Detection of target-binding small regulatory oligonucleotides

Abstract

Disclosed herein are systems, methods, and kits for detecting, quantifying, localizing, and / or determining the binding efficiency or efficacy of one or more small oligonucleotides for a target nucleic acid. The systems, methods, and kits disclosed herein can be used to accurately assess target-binding of, for example, small regulatory oligonucleotides (srONs) in order to characterize their properties for therapeutic use.

Description

DETECTION OF TARGET-BINDING SMALL REGULATORY OLIGONUCLEOTIDESFIELD

[0001] Embodiments of the present disclosure include systems, methods, and kits for detecting, quantifying, localizing, and / or determining the efficiency of one or more small oligonucleotides (e.g., small regulatory oligonucleotides, or srONs) binding to a target nucleic acid.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 671,561, filed July 15, 2024, the content of which is herein incorporated by reference in its entirety.BACKGROUND

[0003] Treating disease at a genetic level using nucleic acid therapeutics (e.g., gene silencing or downregulating oligonucleotides) is a fast-growing field, with numerous FDA-approved oligonucleotide therapeutics and many more under development in clinical or preclinical stages. Although there are several methods to assess the expression level of these oligonucleotides in tissues and cells, free oligonucleotide concentrations do not inform functionality or suitability, as stable target binding facilitates the downstream mechanisms that lead to modulation of gene expression. Accordingly, the current assays do not provide any information to evaluate the oligonucleotides that bind to the targets at a given timepoint or treatment condition. Thus, systems and methods are needed to accurately assess target-binding of small regulatory oligonucleotides in order to characterize their efficacy for therapeutic uses.SUMMARY

[0004] Provided herein are systems, methods and kits for detecting, quantifying, localizing, and / or determining the efficiency of one or more small regulatory oligonucleotides (srONs) binding to a target nucleic acid.

[0005] In one aspect, the systems comprise at least one anchor probe. In some embodiments, each anchor probe comprises a target nucleic acid binding region complementary to one or more regions of a target nucleic acid flanking a target srON binding site on the target nucleic acid and asignal-generating binding region complementary to a section of one or more linker oligonucleotides or a nucleic acid component of a signal-generating complex.

[0006] In some embodiments, the systems comprise a single anchor probe. In some embodiments, the systems comprise a first anchor probe and a second anchor probe. In some embodiments, the first anchor probe and the second anchor probe are complementary to adjacent regions upstream or downstream of the target srON binding site. In some embodiments, the first anchor probe is complementary to a region upstream of the target srON binding site, and the second anchor probe is complementary to a region downstream of the target srON binding site.

[0007] In some embodiments, the signal-generating binding region of each of the at least one anchor probe is individually 5’ or 3’ of the target nucleic acid binding region. In some embodiments, the signal-generating binding region is 10 to 40 nucleotides in length. In some embodiments, the target nucleic acid binding region is 10 to 50 nucleotides in length.

[0008] In some embodiments, each anchor probe further comprises a non-targeting region separating the signal-generating binding region from the target nucleic acid binding region.

[0009] In some embodiments, the system further comprises one or more linker oligonucleotides. In some embodiments, the systems comprise a single linker oligonucleotide. In some embodiments, the linker oligonucleotide is 50 to 120 nucleotides in length. In some embodiments, the systems comprise a first linker oligonucleotide and a second linker oligonucleotide. In some embodiments, each of the first and second linker oligonucleotides are 25 to 60 nucleotides in length.

[0010] In some embodiments, the systems further comprise a signal-generating complex comprising a nucleic acid component capable of hybridizing to the one or more linker oligonucleotides or the signal-generating binding region of the at least one anchor probe. In some embodiments, the signal-generating complex comprises: a pre-amplifier complementary to a section of the one or more linker oligonucleotides or the signal-generating binding region of the at least one anchor probe; one or more amplifiers complementary to a portion of the pre-amplifier; and a plurality of label probes each complementary to a portion of the one or more amplifiers and each comprising a detectable label.

[0011] In some embodiments, the detectable label comprises a fluorescent moiety or a chromogenic moiety. In some embodiments, the detectable label comprises an enzyme or an enzyme substrate. In some embodiments, the detectable label comprises a cleavable label.

[0012] In some embodiments, the systems further comprise a signal blocking probe configured to hybridize with: the linker oligonucleotide or the signal-generating binding region of the at least one anchor probe; and the target srON binding site in the absence of a srON bound to the target nucleic acid. In some embodiments, the signal blocking probe comprises: a srON region complementary to the target srON binding site; and at least one signal-generating blocking region wherein each signal-generating blocking region is complementary to a region of the linker oligonucleotide configured to hybridize to the signal-generating complex or the signal-generating binding region of the at least one anchor probe. In some embodiments, the srON region and the at least one signal-generating blocking region are separated by one or more nucleotides.

[0013] In some embodiments, the signal blocking probe comprises two signal-generating blocking regions flanking the srON region. In some embodiments, each of the two signalgenerating blocking regions are separated from the srON region by one or more nucleotides.

[0014] In some embodiments, the signal-generating blocking region comprises one or more locked nucleic acid (LNA) nucleotides.

[0015] In some embodiments, the systems further comprise the target nucleic acid. In some embodiments, the target nucleic acid comprises or is suspected of comprising the target srON binding site.

[0016] In some embodiments, the systems further comprise a target srON. In some embodiments, the target srON is an RNA molecule. In some embodiments, the target srON is a microRNA (miRNA) molecule, a small non-coding RNA (sncRNA) molecule, a PlWI-interacting RNA (piRNA) molecule, a small interfering RNA (siRNA) molecule, or an antisense oligonucleotide (ASO).

[0017] In one aspect, the methods comprise: (i) contacting a sample comprising a target srON and a target nucleic acid with at least one anchor probe complementary to one or more regions of the target nucleic acid flanking a target srON binding site; (ii) contacting the sample with: a) one or more linker oligonucleotides complementary to a region in each of the least one anchor probes and a signal-generating complex comprising a nucleic acid component capable of hybridizing to the one or more linker oligonucleotides in the presence of a srON bound to the target nucleic acid, or b) a signal-generating complex comprising a nucleic acid component capable of hybridizing to the at least one anchor probe oligonucleotides in the presence of a srON bound to the target nucleic acid; and (iii) detecting a signal from the signal-generating complex.

