Systems, methods, and kits for detecting nucleic acid modifications

Abstract

Disclosed herein are systems, methods, and kits for detecting or visualizing nucleic acid modifications. Particularly, the system, methods, and kits comprise at least one target probe comprising a region that is complementary to a target nucleic acid in the biological sample and at least one binding agent specific to the nucleic acid modification comprising oligonucleotide sequences complementary to a component of a signal generating complex which generates a detectable signal upon binding to the target probe and the binding agent.

Description

Attorney Docket No. ACDX-43932.601SYSTEMS, METHODS, AND KTTS FOR DETECTING NUCLEIC ACID MODIFICATIONSFIELD

[0001] Embodiments of the present disclosure include systems, methods, and kits for detecting or visualizing nucleic acid modifications. Particularly, the system, methods, and kits comprise at least one target probe comprising a region that is complementary to a target nucleic acid in the biological sample and at least one binding agent specific to the nucleic acid modification comprising oligonucleotide sequences complementary to a component of a signal generating complex.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 708,108, filedOctober 16, 2024, the content of which is herein incorporated by reference in its entirety.BACKGROUND

[0003] Immunohistochemistry (1HC) and immunocytochemistry (ICC) are powerful techniques that are used to detect and localize specified proteins within tissue sections and cells, while maintaining spatial resolution and cytological context. However, complete characterization of complex cellular interactions and modifications of nucleic acids within a tissue or cell requires methods to characterize and visualize nucleic acid modifications on a gene level, rather than a whole genome or per cell basis. Imaging nucleic acid modifications, e.g., methylated or hydroxymethylated regions at the genomic level can be accomplished using antibodies to the modified nucleotides. But these methods globally assess nucleic acid modifications at a genomic level without assessing regions associated with a target gene. Furthermore, no methods exist to correlate nucleic acid modifications to transcription or translation events of a target gene. To fully and accurately characterize cells and tissues, nucleic acid modifications need to be detected in a tissue sample in a spatially resolved and gene-specific manner.Attorney Docket No. ACDX-43932.601SUMMARY

[0004] Provided herein are systems, methods, and kits for detecting nucleic acid modifications in a biological sample.

[0005] In one aspect, the methods comprise contacting the biological sample with at least one target probe comprising a region that is complementary to a target nucleic acid in the biological sample and a region that is complementary to a first region of a signal generating complex; contacting the biological sample with at least one binding agent specific to the nucleic acid modification, wherein the at least one binding agent is covalently attached to an oligonucleotide comprising a region that is complementary to a second region of a signal generating complex; and detecting a signal from the signal generating complex.

[0006] In some embodiments, binding of the at least one target probe to the target nucleic acid and the first region of the signal generating complex, and binding of the at least one binding agent to the nucleic acid modification and the second region of the signal generating complex facilitates formation of the signal generating complex, and thereby generates a detectable signal indicative of the nucleic acid modification.

[0007] In some embodiments, the target probe binding region of the target nucleic acid is adjacent to the nucleic acid modification.

[0008] In some embodiments, the first region of the signal generating complex and the second region of the signal generating complex are adjacent.

[0009] In some embodiments, the nucleic acid modification comprises a chemical modification of at least one nucleotide in the target nucleic acid. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the chemical modification comprises cytosine methylation or hydroxymethylation. In some embodiments, the at least one binding agent comprises an antibody that specifically binds methylated or hydroxymethylated cytosine. In some embodiments, the at least one binding agent comprises a secondary antibody that specifically binds to the antibody bound to a methylated or hydroxymethylated cytosine, wherein the secondary antibody is covalently attached to the oligonucleotide comprising a region that is complementary to a second region of a signal generating complex.

[0010] In some embodiments, the target nucleic acid is mRNA. In some embodiments, the methods further comprise performing in situ hybridization to detect a region of the mRNA.Attorney Docket No. ACDX-43932.601

[0011] In some embodiments, the at least one binding agent comprises a monoclonal antibody, a multispecific antibody, a bispecific antibody, a trispecific antibody, a tetravalent antibody, a single-domain antibody, and a chimeric antibody. In some embodiments, the at least one binding agent comprises a Fab, a scFv, a Fv, a scFv-Fc, a Fab', a Fab'-SH, a F(ab')2, a diabody, a minibody, a tribody, a nanobody, and an affibody . In some embodiments, the at least one binding agent further comprises a secondary antibody.

[0012] In some embodiments, the at least one binding agent comprises a primary or secondary antibody covalently attached to the oligonucleotide via a linker. In some embodiments, the linker comprises at least one of 6-hydrazinonicotinate (HyNic), succinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (SMCC), and / or polyethylene glycol (PEG). In some embodiments, the linker comprises at least one reactive moiety. In some embodiments, the at least one reactive moiety comprises at least one of a succinimidyl ester, a sulfosuccinimidyl ester, a pentafluorophenyl ester, a maleimide, an azide, an alkyne, a hydrazine, an isocyanate, an isothiocyanate, and / or a haloacetamide. In some embodiments, the linker comprises an oligonucleotide sequence.

[0013] In some embodiments, the biological sample is a fixed biological sample. In some embodiments, the target nucleic acid is immobilized in the biological sample. In some embodiments, the fixed biological sample is a formalin-fixed paraffin-embedded (FFPE) sample. In some embodiments, the fixed biological sample is a tissue sample or is derived from a tissue sample; a blood sample or is derived from a blood sample; a cytological sample or is derived from a cytological sample; a sample comprising cultured cells; or a sample comprising exosomes.

[0014] In some embodiments, the methods further comprise contacting the biological sample with a blocking agent comprising at least one of tRNA, salmon sperm DNA, herring DNA, calf thymus DNA, bovine serum albumin, casein, heparin, normal goat serum, normal swine serum, normal chicken serum, and / or fish serum.

[0015] In some embodiments, the methods further comprise contacting the biological sample with a crosslinking agent comprising at least one of formalin, bis(succinimidyl) polyethylene glycol, di(N-succinimidy) glutarate, and / or glutaraldehyde.

[0016] In some embodiments, the signal generating complex comprises at least one amplifier and / or pre-amplifier. In some embodiments, the signal generating complex comprises at least one detectable label. In some embodiments, the at least one detectable label comprises a fluorescentAttorney Docket No. ACDX-43932.601 moiety or a chromogenic moiety. In some embodiments, the at least one detectable label is cleavable.

[0017] In some embodiments, contacting the biological sample with at least one target probe is performed before contacting the biological sample with at least one binding agent specific to the nucleic acid modification. In some embodiments, contacting the biological sample with at least one target probe is performed after contacting the biological sample with at least one binding agent specific to the nucleic acid modification. In some embodiments, contacting the biological sample with at least one target probe is performed simultaneously with contacting the biological sample with at least one binding agent specific to the nucleic acid modification.

[0018] In some embodiments, the region of the at least one target probe that is complementary to the target nucleic acid is from about 10 to about 50 nucleotides in length. In some embodiments, the region of the at least one target probe that is complementary to the first region of the signal generating complex is from about 10 to about 50 nucleotides in length.

[0019] In some embodiments, the at least one target probe comprises a non-targeting region separating the target nucleic acid binding region from the region that is complementary to the first region of the signal generating complex. In some embodiments, the region of the at least one target probe that is complementary to the target nucleic acid is 5’ of the region of the at least one target probe that is complementary to the first region of the signal generating complex. In some embodiments, the region of the at least one target probe that is complementary to the target nucleic acid is 3’ of the region of the at least one target probe that is complementary to the first region of the signal generating complex.

[0020] In another aspect provided herein are kits comprising at least one target probe and at least one binding agent as described herein. In some embodiments, the kits further comprise one or more permeabilizing and / or antigen retrieval reagents. In some embodiments, the kits further comprise at least one amplifier, at least one pre-amplifier, and / or at least one detectable label. In some embodiments, the kits further comprise a control or reference sample.

[0021] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.Attorney Docket No. ACDX-43932.601BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIGS. 1 A-1D are exemplary schematics for the detection of a nucleic acid modification, 5 -hydroxymethyl cytosine (5hmC), using the system and methods disclosed herein. In the presence of the target probe and binding agent (oligonucleotide conjugated anti-5hmC antibody), the signal generation complex will bind and allow for detection (FIG. 1 A). In the absence of either the target probe or binding agent, the signal generation complex will not bind (FIG. IB). Use of multiple target probes to a target nucleic acid (e.g., a specific gene) over a long sequence (e.g., a specific gene) can maximize detection (FIG. 1C). FIG. ID shows two exemplary orientations of the target nucleic acid binding region and a single generating complex binding region in the target probe and the ability to detect nucleic acid modifications up and downstream of the region of the target nucleic acid bound by the target probe.

[0023] FIGS. 2A-2D show detection of 5hmC modifications in mouse TNFa genomic DNA in mouse brain and kidney tissue samples, as indicated. FIGS. 2A and 2B show a comparison of methods using a 5hmC antibody binding agent with a population of target probes directed to bind within the TNFa coding and promoter gene regions to those methods lacking the 5hmC antibody binding agent. FIGS. 2C and 2D show detection of TNFa mRNA in comparison to detection of 5hmC modifications within the TNFa gene and promoter regions.

[0024] FIGS. 3A-3E show detection of 5hmC modifications in human TNFa gene (FIGS. 3A- 3E) and TNFa mRNA (FIGS. 3A-3D) in normal and cancerous human tissues; breast (FIG. 3A), lung (FIG. 3B), colon (FIG. 3C), and brain (FIGS. 3D-3E).

[0025] FIG. 4 are exemplary schematics for the detection of a nucleic acid modification, 5- hydroxymethyl cytosine (5hmC), using a binding agent having an oligonucleotide conjugated secondary antibody combined with an anti-5hmC antibody and target probe. In the presence of the target probe and binding agent (oligonucleotide conjugated secondary antibody and anti-5hmC antibody), the signal generation complex will bind and allow for detection.

[0026] FIG. 5 shows representative fluorescent microscopic images of the 5-methyl cytosine (5mC) assay background and positive signals targeting GSTP1 captured from FFPE sections containing a DU145 prostate cancer cell line pellet. Minimal negative background was found in the control slide with only the GSTP1 target probe, without the primary or secondary antibody (left). Negative background was also at a minimal level without GSTP1 target probe while the secondary antibody was present (middle). An assay containing both the GSTP1 target probe andAttorney Docket No. ACDX-43932.601 binding agent comprising a primary antibody / oligonucleotide conjugated secondary antibody complex, as schematized in FIG. 4, showed specific and robust fluorescent puncta under 20x objective in the cell nuclei. Multiplex dots can be observed in some cells. (20X obj, Green= GSTP1 genomic locus 5mC signals, DAPI=nuclear counterstain.)

[0027] FIGS. 6A and 6B show representative fluorescent microscopic images of GSTPl-5mC signals in three prostate cancer cell lines (FIG. 6A) and across three different cancer types (FIG. 6B) using the assay as in FIG. 5.DETAILED DESCRIPTION

[0028] The present disclosure is directed to systems and methods for detecting or visualizing nucleic acid modifications, suitable for use in situ. The methods utilize target probe(s) which interrogate a target nucleic acid, e.g., a gene or gene promoter, and binding agents which interrogate the nucleic acid modification(s) of interest. In the presence of a nucleic acid modification in proximity to the target probe, a signal is generated via formation of a signal generating detection complex. In the absence of a nucleic acid modification in proximity to the target probe a detectable signal is not produced. The disclosed system and methods can provide spatial information and detection of nucleic acid modifications for a desired target nucleic acid rather than at a whole cell or tissue level. The disclosed methods and systems allow for the correlation of specific epigenetic events to various cellular processes (e.g., transcription, translation, etc.) in situ, and facilitate interrogation of abnormal methylation patterns and specific methylation signatures shown to be critical in many disease conditions including cancer.

