), where “Cy” stands for 'cyanine', and the first digit identifies the number of carbon atoms between two indolenine groups. Cy2 which is an oxazole derivative rather than indolenin, and the benzo-derivatized Cy3.5, Cy5.5 and Cy7.5 are exceptions to this rule.

[00214] In some embodiments, the detection label may be a FRET pair, such that multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET may comprise excitation exchange (Forster) transfers, or electron-exchange (Dexter) transfers.

[00215] In some embodiments, the one or more nucleotide conjugates (e.g. detectable nucleotide conjugates) are labeled with fluorophores and the detecting step comprises use of fluorescence imaging; and especially wherein the fluorescence imaging comprises dual wavelength excitation / fourwavelength emission fluorescenceimaging. In some embodiments, the four different nucleotide conjugates (e.g. detectable nucleotide conjugates), each comprising a different nucleotide or nucleotide analog, are used to determine the identity of the terminal nucleotide, wherein the four different nucleotide conjugates (e.g. detectable nucleotide conjugates) are labeled with separate respective fluorophores, and wherein the detecting step comprises simultaneous excitation at a wavelength sufficient to excite all four fluorophores and imaging of fluorescence emission at wavelengths sufficient to detect each respective fluorophore. In some embodiments, four different nucleotide conjugates (e.g. detectable nucleotide conjugates), each comprising a different nucleotide or nucleotide analog, are used to determine the identity of the terminal nucleotide, wherein the four different nucleotide conjugates (e.g detectable nucleotide conjugates) are labeled with Cy3, Cy3.5, Cy5, and Cy5.5 respectively, and wherein the detecting step comprises simultaneous excitation at any two of 532 nm, 568 nm and 633 nm, and imaging of fluorescence emission at about 570 nm, 592 nm, 670 nm, and 702 nm respectively; and / or wherein the fluorescence imaging comprises dual wavelength excitation / dual wavelength emission fluorescence imaging. In some embodiments, the four different nucleotide conjugates (e.g. detectable nucleotide conjugates), each comprising a different nucleotide or nucleotide analog, are used to determine the identity of the terminal nucleotide, wherein one, two, three, or four different nucleotide conjugates (e.g. detectable nucleotide conjugates) are respectively labeled, each with a with distinct fluorophore or set of fluorophores, and wherein the detecting step comprises simultaneous excitation at a wavelength sufficient to excite one, two, three, or four fluorophores or sets of fluorophores, and imaging of fluorescence emission at wavelengths sufficient to detect each respective fluorophore. In some embodiments, the three different nucleotide conjugates (e.g. detectable nucleotide conjugates), each comprising a different nucleotide or nucleotide analog, are used to determine the identity of the terminal nucleotide, wherein one, two, or three different nucleotide conjugates (e.g. detectable nucleotide conjugates) are respectively labeled, each with a with distinct fluorophore or set of fluorophores, and wherein the detecting step comprises simultaneous excitation at a wavelength sufficient to excite one, two, or three, fluorophores or sets of fluorophores, and imaging of fluorescence emission at wavelengths sufficient to detect each respective fluorophore, and wherein detection of the fourth nucleotide is determined or determinable with reference to the location of “dark” or unlabeled spots ortargetnucleotides. In some embodiments, the binding complex consists ofthree types of nucleotide conjugates (e.g. detectable nucleotide conjugates) and wherein each type of the three types of nucleotide conjugates (e.g. detectable nucleotide conjugates) comprises a WO 2025 / 170937                                   PCT / US2025 / 014502 different type of nucleotide. In some embodiments, the detection of the binding complex is performed in the absence of unbound or solution-borne polymer nucleotide conjugates.

[00216] The non-limiting example of the nucleotide conjugate (e.g. detectable nucleotide conjugatejin FIG. ID comprises four nucleotide conjugates (e.g. detectable nucleotide conjugates) 105a, 105b, 105c, and 105d. Each nucleotide conjugate (e.g. detectable nucleotide conjugatejhas multiple copies of a nucleotide attached to the particle, and the four nucleotide conjugates (e.g. detectable nucleotide conjugates) cover fourtypes of nucleotiderespectively. The nucleotide conjugate (e.g. detectable nucleotide conjugate)having a nucleotide that is complementary to the next base on the target may form a binding complex with the polymerase and the target nucleic acid. In some instances, 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 conjugatejean be labeled with a separate label. In some embodiments, three of four types of nucleotide conjugates can be labeled, with a fourth either unlabeled or conjugated to an undetectable label. In some embodiments, 1, 2, 3, or 4 nucleotide conjugates (e.g. detectable nucleotide conjugates) can be labeled, either with the same label, or each with a label corresponding to the identity of its conjugated nucleotide, with, respectively, 3, 2, 1, or no nucleotide conjugates (e.g. detectable nucleotide conjugates) that may be either left unlabeled or conjugated to an undetectable label. In some embodiments, detection of a polymerase complex incorporating a nucleotide conjugate (e.g. detectable nucleotide conjugatejmay be carried out using four-color detection, such that conjugates corresponding to all four nucleotides are present in a sample, each conjugate having a separate label corresponding to the nucleotide conjugated thereto. In some embodiments, the four nucleotide conjugates (e.g. detectable nucleotide conjugates) may be exposed to or contacted with the target at the same time; in some other embodiments, the four nucleotide conjugates (e.g detectable nucleotide conjugates) may be exposed to or contacted with the target sequentially, either individually, or in groups of two or three. In some embodiments, detection of a polymerase complex incorporating a nucleotide conjugate (e.g. detectable nucleotide conjugatejmay be carried out using three-color detection, such that conjugates corresponding to three of the four nucleotides are present in a sample, with three conjugates having a separate label corresponding to the nucleotide conjugated thereto and one conjugate having no label or being conjugated to an undetectable label. In some embodiments, only three types of conjugates are provided, such that conjugates corresponding to three of the four nucleotides are present in a sample, with three conjugates having a separate label corresponding to the nucleotide conjugated thereto and one conjugate being absent. In some embodiments, the identity of nucleotides corresponding to an unlabeled or absent nucleotide conjugate can be determined with respect to the location and / or WO 2025 / 170937                                   PCT / US2025 / 014502 identity of “dark” spots or locations of known targets showing no fluorescence signal. In some embodiments, the detection of the binding complex is performed in the absence of unbound or solution-borne polymer nucleotide conjugates.

[00217] In some embodiments, the nucleotide conjugate (e.g. detectable nucleotide conjugate)comprises one or more nucleotides or nucleotide analogs. In some embodiments, the nucleotide conjugate (e.g. detectable nucleotide conjugate)may comprise a single type of nucleotide. In some embodiments, the nucleotide conjugate (e.g. detectable nucleotide conjugate)comprise 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.