[0018] In some embodiments, step (i) and step (ii) are performed simultaneously. In some embodiments, step (i) is performed before step (ii).

[0019] In some embodiments, methods comprise contacting the sample with a single anchor probe. In some embodiments, the methods comprise contacting the sample with a first anchor probe and a second anchor probe. In some embodiments, the first anchor probe and the second anchor probe are complementary to adjacent regions upstream or downstream of the target srON binding site. In some embodiments, the first anchor probe is complementary to a region upstream of the target srON binding site and the second anchor probe is complementary to a region downstream of the target srON binding site.

[0020] In some embodiments, each anchor probe comprises: a target nucleic acid binding region complementary to one or more regions of a target nucleic acid flanking a target srON binding site on the target nucleic acid; and a signal-generating binding region complementary to a section of one or more linker oligonucleotides or a nucleic acid component of a signal-generating complex.

[0021] In some embodiments, the signal-generating binding region of each of the at least one anchor probe is individually 5’ or 3’ of the target nucleic acid binding region. In some embodiments, the signal-generating binding region is 10 to 40 nucleotides in length. In some embodiments, the target nucleic acid binding region is 10 to 50 nucleotides in length.

[0022] In some embodiments, each anchor probe further comprises a non-targeting region separating the signal-generating binding region from the target nucleic acid binding region.

[0023] In some embodiments, the one or more linker oligonucleotides are each 25 to 120 nucleotides in length.

[0024] In some embodiments, the signal-generating complex comprises: a pre-amplifier complementary to a section of the one or more linker oligonucleotides or complementary to the signal-generating binding region of the at least one anchor probe; one or more amplifiers complementary to a portion of the pre-amplifier; and a plurality of label probes each complementary to a portion of the one or more amplifiers and each comprising a detectable label. In some embodiments, the detectable label comprises a fluorescent moiety or a chromogenic moiety. In some embodiments, the detectable label comprises an enzyme or an enzyme substrate. In some embodiments, the detectable label comprises a cleavable label.

[0025] In some embodiments, the methods further comprise contacting the sample with a signal blocking probe configured to hybridize with: the one or more linker oligonucleotides or the signalgenerating binding region of the at least one anchor probe; and the target srON binding site, in the absence of a srON bound to the target nucleic acid before step (i) or with step (i) and / or step (ii). In some embodiments, the signal blocking probe comprises: a srON region complementary to the target srON binding site; and at least one signal-generating blocking region complementary to a region of the linker oligonucleotide configured to hybridize to the signal-generating complex or the signal-generating binding region of the at least one anchor probe. In some embodiments, the srON region and the at least one signal-generating blocking region are separated by one or more nucleotides. In some embodiments, the signal blocking probe comprises two signal-generating blocking regions flanking the srON region, each separated from the srON region by one or more nucleotides. In some embodiments, the at least one signal-generating blocking region comprises one or more locked nucleic acid (LNA) nucleotides.

[0026] In some embodiments, the methods further comprise contacting the biological sample with one or more nucleic acid or protein detection agents.

[0027] In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is a tissue specimen or is derived from a tissue specimen, a blood sample or is derived from a blood sample, a cytological sample or is derived from a cytological sample, or cultured cells.

[0028] In some embodiments, the method further comprises contacting the sample with a permeabilizing and / or antigen retrieval reagent or condition.

[0029] In some embodiments, the method further comprises contacting the sample with a target srON and / or a target nucleic acid.

[0030] In another aspect provided herein are kits comprising one or more components from a system as disclosed herein. In some embodiments, the kit further comprises one or more permeabilizing and / or antigen retrieval reagents.

[0031] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIGS. 1A-1C arc exemplary schematics for detection of srON-target nucleic acid (c.g., mRNA) binding using two anchor probes which bind to regions of the target nucleic acid flanking the srON binding site (FIG. 1A), two anchor probes which bind to adjacent region of the target nucleic acid upstream of the srON binding site (FIG. IB), and a single anchor probe flanking the srON binding site (FIG. 1C). When the srON is bound to the target site, the signal blocking probe does not associate with the complex and the pre-amplifier from the signal-generating complex can bind the linker oligonucleotide bound to the one or two anchor probes. In the absence of an srON bound to the target site, the signal blocking probe binds to the srON binding site and the linker oligonucleotide, preventing the signal-generating complex from associating with the linker oligonucleotide.

[0033] FIGS. 2A-2D are representative images showing high signal-to-noise ratio detection of anti-sense oligonucleotides (ASOs) that bind to Malatl transcript in FFPE mouse brain in Hippocampal Formation (A-B) and Cerebellum (C-D) using the disclosed system and methods. Following sequential incubation with the anchor probes, linker oligonucleotide, and signalgenerating complex, positive signals (red) were detected in the two regions of mouse brain tissue.

[0034] FIGS. 3A-3H are representative images showing high signal-to-noise ratio detection of ASOs that bind to Malatl transcript in FFPE mouse brain (A-B), lung (C-D), liver (E-F), and intestine (G-H).

[0035] FIGS. 4A-4E are representative fluorescent images showing detection of O-Lamp5 transcript binding in FFPE mouse brain. FIG. 4A is an overview of the staining pattern in mouse brain that shows enriched ASO-Lamp5 mRNA duplex in specific layers of cortex and hindbrain, which is consistent with data from other resources. FIGS. 4B-4E are zoomed-in images showing high signal-to-noise ratio in both cortex and hindbrain. Blue: DAPI, Red: ASO-Lamp5 duplex (Opal 650, 1:1000).

[0036] FIG. 5 shows a comparison of background generated from linker oligos in FFPE mouse tissues. Tissues were incubated with linker oligos only before miRNAscope amplification. Background staining in FFPE mouse brain and lung from full linker (left) and dual linkers (middle). Tissue sections with no linker oligo incubation (right) were used as negative controls. Replacement of full linker with dual linkers largely reduced background staining, which was comparable tobackground in the negative control (Signal Amplification Only) in both tissue types. Blue: DAPI. Red: Opal650 1:1000.