[0029] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.Definitions

[0030] As used in the present disclosure and claims, the singular forms “a,” “an” and “the” include plural forms unless the context clearly dictates otherwise.

[0031] It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. It is also understood that wherever embodiments are described herein with the phrase “consisting essentially of’ otherwise analogous embodiments described in terms of “consisting of’ are also provided.Attorney Docket No. ACDX-43932.601

[0032] The term “between” as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B. For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0033] As used herein, the term “one or more” refers to, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or a greater number, if desired for a particular use.

[0034] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0035] “Antibody” and “antibodies” as used herein refers to monoclonal antibodies, monospecific antibodies (e.g., which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), multi-specific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab’) fragments, F(ab’) fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, dualdomain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dualvariable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25(11): 1290-1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are herein incorporated by reference), or domain antibodies (dAbs) (e.g., such as described in Holt et al., Trends in Biotechnology 21 :484-490 (2014)), and including single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camelid, or which are synthetic, e.g., nanobodies, VHH, or other domainAttorney Docket No. ACDX-43932.601 structure), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. For simplicity’s sake, an antibody against an analyte is frequently referred to herein as being either an “anti-analyte antibody” or merely an “analyte antibody.”

[0036] “Antibody fragment” as used herein refers to a portion of an intact antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)) (e.g., comprises the antigen-binding site or variable region). Any antigenbinding fragment of the antibody described herein is within the scope of the present disclosure. The antibody may not include the constant heavy chain domains (e.g., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab’ fragments, Fab’-SH fragments, F(ab’)2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

[0037] Typically, an immunoglobulin or antibody is a protein that comprises at least one complementarity determining region (CDR). The CDRs form the “hypervariable region” of an antibody, which is responsible for antigen binding (discussed further below). A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N- terminal variable (VH) region and three C-terminal constant (CHI, CH2, and Cm) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, either kappa (K) or lambda (X), based upon the amino acid sequences of their constant domains. In a typical antibody, each light chain is linked to a heavy chain by disulfide bonds, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the variable regionAttorney Docket No. ACDX-43932.601 of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

[0038] The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework (FW or FR) regions. The term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located between the CDRs. There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the 0 sheets that provide the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)).

[0039] As used herein, the term “primary antibody” refers to an antibody that binds directly to the antigen of interest. As used herein, the term “secondary antibody” refers to an antibody that binds to the primary antibody. The second antibody may be conjugated to a moiety that can be used for detection, such as a detectable label, or a moiety to which a detectable label can bind. In some embodiments, a secondary antibody is conjugated to an oligonucleotide that is capable of hybridizing to a nucleic acid component of a signal generating complex. In some embodiments, the secondary antibody provided herein binds directly to the primary antibody. In other embodiments, the secondary antibody provided herein binds indirectly to the primary antibody, e.g., by binding to another antibody that recognizes the primary antibody.

[0040] The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen. The term “bispecific” antibody as used herein denotes an antibody that has at least two binding sites each of which bind to different epitopes of the same antigen or a different antigen. The term “multi specific” antibody as used herein denotes an antibody that has binding specificities for at least two different sites (e.g., bispecific, trispecific, tetraspecific).

[0041] The term “valent” as used within the current application denotes the presence of a specified number of binding sites in an antibody molecule. As such, the terms “bivalent,” “tetravalent,” and “hexavalent” denote the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antibody molecule. The bispecific antibodies according to the invention are at least “bivalent” and may be “trivalent” or “multivalent” (e.g., “tetravalent” orAttorney Docket No. ACDX-43932.601“hexavalent”). That is, the antibodies may be bispecific even in cases where there are more than two binding sites (e.g., that the antibody is trivalent or multivalent).

[0042] The term “detecting” as used herein generally refers to any form of measurement, and includes determining whether an element is present or not. This term includes quantitative and / or qualitative determinations.

[0043] As used herein, the term “fixation” or “fixing” when made in reference to fixing a biological sample refers to a procedure to preserve a biological sample from decay due to, e.g., autolysis or putrefaction. It terminates any ongoing biochemical reactions and may also increase the treated tissues' mechanical strength or stability.

[0044] As used herein, the term “immunohistochemistry” or “IHC” generally refers to a technique for detecting proteins of interest in source samples utilizing antibodies, with the preservation of morphology of the source samples (e.g., tissue samples). As used herein, the term “immunocytochemistry” or “ICC” generally refers to a technique for detecting proteins of interest in source samples utilizing antibodies, with the preservation of morphology of the source samples (e.g., isolated or cultured intact cells, including tissue culture cell lines, either adherent or in suspension). Immunofluorescence (IF) refers to fluorescent labeling, thus it is also encompassed in the terms IHC and ICC. ICC, IHC, and IF assays can be used in conjunction with the imaging processing methods of the present disclosure, as described further herein, including facilitating quantitative and / or qualitative assessments of a target-of-interest in a sample. ICC, IHC, and IF assays can also be performed in conjunction with an in situ hybridization as part of an integrated co-detection process to detect targets-of-interest, which can also include performing the imaging processing methods of the present disclosure.

[0045] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer of any length composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or compounds produced synthetically, which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. As used herein in the context of a polynucleotide sequence, the term “bases” (or “base”) is synonymous with “nucleotides” (or “nucleotide”), the monomer subunit of a polynucleotide. The terms “nucleoside” and “nucleotide” are intended to include those moi eties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modificationsAttorney Docket No. ACDX-43932.601 include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. In addition, the terms “nucleoside” and “nucleotide” include those moieties that contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like. “Analogues” refer to molecules having structural features that are recognized in the literature as being mimetics, derivatives, having analogous structures, or other like terms, and include, for example, polynucleotides incorporating non-natural nucleotides, nucleotide mimetics such as 2’ -modified nucleosides, peptide nucleic acids, oligomeric nucleoside phosphonates, and any polynucleotide that has added substituent groups, such as protecting groups or linking moieties. Accordingly, nucleic acids and polynucleotides can include other hybridizing nucleic-acid-like molecules such as those with substituted backbones e.g., peptide nucleic acids (PNAs), morpholino backboned nucleic acids, locked nucleic acids, or other nucleic acids with modified bases and sugars.

[0046] The term “complementary” refers to specific binding between polynucleotides based on the sequences of the polynucleotides. As used herein, a first polynucleotide and a second polynucleotide are complementary if they bind to each other in a hybridization assay under stringent conditions, e.g., if they produce a given or detectable level of signal in a hybridization assay. Portions of polynucleotides are complementary to each other if they follow conventional base-pairing rules, e.g., A pairs with T (or U) and G pairs with C, although small regions (e.g., fewer than about 3 bases) of mismatch, insertion, or deleted sequence may be present.

[0047] The term “probe” refers herein to an agent that is directed to a specific sequence. In some embodiments, a probe can be used individually. In other embodiments, a probe can be used among a probe set (e.g., two or more probes). Each probe of a probe set has a respective target sequence. In some embodiments, the probe is a “nucleic acid probe” or “oligonucleotide probe” which refers to a nucleic acid capable of binding to a target nucleic acid of complementary sequence, usually through complementary base pairing by forming hydrogen bond. As used herein, a probe may include natural (e.g., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc. . In addition, the bases in a probe may bejoinedby a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.Attorney Docket No. ACDX-43932.601

[0048] The term “sample” as used herein relates to a material or mixture of materials containing one or more components of interest. The term “sample” includes “biological sample” which refers to a sample obtained from a biological subject, including a sample of biological tissue or fluid origin, obtained, reached, or collected in vivo or in situ. A biological sample also includes samples from a region of a biological subject containing precancerous or cancer cells or tissues. Such samples can be, but are not limited to, organs, tissues, cells, and exosomes isolated from a mammal. Exemplary biological samples include but are not limited to cell lysate, a cell, a cell culture, a cell line, a tissue, oral tissue, gastrointestinal tissue, an organ, an organoid, a biological fluid, a blood sample, a urine sample, a skin sample, and the like. Preferred biological samples include, but are not limited to, whole blood, partially purified blood, PBMC, tissue biopsies, and the like.

[0049] As used herein, when a binding agent “specifically recognizes” or “specifically binds” an indicated entity, it preferentially recognizes the indicated entity in a complex mixture of proteins and / or macromolecules, and binds the indicated entity with an affinity which is substantially higher than to other entities. In this regard, “affinity which is substantially higher” means affinity that is high enough to enable detection of an indicated entity which is distinguished from entities. Typically, it means binding affinity having a binding constant (Ka) of at least 107M'1(e.g., >107M’1, >108M’1, >109M’1, >1O10M’1, >10nM’1, >1012NT1, >1013M’1, etc.). In certain such embodiments, the binding agent is an antibody is capable of binding a particular epitope found in the indicated entity.Methods of Detecting Nucleic Acid Modifications in a Sample a. Nucleic Acid Modifications

[0050] In one aspect, provided herein are methods for detecting nucleic acid modifications in a biological sample. In some embodiments, the methods comprise: (i) contacting the biological sample with at least one target probe comprising a region that is complementary to a target nucleic acid in the biological sample and a region that is complementary to a first region of a signal generating complex; (ii) contacting the biological sample with at least one binding agent specific to the nucleic acid modification, wherein the at least one binding agent is covalently attached to an oligonucleotide comprising a region that is complementary to a second region of a signal generating complex; and (iii) detecting a signal from the signal generating complex.

[0051] Any manner of nucleic acid modifications can be detected by the methods disclosed herein. In some embodiments, the nucleic acid modification comprises a covalent modification,Attorney Docket No. ACDX-43932.601 e.g., modifications through the addition of chemical groups not found in standard versions of a nucleotide. For example, the modification can be to the base, sugar, and or phosphate of the nucleotides in the target nucleic acid. Non-limiting, exemplary nucleic acid modifications include methylation, hydroxymethylation, formylation, carboxylation, alkylation, acetylation, phosphorylation, and the like.

[0052] Oftentimes, these types of covalent modifications are an indication of the state of the nucleic acid or, more broadly, the overall health of the cell or subject. For example, abnormal methylation patterns have been shown to be critical in many disease conditions including cancer, and specific methylation signatures have been shown to differentiate cancer types and provide diagnostic clarity. Thus, the disclosed methods can be used to assist in determining a disease state of a cell or subject.

[0053] In some embodiments, the nucleic acid modification is an epigenetic mark. Epigenetic marks include any changes in the underlying DNA structure which result in changes in gene expression or cellular phenotype unrelated to a change in sequence. In some embodiments, the nucleic acid modification being interrogated is a change in methylation status of a target nucleic acid, e.g., genomic DNA. Methylation predominately involves the addition of a methyl group to cytosine residues (e.g., at the C3 or C5 positions) of the dinucleotide CpG and is associated with repression or inhibition of transcriptional activity. Hydroxymethylation, in which the hydrogen atom at the C5-position in cytosine is replaced by a hydroxymethyl group, is relatively less understood as an epigenetic mark as compared to methylation but its enrichment at enhancer and coding regions is usually positively correlated with transcriptional activity and dysregulation has been shown to contribute to the etiology of various diseases including cancer ang age-related disorders.