[00218] In some embodiments, the present disclosure provides a reagent comprising one or more nucleotide conjugates (e.g. detectable nucleotide conjugates) as disclosed herein and a buffer. For example, in some embodiments, the present disclosure provides a reagent, wherein said reagent comprises 1, 2, 3, 4, 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, a reagent of the present disclosure comprises 1, 2, 3, 4, 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 analog, and wherein said nucleotide or nucleotide moiety may respectively correspond to one or more from the group consisting of ATP, ADP, AMP, dATP, dADP, and dAMP; one or more from the group consisting of TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, and dUMP; one or more from the group consisting of CTP, CDP, CMP, dCTP, dCDP, and dCMP; and one or more from the group consisting of GTP, GDP, GMP, dGTP, dGDP, and dGMP. In some other examples or some further examples, the present disclosure provides a reagent comprising or further comprising 1, 2, 3, 4, or more nucleotide conjugates (e.g. detectable nucleotide conjugates), wherein each nucleotide conjugate (e.g. detectable nucleotide conjugate)comprisesa single type of nucleotide or nucleotide moiety, and wherein said nucleotide or nucleotide moiety may respectively correspond to one or more 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.

[00219] In some embodiments, the nucleotide conjugate (e.g. detectable nucleotide conjugate)is included in a mixture of a plurality of nucleotide conjugates (e.g. detectable nucleotide conjugates), wherein each of the plurality of nucleotide conjugates (e.g. detectable WO 2025 / 170937                                   PCT / US2025 / 014502 nucleotide conjugates) comprises different types of nucleotide moi eties from each other. The type of nucleotide 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 a combination thereof.

[00220] In some embodiments, the nucleotide conjugate (e.g. detectable nucleotide conjugate)comprises a polymer having a plurality of branches. In some embodiments, the plurality of branches comprises copies of a nucleotide coupled thereto. In some embodiments, the polymer has a star, comb, cross-linked, bottlebrush, or dendrimer configuration. In some embodiments, the nucleotide conjugate (e.g. detectable nucleotide conjugate)comprises one or more binding groups comprising avidin, biotin, affinity tag, or any combination thereof. In some embodiments, a binding group is interposed between a branch of the polymer and a nucleotide.

[00221] In some embodiments, the nucleotide conjugate (e.g. detectable nucleotide conjugate)may comprise a common core. The common core may comprise multiple copies of the same nucleotide covalently bound to or noncovalently bound to the common core. Examples of the common core can include a polymer particle; a metal particle; a quantum dot; a liposome; an emulsion particle, or any other particle (e.g., nanoparticles, microparticles, orthe like). In some embodiments, the polymer particle is or comprises a branched polymer; a dendrimer; a cross linked polymer particle such as an agarose, polyacrylamide, acrylate, methacrylate, cyanoacrylate, methyl methacrylate particle; a glass particle; a ceramic particle; or a protein particle. In some embodiment, 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. detectable nucleotide conjugate)comprises multiple polymer branches radiating from the common core, wherein some branches are attached to a nucleotide and / or some branches are attached to an oligonucleotide.

[00222] In some embodiments, the present disclosure provides a nucleotide conjugate (e.g. detectable nucleotide conjugate)comprising a branched polymer having two or more branches and two or more copies of a nucleotide, wherein said nucleotide is attached to a first plurality of said branches or arms, and optionally, wherein one or more interaction moieties are attached to a second plurality of said branches or arms. In some embodiments, said nucleotide conjugate (e.g. detectable nucleotide conjugate)may further comprise one or more labels on the polymer. In some embodiments, the present disclosure provides said nucleotide conjugate (e.g detectable nucleotide conjugate)wherein the nucleoside has a surface density of at least 4 nucleotides per polymer. In some embodiments, the present disclosure provides said nucleotide conjugate (e.g. detectable nucleotide conjugate)comprising or incorporating a nucleotide or nucleotide analog that is modified so as to prevent its incorporation into an extending nucleic acid chain during a polymerase reaction. In some embodiments, said nucleotide conjugate (e.g. detectable nucleotide conjugate)may comprise or incorporate a nucleotide or nucleotide analog that is reversibly modified so as to preventits incorporation into an extending nucleic acid chain during a polymerase reaction. In some embodiments, the present disclosure provides said nucleotide conjugate (e.g. detectable nucleotide conjugatejwherein one or more labels comprise a fluorescent label, a FRET donor, and / or a FRET acceptor. In some embodiments, said nucleotide conjugate (e.g. detectable nucleotide conjugatejmay comprise2, 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 moiety. In some embodiments, said nucleotide conjugate (e.g. detectable nucleotide conjugatejmay comprise one or more interaction moieties, which interaction moieties may comprise avidin or streptavidin; a biotin moiety; an affinity tag; an enzyme, antibody, minibody, receptor, or other protein; a non-protein tag; a metal affinity tag, or any combination thereof. In some embodiments, the present disclosure provides said nucleotide conjugate (e.g. detectable nucleotide conjugate jwherein the polymer comprises polyethylene glycol, polypropylene glycol, polyvinyl acetate, polylactic acid, or polyglycolic acid. In some embodiments, the present disclosure provides said nucleotide conjugate (e.g. detectable nucleotide conjugatejwherein the nucleotide or nucleotide analog is attached to the branch or arm through a linker; and especially wherein the linker comprises PEG, and wherein the PEG moiety has an average molecular weight of about IK, about 2K, about 3K, about4K, about5K, about 10K, about 15K, about20K, about50K, about 100K, about 150K,or about 200K, or greater than about 200K. In some embodiments, the present disclosure provides said nucleotide conjugate (e.g. detectable nucleotide conjugatejwherein the linker comprises PEG, and wherein the PEG moiety has an average molecular weight of between about 5K and about 20K. In some embodiments, the present disclosure provides said nucleotide conjugate (e.g. detectable nucleotide conjugatejwherein at least one nucleotide or nucleotide analog comprises a deoxyribonucleotide, a ribonucleotide, a deoxyribonucleoside, or a ribonucleoside; and / or wherein the nucleotide or nucleotide analog is conjugated to the linker through the 5’ end of the nucleotide or nucleotide analog. In some embodiments, the present disclosure provides said nucleotide conjugate (e.g. detectable nucleotide conjugatejwherein one of the nucleotides or nucleotide analogs comprises deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxy cytidine, adenosine, guanosine, 5-methyl-uridine, and / or cytidine; and wherein the length of the linker is between 1 and l,000nm.

[00223] In any of the methods described herein, any of the sequencing methods described herein can employ at least one nucleotide. The nucleotides comprise a base, sugar and at least one phosphate group. In some embodiments, at least one nucleotide in the plurality comprises an aromatic base, a five carbon sugar (e.g., ribose or deoxyribose), and one or more phosphate groups (e.g., 1-10 phosphate groups). The plurality of nucleotides can comprise at least one type of nucleotide selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. The plurality of nucleotides can comprise at a mixture of any combination of two or more types of nucleotides selected from a group consisting of dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, at least one nucleotide in the plurality is not a nucleotide analog. In some embodiments, at least one nucleotide in the plurality comprises a nucleotide analog.

[00224] In some embodiments, in any of the methods for sequencing described herein, at least one nucleotide in the plurality of nucleotides comprise a chain of one, two or three phosphorus atoms where the chain is typically attached to the 5’ carbon of the sugar moiety via an ester or phosphoramide linkage. In some embodiments, at least one nucleotide in the plurality is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene or ethylene. In some embodiments, the phosphorus atoms in the chain include substituted side groups including O, S or BH3. In some embodiments, the chain includes phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphordithioate, and O-methylphosphoroamidite groups.