[0037] FIG. 6 shows ASO-mRNA duplex detection using full linker and dual linkers. ASO targeting mouse Lamp5 mRNA was applied to FFPE mouse brain sections before the ASO detection assay was performed with either full linker or dual linkers. Background staining in FFPE mouse brain was largely reduced when dual linker was used, while a high signal-to-noise ratio was maintained. Blue: DAPI. Red: ASO-Lamp5 mRNA duplex (Opal650 1:1000).

[0038] FIG. 7 shows ASQ-Lamp5 mRNA duplex detection using blocking probes with or without LNAs, or with LNA-containing blocking probes incubated prior to anchoring probe hybridization (top half). Background staining in FFPE mouse brain was largely reduced when LNA blocking probe was used. The signal-to-noise ratio was further improved when LNA blocking probes were pre-incubated. Same images without DAPI were shown in the lower half. White arrows showed background generated by insufficient blocking when blocking probes without LNA were used. Blue: DAPI. Red: ASO-Lamp5 mRNA duplex (Opal650 1:1000).

[0039] FIG. 8 shows improved signal-to-noise ratio when comparing ASO-Lamp5 mRNA duplex detection using different linkers and blocking probes in FFPE mouse brain cortex. Blue: DAPI. Red: ASO-Lamp5 mRNA duplex (Opal650 1:1000).

[0040] FIGS. 9A-9Dshow ASO- Lainp5 mRNA binding in FFPE Mouse brain at different ASO dosages: no ASO (FIG. 9A), 0.2 nM ASO (FIG. 9B), 2 nM ASO (FIG. 9C) and 20 nM ASO (FIG. 9D). Blue: DAPI, Red: ASO-Lamp5 complex (Opal650 1:1000).

[0041] FIG. 10 shows a schematic of the components of a second exemplary system for srON detection. The exemplary system includes two anchor probes and one blocking probe. Each anchor probe contains a target binding sequence, a spacer, and a region for binding a component of a signal-generating complex (e.g., pre-amplifier). The anchor probes flank the srON binding region on the target sequence. The anchor probes are also configured to hybridize to the pre-amplifier oligo in the absence of a blocking probe to facilitate signal amplification. When there is no srON- target binding, the blocking probe can hybridize to both the srON-binding region on the target sequence and the anchor probes, preventing signal amplification.

[0042] FIG. 11 provides schematics of two different exemplary blocking probe designs. The Ilshaped blocking probe contains a sequence that can hybridize to the srON-binding region and two arms that can hybridize to the signal-generating region of the anchor probes. The C-shapedblocking probe contains a sequence that can hybridize to the srON-binding region and another sequence that can hybridize to the signal-generating binding region of the anchor probes. At the junction between two anchor probes, the blocking probe may contain 6 alternating LNAs, e.g., to enhance the binding affinity.

[0043] FIG. 12 are exemplary schematics for detection of srON-target nucleic acid binding using the components of the system outlined in FIG. 10. The blocking probe may or may not bind to the target sequence, depending on whether srON is present. Anchor probes hybridize to the target sequence. If the blocking probe is present, the blocking probe will hybridize with the anchor probes and form double strands with the signal-generating binding regions. A signal will be generated and amplified if the anchor probe is available for binding by the signal-generating complex (e.g., a preamplifier). Readouts can be either chromogenic or fluorescent images, depending on the signal development methods.

[0044] FIG. 13 shows representative fluorescent images of ASO-target binding in HCT116 cells treated by an ASO at different dosages (3nM. lOnM, 30nM), using systems and methods outlined as in FIGS. 10 and 12, respectively. HCT116 cells treated with higher dose of ASO-X showed increased signal. Blue: DAPI. Green: Target-binding ASO (iFluor546 Styramide, 1:1000).DETAILED DESCRIPTION

[0045] The present disclosure is directed to systems and methods of detecting target nucleic acid binding oligonucleotides, suitable for use in situ, regardless of chemical modifications (e.g., 2’-M0E, PS, LNA, etc.). The system comprises probes which interrogate oligonucleotide- target nucleic acid binding via formation of a signal-generating detection complex. In the absence of an oligonucleotide binding to its target nucleic acid, a blocked complex is formed which does not produce a detectable signal. The disclosed system and methods can provide spatial information of oligonucleotides that specifically bind to their target sequences as well as determination of efficiencies of oligonucleotide binding, oligonucleotide half-life, oligonucleotide binding strength, and dosing requirements of oligonucleotides. As such, the disclosed systems and methods can be readily used in pharmacokinetics and pharmacodynamics studies in nucleic acid therapeutic development.

[0046] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.a. Definitions

[0047] As used in the present disclosure and claims, the singular forms “a,” “an” and “the” include plural forms unless the context clearly dictates otherwise.

[0048] 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.

[0049] 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 therebetween 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.

[0050] 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.

[0051] 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).

[0052] The terms “detecting” as used herein generally refer to any form of measurement and include determining whether an element is present or not. This term includes quantitative and / or qualitative determinations.

[0053] 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.

[0054] As used herein, the term “immunohistochemistry" or “IHC” generally refers to a technique for detecting proteins of interest in source samples utilizing antibodies, with thepreservation 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.

[0055] 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 moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications 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 caninclude other hybridizing nucleic-acid-like molecules such as those with substituted backbones c.g., peptide nucleic acids (PNAs), morpholino backboned nucleic acids, locked nucleic acids, or other nucleic acids with modified bases and sugars.

[0056] 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.

[0057] The term “probe” as used herein refers 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 (7deazaguanosine, inosine, etc. ) . In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.

[0058] 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.b. Small regulatory oligonucleotide (srON) Detection System

[0059] In one aspect, provided herein arc systems for detecting small regulatory oligonucleotides (srONs). Small regulatory oligonucleotides include any oligonucleotides which bind to a target sequence. In some embodiments, the srON is DNA. In some embodiments, the srON is RNA. In some embodiments, the srON comprises DNA and / or RNA nucleotides.