[0054] In some embodiments, the nucleic acid modification is selected from cytosine methylation, cytosine hydroxymethylation, cytosine formylation, cytosine carboxylation, adenosine methylation, and alkylation (alkylation can occur at oxygen and nitrogen atoms of all nucleobases, as well as on the oxygen atoms in the phosphodi ester backbone). In such embodiments, the binding agent is an agent that specifically binds to methylated cytosine, hydroxymethylated cytosine, formylated cytosine, carboxylated cytosine, and methylated adenosine. b. Binding AgentsAttorney Docket No. ACDX-43932.601

[0055] The methods described above make use of binding agents directed to the nucleic acid modification. The binding agents may include any moiety (e.g., nucleic acid, peptide, protein, or small molecule compound) which specifically binds to the nucleic acid modification. The binding agent may comprise any combination of moi eties which facilitate specific binding to the nucleic acid modification. The binding agents are covalently attached to an oligonucleotide comprising a region that is complementary to a second region of a signal generating complex.

[0056] In some embodiments, the binding agent is a peptide- or protein-based agent covalently attached to the oligonucleotide. The peptide- or protein-based agent (e.g., antibody or fragment thereof) binds to the nucleic acid modification, and their respective oligonucleotides provide a binding site for a signal generating complex, as will be further discussed below. In some embodiments, the binding agent is an aptamer. In some embodiments, the aptamer is a singlestranded nucleic acid molecule (DNA or RNA) that can selectively bind to a specific target molecule.

[0057] In select embodiments, the binding agent is an antibody or fragment thereof directed to the nucleic acid modification. Any suitable antibody can be used. In some embodiments, the binding agent(s) comprise a monoclonal antibody, a multispecific antibody, a bispecific antibody, a trispecific antibody, a tetravalent antibody, a single-domain antibody, a chimeric antibody, and a polyclonal antibody mixture. In some embodiments, the binding agent(s) comprise a Fab, a scFv, a Fv, a scFv-Fc, a Fab', a Fab'-SH, a F(ab')2, a diabody, a minibody, and a tribody. In some embodiments the binding agents comprise a secondary antibody.

[0058] In some embodiments, the binding agent(s) may comprise a composition of polyclonal antibodies, in which a plurality of antibodies in the composition are conjugated to the oligonucleotide.

[0059] In some embodiments, the binding agent(s) comprise a primary antibody covalently attached to the oligonucleotide. In some embodiments, the binding agent(s) comprise a secondary antibody covalently attached to the oligonucleotide. In select embodiments, the binding agent comprises a primary antibody or fragment thereof, as described above, directed to the nucleic acid modification and a secondary antibody comprising an oligonucleotide comprising a region that is complementary to a second region of a signal generating complex directed to the primary antibody or fragment thereof.Attorney Docket No. ACDX-43932.601

[0060] The oligonucleotide covalently attached to the binding agent can have a length of about 5 to about 100 nucleotides. In some embodiments, the first and / or the second oligonucleotide has a length of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides. In some embodiments, the oligonucleotide has a length of about 5 to about 50 nucleotides. In some embodiments, the oligonucleotide has a length of about 12 to about 16 nucleotides (e.g., 14 nucleotides). In some embodiments, the oligonucleotide has a length of about 26 to about 30 oligonucleotides (e.g., 28 nucleotides). In some embodiments, the oligonucleotide has a length of about 40 to about 60 nucleotides (e.g., 50 nucleotides).

[0061] As will be further discussed below, the oligonucleotide is selected such that a nucleic acid component of the signal generating complex is capable of hybridizing to the oligonucleotide. In some embodiments, the oligonucleotide has a sequence that is complementary to a sequence of a nucleic acid component of the signal generating complex. For example, in some embodiments, the oligonucleotide has a sequence that is complementary to a sequence of a nucleic acid component of the signal generating complex over a sequence of about 5 to about 100 nucleotides, e.g., a sequence of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides. In some embodiments, the second oligonucleotide has a sequence that is complementary to a sequence of a nucleic acid component of the signal generating complex over a sequence of about 5 to about 50 nucleotides, about 12 to about 16 nucleotides (e.g., 14 nucleotides, about 26 to about 30 oligonucleotides (e.g., 28 nucleotides), or about 40 to about 60 nucleotides (e.g., 50 nucleotides). In some embodiments, the portion of the oligonucleotide which hybridizes to the signal generating complex hybridize is complementary to non-overlapping sections of the nucleic acid component of the signal generating complex which hybridizes to the target probe, as described further below.

[0062] The oligonucleotide is covalently attached to the binding agent. In some embodiments, the covalent attachment is via a direct bond between the binding agent and the oligonucleotide. In some embodiments, the oligonucleotide is covalently attached via a linker. In some embodiments,Attorney Docket No. ACDX-43932.601 the binding agent comprises a primary or secondary antibody covalently attached to the oligonucleotide via a linker.

[0063] A variety of methods of conjugating oligonucleotides to peptides and proteins (e.g., antibodies) are known to those skilled in the art. For example, a typical conjugation method includes use of a linker compound that includes two distinct reactive moi eties, which react with different types of functional groups (e.g., one group that reacts with an amine, such as an activated ester group, and one group that reacts with a thiol, such as a maleimide group). Such reactive moi eties used in conjugation reactions are well-known to those skilled in the art, and include activated esters such as succinimidyl and sulfosuccinimidyl esters and pentafluorophenyl esters, maleimides, azides, alkynes, hydrazines, isocyanates, isothiocyanates, haloacetamides, and the like. Methods of installing such reactive groups are well-known to those skilled in the art. As one non-limiting example, an amino group can be installed at the 5’-end of an oligonucleotide via phosphoramidite chemistry.

[0064] In some embodiments, a binding agent is first reacted with the linker compound to provide a functionalized binding agent, which is subsequently reacted with an oligonucleotide to provide the oligonucleotide-labeled binding agent. In other embodiments, an oligonucleotide is first reacted with the linker compound to provide a functionalized oligonucleotide, which is subsequently reacted with a binding agent to provide the oligonucleotide-labeled binding agent.

[0065] Some oligonucleotide conjugation reagents or linkers are commercially available, and some are sold as parts of commercial kits. Exemplary commercially available linker compounds include those shown in Scheme 1, such as sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate (sulfo-SMCC), succinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate (SMCC), PEGylated crosslinkers such as SM(PEG)ncompounds (succinimidyl-([N- maleimidopropionamidoXCFbCFEOjn) esters), and 6-hydrazinonicotinate (HyNic) containing linkers such as S-HyNic.

[0066] In some embodiments, the commercial linkers can be used directly to conjugate the binding agent to the oligonucleotide. In other embodiments, the binding agent and / or the oligonucleotide must be first derivatized with a specific functional group prior to reaction with the linker compound. For example, the binding agent can be reacted with a 2-iminothiolane to install a thiol group for reaction with a maleimide group. As another example, the oligonucleotide orAttorney Docket No. ACDX-43932.601 binding agent can be reacted with succinimidyl-4-formylbenzamide to install an aldehyde group for reaction with a HyNic-containing linker compound.Scheme 1. Linker CompoundsNHS-(PEG)n-MaleimideR = H or SO3Na; n = 0-24

[0067] Other known linker chemistries involve separate functionalizations of the binding agent and the oligonucleotide followed by a reaction to generate the linker moiety. Examples include installation of an azide-containing moiety on one compound and an alkyne-containing moiety on the other, for linkage via click chemistry (e.g., either copper-catalyzed or copper-free click chemistry).

[0068] Accordingly, in some embodiments, the linker comprises a moiety selected from:wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0069] In some embodiments, the linker can include one or more nucleotides. For example, the linker can comprise an oligonucleotide sequence. Such a sequence may be considered separate from the oligonucleotide sequence to which the nucleic acid component of the signal generatingAttorney Docket No. ACDX-43932.601 complex can hybridize. For example, in some embodiments, the linker can include one or more thymine groups. In some embodiments, the linker is a 5T linker.

[0070] The linker can include additional atoms or groups; for example, if the antibody is reacted with 2-iminothiolane, it is understood that the linker will further include atoms derived from such reaction. Accordingly, in some embodiments, the linker further comprises one or more additional groups selected from -CH2-, -O-, -NH-, -S-, -C(=O)-, -C(=NH)-, and any combination thereof (e.g., combinations of such moieties could include ester groups (-C(=O)O-), amide groups (-C(=O)NH-), carbamate groups (-NHC(=O)O-), ethylene glycol groups (-CH2CH2O-), and the like.

[0071] In some embodiments, the linker comprises an antibody-binding domain. In some embodiments, an antibody binding domain (AbBD) comprises Protein A, Protein G, Protein L, CD4, or a fragment thereof. In some embodiments, the antibody-binding domain is an engineered antibody -binding domain, such as to include a non-natural amino acid, a photoreactive group, or a crosslinker. In some embodiments, the antibody binding domain is operably linked to a photoreactive amino acid group, for example, benzoylphenylalanine (BPA), resulting in a photoreactive antibody binding domain (pAbBD). In some embodiments, the antibody-binding domain (AbBD) is operably linked to a photoreactive amino acid which is operably linked to an antibody or a fragment thereof. See, for example, United States Patent Nos. 11, 156,608 and 11,123,440.

[0072] In some embodiments, the component of the binding agent conjugated to the oligonucleotide binds directly to the target in the biological sample. In other embodiments, component of the binding agent conjugated to the oligonucleotide binds indirectly to the target in the biological sample. For binding agents comprising multiple components (e.g., primary and secondary antibodies), the components can be added to the sample concurrently or sequentially, in any order. Either component may be added to the sample concurrently with the at least one target probe. For example, a primary antibody may be contacted with the biological sample prior to or concurrently with a secondary antibody that is covalently attached to an oligonucleotide. In such embodiments, the method can further comprise a step of contacting the sample with a first component of the binding agent (e.g., a primary antibody) prior to or concurrently with step (i) and / or step (ii), wherein the first component of the binding agent binds directly to the target nucleic acid modification; and the second component of the binding agent (e.g., secondary antibody) that is covalently attached to an oligonucleotide then binds to the first component of the binding agent.Attorney Docket No. ACDX-43932.601 c. Target Probes

[0073] The methods described above make use of target probes directed to the target nucleic acid. In some embodiments, the methods utilize a single target probe directed to a target nucleic acid. In some embodiments, the methods utilize two or more target probes, each directed to a similar region of the target nucleic acid, e.g., two or more target probes directed to a specific gene or gene promoter region.

[0074] Each of the target probes comprises a target nucleic acid binding region complementary to a target nucleic acid. The target nucleic acid binding region need not exhibit complete complementarity to the one or more regions of a target nucleic acid, provided that there is sufficient complementarity to result in specific hybridization / binding.

[0075] The region of the target probes responsible for target nucleic acid binding may be any size necessary to specifically and sensitively bind the target nucleic acid. In some embodiments, the target nucleic acid binding region is at least 10 nucleotides in length. For example, the target nucleic acid binding region may be about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, or more nucleotides in length. In some embodiments, the target nucleic acid binding region is 10 to 50 nucleotides in length (e.g., 10 to 40 nucleotides, 10 to 30 nucleotides, 10 to 20 nucleotides, 20 to 50 nucleotides, 20 to 40 nucleotides, 20 to 30 nucleotides, 30 to 50 nucleotides, 30 to 40 nucleotides, or 40 to 50 nucleotides in length).

[0076] Each of the target probes also comprises a signal generating complex binding region complementary to a region of a signal generating complex. The signal generating complex binding region need not exhibit complete complementarity to the region of the signal generating complex to which it binds, provided that there is sufficient complementarity to cause hybridization / binding.

[0077] The region of the target probes responsible for signal generating complex binding may be any size necessary to specifically and sensitively bind the signal generating complex. In some embodiments, the region of the target probes responsible for signal generating complex binding is at least 10 nucleotides in length. For example, the linker oligonucleotide binding region may be about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, or more nucleotides in length. In some embodiments, the linker oligonucleotide binding region is 10 to 50 nucleotides in length (e.g., 10 to 40 nucleotides, 10 to 30 nucleotides, 10 to 20 nucleotides, 20 toAttorney Docket No. ACDX-43932.60150 nucleotides, 20 to 40 nucleotides, 20 to 30 nucleotides, 30 to 50 nucleotides, 30 to 40 nucleotides, or 40 to 50 nucleotides in length).