[00225] In some embodiments, in any of the methods for sequencing described herein, at least one nucleotide in the plurality of nucleotides comprises a terminator nucleotide analog having a chain terminating moiety (e.g., blocking moiety) at the sugar 2’ position, at the sugar 3 ’ position, or at the sugar 2’ and 3 ’ position. In some embodiments, the chain terminating moiety can inhibit polymerase-catalyzed incorporation of a subsequent nucleotide moiety or free nucleotide in a nascent strand during a primer extension reaction. In some embodiments, the chain terminating moiety is attached to the 3 ’ sugar hydroxyl position where the sugar comprises a ribose or deoxyribose sugar moiety. In some embodiments, the chain terminating moiety is removable / cleavable from the 3 ’ sugar hydroxyl position to generate a nucleotide having a 3 ’OH sugar group which is extendible with a subsequent nucleotide in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group. In some embodiments, the chain terminating moiety is cleavable / removable from the nucleotide, for example by reacting the chain terminating moiety with a chemical agent, pH change, light or heat. In some embodiments, the chain terminating moieties alkyl, alkenyl, alkynyl and allyl are cleavable with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano-l,4-benzo-quinone (DDQ). In some embodiments, the chain terminating moieties aryl and benzyl are cleavable with H2 Pd / C. In some embodiments, the chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are cleavable with phosphine or with a thiol group including beta-mercaptoethanol or dithiothritol (DTT). In some embodiments, the chain terminating moiety carbonate is cleavable with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). In some embodiments, the chain terminatingmoieties urea and silyl are cleavable with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride.

[00226] In some embodiments, in any of the methods for sequencing described herein, at least one nucleotide in the plurality of nucleotides comprises a terminator nucleotide analog having a chain terminating moiety (e.g., blocking moiety) at the sugar 2’ position, at the sugar 3’ position, or at the sugar 2’ and 3’ position. In some embodiments, the chain terminating moiety comprises an azide, azido or azidomethyl group. In some embodiments, the chain terminating moiety comprises a 3’-O-azido or 3’-O-azidomethyl group. In some embodiments, the chain terminating moieties azide, azido and azidomethyl group are cleavable / removable with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized tri-alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound comprises Tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[00227] In some embodiments, in any of the methods for sequencing described herein, the nucleotide comprises a chain terminating moiety which is selected from a group consisting of 3’-deoxy nucleotides, 2’,3’-dideoxynucleotides, 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’-sulfhydral, 3’-aminomethyl, 3’-ethyl, 3’butyl, 3’-tertbutyl, 3’- Fluorenylmethyloxycarbonyl, 3’ tert-Butyloxycarbonyl, 3’-O-alkyl hydroxylamino group, 3’-phosphorothioate, and 3-O-benzyl, or derivatives thereof.

[00228] In some embodiments, in any of the methods for sequencing described herein, the plurality of nucleotides comprises a plurality of nucleotides labeled with detectable reporter moiety. The detectable reporter moiety comprises a fluorophore. In some embodiments, the fluorophore is attached to the nucleotide base. In some embodiments, the fluorophore is attached to the nucleotide base with a linker which is cleavable / removable from the base. In some embodiments, at least one of the nucleotides in the plurality is not labeled with a detectable reporter moiety. In some embodiments, a particular detectable reporter moiety (e.g., fluorophore) WO 2025 / 170937                                   PCT / US2025 / 014502 that is attached to the nucleotide can correspond to the nucleotide base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) to permit detection and identification of the nucleotide base.

[00229] In some embodiments, in any of the methods for sequencing nucleic acid molecules described herein, the cleavable linker on the nucleotide base comprises a cleavable moiety comprising an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group. In some embodiments, the cleavable linker on the base is cleavable / removable from the base by reacting the cleavable moiety with a chemical agent, pH change, light or heat. In some embodiments, the cleavable moieties alkyl, alkenyl, alkynyl and allyl are cleavable with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone(DDQ). In some embodiments, the cleavable moieties aryl and benzyl are cleavable with H2 Pd / C. In some embodiments, the cleavable moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are cleavable with phosphine or with a thiol group including beta-mercaptoethanol or dithiothritol (DTT). In some embodiments, the cleavable moiety carbonate is cleavable with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). In some embodiments, the cleavable moieties urea and silyl are cleavable with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with tri ethylamine trihydro fluoride.

[00230] In some embodiments, in any of the methods for sequencing described herein, the cleavable linker on the nucleotide base comprises cleavable moiety including an azide, azido or azidomethyl group. In some embodiments, the cleavable moieties azide, azido and azidomethyl group are cleavable / removable with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized tri-alkyl phosphine moiety or a derivatized triaryl phosphine moiety. In some embodiments, the phosphine compound comprises Tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[00231] In some embodiments, in any of the methods for sequencing described herein, the chain terminating moiety (e.g., at the sugar 2’ and / or sugar 3’ position) and the cleavable linker on the nucleotide base have the same or different cleavable moieties. In some embodiments, the chain terminating moiety (e.g., at the sugar 2’ and / or sugar 3’ position) and the detectable reporter moiety linked to the base are chemically cleavable / removable with the same chemical agent. In some embodiments, the chain terminating moiety (e.g., atthe sugar 2’ and / or WO 2025 / 170937                                   PCT / US2025 / 014502 sugar 3’ position) and the detectable reporter moiety linked to the base are chemically cleavable / removable with different chemical agents.

[00232] The nucleotide can be linked to the common core through a linker, and the nucleotide can be attached to one end or location of a polymer. The nucleotide can be conjugated to the common core through the 5’ end of the nucleotide. In some common core-nucleotide conjugates, one nucleotide attached to one end or location of a polymer. In some common core-nucleotide conjugate, multiple nucleotides are attached to one end or location of a polymer. The conjugated nucleotide is sterically accessible to one or more proteins, one or more enzymes, and nucleotide bindingmoieties. In some embodiments, a nucleotide may be provided separately from a nucleotide binding moiety such as a polymerase. In some embodiments, the linker does not comprise a photo emitting or photo absorbing group.

[00233] The common core can also have a binding moiety. In some embodiments, common cores may self-associate without the use of a separate interaction moiety. In some embodiments, common cores may self-associate due to buffer conditions or salt conditions, e.g, 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 metallic (such as iron or gold) nanoparticles.

[00234] The nucleotide conjugate (e.g. detectable nucleotide conjugate)can have one or more labels. Examples of the labels include but are not limited to fluorophores, spin labels, metals or metal ions, colorimetric labels, nanoparticles, PET labels, radioactive labels, or other such label as may render said nucleotide conjugate (e.g. detectable nucleotide conjugate)detectable by such methods as are known in the art of the detection of macromolecules or molecular interactions. The label may be attached to the nucleotide (e.g. by attachment to the 5’ phosphate moiety of a nucleotide), to the common core itself (e.g., to the PEG subunits), to an end of the polymer, to a central moiety, or to any other location within said polymer-nucleotide conjugate which would be recognized by one of skill in the art to be sufficient to render said detectable nucleotide conjugate, such as a common core, detectable by such methods as are known in the art or described elsewhere herein. In some embodiments, one or more labels are provided so as to correspond to or differentiate a particular detectable nucleotide conjugate.