[0060] hi some embodiments, the small regulatory oligonucleotides affect a change in the target sequence function. For example, small regulatory oligonucleotides include gene silencing or downregulating oligonucleotides (e.g., small interfering RNAs (siRNAs), an antisense oligonucleotides, short-hairpin RNAs, microRNAs (miRNA), PlWI-interacting RNAs (piRNA), dicer-substrate RNAs, DNAzymes, small circular RNAs, aptamers targeting a gene or messenger RNAs), which can be used to modulate gene expression. Small regulatory oligonucleotides also include oligonucleotides used for targeting effector molecules to a target sequence to facilitate structural or functional modifications (e.g., insertion, deletions, mutations, chemical modifications) on a target sequence which does not necessarily result in gene silencing or downregulation (e.g., guide RNAs, snoRNAs, snoRNA-like RNAs). In select embodiments, the srON is a microRNA (miRNA) molecule, a small non-coding RNA (sncRNA) molecule, a PlWI-interacting RNA (piRNA), a small interfering RNA (siRNA) molecule, or an antisense oligonucleotide (ASO). In some embodiments, the system further comprises a small regulatory oligonucleotide.

[0061] The small regulatory oligonucleotides are not limited by size. In some embodiments, the small regulatory oligonucleotides are about 10 to about 50 nucleotides in length. For example, the small regulatory oligonucleotides may be 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, 45, 46, 47, 48, 49 or 50 nucleotides in length. The small regulatory oligonucleotides can be greater than 50 nucleotides in length but have a target binding or antisense region of less than 50 nucleotides in length. For example, guide RNAs and snoRNAs have antisense elements of less than 50 nucleotides in length but the entire length of the molecule can be greater than 50 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, or more in length.

[0062] In particular, the disclosed systems facilitate detection of binding between the small regulatory oligonucleotides (srONs) and the target nucleic acid. In some embodiments, the systems facilitate detection and characterization of the binding between the small regulatory oligonucleotides (srONs) and the target nucleic acid in situ.

[0063] The systems and 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 comprises or is suspected of comprising the srON binding site. In some embodiments, the system further comprises a target nucleic acid.

[0064] 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.

[0065] In some embodiments, the systems comprise at least one anchor probe. In some embodiments, the systems comprise a single anchor probe. In some embodiments, the systems comprise one or more anchor probes (e.g., a first anchor probe, a second anchor probe, etc.). Each of the anchor probes comprise a target nucleic acid binding region complementary to one or more regions of a target nucleic acid flanking a target srON binding site on the 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 flanking a target srON binding site, provided that there is sufficient complementarity to result in specific hybridization / binding.

[0066] In systems which comprise a single anchor probe, the anchor probe can bind and be complementary to a region upstream or downstream of the target srON binding site on the target nucleic acid. In systems which comprise two or more anchor probes, the two or more anchor probes can be configured to bind in any arrangement around the target srON binding site on the target nucleic acid. For example, all of the two or more anchor probes bind and are complementary to adjacent regions upstream of the srON binding site on the target nucleic acid. Alternatively, all of the two or more anchor probes bind and are complementary to adjacent regions downstream of the srON binding site on the target nucleic acid. Thus, in some embodiments, a first anchor probe and a second anchor probe are complementary to adjacent regions upstream or downstream of the target srON binding site. Alternatively, at least one of the two or more anchor probes binds and is complementary to a region downstream of the srON binding site on the target nucleic acid and at least one of the two or more anchor probes binds and is complementary to a region upstream ofthe srON binding site on the target nucleic acid. Thus, in some embodiments, a first anchor probe is complementary to a region upstream of the target srON binding site and a second anchor probe is complementary to a region downstream of the target srON binding site.

[0067] The anchor probe(s) can bind and be complementary to a region immediately adjacent to a target srON binding site, a region separated from a target srON binding site on the target nucleic acid by one or more nucleotides, or a region having a degree of overlap (e.g., 1 nucleotide, 2 nucleotides, 3 nucleotides) with a portion of a target srON binding site.

[0068] The target nucleic acid binding region 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).

[0069] Each of the anchor probes also comprises a signal-generating binding region complementary to a section of one or more linker oligonucleotides or a nucleic acid component of a signal-generating complex. The signal-generating binding region need not exhibit complete complementarity to the one or more linker oligonucleotides or the nucleic acid component of the signal-generating complex, provided that there is sufficient complementarity to cause hybridization / b inding .

[0070] The signal-generating binding region may be any size necessary to specifically and sensitively bind the one or more linker oligonucleotides or the nucleic acid component of the signal-generating complex. In some embodiments, the signal-generating region is at least 10 nucleotides in length. For example, the signal-generating 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 signal-generating binding region is 10 to 40 nucleotides in length (e.g., 10 to 30 nucleotides, 10 to 20 nucleotides, 20 to 40 nucleotides, 20 to 30 nucleotides, or 30 to 40 nucleotides in length).

[0071] The anchor probes are also not limited by the length of the target nucleic acid binding region with respect to the signal-generating 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 linker oligonucleotide(s) or nucleic acid component of the signalgenerating complex.

[0072] Accordingly, each anchor probe comprises a target nucleic acid binding region and a signal-generating binding region. The target nucleic acid binding region and signal-generating binding region may be arranged in any configuration about the anchor probe. The anchor probes are not limited by the orientation of target nucleic acid binding region with respect to the signalgenerating binding region. In some embodiments, the target nucleic acid binding region is 5’ of the signal-generating binding region. In some embodiments, the target nucleic acid binding region is 3’ of the signal-generating binding region. The orientation of target nucleic acid binding region with respect to the signal-generating binding region in each of the at least one anchor probes does not need to be the same. Accordingly, the signal-generating binding region of each of the at least one anchor probe is individually 5’ or 3’ of the target nucleic acid binding region. For example, in systems comprising two or more anchor probes, a first anchor probe may have the target nucleic acid binding region is 5’ of the signal-generating binding region and the second anchor probe may have the target nucleic acid binding region is 3’ of the signal-generating binding region. The anchor probe is generally single stranded so that the anchor probe is available to hybridize with a corresponding target nucleic acid and linker oligonucleotide(s) or nucleic acid component of the signal-generating complex.