[0078] The target probes are also not limited by the length of the target nucleic acid binding region with respect to the signal generating complex binding region. The length will be partially based upon the sequence and the number of nucleotides necessary for specific and / or selective binding to the target nucleic acid and the single generating complex.

[0079] Accordingly, each target probe comprises a target nucleic acid binding region and a signal generating complex binding region. The target nucleic acid binding region and signal generating complex binding region may be arranged in any configuration about the target probe(s). The target probe(s) are not limited by the orientation of target nucleic acid binding region with respect to the signal generating complex binding region. In some embodiments, the target nucleic acid binding region is 5’ of the signal generating complex binding region. In some embodiments, the target nucleic acid binding region is 3’ of the signal generating complex binding region. The target probe is generally single stranded so that the target probe is available to hybridize with a corresponding target nucleic acid and signal generating complex.

[0080] The target nucleic acid binding region and the signal generating complex binding region may be consecutive or separated by any number of nucleotides to facilitate independent and accessible binding to the target nucleic acid and the signal generating complex. The target nucleic acid binding region and signal generating complex binding region may be separated by a nontargeting region. The non-targeting region does not have any complementarity to the target nucleic acid or the signal generating complex. The non-targeting region does not participate in hybridization or binding interactions with the other components of the system.

[0081] The non-targeting region is not limited by a specific sequence. In some embodiments, the non-targeting region comprises a random sequence. In some embodiments, the non-targeting region comprises a poly-thymidine (poly(T)) sequence.

[0082] The non-targeting region may be any length which facilitates the target probe interaction with both the target nucleic acid and the signal generating complex. The non-targeting region may be at least 1 nucleotide in length, for example, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or more in length. In some embodiments, the non-targeting region is 1 to 20, 1 to 15, or 1 to 10 nucleotides in length.Attorney Docket No. ACDX-43932.601

[0083] The methods are not limited by the type or location of the target nucleic acid. Target nucleic acid refers to the polynucleotide (nucleic acid, gene, chromosome, genome, etc.) which has or is suspected of having a DNA modification.

[0084] The target nucleic acid can be an endogenous nucleic acid or an exogenous nucleic acid. The target nucleic acid can be from a host organism or, alternatively, can be a nucleic acid acquired or transformed into a host cell or organism, e.g., from a pathogen or genetic modification. The target nucleic acid can be from organelles outside the nucleus. In some embodiments, the target nucleic acid is DNA. The target nucleic acid can be genomic DNA. In some embodiments, the target nucleic acid is RNA. Exemplary target RNAs include, but are not limited to, messenger RNAs (primary and mature mRNAs), ribosomal RNAs, transfer RNAs and small nuclear RNAs. d. Signal Generation

[0085] The methods may further include contacting the sample with a signal generating complex (SGC). In some embodiments, the signal generating complex comprises a nucleic acid component capable of hybridizing to a portion of the target probe bound to the target nucleic acid and an oligonucleotide covalently attached to a binding agent bound to a nucleic acid modification. When the target probe binding region of the target nucleic acid is adjacent to the nucleic acid modification a detectable signal is generated.

[0086] In some embodiments, the SGC is the same or similar SGC used in RNAscope™, which is described in more detail in, e.g., U.S. Patent Nos. 7709198, 8604182, and 8951726. Specifically, RNAscope™ uses specially designed oligonucleotide probes in combination with a branched- DNA-like SGC to reliably detect RNA under standard bright-field microscopy (Anderson el al., J. Cell. Biochem. 117(10):2201-2208 (2016); Wang etal., J. Mol. Diagn. 14(l):22-29 (2012)). When used in methods described herein, rather than binding to one or more target probe(s) that bind to a target nucleic acid as in RNAscope™, the SGC instead binds to a portion of the target probe that binds to a target nucleic acid and an oligonucleotide covalently attached binding agent that binds to a nucleic acid modification.

[0087] In some embodiments, the SGC includes a pre-pre-amplifier, a pre-amplifier, and / or an amplifier, and one or more label probes, wherein each label probe comprises a detectable label. In some embodiments, the SGC comprises a pre-pre-amplifier, a pre-amplifier, an amplifier, and one or more label probes, wherein each label probe comprises a detectable label. Accordingly, the methods may comprise contacting the biological sample with a pre-amplifier, an amplifier, and / orAttorney Docket No. ACDX-43932.601 one or more label probes simultaneously, sequentially in any order, or a combination thereof where some of the SGC components are provided simultaneously before or after another component or components.

[0088] Any nucleic acid portion of the SGC may hybridize to the to the target probe and the oligonucleotide covalently attached to a binding agent, preferably simultaneously. In some embodiments, the pre-pre-amplifier hybridizes to the target probe and the oligonucleotide covalently attached to a binding agent. In some embodiments, the pre-amplifier hybridizes to the target probe and the oligonucleotide covalently attached to a binding agent. In some embodiments, the amplifier hybridizes to the target probe and the oligonucleotide covalently attached to a binding agent.

[0089] The methods may further comprise: contacting the biological sample with a preamplifier capable of hybridizing to the target probe and the oligonucleotide covalently attached to a binding agent simultaneously, wherein the pre-amplifier comprises binding sites for a plurality of amplifiers; contacting the biological sample with the plurality of amplifiers capable of hybridizing to the pre-amplifier, wherein the plurality of amplifiers comprises binding sites for a plurality of label probes; contacting the biological sample with the plurality of label probes capable of hybridizing to the plurality of amplifiers, wherein each label probe comprises a detectable label; and detecting a signal generated from the plurality of label probes when the target nucleic acid and the nucleic acid modification are in sufficiently close proximity to allow binding of the preamplifier to the target probe and the oligonucleotide covalently attached to a binding agent simultaneously.

[0090] Alternatively, the methods may further comprise: contacting the biological sample with a pre-pre-amplifier capable of hybridizing to the target probe and the oligonucleotide covalently attached to a binding agent simultaneously, wherein the pre-pre-amplifier comprises binding sites for a plurality of pre-amplifiers; contacting the biological sample with a plurality of pre-amplifiers capable of hybridizing pre-amplifier simultaneously; contacting the biological sample with the plurality of amplifiers capable of hybridizing to the pre-amplifiers, wherein the plurality of amplifiers comprises binding sites for a plurality of label probes; contacting the biological sample with the plurality of label probes capable of hybridizing to the plurality of amplifiers, wherein each label probe comprises a detectable label; and detecting a signal generated from the plurality of label probes when the target nucleic acid and the nucleic acid modification are in sufficiently closeAttorney Docket No. ACDX-43932.601 proximity to allow binding of the pre-pre-amplifier to the target probe and the oligonucleotide covalently attached to a binding agent simultaneously.

[0091] As used herein, an “amplifier” is a molecule, typically a polynucleotide, that is capable of hybridizing to multiple label probes. Typically, the amplifier hybridizes to multiple identical label probes. The amplifier can also hybridize directly to the target, or to another nucleic acid bound to the target such as a pre-amplifier. For example, the amplifier can hybridize to the target and to a plurality of label probes, or to a pre-amplifier and a plurality of label probes. The amplifier can be, for example, a linear, forked, comb-like, or branched nucleic acid. As described herein for all polynucleotides, the amplifier can include modified nucleotides and / or nonstandard internucleotide linkages as well as standard deoxyribonucleotides, ribonucleotides, and / or phosphodiester bonds. Suitable amplifiers are described, for example, in U.S. Patent Nos. 5635352, 5124246, 5710264, 5849481, and 7709198, and U.S. Publication Nos. 2008 / 0038725 and 2009 / 0081688, each of which is incorporated herein by reference.

[0092] As used herein, a “pre-amplifier” is a molecule, typically a polynucleotide, that serves as an intermediate binding component between the target and one or more amplifiers. Typically, the pre-amplifier hybridizes simultaneously to the target and to a plurality of amplifiers. Exemplary pre-amplifiers are described, for example, in U.S. Patent Nos. 5635352, 5681697, 7709198, and 11078528, and U.S. Publication Nos. 2008 / 0038725 and 2009 / 0081688, each of which is incorporated by reference.

[0093] As used herein, a “pre-pre-amplifier” is a molecule, typically a polynucleotide, that serves as an intermediate binding component between the target and one or more pre-amplifiers. Typically, the pre-pre-amplifier hybridizes simultaneously to the target and to a plurality of preamplifiers. Exemplary pre-pre-amplifiers are described, for example, in U.S. Patent No. 11078528, which is incorporated by reference.

[0094] As used herein, the term “label probe” refers to an entity that binds to a target molecule, directly or indirectly, generally indirectly, and allows detection. A label probe (or “LP”) contains a nucleic acid binding portion that is typically a single stranded polynucleotide or oligonucleotide that comprises one or more labels which directly or indirectly provides a detectable signal. The label can be covalently attached to the polynucleotide, or the polynucleotide can be configured to bind to the label. For example, a biotinylated polynucleotide can bind a streptavidin-associated label. In general, the label probe can hybridize to a nucleic acid that is in turn hybridized to theAttorney Docket No. ACDX-43932.601 target, or to one or more other nucleic acids that are hybridized to the target. Thus, the label probe can comprise a polynucleotide sequence that is complementary to a polynucleotide sequence, particularly a portion, of the target. Alternatively, the label probe can comprise at least one polynucleotide sequence that is complementary to a polynucleotide sequence in an amplifier, preamplifier, or pre-pre-amplifier in an SGC.

[0095] As used herein, a “detectable label” is a moiety that facilitates detection of a molecule. Common labels include fluorescent, luminescent, light-scattering, and / or colorimetric labels. Suitable labels include enzymes, and fluorescent and chromogenic moieties, as well as radionuclides, substrates, cofactors, inhibitors, chemiluminescent moieties, magnetic particles, rare earth metals, metal isotopes, and the like. In particular embodiments, the label comprises a fluorescent moiety or a chromogenic moiety. In a particular embodiment, the label is an enzyme. Exemplary enzyme labels include, but are not limited to horseradish peroxidase (HRP), alkaline phosphatase (AP), 0-galactosidase, glucose oxidase, and the like, as well as various proteases. Other labels include, but are not limited to, fluorophores, dinitrophenyl (DNP), and the like. Labels are well known to those skilled in the art, as described, for example, in Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996), and U.S. PatentNos. 3817837, 3850752, 3939350, 3996345, 4277437, 4275149, and 4366241. Many labels are commercially available and can be used in methods and assays of the disclosure, including detectable enzyme / substrate combinations (Pierce, Rockford IL; Santa Cruz Biotechnology, Dallas TX; Life Technologies, Carlsbad CA). In a particular embodiment of the disclosure, the enzyme can utilize a chromogenic or fluorogenic substrate to produce a detectable signal, as described herein. Exemplary labels are described herein.