[00235] One example of the nucleotide conjugate (e.g. detectable nucleotide conjugate)is a polymer-nucleotide conjugate. Some non-limiting examples of the nucleotide conjugates (e.g. detectable nucleotide conjugates) are shown in FIG. 5A-5C. For example, FIG. 5A shows nucleotide conjugates (e.g. detectable nucleotide conjugates) having various configurations; FIG. 5B shows a nucleotide conjugate (e.g. detectable nucleotide WO 2025 / 170937                                   PCT / US2025 / 014502 conjugate)having a polymer branch radiating from the center; and FIG. 5C shows nucleotide conjugates (e.g. detectable nucleotide conjugates) having a binding moiety such as a biotin.

[00236] Examples of the branched polymer include polyethylene glycol (PEG), polypropylene glycol, polyvinyl alcohol, polylactic acid, polyglycolic acid, polyglycine, polyvinyl acetate, a dextran, or other such polymers, or copolymers incorporating any two or more of the foregoing or incorporating other polymers as are known in the art. In some embodiment, the polymer is a PEG. In another embodiment, the polymer may have PEG branches.

[00237] Suitable polymers may be characterized by a repeating unit incorporating a functional group suitable for derivatization such as an amine, a hydroxyl, a carbonyl, or an allyl group. The polymer can also have one or more pre-derivatized substituents such that one or more particular subunits will incorporate a site of derivatization or a branch site, whether or not other subunits incorporate the same site, substituent, or moiety. A pre-derivatized substituent may comprise or may further comprise, for example, a nucleotide, a nucleoside, a nucleotide analog a label such as a fluorescent label, radioactive label, or spin label, an interaction moiety, an additional polymer moiety, or the like, or any combination of the foregoing.

[00238] In some embodiments, the nucleotide conjugate (e.g. detectable nucleotide conjugate)may comprise a polymer. The polymer can have a plurality of branches. The branched polymer can have various configurations, including but are not limited to stellate (“starbursf’) forms, aggregated stellate (“helter skelter”) forms, bottle brush, or dendrimer. The branched polymer can radiate from a central attachment point or central moiety, or may incorporate multiple branch points, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more branch points. In some embodiments, each subunit of a polymer may optionally constitute a separate branch point.

[00239] The length and size of the branch can differ based on the type of polymer. In some branched polymers, the branch may have a length of between 1 and 1,000 nm, between 1 and 100 nm, between 1 and 200 nm, between 1 and 300 nm, between 1 and 400 nm, between 1 and 500 nm, between 1 and 600 nm, between 1 and 700 nm, between 1 and 800 nm, or between 1 and 900 nm, or more, or having a length falling within or between any of the values disclosed herein. In some branched polymers, the branch may have a size corresponding to an apparent molecular weight of IK, 2K, 3K, 4K, 5K, 10K, 15K, 20K, 3OK, 5OK, 80K, 100K, or any value within a range defined by any two of the foregoing. The apparent molecular weight of a polymer may be calculated from the known molecular weight of a representative number of subunits, as determined by size exclusion chromatography, as determined by mass spectrometry, or as determined by any other method as is known in the art. The polymer can have multiple branches. The number of branches in the polymer can be 2, 3, 4, 5, 6, 7, 8, 12, 16, 24, 32, 64, 128 or more, or a number falling within a range defined by any two of these values.

[00240] For the detectable nucleotide conjugate, the branched polymer of 4, 8, 16, 32, or 64 branches can have nucleotides attached to the ends of PEG branches, such that each end has attached thereto 0, 1, 2, 3, 4, 5, 6 or more nucleotides. In one non-limiting example, the branched polymer of between 3 and 128 PEG arms having attached to the polymer branches ends one or more nucleotides, such that each end has attached thereto 0, 1, 2, 3, 4, 5,6 or more nucleotides or nucleotide analogs. In some embodiments, a branched polymer or dendrimer has an even number of arms. In some embodiments, a branched polymer or dendrimer has an odd number of arms.

[00241] In some embodiments, the nucleotide conjugate (e.g. detectable nucleotide conjugate)may comprisea polymer where each branch or a subset ofbranchesofthe polymer may have attached thereto a moiety comprising a nucleotide (e.g., an adenine, a thymine, an uracil, a cytosine, or a guanine residue or a derivative or mimetic thereof), and the moiety is capable of binding to a polymerase, reverse transcriptase, or other nucleotide binding domain. Optionally, the moiety may be capable of being incorporated into an elongating nucleic acid chain during a polymerase reaction. In some instances, said moiety may be blocked such that it is not capable of being incorporated into an elongating nucleic acid chain during a polymerase reaction. In some other instances, said moiety may be reversibly blocked such that it is not capable of being incorporated into an elongating nucleic acid chain during a polymerase reaction until such block is removed, after which said moiety is then capable of being incorporated into an elongating nucleic acid chain during a polymerase reaction.

[00242] The nucleotide can be conjugated to the polymer branch through the 5’ end of the nucleotide. In some instances, the nucleotide may be modified so as to inhibit or prevent incorporation of the nucleotideinto an elongating nucleic acid chain during a polymerase reaction. By way of example, the nucleotide may include a 3 ’ deoxyribonucleotide, a 3 ’ azidonucleotide, a 3 ’-methyl azido nucleotide, or another such nucleotide as is or may be known in the art, so as to not be capable of being incorporated into an elongating nucleic acid chain during a polymerase reaction. In some embodiments, the nucleotide can include a 3’-O-azido group, a 3’-O-azidomethyl group, a 3’-phosphorothioate group, a 3’-O-malonyl group, a 3’-O-alkyl hydroxylamino group, or a 3’-O-benzyl group. In some embodiments, the nucleotide lacks a 3’ hydroxyl group.

[00243] The polymer can further have a binding moiety in each branch or a subset of branches. Some examples of the binding moiety include but are not limited to biotin, avidin, streptavidin or the like, polyhistidine domains, complementary paired nucleic acid domains, G-quartet forming nucleic acid domains, calmodulin, maltose-binding protein, cellulase, maltose, sucrose, glutathione-S-transferase, glutathione, O-6-methylguanine-DNA methyltransferase, benzylguanine and derivatives thereof, benzyl cysteine and derivatives thereof, an antibody, an epitope, a protein A, a protein G. The binding moiety can be any interactive molecules or fragment thereof known in the art to bind to or facilitate interactions between proteins, between proteins and ligands, between proteins andnucleic acids, between nucleic acids, orbetween small molecule interaction domains or moieties.