[0073] The target nucleic acid binding region and the signal-generating 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 linker oligonucleotide(s) or nucleic acid component of the signal-generating complex. The target nucleic acid binding region and signal-generating binding region may be separated by a non-targeting region. The non-targeting region does not have any complementarity to the target nucleic acid, the one or more linker oligonucleotides, or the nucleic acid component of the signal-generating complex. The non-targeting region does not participate in hybridization or binding interactions with the other components of the system.

[0074] 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.

[0075] The non-targeting region may be any length which facilitates the anchor probe interaction with both the target nucleic acid and the linker oligonucleotide. 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.

[0076] In some embodiments, the systems comprise one or more linker oligonucleotides. A linker oligonucleotide or linker oligonucleotides refers to an oligonucleotide that is capable of hybridizing to one or more anchor probes and is capable of at least partially hybridizing to a component of a signal-generating complex or a signal blocking probe, as described below. The linker oligonucleotide(s) are not limited in sequence length but will be influenced by the number of anchor probes and the length of the signal-generating binding region of each of those anchor probes.

[0077] In some embodiments, the system comprises a single linker oligonucleotide. In some embodiments, the single linker oligonucleotide is at least 50 nucleotides in length. For example, the single linker oligonucleotide may be at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 110 nucleotides, at least 120 nucleotides, at least 130 nucleotides, at least 140 nucleotides, at least 150 nucleotides, or more nucleotides in length. In some embodiments, the linker oligonucleotide is 50 to 120 nucleotides in length. For example, the single linker oligonucleotide may be 60 to 120 nucleotides in length, 70 to 120 nucleotides in length, 80 to 120 nucleotides in length, 90 to 120 nucleotides in length, 100 to 120 nucleotides in length, 110 to 120 nucleotides in length, 50 to 110 nucleotides in length, 60 to 110 nucleotides in length, 70 to 110 nucleotides in length, 80 to 110 nucleotides in length, 90 to 110 nucleotides in length, 100 to 110 nucleotides in length, 50 to 100 nucleotides in length, 60 to 100 nucleotides in length, 70 to 100 nucleotides in length, 80 to 100 nucleotides in length, 90 to 100 nucleotides in length, 50 to 90 nucleotides in length, 60 to 90 nucleotides in length, 70 to 90 nucleotides in length, 80 to 90 nucleotides in length, 50 to 80nucleotides in length, 60 to 80 nucleotides in length, 70 to 80 nucleotides in length, 50 to 70 nucleotides in length, 60 to 70 nucleotides in length, or 50 to 60 nucleotides in length.

[0078] The system may comprise more than one linker oligonucleotide. In some embodiments, the system comprises a first linker oligonucleotide and a second linker oligonucleotide. In some embodiments, each of the first and second linker oligonucleotides are at least 25 nucleotides length. For example, the first and second linker oligonucleotides may be at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, or more nucleotides in length. In some embodiments, each of the first and second linker oligonucleotides are 25 to 60 nucleotides in length. For example, the first and second linker oligonucleotides may be 25 to 55 nucleotides, 25 to 50 nucleotides, 25 to 45 nucleotides, 25 to 40 nucleotides, 25 to 35 nucleotides, 25 to 30 nucleotides, 30 to 60 nucleotides, 30 to 55 nucleotides, 30 to 50 nucleotides, 30 to 45 nucleotides, 30 to 40 nucleotides, 30 to 35 nucleotides, 35 to 60 nucleotides, 35 to 55 nucleotides, 35 to 50 nucleotides, 35 to 45 nucleotides, 35 to 40 nucleotides, 40 to 60 nucleotides, 40 to 55 nucleotides, 40 to 50 nucleotides, 40 to 45 nucleotides, 45 to 60 nucleotides, 45 to 55 nucleotides, 45 to 50 nucleotides, 50 to 60 nucleotides, 50 to 55 nucleotides, or 55 to 60 nucleotides in length.

[0079] The system may comprise one or more sets of at least one anchor probes useful to detect one or more srONs. Accordingly, the system may comprise one or more corresponding sets of one or more linker oligonucleotides and / or one or more corresponding signal-generating complexs.

[0080] In some embodiments, the system further comprises a signal-generating complex comprising a nucleic acid component capable of hybridizing to the one or more linker oligonucleotides or the signal-generating binding region of the at least one anchor probe.

[0081] In some embodiments, the signal-generating complex is the same or similar signalgenerating complex 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 signal-generating complex to reliably detect RNA under standard bright-field microscopy (Anderson et al., J. Cell. Biochem. 117(10):2201-2208 (2016); Wang et al., 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 targetnucleic acid as in RNAscope™, the signal-generating complex instead binds to the one or more linker oligonucleotides.

[0082] In some embodiments, the signal-generating complex is the same or similar signalgenerating complex used in the BaseScope™ signal amplification system, which is described in more detail in, e.g., U.S. Patent No. 11,078,528. BaseScope™ exploits collaborative hybridization of one or more components of the signal-generating complex to form more stable branched-DNA- like signal amplification complexes and reduce background in in situ detection, which can be applied to detect short nucleotide sequences and single molecule analysis (Baker el al., Nat. Commun. 8(1): 1998 (2017)).

[0083] In some embodiments, the signal-generating complex includes one or all of: a preamplifier, one or more amplifiers, and a plurality of label probes, wherein each label probe comprises a detectable label. In some embodiments, the signal-generating complex comprises a pre-amplifier, one or more amplifiers, and a plurality of label probes, wherein each label probe comprises a detectable label.

[0084] 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 to another nucleic acid bound to the target such as a pre-amplifier or linker oligonucleotide(s). For example, the amplifier can hybridize to the linker oligonucleotide(s) 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 intemucleotide 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.

[0085] As used herein, a “pre-amplifier” is a molecule, typically a polynucleotide, that serves as an intermediate binding component between the one or more linker oligonucleotides or the at least one anchor probe and one or more amplifiers. Typically, the pre-amplifier hybridizes simultaneously to the one or more linker oligonucleotides or the at least one anchor probe and to a plurality of amplifiers. Exemplary pre-amplifiers are described, for example, in U.S. Patent Nos.5635352, 5681697, and 7709198, and U.S. Publication Nos. 2008 / 0038725, 2009 / 0081688 and 2017 / 0101672, each of which is incorporated by reference.