[0096] Any of a number of enzymes or non-enzyme labels can be utilized so long as the enzymatic activity or non-enzyme label, respectively, can be detected. The enzyme thereby produces a detectable signal, which can be utilized to detect a target. Particularly useful detectable signals are chromogenic or fluorogenic signals. Accordingly, particularly useful enzymes for use as a label include those for which a chromogenic or fluorogenic substrate is available. Such chromogenic or fluorogenic substrates can be converted by enzymatic reaction to a readily detectable chromogenic or fluorescent product, which can be readily detected and / or quantified using microscopy or spectroscopy. Such enzymes are well known to those skilled in the art, including but not limited to, horseradish peroxidase, alkaline phosphatase, |3-galactosidase, glucose oxidase, and the like (see Hermanson, Bioconjugate Techniques, Academic Press, SanAttorney Docket No. ACDX-43932.601Diego (1996)). Other enzymes that have well known chromogenic or fluorogenic substrates include various peptidases, where chromogenic or fluorogenic peptide substrates can be utilized to detect proteolytic cleavage reactions. The use of chromogenic and fluorogenic substrates is also well known in bacterial diagnostics, including but not limited to the use of a- and 0-galactosidase, P-glucuronidase, 6-phospho-P-D-galactoside 6-phosphogalactohydrolase, P-glucosidase, a- glucosidase, amylase, neuraminidase, esterases, lipases, and the like (Manafi et al., Microbiol. Rev. 55:335-348 (1991)), and such enzymes with known chromogenic or fluorogenic substrates can readily be adapted for use in methods provided herein.

[0097] Various chromogenic or fluorogenic substrates to produce detectable signal are well known to those skilled in the art and are commercially available. Exemplary substrates that can be utilized to produce a detectable signal include, but are not limited to, 3,3'-diaminobenzidine (DAB), 3,3’,5,5’-tetramethylbenzidine (TMB), chloronaphthol (4-CN)(4-chl oro-1 -naphthol), 2,2'-azino- bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), o-phenylenediamine dihydrochloride (OPD), and 3-amino-9-ethylcarbazole (AEC) for horseradish peroxidase; 5-bromo-4-chloro-3- indolyl-1 -phosphate (BCIP), nitroblue tetrazolium (NBT), Fast Red (Fast Red TR / AS-MX), and p-nitrophenyl phosphate (PNPP) for alkaline phosphatase; l-methyl-3-indolyl-P-D- galactopyranoside and 2-methoxy-4-(2-nitrovinyl)phenyl -D-galactopyranoside for P- galactosidase; 2-methoxy-4-(2-nitrovinyl)phenyl P-D-glucopyranoside for P-glucosidase; and the like. Exemplary fluorogenic substrates include, but are not limited to, 4- (trifluoromethyl)umbelliferyl phosphate for alkaline phosphatase; 4-methylumbelliferyl phosphate bis (2-amino- 2-methyl-l,3-propanediol), 4-methylumbelliferyl phosphate bis (cyclohexylammonium) and 4-methylumbelliferyl phosphate for phosphatases; QuantaBlu™ and Quintolet for horseradish peroxidase; 4-methylumbelliferyl P-D-galactopyranoside, fluorescein di (P-D-galactopyranoside) and naphthofluorescein di-(P-D-galactopyranoside) for P-galactosidase; 3-acetylumbelliferyl P-D-glucopyranoside and 4-methylumbelliferyl-P- D-glucopyranoside for P- glucosidase; and 4-methylumbelliferyl-a- D-galactopyranoside for a-galactosidase. Exemplary enzymes and substrates for producing a detectable signal are also described, for example, in U.S. Publication No. 2012 / 0100540. Various detectable enzyme substrates, including chromogenic or fluorogenic substrates, are well known and commercially available (Pierce, Rockford IL; Santa Cruz Biotechnology, Dallas TX; Invitrogen, Carlsbad CA; 42 Life Science; Biocare). Generally, the substrates are converted to products that form precipitates that are deposited at the site of theAttorney Docket No. ACDX-43932.601 target. Other exemplary substrates include, but are not limited to, HRP-Green (42 Life Science), Betazoid DAB, Cardassian DAB, Romulin AEC, Bajoran Purple, Vina Green, Deep Space Black™, Warp Red™, Vulcan Fast Red and Ferangi Blue from Biocare (Concord CA; bi ocare .net / products / detecti on / chromogens) .

[0098] Exemplary rare earth metals and metal isotopes suitable as a detectable label include, but are not limited to, lanthanide (III) isotopes such as141Pr,142Nd,143Nd,144Nd,145Nd,146Nd,147Sm,148Nd,149Sm,150Nd,151Eu,152Sm,153Eu,154Sm,155Gd,156Gd,158Gd,159Tb,160Gd,161Dy,162Dy,163Dy,164Dy,165Ho,166Er,167Er,168Er,169Tm,170Er,171Yb,172Yb,173Yb,174Yb,175Lu, and176Yb. Metal isotopes can be detected, for example, using time-of-flight mass spectrometry (TOF- MS) (for example, Fluidigm Helios and Hyperion systems, fluidigm.com / systems; South San Francisco, CA).

[0099] Biotin-avidin (or biotin-streptavidin) is a well-known signal amplification system based on the fact that the two molecules have extraordinarily high affinity to each other, and that one avidin / streptavidin molecule can bind four biotin molecules. Antibodies are widely used for signal amplification in immunohistochemistry and ISH. Tyramide signal amplification (TSA) is based on the deposition of a large number of haptenized tyramide molecules by peroxidase activity. Tyramine is a phenolic compound. In the presence of small amounts of hydrogen peroxide, immobilized horseradish peroxidase (HRP) converts the labeled substrate into a short-lived, extremely reactive intermediate. The activated substrate molecules then very rapidly react with and covalently bind to electron-rich moi eties of proteins, such as tyrosine, at or near the site of the peroxidase binding site. In this way, many hapten molecules conjugated to tyramide can be introduced at the hybridization site in situ. Subsequently, the deposited tyramide-hapten molecules can be visualized directly or indirectly. Such a detection system is described in more detail, for example, in U.S. publication 2012 / 0100540.

[0100] Embodiments described herein can utilize enzymes to generate a detectable signal using appropriate chromogenic or fluorogenic substrates. It is understood that, alternatively, a label probe can have a detectable label directly coupled to the nucleic acid portion of the label probe. Exemplary detectable labels are well known to those skilled in the art, including but not limited to chromogenic or fluorescent labels (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Exemplary fluorophores useful as labels include, but are not limited to, rhodamine derivatives, for example, tetramethylrhodamine, rhodamine B, rhodamine 6G,Attorney Docket No. ACDX-43932.601 sulforhodamine B, Texas Red (sulforhodamine 101), rhodamine 1 10, and derivatives thereof such as tetramethylrhodamine-5-(or 6), lissamine rhodamine B, and the like; 7 -nitrob enz-2 -oxa- 1,3- diazole (NBD); fluorescein and derivatives thereof; napthalenes such as dansyl (5- dimethylaminonapthalene-1 -sulfonyl); coumarin derivatives such as 7-amino-4-methylcoumarin- 3-acetic acid (AMCA), 7-diethylamino-3-[(4'-(iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA), Alexa fluor dyes (Molecular Probes), and the like; 4,4-difluoro-4-bora-3a,4a-diaza-s- indacene (BODIPY™) and derivatives thereof (Molecular Probes; Eugene, OR); pyrenes and sulfonated pyrenes such as Cascade Blue™ and derivatives thereof, including 8-methoxypyrene- 1,3,6-trisulfonic acid, and the like; pyridyl oxazole derivatives and dapoxyl derivatives (Molecular Probes); Lucifer Yellow (3,6-disulfonate-4-amino-naphthalimide) and derivatives thereof; CyDye™ fluorescent dyes (Amersham / GE Healthcare Life Sciences; Piscataway NJ), ATTO 390, DyLight 395XL, ATTO 425, ATTO 465, ATTO 488, ATTO 490LS, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol l, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO 590, ATTO 594, ATTO Rhol3, ATTO 610, ATTO 620, ATTO Rhol4, ATTO 633, ATTO 643, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxal2, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740, Cyan 500 NHS- Ester (ATTO-TECH, Siegen, Germany), and the like. Exemplary chromophores include, but are not limited to, phenolphthalein, malachite green, nitroaromatics such as nitrophenyl, diazo dyes, dabsyl (4-dimethylaminoazobenzene-4'-sulfonyl), and the like.

[0101] The methods are not limited by the order in which steps (i) and (ii) are performed. In some embodiments, step (i) and step (ii) are performed simultaneously. In some embodiments, step (i) is performed before step (ii). In some embodiments, step (ii) is performed before step (i).

[0102] In some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the target probe and binding agent, respectively, for about 10 minutes to about 48 hours, or about 15 minutes to about 120 minutes. For example, in some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the target probe and binding agent, respectively, for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 3 hours, about 4 hours,Attorney Docket No. ACDX-43932.601 about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about47 hours, or about 48 hours.

[0103] In some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the target probe and binding agent, respectively, at a temperature of about 4 °C to about 75 °C, or about 4 °C to about 25 °C. For example, in some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the target probe and binding agent, respectively, at a temperature of about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14 °C, about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, about 50 °C, about 51 °C, about 52 °C, about 53 °C, about 54 °C, about 55 °C, about 56 °C, about 57 °C, about 58 °C, about 59 °C, about 60 °C, about 61 °C, about 62 °C, about 63 °C, about 64 °C, about 65 °C, about 66 °C, about 67 °C, about 68 °C, about 69 °C, about 70 °C, about 71 °C, about 72 °C, about 73 °C, about 74 °C, or about 75 °C. In some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the target probe and binding agent, respectively, at room temperature.

[0104] The methods may utilize other components in conjunction with those components described above. For example, the methods may utilize epitope retrieval or target unmasking. The methods may utilize reagents for use in the detection of other targets of interest in the sample. For example, the methods may detect one or more protein targets, or nucleic acids targets. The methods may include immunohistochemistry or immunocytochemistry for other targets (e.g., protein or other nucleic acid targets).Attorney Docket No. ACDX-43932.601

[0105] In some embodiments, the method further comprises contacting the sample with a blocking agent prior to step (i) and / or step (ii), to minimize non-specific binding that can result in unwanted background signals. Suitable blocking agents include those that comprise DNA, RNA, or protein. For example, in some embodiments, the blocking agent comprises DNA, such as salmon sperm DNA, herring sperm DNA, or calf thymus DNA. In some embodiments, the blocking agent comprises RNA, such as tRNA. In some embodiments, the blocking agent comprises a protein or polypeptide; for example, in some embodiments, the blocking agent comprises bovine serum albumin (BSA), casein, heparin, an animal serum such as normal goat serum, normal swine serum, normal chicken serum, or a fish serum such as steelhead salmon serum. In some embodiments, the blocking agent is a non-animal protein blocking agent, such as one comprising a plant protein. Non-animal protein blocking agents are commercially available, e.g., from G-Biosciences® (NAP -BLOCKER™) and Vector Laboratories (Animal-Free Blocker®).

[0106] In some embodiments, the method further comprises contacting the sample with a crosslinking agent after steps (i) and (ii) but before step (iii). Such a step has been found to preserve and even improve signals in IHC assays when conducted after incubation with a primary antibody and before incubation with a secondary antibody, such as when samples are exposed to protease treatment (see WO 2021 / 226311). In certain embodiments, the crosslinking agent is a fixative. In some embodiments, the crosslinking agent is selected from neutral-buffered formalin (NBF), formaldehyde, glutaraldehyde, acrolein, osmium tetroxide, a permanganate fixative (e.g., potassium permanganate), a dichromate fixative (e g., potassium dichromate), chromic acid, and a mixture of any thereof. In particular embodiments, the crosslinking agent is NBF, such as about 1% to about 20% NBF (e.g., 10% NBF). In some embodiments, the crosslinking agent is a mixture of any of the above fixatives, with or without additional compounds. For example, in some embodiments, the crosslinking agent is selected from: Bouin’s fixative (picric acid, formaldehyde, and acetic acid), a mixture of formaldehyde and glutaraldehyde; FAA (ethanol, acetic acid, and formaldehyde); periodate-lysine-paraformaldehyde (PLP) (paraformaldehyde, L-lysine, and INaCL); phosphate buffered formalin (PBF); formal calcium (formaldehyde and calcium chloride); formal saline (formaldehyde and sodium chloride); zinc formalin (formaldehyde and zinc sulfate); Helly’s fixative (formaldehyde, potassium di chromate, sodium sulphate, and mercuric chloride); Hollande’s fixative (formaldehyde, copper acetate, picric acid, and acetic acid); Gendre’s solution (formaldehyde, ethanol, picric acid, and glacial acetic acid); alcoholic formalin (formaldehyde,Attorney Docket No. ACDX-43932.601 ethanol, and calcium acetate); and formol acetic alcohol (formaldehyde, glacial acetic acid, and ethanol). In some embodiments, the crosslinking agent comprises a polymer with at least two reactive functional groups, such as succinimidyl esters. In some embodiments, the crosslinking agent is a bis( succinimidyl) polyethylene glycol. In some embodiments, the crosslinking agent is di(N-succinimidy) glutarate. In some embodiments, the crosslinking agent is provided as an aqueous solution at a pH of about 6 to about 9, e.g., about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In embodiments in which two or more crosslinking agents are used, the sample may be contacted with the two or more crosslinking agents either simultaneously or consecutively.