[00244] In some embodiments, a nucleotide conjugate (e.g. detectable nucleotide conjugate)as provided herein may comprise one or more elements of a complementary interaction moiety. Example complementary interaction moieties include, for example, biotin and avidin; SNAP-benzylguanosine; antibody or FAB and epitope; IgG FC and Protein A, Protein G, Protein A / G, or Protein L; maltose binding protein and maltose; lectin and cognate polysaccharide; ion chelation moieties, complementary nucleic acids, nucleic acids capable of forming triplex or triple helical interactions; nucleic acids capable of forming G-quartets, and the like. One of skill in the art will readily recognize that many pairs of moieties exist and are commonly used for their property of interacting strongly and specifically with one another; and thus any such complementary pair or set is considered to be suitable for this purpose in constructing or envisioning the nucleotide conjugates (e.g. detectable nucleotide conjugates) of the present disclosure. In some embodiments, a nucleotide conjugate (e.g. detectable nucleotide conjugate)as disclosed herein may comprise nucleotide conjugates (e.g. detectable nucleotide conjugates) in which one element of a complementary interaction moiety is attached to one molecule or multivalent ligand, and the other element of the complementary interaction moiety is attached to a separate molecule or multivalent ligand. In some embodiments, a nucleotide conjugate (e.g detectable nucleotide conjugate)as disclosed herein may comprise nucleotide conjugates (e.g. detectable nucleotide conjugates) in which both or all elements of a complementary interaction moiety are attached to a single molecule or multivalent ligand. In some embodiments, a nucleotide conjugate (e.g. detectable nucleotide conjugate)as disclosed herein may comprise nucleotide conjugates (e.g. detectable nucleotide conjugates) in which both or all elements of a complementary interaction moiety are attached to separate arms of, or locations on, a single molecule or multivalent ligand. In some embodiments, a nucleotide conjugate (e.g. detectable nucleotide conjugate)as disclosed herein may comprise nucleotide conjugates (e.g. detectable nucleotide conjugates) in which both or all elements of a complementary interaction moiety are attached to the same arm of, or locations on, a single molecule or multivalent ligand. In some embodiments, nucleotide conjugates (e.g. detectable nucleotide conjugates) comprising one element of a complementary interaction moiety and nucleotide conjugates (e.g. detectable nucleotide conjugates) comprising another element of a complementary interaction moiety may be simultaneously or sequentially mixed. In some embodiments, interactions between molecules or particles as disclosed herein allow for the association or aggregation of multiple molecules or particles such that, for example, detectable signals are increased. In some embodiments, fluorescent, colorimetric, or radioactive signals are enhanced. In other embodiments, other interaction moieties as disclosed herein or as are known in the art are contemplated. In some embodiments, a nucleotide conjugate (e.g. detectable nucleotide conjugate)as provided herein may be provided such that one or more molecules comprising a first interaction moiety such as, for example, one or more imidazole or pyridine moieties, and one or more additional molecules comprising a second interaction moiety such as, for example, histidine residues, are simultaneously or sequentially mixed. In some embodiments, said nucleotide conjugate (e.g. detectable nucleotide conjugate)comprises 1, 2, 3, 4, 5, 6, or more imidazole or pyridine moieties. In some embodiments, said nucleotide conjugate (e.g. detectable nucleotide conjugatejcomprises 1,2, 3,4,5, 6, or more histidine residues. In such embodiments, interaction between the molecules or particles as provided may be facilitated by the presence of a divalent cation such as nickel, manganese, magnesium, calcium, strontium, or the like. In some embodiments, for example, a (His)3 group may interact with a (His)3 group on another molecule or particle via coordination of a nickel or manganese ion. ix. Binding complexes

[00245] The methods described herein may involve detecting a target by forming a binding complex between a primed index sequence described herein and a nucleotide conjugate (e.g. detectable nucleotide conjugate)as described herein. The target may be a ribonucleic acid. The binding complex may include one or more non-covalent interactions.

[00246] In any of the methods described herein, the sequencing employs at least one multivalent molecule which comprises a plurality of nucleotide arms attached to a core and having any configuration including a starburst, helter skelter, or bottle brush configuration (e.g, FIGs. 5A-5C). In some embodiments, the multivalent molecule comprises: (1) a core; and (2) a plurality of nucleotide arms which comprise (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide moiety, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, wherein the linker is attached to the nucleotide moiety (e.g., FIGs. 16-18). In some embodiments, the nucleotidemoiety comprises a base, sugar and at least one phosphate group, and the linker is attached to the nucleotide moiety through the base. In some embodiments, the linker comprises an aliphatic chain or an oligo ethylene glycol chain where both linker chains having 2-6 subunits. In some embodiments, the linker also includes an aromatic moiety. An example nucleotide arm is shown in FIG. 19. Example multivalent molecules are shown in FIGs. 5A-C and 16-18. An example WO 2025 / 170937                                   PCT / US2025 / 014502 spacer is shown in Figure 6 (top) and example linkers are shown in FIG. 20 (bottom) and FIG. 21. Example nucleotides attached to a linker are shown in FIGs. 22-24. An example biotinylated nucleotide arm is shown in FIG. 25.

[00247] The formation of a binding complex in the context of the methods and systems described herein may have certain advantages. An increase in binding of a nucleotide to an enzyme (e.g., polymerase) or an enzyme complex can be affectedby increasingthe effective concentration of the nucleotide. The increase can be achieved by increasingthe concentration of the nucleotide in free solution, or by increasingthe amount of the nucleotide in proximity to the relevant binding site. The increase can also be achieved by physically restricting a number of nucleotides into a limited volume thus resulting in a local increase in concentration, and such as structure may thus bind to the binding site with a higher app arent avidity than would be observed with unconjugated, untethered, or otherwise unrestricted individual nucleotide. One example means of effecting such restriction is by providing a binding complex in which multiple nucleotides are bound to a particle such as a polymer, a branched polymer, a dendrimer, a micelle, a liposome, a microparticle, a nanoparticle, a quantum dot, or other suitable particle known in the art.

[00248] The binding complex can be used to localize detectable signals to active regions of biochemical interactions, such as sites of protein-nucleic acid interactions, nucleic acid hybridization reactions, or enzymatic reactions, such as polymerase reactions. For instance, the binding complex described herein can be utilized to identify sites of base incorporation in elongating nucleic acid chains during polymerase reactions and to provide base discrimination 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 enhanced signal that greatly improve base call accuracy and shorten imaging time.

[00249] In addition, the formation of a binding complex as described herein allows sequencing signals from a given sequence to originate within cluster regions containing multiple copies of the target sequence. Sequencing methods incorporating multiple copies of a target sequence have the advantage that signals can be amplified due to the presence of multiple simultaneous sequencing reactions within the defined region, each providing its own signal. The presence of multiple signals within a defined area also reduces the impact of any single skipped cycle, due to the fact that the signal from a large number of correct base calls can overwhelm the signal from a smaller number of skipped or incorrect base calls, therefore providing methods for reducing phasing errors and / or to improve read length in sequencing reactions.

[00250] The formation of a binding complex and the compositions associated with it, as described herein may lead to one or more of: (i) stronger signal for better base-calling accuracy compared to conventional nucleic acid amplification and sequencing methodologies; ii) allow greater discrimination of sequence-specific signal from background signals; (iii) reduced requirements for the amount of starting material necessary, (iv) increased sequencing rate and shortened sequencing time; (v) reducing phasing errors, and (vi) improving read length in sequencing reactions.

[00251] In some embodiments, the nucleotide moiety comprises a chain of one, two or three phosphorus atoms where the chain is typically attached to the 5’ carbon of the sugar moiety via an ester or phosphoramide linkage. In some embodiments, at least one nucleotide moiety is a nucleotide analog having a phosphorus chain in which the phosphorus atomsare linked together with interveningO, S, NH, methylene or ethylene. In some embodiments, the phosphorus atoms in the chain include substituted side groups including O, S or BH3. In some embodiments, the chain includes phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphordithioate, and O-methylphosphoroamidite groups.