[0086] As used herein, the term “label probe” refers to an entity that binds to a target molecule, generally indirectly, and allows the target to be detected. A label probe 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 the 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 from the signalgenerating complex, e.g., an amplifier or pre-amplifier. In some embodiments, the label probe comprises a polynucleotide sequence that is complementary to the one or more amplifiers.

[0087] 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.

[0088] Exemplary enzyme labels include but are not limited to horseradish peroxidase (HRP), alkaline phosphatase (AP), |3-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. Patent Nos. 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.

[0089] 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 ail, including but not limited to, horseradish peroxidase, alkaline phosphatase, [3-galactosidase, glucose oxidase, and the like (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (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 [3-galactosidase, |3-glucuronidase, 6-phospho-|3-D-galactoside 6-phosphogalactohydrolase, |3-glucosidase, a- glucosidase, amylase, neuraminidase, esterases, lipases, and the like (Manafi etal., 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.

[0090] 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-chloro-l-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-[3-D- galactopyranoside and 2-methoxy-4-(2-nitrovinyl)phenyl [3-D-galactopyranoside for [3- galactosidase; 2-methoxy-4-(2-nitrovinyl)phenyl [3-D-glucopyranoside for |3-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 Quintolct for horseradish peroxidase; 4-mcthylumbcllifcryl P-D-galactopyranosidc, 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 Anorogenic 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 the 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; biocare.net / products / detection / chromogens).

[0091] 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-Aight mass spectrometry (TOF- MS) (for example, Fluidigm Helios and Hyperion systems, Auidigm.com / systems; South San Francisco, CA).

[0092] 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).

[0093] 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 et al., 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 et al., FEBS Lett. 582(3):410- 404 (2008)).

[0094] 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. Sci. USA 113:11046- 11051 (2016)). In one embodiment, TCEP is used as an agent to cleave a label from a label probe.

[0095] In some embodiments, the system further comprises a signal blocking probe. The signal blocking probe is configured to hybridize with: the linker oligonucleotides or the at least one anchor probe (e.g., in the signal-generating binding region); and the target srON binding site in the absence of a srON bound to the target nucleic acid. In some embodiments, the signal blocking probe comprises a srON region complementary to at least a portion of the target srON binding site and at least one signal-generating blocking region complementary to a region of the linkeroligonucleotide or the at least one anchor probe (e.g., the signal-generating binding region) configured to hybridize to the signal-generating complex. Thus, when the signal blocking probe is bound to the target srON binding site, it prevents signal-generating complex binding, thereby preventing a positive readout of presence of the srON bound to the target nucleic acid.

[0096] The srON region need not exhibit complete complementarity to the target srON binding site of a target nucleic acid, provided that there is sufficient complementarity to cause hybridization / binding. In some embodiments, the srON region does not provide complete complementarity to the target srON binding site. The sequence of the srON region is sufficient for hybridization but not sufficient to compete with the target srON for the target srON binding site. Thus, in some embodiments, the srON region may comprise a sequence that is less than 100% complementary to the target srON binding site. The srON region may be less than 99%, less than 98%, less than 97%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75% or less than 70% complementary to the target srON binding site.

[0097] The srON region may be any size necessary to bind the target srON binding site. In some embodiments, the srON region is at least 10 nucleotides in length. For example, the srON region may be about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, or more nucleotides in length. In some embodiments, the srON region is approximately the same length as the target srON or the antisense region of the target srON (e.g., the sequence of the srON which interacts with the target nucleic acid). For example, the srON region can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides shorter or longer than the target srON or the antisense region of the target srON. In some embodiments, the srON region is the same length as the target srON or the antisense region of the target srON.

[0098] The signal blocking probe also comprises at least one signal-generating blocking region complementary to a section of the one or more linker oligonucleotides which interacts with the signal-generating complex or the signal-generating binding region of the at least one anchor probe. The signal-generating blocking region(s) need not exhibit complete complementarity to the one or more linker oligonucleotides or the signal-generating binding region of the at least one anchor probe, provided that there is sufficient complementarity to cause hybridization / binding. In some embodiments, the signal-generating blocking region exhibits sufficient complementarity to prevent competition with the oligonucleotides of the signal-generating complex.

[0099] The srON region and the signal-generating blocking region may be arranged in any configuration about the signal blocking probe. The signal blocking probe is not limited by the orientation of the srON region with respect to the signal-generating blocking region. In some embodiments, the srON region is 5’ of the signal-generating blocking region. In some embodiments, the srON region is 3’ of the signal-generating blocking region. The signal blocking probe is generally single stranded so that the signal blocking probe is available to hybridize with a corresponding target srON binding site and linker oligonucleotide(s). The signal blocking probe may have more than one signal-generating blocking region. In some embodiments, the signal blocking probe comprises two signal-generating blocking regions. The two signal-generating blocking regions may be arranged in any orientation in the signal blocking probe. In some embodiments, the signal blocking probe may have a first signal-generating blocking region 5’ of the srON region and a second signal-generating blocking region 3’ of the srON region. Thus, in some embodiments, the signal blocking probe comprises two signal-generating blocking regions flanking the srON region.

[0100] The signal-generating blocking region(s) may be any size necessary to specifically and sensitively bind the one or more linker oligonucleotides. In some embodiments, the signalgenerating blocking region is at least 10 nucleotides in length. For example, the signal-generating blocking 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 signal-generating blocking region is 10 to 40 nucleotides in length (e.g., 10 to 30 nucleotides, 10 to 20 nucleotides, 20 to 40 nucleotides, 20 to 30 nucleotides, or 30 to 40 nucleotides in length). In embodiments in which the signal blocking probe has more than one signal-generating blocking region, each individual signalgenerating blocking region may be the same or different lengths.