[0107] In some embodiments, the step of contacting the sample with a crosslinking agent is conducted at a temperature of about 0 °C to about 100 °C, about 1 °C to about 90 °C, about 2 °C to about 80 °C, about 3 °C to about 70 °C, or about 4 °C to about 60 °C, e g., about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14 °C, about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C, about 65 °C, about 70 °C, about 75 °C, about 80 °C, about 85 °C, about 90 °C, about 95 °C, about or 100 °C.

[0108] In some embodiments, the step of contacting the sample with a crosslinking agent is conducted for about 5 minutes to about 48 hours, about 5 minutes to about 24 hours, about 15 minutes to about 24 hours, or about 15 minutes to about 18 hours, e.g., about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, or about 48 hours.Attorney Docket No. ACDX-43932.601

[0109] In some embodiments, the method further comprises treating the biological sample with a protease after treating the biological sample with the crosslinking agent and before step (iii). This step can be used to digest certain proteins that surround the target. In some embodiments, the protease is selected from trypsin, proteinase K, pepsin, pronase, endoproteinase AspN, and endoproteinase GluC. In some embodiments, the method further comprises treating the biological sample with hydrogen peroxide after treating the biological sample with the crosslinking agent and before step (ii). This step is particularly useful when horseradish peroxidase (HRP) will be used as detection enzyme in the later steps, as the hydrogen peroxide inactivates endogenous HRP activity in the sample, thus reducing assay background.

[0110] The methods disclosed herein can be used for concurrent or sequential detection of multiple nucleic acid modifications in the same sample. Thus, the disclosed methods can be multiplexed. For example, in some embodiments, the methods comprise detecting two or more nucleic acid modifications in the same sample. In some embodiments, the methods comprise detecting 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more different nucleic acid modifications in the same sample.

[0111] For example, in some embodiments, the methods may comprise contacting the biological sample with an additional one or more, or one or more sets, of target probes and contacting the biological sample with one or more additional binding agents comprising binding agents directed to the nucleic acid modification, wherein each binding agent is covalently attached to an oligonucleotide. Accordingly, using one or more, or one or more sets, of target probes with coordinating additional binding agents allows detection of detecting two or more nucleic acid modifications. These additional steps may be performed simultaneously or sequentially, in any order, in relationship to each other, as described above for steps (i) and (ii).

[0112] Embodiments in which higher numbers of nucleic acid modifications are detected in the same sample may involve the use of cleavable labels, which are further described elsewhere herein. In the case of using fluorophores as labels, the fluorophores to be used for detection of multiple nucleic acid modifications are selected so that each of the fluorophores are distinguishable and can be detected concurrently in a fluorescence microscope. Such fluorophores are selected to have spectral separation of the emissions so that distinct labeling can be detected concurrently. Methods of selecting suitable distinguishable fluorophores for use in methods of the disclosure are wellAttorney Docket No. ACDX-43932.601 known in the art (see, e.g., Johnson and Spence, “Molecular Probes Handbook, a Guide to Fluorescent Probes and Labeling Technologies, 11th ed., Life Technologies (2010)).

[0113] The label can be designed such that the labels are optionally cleavable. As used herein, a “cleavable label” refers to a label that is attached or conjugated to a label probe so that the label can be removed, for example, in order to use the same label in a subsequent round of labeling and detecting of targets. Methods for multiplex detection of nucleic acids of using cleavable labels have been described, e.g., in WO 2020 / 168162, which is incorporated herein by reference in its entirety, and are commercially available as RNAscope™ HiPlex assays (e.g., RNAscope™ HiPlex and RNAscope™ HiPlex v2).

[0114] Generally, the labels are conjugated to the label probe by a chemical linker that is cleavable. Methods of conjugating a label to a label probe so that the label is cleavable are well known to those skilled in the art (see, e.g., Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996); Daniel et al., BioTechniques 24(3):484-489 (1998)). One particular system of labeling oligonucleotides is the FastTag™ system (Daniel etal., supra, 1998; Vector Laboratories, Burlingame CA). Various cleavable moieties can be included in the linker so that the label can be cleaved from the label probe. Such cleavable moieties include groups that can be chemically, photochemically, or enzymatically cleaved. Cleavable chemical linkers can include a cleavable chemical moiety, such as disulfides, which can be cleaved by reduction, glycols, or diols, which can be cleaved by periodate, diazo bonds, which can be cleaved by dithionite, esters, which can be cleaved by hydroxylamine, sulfones, which can be cleaved by base, and the like (see Hermanson, supra, 1996). One particularly useful cleavable linker is a linker containing a disulfide bond, which can be cleaved by reducing the disulfide bond. In other embodiments, the linker can include a site for cleavage by an enzyme. For example, the linker can contain a proteolytic cleavage site. Generally, such a cleavage site is for a sequence-specific protease. Such proteases include, but are not limited to, human rhinovirus 3C protease (cleavage site LEVLFQ / GP), enterokinase (cleavage site DDDDK / ), factor Xa (cleavage site IEGR / ), tobacco etch virus protease (cleavage site ENLYFQ / G), and thrombin (cleavage site LVPR / GS) (see, e.g., Oxford Genetics, Oxford, UK). Another cleavable moiety can be, for example, uracil-DNA (DNA containing uracil), which can be cleaved by uracil-DNA glycosylase (UNG) (see, e.g., Sidorenko etal., FEBSLett. 582(3):410 404 (2008)).Attorney Docket No. ACDX-43932.601

[0115] The cleavable labels can be removed by applying an agent, such as a chemical agent or light, to cleave the label and release it from the label probe. As discussed above, useful cleaving agents for chemical cleavage include, but are not limited to, reducing agents, periodate, dithionite, hydroxylamine, base, and the like (see Hermanson, supra, 1996). One useful method for cleaving a linker containing a disulfide bond is the use of tris(2-carboxyethyl)phosphine (TCEP) (see Moffitt et al., Proc. Natl. Acad. Set. USA 113: 11046-11051 (2016)). In one embodiment, TCEP is used as an agent to cleave a label from a label probe.

[0116] Step (iii) of the disclosed method comprises detecting a signal from the signal generating complex. Well-known methods such as microscopy, cytometry (e.g., mass cytometry, cytometry by time of flight (CyTOF), flow cytometry), or spectroscopy can be utilized to detect chromogenic, fluorescent, or metal detectable signals associated with the respective targets. In general, either chromogenic substrates or Anorogenic substrates, or chromogenic or Auorescent labels, or rare earth metal isotopes, will be utilized for a particular assay, if different labels are used in the same assay, so that a single type of instrument can be used for detection in the same sample.

[0117] The biological sample used in the disclosed methods can be derived from various sources. In one embodiment, the biological sample is a tissue specimen or is derived from a tissue specimen. In one embodiment, the biological sample is a blood sample or is derived from a blood sample. In one embodiment, the biological sample is a cytological sample or is derived from a cytological sample. In one embodiment, the biological sample is cultured cells. In another embodiment, the biological sample is a sample containing exosomes.

[0118] Tissue specimens include, for example, tissue biopsy samples. Blood samples include, for example, blood samples taken for diagnostic purposes. In the case of a blood sample, the blood can be directly analyzed, such as in a blood smear, or the blood can be processed, for example, lysis of red blood cells, isolation of PBMCs or leukocytes, isolation of target cells, and the like, such that the cells in the sample analyzed by methods of the disclosure are in a blood sample or are derived from a blood sample. Similarly, a tissue specimen can be processed, for example, the tissue specimen minced and treated physically or enzymatically to disrupt the tissue into individual cells or cell clusters. Additionally, a cytological sample can be processed to isolate cells or disrupt cell clusters, if desired. Thus, the tissue, blood and cytological samples can be obtained and processed using methods well known in the art. The methods of the disclosure can be used inAttorney Docket No. ACDX-43932.601 diagnostic applications to identify the presence or absence of pathological cells based on the presence or absence of a target that is a biomarker indicative of a pathology.

[0119] The biological sample can be obtained from a subject, including a sample of biological tissue or fluid origin that is collected from an individual or some other source of biological material such as biopsy, autopsy, or forensic materials. A biological sample also includes samples from a region of a biological subject containing or suspected of containing precancerous or cancer cells or tissues, for example, a tissue biopsy, including fine needle aspirates, blood sample, or cytological specimen. Such samples can be, but are not limited to, organs, tissues, tissue fractions, cells, and / or exosomes isolated from an organism such as a mammal. Exemplary biological samples include, but are not limited to, a cell culture, including a cell, a primary cell culture, a cell line, a tissue, an organ, an organoid, a biological fluid, and the like. Additional biological samples include but are not limited to a skin sample, tissue biopsies, including fine needle aspirates, cytological samples, stool, bodily fluids, including blood and / or serum samples, saliva, semen, and the like. Such samples can be used for medical or veterinary diagnostic purposes.

[0120] Collection of cytological samples for analysis by methods provided herein are well known in the art (see, e.g., Dey, “Cytology Sample Procurement, Fixation and Processing” in Basic and Advanced Laboratory Techniques in Histopathology and Cytology pp. 121-132, Springer, Singapore (2018); “Non-Gynecological Cytology Practice Guideline” American Society of Cytopathology, Adopted by the ASC executive board March 2, 2004).

[0121] For example, methods for processing samples for analysis of cervical tissue, including tissue biopsy and cytology samples, are well known in the art (see, e.g., Cecil Textbook of Medicine, Bennett and Plum, eds., 20th ed., WB Saunders, Philadelphia (1996); Colposcopy and Treatment of Cervical Intraepithelial Neoplasia: A Beginner ’s Manual, Sellers and Sankaranarayanan, eds., International Agency for Research on Cancer, Lyon, France (2003); Kalaf and Cooper, J. Clin. Pathol. 60:449-455 (2007); Brown and Trimble, Best Pract. Res. Clin. Obstet. Gynaecol. 26:233- 242 (2012); Waxman et al., Obstet. Gynecol. 120: 1465-1471 (2012); Cervical Cytology Practice Guidelines TOC, Approved by the American Society of Cytopathology (ASC) Executive Board, November 10, 2000)).

[0122] In particular embodiments, the sample is a tissue specimen or is derived from a tissue specimen. In some embodiments, the tissue specimen is a formalin-fixed paraffin-embedded (FFPE) sample. In some embodiments, the tissue specimen is fresh frozen. In some embodiments,Attorney Docket No. ACDX-43932.601 the tissue specimen is prepared with a fixative. Tn some embodiments, the tissue specimen is prepared with a crosslinking fixative. In other particular embodiments, the sample is a blood sample or is derived from a blood sample. In still other particular embodiments, the sample is a cytological sample or is derived from a cytological sample.