[00252] In some embodiments, the multivalent molecule comprises a coreattached to multiple nucleotide arms, and wherein individual nucleotide arms comprise a nucleotide moiety which is a nucleotide analog havinga chain terminating moiety (e.g., blockingmoiety) atthe sugar 2’ position, atthe sugar3’ position, or atthe sugar 2’ and 3’ position. In some embodiments, the nucleotide moiety comprises a chain terminating moiety (e.g., blocking moiety) at the sugar 2’ position, at the sugar 3’ position, or at the sugar 2’ and 3’ position. In some embodiments, the chain terminating moiety can inhibit polymerase-catalyzed incorporation of a subsequent nucleotide moiety or free nucleotide in a nascent strand during a primer extension reaction. In some embodiments, the chain terminating moiety is attached to the 3’ sugar hydroxyl position where the sugar comprises a ribose or deoxyribose sugar moiety. In some embodiments, the chain terminating moiety is removable / cleavable from the 3’ sugar hydroxyl position to generate a nucleotide having a 3’OH sugar group which is extendible with a subsequent nucleotide in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group. In some embodiments, the chain terminating moiety is cleavable / removable from the nucleotide moiety, for example by reacting the chain terminating moiety with a chemical agent, pH change, light or heat. In some embodiments, the chain terminating moieties alkyl, alkenyl, alkynyl and allyl are cleavable with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano-l,4-benzo-quinone (DDQ). In some embodiments, the chain terminating moieties aryl and benzyl are cleavable with H2 Pd / C. In some embodiments, the chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are cleavable with phosphine or with a thiol group including beta-mercaptoethanol or dithiothritol (DTT). In some embodiments, the chain terminating moiety carbonate is cleavable with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). In some embodiments, the chain terminating moieties urea and silyl are cleavable with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride.

[00253] In some embodiments, the nucleotide moiety comprises a chain terminating moiety (e.g., blocking moiety) at the sugar 2’ position, at the sugar 3’ position, or at the sugar 2’ and 3’ position. In some embodiments, the chain terminating moiety comprises an azide, azido or azidomethyl group. In some embodiments, the chain terminating moiety comprises a 3’-O-azido or 3’-O-azidomethyl group. In some embodiments, the chain terminating moieties azide, azido and azidomethyl group are cleavable / removable with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized tri-alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound comprises Tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[00254] In some embodiments, the nucleotide moiety comprising a chain terminating moiety which is selected from a group consisting of 3’-deoxy nucleotides, 2’,3’-dideoxynucleotides, 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’-sulfhydral, 3’-aminomethyl, 3’-ethyl, 3’butyl, 3’-tertbutyl, 3’-Fluorenylmethyloxycarbonyl, 3’ / c / 7-Butyloxycarbonyl, 3’-O-alkyl hydroxylamino group, 3’-phosphorothioate, and 3-O-benzyl, or derivatives thereof.

[00255] In some embodiments, the multivalent molecule comprises a coreattached to multiple nucleotide arms, wherein the nucleotide arms comprise a spacer, linker and nucleotide moiety, and wherein the core, linker and / or nucleotide moiety is labeled with detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, a particular detectable reporter moiety (e.g., fluorophore) that is attached to the multivalent molecule can correspond to the base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) of the nucleotide moiety to permit detection and identification of the nucleotide base.

[00256] In some embodiments, at least one nucleotide arm of a multivalent molecule has a nucleotide moiety that is attached to a detectable reporter moiety. In some embodiments, the detectable reporter moiety is attached to the nucleotide base. In some WO 2025 / 170937                                   PCT / US2025 / 014502 embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, a particular detectable reporter moiety (e.g., fluorophore) that is attached to the multivalent molecule can correspond to the base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) of the nucleotide moiety to permit detection and identification of the nucleotide base.

[00257] In some embodiments, the core of a multivalent molecule comprises an avidin-like or streptavidin-like moiety and the core attachment moiety comprises biotin. In some embodiments, the core comprises a streptavidin-type or avidin-type moiety which includes an avidin protein, as well as any derivatives, analogs and other non-nativeforms of avidin that can bind to at least one biotin moiety. Other forms of avidin moieties include native and recombinant avidin and streptavidin as well as derivatized molecules, e.g. nonglycosylated avidin and truncated streptavidin . For example, avidin moiety includes deglycosylated forms of avidin,bacterial streptavidin produced by Streptomyces (e.g., Streptomyces avidinii\ as well as derivatized forms, for example, N-acyl avidins, e.g., N-acetyl, N-phthalyl and N-succinyl avidin, and the commercially-available products EXTRA VIDIN, CAPTAVIDIN, NEUTRAVIDIN and NEUTRALITE AVIDIN.

[00258] In some embodiments, the binding complex may be formed between one or more nucleotides of a detectable nucleotide conjugate, as described herein, and a nucleotide of a primed index sequence, as described herein. In some embodiments, the binding complex may be formed between one or more nucleotides of the detectable nucleotide conjugate, a nucleotide of a primed index sequence and a polymerizing enzyme (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 type 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, a 3 ’ -O-alkyl hydroxylamino or 3 ’ -O-methyl nucleotide). In some embodiments, the blocked nucleotide is atthe 3 ’ position of the primer nucleic acid sequence of the primed index sequence. In some embodiments, the nucleotide of the nucleotide conjugate (e.g. detectable nucleotide conjugate)is the blocked nucleotide.

[00259] 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, as described herein, and a nucleotide of a primed index sequence, as described herein. In some embodiments, the binding complex may be formed between two or more nucleotides of the detectable nucleotide conjugate, a nucleotide of a primed index sequence and a polymerizing enzyme (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 type 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, a 3’-O-alkyl hydroxylamino or 3 ’ -O-methyl nucleotide). In some embodiments, the blocked nucleotide is at the 3’ position of the primer nucleic acid sequence of the primed index sequence. In some embodiments, the nucleotide of the nucleotide conjugate (e.g. detectable nucleotide conjugate)is the blocked nucleotide.

[00260] In some embodiments, the nucleotide moieties involved in the formation of the binding complex are not incorporated. In some embodiments, the binding complex further comprises a polymerase. In some embodiments, the present disclosure provides said method wherein the polymerase molecule is catalytically inactive, such as where the polymerase molecule been rendered catalytically inactive by mutation, by chemical modification, or by the absence of a necessary ion or cofactor. In some embodiments, the present disclosure also provides said method wherein the polymerase molecule is catalytically active, and / or wherein the binding complex does not comprise a blocked nucleotide.

[00261] In some embodiments, any of the methods for sequencing nucleic acid molecules described herein can include forming a binding complex, where the binding complex comprises (i) a polymerase, a nucleic acid concatemer molecule duplexed with a primer, and a nucleotide, or the binding complex comprises (ii) a polymerase, a nucleic acid concatemer molecule duplexed with a primer, and a nucleotide moiety of a multivalent molecule. In some embodiments, the binding complex has a persistence time of 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 persistence time of greater than about 0.1-0.25 seconds, or about 0.25-0.5 seconds, or about 0.5-0.75 seconds, or about 0.75-1 second, or about 1-2 seconds, or about 2-3 seconds, or about 3-4 second, or about 4-5 seconds, and / or wherein the method is or may be carried out at a temperature of at or above 15 °C, at or above 20 °C, at or above 25 °C, at or above 3 5 °C, at or above 37 °C, at or above 42 °C at or above 55 °C at or above 60 °C, orator above 72 °C, orator above 80 °C, or within a range defined by any of the foregoing. The binding complex (e.g., ternary complex) remains stable until subjected to a condition that causes dissociation of interactions between any of the polymerase, template molecule, primer and / or the nucleotide moiety or the nucleotide. For example, a dissociating condition comprises contacting the binding complex with any one or any combination of a detergent, EDTA and / or water. In some embodiments, the present disclosure provides said method wherein the binding complex is deposited on, attached to, or hybridized to, a surface showing a contrast to noise ratio in the detecting step of greater than 20. In some embodiments, the present disclosure provides said method wherein the contacting is performed under a condition that stabilizes the binding complex when the nucleotide or nucleotide moiety is complementary to a next base of the template nucleic acid, and destabilizes the binding complex when the nucleotide or nucleotide moiety is not complementary to the next base of the template nucleic acid.