[0101] The srON region and the signal-generating blocking region(s) may be consecutive or separated by any number of nucleotides to facilitate independent and accessible binding to the target srON binding site acid and the linker oligonucleotide(s). In some embodiments, the srON region and the signal-generating blocking region(s) are separated by one or more nucleotides. In embodiments in which the signal blocking probe comprises two signal-generating blocking regions flanking the srON region, as described above, each of the blocking regions may be individually separated by any number of nucleotides from the srON binding region. The number of nucleotidesseparating a first signal-generating blocking region and a second signal-generating blocking region from the srON region may be the same or different. In some embodiments, the signal blocking probe may have a first signal-generating blocking region 5’ of the srON region and a second signalgenerating blocking region 3’ of the srON region, each separated from the srON region by the same number of nucleotides.

[0102] The signal blocking probe may comprise one or more locked nucleic acid (LNA) nucleotides in any region of the signal blocking probe (e.g., srON region and the signal-generating blocking region(s)). In some embodiments, the signal blocking probe comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (LNA) nucleotides in the signal-generating blocking region(s). The LNA may be incorporated at any nucleotide position within the signal blocking probe or signal-generating blocking region(s).

[0103] Locked nucleic acid (LNA) nucleotides are nucleotides where a linkage has been engineered between the 2' and 4' carbons of the pentose sugar ring. The pentose sugar ring structure with this bridge is a bicyclic structure. The bridge “locks” the pentose sugar ring in a 3'-endo structural conformation, which is often found in the A-form of DNA or RNA. The locked conformation enhances base stacking and backbone pre -organization and has the effect of significantly increasing the thermal stability (melting temperature) of a DNA duplex. Exemplary LNAs include, but are not limited to, an LNA which has a single carbon (methylene) bridge between the pentose sugar ring 2' and 4' carbons and ENA, a type of LNA which has an ethylene bridge between the pentose sugar ring 2' and 4' carbons.

[0104] The system may comprise other components used in conjunction with those components described above. For example, the system may comprise reagents for use in epitope retrieval or target unmasking. The system may also comprise reagents for use in the detection of other targets of interest in the sample. For example, the system may comprise one or more reagents for the detection of protein targets, or other nucleic acids targets. The system may comprise reagents to carry out immunohistochemistry or immunocytochemistry for other targets (e.g., protein or other nucleic acid targets). c. Methods of Detecting srONs in a Sample

[0105] Additionally provided herein are methods for detecting, quantifying, localizing, and / or determining the efficiency of one or more small regulatory oligonucleotides (srONs) binding to a target nucleic acid. In some embodiments, the methods comprise (i) contacting a samplecomprising a target srON and a target nucleic acid with at least one anchor probe complementary to one or more regions of the target nucleic acid flanking a target srON binding site; (ii) contacting the sample with: a) one or more linker oligonucleotides complementary to a region in each of the least one anchor probes and a signal-generating complex comprising a nucleic acid component capable of hybridizing to the one or more linker oligonucleotides in the presence of a srON bound to the target nucleic acid, or b) a signal-generating complex comprising a nucleic acid component capable of hybridizing to the at least one anchor probe oligonucleotides in the presence of a srON bound to the target nucleic acid; and (iii) detecting a signal from the signal-generating complex.

[0106] The method is 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).

[0107] In some embodiments, the methods comprise contacting the biological sample with a single anchor probe capable of hybridizing to a region of the target nucleic acid flanking a target srON binding site. In some embodiments, the methods comprise contacting the biological sample with a first anchor probe and a second anchor probe, each capable of hybridizing to separate regions of the target nucleic acid flanking a target srON binding site. In some embodiments, the first anchor probe and the second anchor probe are complementary to adjacent regions upstream or downstream of the target srON binding site. In some embodiments, the first anchor probe is complementary to a region upstream of the target srON binding site and the second anchor probe is complementary to a region downstream of the target srON binding site.

[0108] In some embodiments, the methods comprise contacting the biological sample with a single linker oligonucleotide. In some embodiments, the methods comprise contacting the biological sample with two or more linker oligonucleotides. The anchor probe(s) (e.g., the single anchor probe or the first anchor probe and the second anchor probe) can hybridize to the single linker oligonucleotide or the two or more linker oligonucleotides.

[0109] The methods may comprise contacting the biological sample with a pre-amplifier. The pre-amplifier can hybridize to a single linker oligonucleotide or two or more linker oligonucleotides simultaneously. Alternatively, the pre-amplifier can hybridize to the signalgenerating binding region of an anchor probe, or simultaneously to the signal-generating binding regions of two anchor probes. The pre-amplifier comprises binding sites for a plurality of amplifiers and the methods may further comprise contacting the biological sample with theplurality 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.

[0110] In some embodiments, step (i) comprises contacting the biological sample with at least one anchor probe, respectively, for about 10 minutes to about 48 hours, or about 30 minutes to about 150 minutes. For example, in some embodiments, step (i) comprises contacting the biological sample with the at least one anchor probe for about 30 minutes, about 35 minutes, about40 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 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, or about 150 minutes.

[0111] In some embodiments, step (i) comprises contacting the biological sample with at least one anchor probe at a temperature of about 4 °C to about 75 °C, or about 30 °C to about 50 °C. For example, in some embodiments, step (i) comprises contacting the biological sample with the at least one anchor probe, at a temperature of 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, about41 °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, or about 50 °C.

[0112] In some embodiments, step (ii) comprises contacting the biological sample with one or more linker oligonucleotides complementary to a region in each of the least one anchor probes and a signal-generating complex comprising a nucleic acid component capable of hybridizing to the one or more linker oligonucleotides in the presence of a srON bound to the target nucleic acid. The linker oligonucleotide can hybridize to a component of the signal-generating complex (e.g., an amplifier, a pre-amplifier, label probe). Contacting the biological sample with the one or more linker oligonucleotides may be carried out simultaneously, before or after contacting the biological sample with the signal-generating complex.

[0113] In select embodiments, the biological sample is contacted with the one or more linker oligonucleotides for a period of time prior to the addition of the signal-generating complex to thebiological sample. In some embodiments, contacting the biological sample with the one or more linker oligonucleotides may be for about 10 minutes to about 48 hours, or about 30 minutes to about 150 minutes. For example, in some embodiments, contacting the biological sample with the one or more linker oligonucleotides may be for 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 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, or about 150 minutes.