[0123] In some embodiments, the method further comprises steps to prepare a sample for detection of the target. For example, if the sample is a FFPE sample, a de-paraffinization step can be used to remove paraffin and rehydrate the sample. In some embodiments, the method further comprises dehydrating the biological sample. In certain embodiments, the dehydration is carried out with ethanol of increasing concentrations, such as in the order of 70%, 95%, and 100% ethanol .

[0124] In some embodiments, the method further comprises an epitope retrieval step, where certain epitope retrieval buffer(s) can be added to unmask the target. In some embodiments, the epitope retrieval step comprises heating the sample. In some embodiments, the epitope retrieval step comprises heating the sample to about 50 °C to about 100 °C. In one embodiment, the epitope retrieval step comprises heating the sample to about 88 °C. Detergents (e.g., Triton X-100 or SDS) and Proteinase K can be used to increase the permeability of the fixed cells. Detergent treatment, usually with Triton X-100 or SDS, is frequently used to permeate the membranes by extracting the lipids. Proteinase K is a nonspecific protease that is active over a wide pH range and is not easily inactivated. It is used to digest proteins that surround the targets. Optimal concentrations and durations of treatment can be experimentally determined as is well known in the art.

[0125] In some embodiments, the method further comprises a depurination step. The depurination step cleaves the guanine and adenine bases from the deoxyribonucleosides. This depurination step may improve the specificity of the binding agent, depending on the nucleic acid modification being detected. In some embodiments, the depurination step comprises treating the sample with acid (e g., HC1). In one embodiment, the depurination step comprises treating the sample with 2-5N HC1 at room temperature.

[0126] The disclosed methods and components can also be used with methods and components for detection of other targets of interest in the sample. For example, the methods may further comprise detecting one or more nucleic acid targets. In some embodiments, the methods further comprise contacting the biological sample with one of more nucleic acid detection agents. The methods are not limited by the type of nucleic acid detection agents. For example, the nucleic acid detection agents may comprise in situ hybridization probes for specific sequences, or probes forAttorney Docket No. ACDX-43932.601 detection of two or more nucleic acid targets (see, e g., International Patent Publication W02007001986).

[0127] In some embodiments, the methods comprise performing in situ hybridization to detect mRNA. The mRNA may be the target nucleic acid or the mRNA may correspond to the region being interrogated for a nucleic acid modification in a target DNA. For example, mRNA detection may be used to correlate nucleic acid modifications to target gene modification in genomic DNA. Image Processing

[0128] In some embodiments, the methods include an image processing method, such as the methods described in International Patent Publication WO2022221635, which is herein incorporated by reference. The method is implemented at least in part with a computer having corresponding instructions stored on a memory (e.g., a non-transitory computer readable medium). The final images, and in some embodiments the intermediate images, from the method are stored in a memory. In some embodiments, the memory is accessible by a network. In some embodiments, user input or instructions are receivable or accessible over the network.

[0129] The method includes imaging a sample with a target signal to create a probe image and imaging a sample with no target signal to create a background image (e.g., a “blank image”). In some embodiments, the imaging utilizes a fluorescent microscope coupled to a computer via a network. In some embodiments, the background image with no target signal is obtained by removing the target signal from the sample (e.g., by a cleaving process). In other embodiments, the background image with no target signal is obtained before the assay is performed. In some embodiments, the target signal comprises a fluorescent label bound to a target nucleic acid. In other embodiments, the target signal comprises a fluorescent label bound to a target peptide or polypeptide.

[0130] The method can also include registering the probe image and the background image. Potential background fluorescence discrepancy between the probe image and the background image creates spatial pattern mismatches that occur due to whole sample movement between different rounds of image acquisition. To remove such discrepancies, image registration techniques (e.g., phase correlation) are utilized. Robust image registration utilizes detection and matching of image features to compensate for any global sample movement (i.e., translation and rotation).

[0131] The method further includes modifying the background image to create an adjusted background image (e.g., transformed, intensity-adjusted blank image) based on at least one imageAttorney Docket No. ACDX-43932.601 metric. As explained further herein, the at least one image metric is a ratio factor, a multiplication factor, a local maximum value transform, and any other suitable metric. In some embodiments, the method includes a single image metric. In other embodiments, the method includes a combination of image metrics.

[0132] In some embodiments, the method further includes subtracting the adjusted background image from the probe image to create a final image comprising an enhanced target signal. In other words, the modified (i.e., transformed, adjusted, scaled, etc.) blank image is used in the subtracting step instead of the original blank image. In some embodiments, the enhanced target signal includes enhanced contrast. In some embodiments, the method includes displaying the final image on a display (e.g., a computer display). The final image may be saved to a memory and may be accessible by a user, for example, over a network. As such, the method provides improved signal detection in the presence of a background with tissue autofluorescence.Kits

[0133] Also provided herein is a kit for performing the methods described herein.

[0134] In some embodiments, provided herein is a kit for detecting one or more nucleic acid modification. In some embodiments, the kit comprises at least one target probe and at least one binding agent as described herein.

[0135] In some embodiments, the kit further comprises one or more permeabilizing and / or antigen retrieval reagents. In some embodiments, the kit further comprises at least one amplifier, at least one pre-amplifier, and / or at least one detectable label. In some embodiments, the kit further comprises a control or reference sample.

[0136] In some embodiments, the kit further comprises a blocking agent, a crosslinking agent, a protease, or any combination thereof. The blocking agent, the crosslinking agent, and the protease can be selected from any of those described above.

[0137] In some embodiments, the kit comprises an agent used for fixing a biological sample. In some embodiments, the kit includes a fixative(s) that is suitable for preserving nucleic acids. In one embodiment, the fixative is FineFix (see Kothmaier et al., Arch. Pathol. Lab. Med. 135:744- 752, 2011). In one embodiment, the fixative is Glyo-fix (see Lykidis et al., Nucleic Acids Res. 35:e85, 2007). In one embodiment, the fixative is Histochoice (see Vince etal., Anal. Cell. Pathol. 15: 119-129, 1997). In one embodiment, the fixative is HOPE (see Kothmaier et al., Arch. Pathol. Lab. Med. 135:744-752, 2011). In one embodiment, the fixative is Neo-Fix (see Paavilainen etal.,Attornev Docket No. ACDX-43932.601Histochem. Cytochem.: Official J. Histochem. Soc. 58:237-246, 2010). In one embodiment, the fixative is the PAXgene Tissue System (see Nietner et al., Int. J. Pathol. 461 :259-269, 2012). In one embodiment, the fixative is RCL2 (see van Essen et al., Clin. Pathol.63: 1090-1094, 2010). In one embodiment, the fixative is Streck’s Tissue Fixative (see Burns et al., Histochem. Cytochem. 57:257-264, 2009). In one embodiment, the fixative is UMFIX (see Nadji et al., Appl. Immunohistochem. Mol. Morphol. 13:277-282, 2005). In one embodiment, the fixative is Z7 (see Lykidis et al., Nucleic Acids Res. 35:e85, 2007). In one embodiment, the fixative is ZBF (see Paavilainen et al., Histochem. Cytochem.: Official J. Histochem. Soc. 58:237-246, 2010).

[0138] In some embodiments, the kit provided herein comprises an aldehyde-containing fixative. In one embodiment, the aldehyde-containing fixative in the kit is formaldehyde. In one embodiment, the aldehyde-containing fixative in the kit is glutaraldehyde. In one embodiment, the aldehyde-containing fixative in the kit is Bouin’s fixative, which is a solution of picric acid, formaldehyde, and acetic acid. In one embodiment, the aldehyde-containing fixative in the kit is a mixture of formaldehyde and glutaraldehyde. In one embodiment, the aldehyde-containing fixative in the kit is FAA, which is a solution of ethanol, acetic acid, and formaldehyde. In one embodiment, the aldehyde-containing fixative in the kit is periodate-lysine-paraformaldehyde (PLP), which is a solution of paraformaldehyde, L-lysine, and INaC . In one embodiment, the aldehyde-containing fixative in the kit is phosphate buffered formalin (PBF). In one embodiment, the aldehyde- containing fixative in the kit is formal calcium, which is a solution of formaldehyde and calcium chloride. In one embodiment, the aldehyde-containing fixative in the kit is formal saline, which is a solution of formaldehyde and sodium chloride. In one embodiment, the aldehyde-containing fixative in the kit is zinc formalin, which is a solution of formaldehyde and zinc sulphate. In one embodiment, the aldehyde-containing fixative in the kit is Helly’s fixative, which is a solution of formaldehyde, potassium dichromate, sodium sulphate, and mercuric chloride. In one embodiment, the aldehyde-containing fixative in the kit is Hollande’s fixative, which is a solution of formaldehyde, copper acetate, picric acid, and acetic acid. In one embodiment, the aldehyde- containing fixative in the kit is Gendre’s solution, which is a solution of formaldehyde, ethanol, picric acid, and acetic acid glacial. In one embodiment, the aldehyde-containing fixative in the kit is alcoholic formalin, which is a solution of formaldehyde, ethanol, and calcium acetate. In one embodiment, the aldehyde-containing fixative in the kit is formol acetic alcohol, which is a solution of formaldehyde, acetic acid glacial, and ethanol. In one embodiment, the aldehyde-Attorney Docket No. ACDX-43932.601 containing fixative in the kit is a mixture of fixatives, wherein at least one fixative of the mixture is formaldehyde or glutaraldehyde. In one embodiment, the aldehyde-containing fixative in the kit is fixatives that are not used at the same time but consecutively, wherein at least one fixative is formaldehyde or glutaraldehyde.

[0139] In some embodiments, the kit further comprises a tool for obtaining a biological sample from a subject. In certain embodiments, the biological sample is a tissue specimen or is derived from a tissue specimen. In certain embodiments, the biological sample is a blood sample or is derived from a blood sample. In certain embodiments, the biological sample is a cytological sample or is derived from a cytological sample.

[0140] The kit may further comprise “packaging material” which refers to a physical structure housing the components of the kit. The packaging material can maintain the components under sterile conditions, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).

[0141] Kits provided herein can include labels or inserts, which can include information for which the kit component may be used. Labels or inserts can include instructions for carrying out any of the methods disclosed herein.EXAMPLES

[0142] The following is a description of methods, materials, and results corresponding to the various embodiments of the present disclosure. These descriptions are provided as examples and are not intended to be limiting. Rather, these examples are intended to provide those of ordinary skill in the art with a description of how to make and use the various embodiments of the present disclosure. These examples are not intended to limit the scope of what the inventors regard as inventive subject matter, nor are they intended to represent all of the experiments that can be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data associated with the teachings of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental error and deviation may be present.Example 1

[0143] Detection of gene-specific 5 -hydroxymethylcytosine (5hmC) sitesAttorney Docket No. ACDX-43932.601

[0144] Formalin-fixed paraffin-embedded (FFPE) mouse tissues (brain and kidney) were analyzed for 5hmCs in TNFa DNA using target probes directed to the mouse TNFa gene (e.g., promoter region) and an oligo-conjugated mouse monoclonal antibody against 5hmC sites as the binding agent.

[0145] Target retrieval on the tissue sample slides was completed at <100°C to denature the chromosome and DNA strands. The tissue slides were later, optionally, treated with 2~5N HC1 at room temperature to cleave the guanine and adenine bases from the deoxyribonucleosides, to improve the specificity of the antibody detections. Tissues were blocked with bovine serum albumin (0.1% w / v), and primary anti-5HMC antibody conjugated with 5pl4mer oligo (C tail) was incubated with the tissues for 2hr at RT. The primary anti-5HMC antibody was mixed with oYo-Link®-modified 5pl4mer oligo and photo-crosslinked for conjugation to the antibody. The antibody -DNA binding was stabilized with post-primary NBF fixation (10% NBF) to prevent the antibody wash-off during the remaining steps. The target probes were designed to detect the negative DNA strand, to avoid the cross-detection of the orthogonal mRNAs. The target probes were incubated for at least 3hrs to ensure sufficient probe hybridization to the DNA. The probeantibody complex was then amplified with Multiplex amplifiers and labeled with a TSA dye. Slides were imaged under a fluorescent microscope, e.g., at 40X magnification.