[00262] In some embodiments, in any of the sequencing methods that employ multivalent molecules, the binding of the plurality of first complexed polymerases with the plurality of multivalent molecules forms at least one avidity complex, the method comprising the steps: (a) binding a first nucleic acid primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of a concatemer template molecule thereby forming a first binding complex, wherein a first nucleotide moiety 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 concatemer template molecule thereby forming a second binding complex, wherein a second nucleotide moiety of the first multivalent molecule binds to the second sequencing polymerase, wherein the firstand second binding complexes which include the same multivalent molecule forms an avidity 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 concatemer template molecule comprises tandem repeat sequences of a sequence of interest and at least one universal sequencingprimer binding site. The first and second nucleic acid primers canbind to a sequencing primer binding site along the concatemer template molecule. Example multivalent molecules are shown in FIGs. 21-24.

[00263] In some embodiments, in any of the sequencing methods that employ multivalent molecules, the method includes binding the plurality of first complexed polymerases with the plurality of multivalent molecules to form at least one avidity complex, the method comprising the steps: (a) contacting the plurality of sequencing polymerases and the plurality of nucleic acid primers with different portions of a concatemer nucleic acid concatemer molecule to form at least first and second complexed polymerases on the same concatemer template molecule; (b) contacting a plurality of multivalent molecules to the at least first and second complexed polymerases on the same concatemer template molecule, under conditions suitable to bind a single multivalent molecule from the plurality to the first and second complexed polymerases, wherein at least a first nucleotide moiety of the single multivalent molecule is bound to the first complexed polymerase which includes a first primer hybridized to a first portion of the concatemer template molecule thereby forming a first binding complex (e.g., first ternary complex), and wherein at least a second nucleotide moiety of the single multivalent molecule is bound to the second complexed polymerase which includes a second primer hybridized to a second portion of the concatemer template molecule thereby forming a second binding complex (e.g., second ternary complex), wherein the contacting is conducted under a condition s...

Claims

What is Claimed:

1. A method for identifying a ribonucleic acid (RNA) in situ, the method comprising:(a) providing a cell or a tissue that has been permeabilized, wherein said cell or said tissue comprises said RNA;(b) directing a probe to said cell or tissue, wherein said probe comprises (i) an RNA-binding sequence and (ii) an index sequence, under conditions sufficient to couple said RNA-binding sequence to at least a portion of said RNA;(c) amplifying said index sequence to generate one or more amplicons, wherein said one or more amplicons comprises multiple copies of said index sequence or derivative thereof;(d) directing a primer nucleic acid sequence to said cell or tissue under conditions sufficient to bind said primer nucleic acid sequence to a portion of said one or more amplicons to produce a primed index sequence;(e) directing said cell or tissue with a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide in said primed index sequence or said derivative thereof, under conditions suitable to form a binding complex between said one or more nucleotide moieties and said nucleotide of said primed index sequence without incorporating said one or more nucleotide moieties into said primer nucleic acid sequence; and(f) detecting said binding complex within said cell or tissue, thereby identifying said RNA.

2. The method of claim 1, wherein said method does not comprise performing a reverse transcription reaction of said RNA.

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

4. The method of claim 1, wherein said cell is a cancer cell.

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

6. The method of claim 5, wherein said tissue is a formalin-fixed paraffin embedded tissue.

7. The method of claim 5, wherein said tissue is a fresh-frozen tissue.

8. The method of any one of claims 1 or 2, wherein said cell or said tissue is extracted from a subject.

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

10. The method of any one of claims 1-9, wherein said index sequence comprises a nucleicacid.

11. The method of claim 10, wherein said index sequence is 4-50 nucleotides in length.WO 2025 / 170937                                   PCT / US2025 / 01450212. The method of any one of claims 1-11, wherein at least a portion of said index sequence or derivative thereof recognizes said primer nucleic acid sequence.

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

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

15. The method of claim 13, wherein said blocking group comprises a 3’-0-azido group, a 3’-0-azidomethyl group, a 3’ -O-alkyl hydroxylamino group, a 3’-phosphorothioate group, a 3’-0-malonyl group, or a 3’-0-benzyl group.

16. The method of any one of claims 1-15, wherein said primer nucleic acid sequence does not include a 3’ hydroxyl group.

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

18. The method of any one of claims 1-17, wherein said primer nucleic acid sequence binds to at least a portion of a reverse complement of said index sequence.

19. The method of any one of claims 1-18, wherein said primed index sequence comprises a double-stranded nucleic acid.

20. The method of any one of claims 1-19, wherein said primed index sequence comprises a single-stranded nucleic acid.

21. The method of any one of claims 1 -20, wherein said cell or said tissue is immobilized on a surface of a substrate.

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

23. The method of claim 21, wherein said surface of said substrate is an interior surface of a flow cell.

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

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

26. The method of claim 24, wherein said one or more hydrophilic polymer layers comprises a polymer comprising polyethylene glycol.

27. The method of any one of claims 1-26, wherein said nucleotide conjugate comprises a common core.

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

29. The method of claim 27 or 28, wherein said common core comprises a polymer, a micelle, a liposome, a microparticle, a nanoparticle, or a quantum dot.

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

31. The method of any one of claims 1-30, wherein in (e) said nucleotide conjugate isincluded in a mixture of a plurality of nucleotide conjugates, wherein each of said plurality of nucleotide conjugates comprises different types of nucleotide moieties from each other.

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

33. The method of any one of claims 1-32, wherein said one or more nucleotide moieties do not comprise a blocking group coupled thereto.

34. The method of any one of claims 1-33, wherein said detecting in (f) comprises imaging said cell or said tissue using one or more image sensors.

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

36. The method of any one of claims 1-35, further comprising washing said cell or said tissue following (f) to remove said nucleotide conjugate from said cell or said tissue.

37. The method of claim 36, further comprising 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 said primed index sequence or derivative thereof.

38. The method of any one of claims 1-37, wherein said detectable label comprises a fluorophore.

39. The method of any one of claims 1-38, wherein said detectable label comprises a quantum dot.

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

41. The method of any one of claims 1 -40, wherein said conditions sufficient to couple said target-binding sequence to at least a portion of said target in (b) comprises a salt.

42. The method of any one of claims 1-41, wherein said RNA is a messenger RNA.

43. The method of any one of claims 1-41, wherein said RNA is a micro RNA.

44. The method of any one of claims 1-41, wherein said RNA is a ribosomal RNA.

45. The method of any one of claims 1-41, wherein said RNA is a transfer RNA.

46. The method of any one of claims 1-41, wherein said RNA is a transfer-messenger RNA.WO 2025 / 170937                                   PCT / US2025 / 01450247. The method of any one of claims 1-46, wherein said RNA comprises a modification.