[0146] As shown in FIGS. 2A-2B, 5hmC modifications were detected in each of the mouse FFPE tissues. TNFot mRNA was separately detected and positively correlated with TNFa DNA 5hmC levels (FIGS. 2C-2D).Example 2

[0147] A similar set of analyses was done on a human tissue array to detect 5hmCs in the human TNFa gene. Formalin-fixed paraffin-embedded (FFPE) normal and cancerous human tissues from a tissue microarray were analyzed for 5hmCs in TNFa DNA using target probes directed to the human TNFa gene and an oligo-conjugated mouse monoclonal antibody against 5hmC sites as the binding agent.

[0148] The workflow for the analysis was as described above with minimal alterations. Briefly, tissues were blocked with bovine serum albumin (0.1% w / v) for 1 hour at RT, and primary anti- 5hmC antibody conjugated with an oligonucleotide was incubated with the tissues for 2hr at RT (1 :750 in PBS+0.1% Triton buffer). The antibody-DNA binding was stabilized with post-primary crosslinking (10% NBF for 10 minutes) to prevent the antibody wash-off during the remainingAttorney Docket No. ACDX-43932.601 steps, followed by hydrogen peroxide blocking. Target probes were added to the tissues for at least 3hrs (40 °C) to ensure sufficient probe hybridization to the DNA. The probe-antibody complex was then amplified with Multiplex amplifiers and labeled with a TSA dye. Slides were imaged under a fluorescent microscope, e.g., at 40X magnification.

[0149] 5hmC modifications were detected in the human tissues (FIGS. 3A-3D, top panels, 3E). mRNA expression was also detected in the tissues (FIGS. 3A-3E, lower panels). Breast cancer cells showed an increase in TNFa 5hmC and TNFa mRNA expression compared to the normal breast tissue (FIG. 3 A). Lung cancer cells and colon cancer cells did not show an increase in TNFa 5hmC or TNFa mRNA expression (FIGS. 3B-3C). Some brain cancer cells showed hyper-TNFa 5hmC and an increase TNFa mRNA expression (FIG. 3D). Overall, there was a positive correlation between human TNFa genomic DNA 5hmC and mRNA expression (Table 1).Example 3

[0150] A set of analyses were used to detect 5hmCs in the GSTP1 genomic locus in FFPE cancerous tissues using dual -component binding agent, a 5mC primary antibody (unconjugated) with an oligo-conjugated goat anti-rabbit secondary antibody, in 3pC6 tail, and target probes directed to the human GSTP1 locus. As for the anti-5HMC antibody in Examples 1 and 2, the oligo-conjugated goat anti-rabbit secondary antibody was also generated by photo-crosslinking an oYo-Link®-modified oligo to the antibody.

[0151] The workflow for the analysis was as described above with some alterations. Briefly, target probes were added to the samples for at least 3hrs (40 °C) to ensure sufficient probe hybridization to the DNA. Samples were blocked with bovine serum albumin (0.1% w / v) for 1 hour at RT, and primary anti-5hmC antibody was incubated with the samples for 3hr atRT (1 : 1000 in Leica Bond primary antibody diluent). Negative controls were incubated with primary antibody diluent only. The oligo-conjugated second antibody was incubated with the samples for Ihr at RT. Following both primary antibody and secondary antibody incubation, the sample was washed in Leica Bond wash solution. The antibody-DNA binding was stabilized with post-primaryAttorney Docket No. ACDX-43932.601 crosslinking (10% NBF for 15 minutes) to prevent the antibody wash-off during the remaining steps, followed by hydrogen peroxide blocking (10 min at RT). The probe-antibody complex was then amplified with Multiplex amplifiers and labeled with a TSA dye. Slides were imaged under a fluorescent microscope, e.g., at 40X magnification.

[0152] As shown in FIGS. 5 and 6, using a primary antibody / oligo-conjugated secondary antibody pair as the binding agent detected 5hmCs in the GSTP1 genomic locus in prostate, liver, and cervical cancer cells. The genomic GSTPl-5mC levels were sensitively differentiated in various prostate cancer cell lines of different pathological and genetic background (FIG. 6A) and distinct GSTPl-locus 5mC spatial patterns were revealed across three different cancer types (FIG. 6B).

[0153] From the foregoing, it will be appreciated that, although specific embodiments have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of what is provided herein. All of the references referred to above are incorporated herein by reference in their entireties.

Claims

Attorney Docket No. ACDX-43932.601CLAIMS1. A method of detecting a nucleic acid modification in a biological sample, the method comprising: contacting the biological sample with at least one target probe comprising a region that is complementary to a target nucleic acid in the biological sample and a region that is complementary to a first region of a signal generating complex; contacting the biological sample with at least one binding agent specific to the nucleic acid modification, wherein the at least one binding agent is covalently attached to an oligonucleotide comprising a region that is complementary to a second region of a signal generating complex; and detecting a signal from the signal generating complex.

2. The method of claim 1, wherein binding of the at least one target probe to the target nucleic acid and the first region of the signal generating complex, and binding of the at least one binding agent to the nucleic acid modification and the second region of the signal generating complex facilitates formation of the signal generating complex, and thereby generates a detectable signal indicative of the nucleic acid modification.

3. The method of claim 2, wherein the target probe binding region of the target nucleic acid is adjacent to the nucleic acid modification.

4. The method of any one of claims 1 to 3, wherein the first region of the signal generating complex and the second region of the signal generating complex are adjacent.

5. The method of any one of claims 1 to 4, wherein the nucleic acid modification comprises a chemical modification of at least one nucleotide in the target nucleic acid.

6. The method of claim 5, wherein the target nucleic acid is genomic DNA.

7. The method of claim 5 or claim 6, wherein the chemical modification comprises cytosine methylation or hydroxymethylation.Attorney Docket No. ACDX-43932.6018. The method of any one of claims 5 to 7, wherein the at least one binding agent comprises an antibody that specifically binds methylated or hydroxymethylated cytosine.

9. The method of claim 8, wherein the at least one binding agent comprises a secondary antibody that specifically binds to the antibody bound to a methylated or hydroxymethylated cytosine, wherein the secondary antibody is covalently attached to the oligonucleotide comprising a region that is complementary to a second region of a signal generating complex.

10. The method of any one of claims 1 to 9, wherein the target nucleic acid is mRNA, and wherein the method further comprises performing in situ hybridization to detect a region of the mRNA.

11. The method of any one of claims 1 to 10, wherein the at least one binding agent comprises a monoclonal antibody, a multispecific antibody, a bispecific antibody, a trispecific antibody, a tetraval ent antibody, a single-domain antibody, and a chimeric antibody.

12. The method of any one of claims 1 to 11, wherein the at least one binding agent comprises a Fab, a scFv, a Fv, a scFv-Fc, a Fab', a Fab'-SH, a F(ab') , a diabody, a minibody, a tribody, a nanobody, and an affibody.

13. The method of claims 11 or claim 12, wherein the at least one binding agent further comprises a secondary antibody.

14. The method of any one of claims 1 to 13, wherein the at least one binding agent comprises a primary or secondary antibody covalently attached to the oligonucleotide via a linker.Attorney Docket No. ACDX-43932.60115. The method of claim 14, wherein the linker comprises at least one of 6- hydrazinonicotinate (HyNic), succinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC), and / or polyethylene glycol (PEG).

16. The method of claim 14, wherein the linker comprises at least one reactive moiety.

17. The method of claim 16, wherein the at least one reactive moiety comprises at least one of a succinimidyl ester, a sulfosuccinimidyl ester, a pentafluorophenyl ester, a maleimide, an azide, an alkyne, a hydrazine, an isocyanate, an isothiocyanate, and / or a haloacetamide.

18. The method of claim 14, wherein the linker comprises an oligonucleotide sequence.

19. The method of any one of claims 1 to 18, wherein the biological sample is a fixed biological sample.

20. The method of any one of claims 1 to 19, wherein the target nucleic acid is immobilized in the biological sample.

21. The method of claim 19, wherein the fixed biological sample is a formalin-fixed paraffin- embedded (FFPE) sample.

22. The method of claim 19, wherein the fixed biological sample is a tissue sample or is derived from a tissue sample; a blood sample or is derived from a blood sample; a cytological sample or is derived from a cytological sample; a sample comprising cultured cells; or a sample comprising exosomes.

23. The method of any one of claims 1 to 22, wherein the method further comprises contacting the biological sample with a blocking agent comprising at least one of tRNA, salmon sperm DNA, herring DNA, calf thymus DNA, bovine serum albumin, casein, heparin, normal goat serum, normal swine serum, normal chicken serum, and / or fish serum.Attorney Docket No. ACDX-43932.60124. The method of any one of claims 1 to 23, wherein the method further comprises contacting the biological sample with a crosslinking agent comprising at least one of formalin, bis(succinimidyl) polyethylene glycol, di(N-succinimidy) glutarate, and / or glutaraldehyde.

25. The method of any one of claims 1 to 24, wherein the signal generating complex comprises at least one amplifier and / or pre-amplifier.

26. The method of any one of claims 1 to 25, wherein the signal generating complex comprises at least one detectable label.

27. The method of claim 26, wherein the at least one detectable label comprises a fluorescent moiety or a chromogenic moiety.

28. The method of claim 26 or claim 27, wherein the at least one detectable label is cleavable.

29. The method of any one of claims 1 to 28, wherein contacting the biological sample with at least one target probe is performed before contacting the biological sample with at least one binding agent specific to the nucleic acid modification.

30. The method of any one of claims 1 to 29, wherein contacting the biological sample with at least one target probe is performed after contacting the biological sample with at least one binding agent specific to the nucleic acid modification.

31. The method of any one of claims 1 to 30, wherein the contacting the biological sample with at least one target probe is performed simultaneously with contacting the biological sample with at least one binding agent specific to the nucleic acid modification.

32. The method of any one of claims 1 to 31, wherein the region of the at least one target probe that is complementary to the target nucleic acid is from about 10 to about 50 nucleotides in length.Attorney Docket No. ACDX-43932.60133. The method of any one of claims 1 to 32, wherein the region of the at least one target probe that is complementary to the first region of the signal generating complex is from about 10 to about 50 nucleotides in length.

34. The method of any one of claims 1 to 33, wherein the at least one target probe comprises a non-targeting region separating the target nucleic acid binding region from the region that is complementary to the first region of the signal generating complex.

35. The method of any one of claims 1 to 34, wherein the region of the at least one target probe that is complementary to the target nucleic acid is 5’ of the region of the at least one target probe that is complementary to the first region of the signal generating complex.

36. The method of any one of claims 1 to 35, wherein the region of the at least one target probe that is complementary to the target nucleic acid is 3’ of the region of the at least one target probe that is complementary to the first region of the signal generating complex.

37. A kit comprising the at least one target probe and the at least one binding agent of any one of claims 1 to 36.

38. The kit of claim 37, wherein the kit further comprises one or more permeabilizing and / or antigen retrieval reagents.

39. The kit of claim 37 or claim 38, wherein the kit further comprises at least one amplifier, at least one pre-amplifier, and / or at least one detectable label.

40. The kit of any one of claims 37 to 39, wherein the kit further comprises a control or reference sample.

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