48. The method of claim 47, wherein said modification is a 5’ phosphorylation.

49. The method of claim 47, wherein said modification is an inter-nucleotide linkage.

50. The method of any one of claims 1-49, wherein said probe comprises nucleic acid.

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

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

53. The method of any one of claims 1-49, wherein said RNA-binding sequence comprisesnucleic acid.

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

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

56. The method of any one of claims 1-55, wherein said probe is a padlock probe.

57. The method of claim 56, further comprising, before (c), ligating a first end of said probeto a second end of said probe using a ligase.

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

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

60. The method of any one of claims 1-59, wherein performing said amplification reaction in (c) comprises performing a rolling circle amplification reaction using a polymerizing enzyme.

61. The method of claim 60, wherein said polymerizing enzyme is a phi29 polymerase.

62. The method of any one of claims 1-61, wherein said cell or said tissue is permeabilizedusing a solvent.

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

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

65. The method of any one of claims 1-61, wherein said cell or said tissue is permeabilizedusing a detergent.

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

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

68. A method for identifying a polypeptide in situ, the method comprising:(a) providing a cell or a tissue that has been permeabilized, wherein said cell or said tissue comprises said polypeptide;(b) directing a polypeptide-binding probe to said cell or tissue, wherein said polypeptide-binding probe comprises a polypeptide binding moiety and an oligonucleotideWO 2025 / 170937                                   PCT / US2025 / 014502coupled thereto, under conditions sufficient to couple said polypeptide-binding probe to said polypeptide;(c) directing a probe to said cell or said tissue, wherein said with a probe comprising (i) an oligonucleotide-binding probe and (ii) an index sequence under conditions sufficient to couple said oligonucleotide-binding probe to at least a portion of said oligonucleotide of said polypeptide-binding probe;(d) amplifying said index sequence to generate one or more amplicons, wherein said one or more amplicons comprises multiple copies of said index sequence or derivative thereof;(e) directing a primer nucleic acid sequence to said cell or said tissue under conditions sufficient to bind said primer nucleic acid sequence to at least a portion of said one or more amplicons to produce a primed index sequence;(f) directing said cell or said tissue with a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide in said primed index sequence or said one or more amplicons thereof, under conditions suitable to form a binding complex between said one or more nucleotide moieties and said nucleotide of said primed index sequence without incorporating said one or more nucleotide moieties into said primer nucleic acid sequence; and(g) detecting said binding complex within said cell or tissue, thereby identifying said polypeptide.

69. A method for identifying a DNA in situ, the method comprising:(a) providing a cell or a tissue that has been permeabilized, wherein said cell or said tissue comprises said DNA, where said DNA is not cDNA;(b) directing a probe to said cell or said tissue, wherein said probe comprises a (i) DNA-binding sequence and (ii) an index sequence, under conditions sufficient to couple said DNA-binding sequence to at least a portion of said DNA;(c) amplifying said index sequence to generate one or more amplicons, wherein said one or more amplicons comprises multiple copies of said index sequence or derivative thereof;(d) directing a primer nucleic acid sequence to said cell or said tissue under conditions sufficient to bind said primer nucleic acid sequence to at least a portion of said one or more amplicons to produce a primed index sequence;(e) directing said cell or said tissue with a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide in said primed index sequence or said derivatives thereof, under conditions suitable to form a binding complex between said one or more nucleotide moieties and said nucleotide of said primed indexWO 2025 / 170937                                   PCT / US2025 / 014502sequence without incorporating said one or more nucleotide moieties into said primer nucleic acid sequence; and(f) detecting said binding complex within said cell or tissue, thereby identifying said DNA.

70. A method for identifying a ribonucleic acid in situ, the method comprising:(a) providing a cell or tissue that has been permeabilized, wherein said cell or said tissue comprises said ribonucleic acid;(b) directing a nucleic acid molecule to said cell or said tissue, wherein said nucleic acid molecule comprises a target-binding sequence, under conditions sufficient to couple said target-binding sequence to at least a portion of said ribonucleic acid, wherein said nucleic acid molecule further comprises an index sequence;(c) directing a primer nucleic acid sequence to said cell or said tissue under conditions sufficient to bind said primer nucleic acid sequence to at least a portion of said one or more amplicons to produce a primed index sequence;(d) directing said cell or said tissue with a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide in said primed index sequence or derivative thereof, under conditions suitable to form a binding complex between said one or more nucleotide moieties and said nucleotide of said primed index sequence without incorporating the one or more nucleotide moieties into said primer nucleic acid sequence;(e) detecting said binding complex within said cell or tissue, thereby identifying said ribonucleic acid.

71. A system for identifying a RNA in situ, the system comprising:a computing device comprising one or more computer processors, an operating system configured to perform executable instructions, a memory, and a computer program including instructions comprising the method of any one of clams 1-67.

72. A system for identifying a polypeptide in situ, the system comprising:a computing device comprising one or more computer processors, an operating system configured to perform executable instructions, a memory, and a computer program including instructions comprising the method of claim 68.

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

74. A system for identifying a ribonucleic acid in situ, the system comprising:WO 2025 / 170937                                   PCT / US2025 / 014502a computing device comprising one or more computer processors, an operating system configured to perform executable instructions, a memory, and a computer program including instructions comprising the method of claim 70.

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

76. A kit for identifying a polypeptide in situ, 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 said oligonucleotide-binding probe recognizes to at least a portion of said oligonucleotide;(c) a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide of a primed index sequence, wherein said nucleotide conjugate is configured to form a binding complex between said one or more nucleotide moieties and said nucleotide of said primed index sequence without incorporating the one or more nucleotide moieties into said primer nucleic acid sequence; and(d) instructions for identifying said polypeptide in situ, wherein said instructions comprise the method of claim 68.

77. A kit for identifying a DNA in situ, the kit comprising:(a) a probe comprising (i) an index sequence, and (ii) an RNA-binding sequence, wherein said RNA-binding sequence recognizes to at least a portion of said target;(b) a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide of a primedWO 2025 / 170937                                   PCT / US2025 / 014502index sequence, wherein said nucleotide conjugate is configured to form a binding complex between said one or more nucleotide moieties and said nucleotide of said primed index sequence without incorporating the one or more nucleotide moieties into said primer nucleic acid sequence; and(c) instructions for identifying said RNA in situ, wherein said instructions comprise the method of claim 69.

78. A kit for identifying a ribonucleic acid in situ, the kit comprising:(a) a nucleic acid molecule comprising (i) an index sequence, and (ii) a target-binding sequence, wherein said target-binding sequence is complementary to at least a portion of said ribonucleic acid sequence;(b) a nucleotide conjugate comprising (i) a detectable label and (ii) one or more nucleotide moieties complementary to a nucleotide in said primed index sequence, wherein said nucleotide conjugate is configured to form a binding complex between said one or more nucleotide moieties and said nucleotide of said primed index sequencewithoutincorporatingthe one or more nucleotide moieties into said primer nucleic acid sequence; and(c) instructions for identifying said ribonucleic acid in situ, wherein said instructions comprise the method of claim 70.

Images