[0114] In some embodiments, contacting the biological sample with the one or more linker oligonucleotides is carried out at a temperature of about 4 °C to about 75 °C, or about 30 °C to about 50 °C. For example, in some embodiments, contacting the biological sample with the one or more linker oligonucleotides is carried out at a temperature of 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, or about 50 °C.

[0115] In other embodiments, step (ii) comprises contacting the biological sample with a signalgenerating complex comprising a nucleic acid component capable of hybridizing to the at least one anchor probe oligonucleotides in the presence of a srON bound to the target nucleic acid.In either embodiment of steps (ii), the method comprises contacting the sample with a signalgenerating complex.

[0116] In some embodiments, the methods further comprise contacting the sample with a signal blocking probe configured to hybridize with the one or more linker oligonucleotides or the signalgenerating binding region of the at least one anchor probe, and the target srON binding site in the absence of a srON bound to the target nucleic acid. The signal blocking probe can be incubated with the sample before step (i) or with step (i) and / or step (ii). In some embodiments, the signal blocking probe is incubated with the sample before step (i) or before contacting the sample with the at least one anchor probe. In some embodiments, the sample is contacted with at least one anchor probe and the signal blocking probe simultaneously, e.g., in step (i). In some embodiments, the sample is contacted with the signal blocking probe after step (i), e.g., before step (ii) orconcomitant with step (ii). In some embodiments, the signal blocking probe is incubated with the same at the same time as the one or more linker oligonucleotides, c.g., in step (ii).

[0117] In some embodiments, the biological sample is contacted with the signal blocking probe for about 10 minutes to about 48 hours, or about 30 minutes to about 150 minutes. For example, in some embodiments, contacting the biological sample with the signal blocking probe is for 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 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, or about 150 minutes.

[0118] In some embodiments, the biological sample is contacted with the signal blocking probe at a temperature of about 4 °C to about 75 °C, or about 30 °C to about 50 °C. For example, in some embodiments, contacting the biological sample with the signal blocking probe is at a temperature of 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, or about 50 °C.

[0119] The methods disclosed herein can be used for concurrent or sequential detection of multiple srONs in the same sample. For example, in some embodiments, the method comprises detecting two or more srONs in the same sample. For example, in some embodiments, the method comprises detecting from 1 to 100 srONs in the sample. In some embodiments, the method comprises detecting from 1 to 50 different srONs in the sample.

[0120] For example, in some embodiments, the methods may comprise contacting the biological sample with at least one second set of anchor probe(s) and, optionally, at least one second set of linker oligonucleotide(s), directed to one or more additional srONs and contacting the biological sample with one or more additional signal-generating complexs comprising a nucleic acid component capable of hybridizing to at least one second set of anchor probe(s) or linker oligonucleotides. Accordingly, using one or more additional sets of anchor probe(s) facilitates detection of two or more additional srONs in the same sample. In some embodiments, the detection of the two or more additional srONs are performed simultaneously with the initial detection. Alternatively, detection of the initial srON and any or all of the two or more additional srONs are performed sequentially.

[0121] Embodiments in which higher numbers of srONs or other target molecules (e.g., nucleic acids) arc detected in the same sample may involve the use of clcavablc labels, described elsewhere herein. In the case of using fluorophores as labels, the fluorophores to be used for detection of multiple srONs 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 of the target srONs can be detected concurrently. Methods of selecting suitable distinguishable fluorophores for use in methods of the disclosure are well known in the ail (see, e.g., Johnson and Spence, “’Molecular Probes Handbook, a Guide to Fluorescent Probes and Labeling Technologies, 11th ed., Life Technologies (2010)).

[0122] Step (iii) of the disclosed method comprises detecting a signal from the signalgenerating 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 of protein targets in the same sample.

[0123] Embodiments described herein can utilize enzymes to generate a detectable signal using appropriate chromogenic or Anorogenic 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 Auorescent labels (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Exemplary Auorophores useful as labels include, but are not limited to, rhodamine derivatives, for example, tetramethylrhodamine, rhodamine B, rhodamine 6G, sulforhodamine B, Texas Red (sulforhodamine 101), rhodamine 110, and derivatives thereof such as tetramethylrhodamine-5-(or 6), lissamine rhodamine B, and the like; 7-nitrobenz-2-oxa-l,3- diazole (NBD); Auorescein 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 Auor 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 andsulfonated pyrenes such as Cascade Blue™ and derivatives thereof, including 8-methoxypyrene- 1,3,6-trisulfonic acid, and the like; pyridyloxazolc 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.

[0124] Descriptions of the target srON, target nucleic acids, anchor probe(s), linker oligonucleotide(s), signal-generating complex, and signal blocking probe provided for the above disclosed systems are equally applicable to the methods described herein.

[0125] 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.

[0126] 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 indiagnostic applications to identify the presence or absence of pathological cells based on the presence or absence of a target that is a biomarkcr indicative of a pathology.

[0127] 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.

[0128] 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).

[0129] 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)).

[0130] 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,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.

[0131] In some embodiments, the methods comprise a pretreatment step before hybridization contacting the sample with the anchor probe(s). In some embodiments, the pretreatment step comprises a blocking step where certain blocking agent(s) are applied to block certain endogenous components of the cell thus reducing assay background. As described above, a blocking step can include contacting a biological sample with a signal blocking probe described herein. In accordance with these methods, the use of signal blocking probes enhances signal efficiency so that the detection of the target nucleic acid is more specific, more sensitive, or otherwise improved compared to not using the RNA blocking molecules.

[0132] In some embodiments, various other blocking agents can be used in addition to the signal blocking probes. For example, hydrogen peroxide is a blocking agent when horseradish peroxidase (HRP) is used as detection enzyme in the later steps. Hydrogen peroxide is added to inactivate the endogenous HRP activity in the sample, thus reducing assay background. In a specific embodiment, this blocking step is added as the first step in the pretreatment right after deparaffinization. In some embodiments, the pretreatment step comprises an epitope retrieval step, where certain epitope retrieval buffer(s) can be added to unmask the target nucleic acid. In some embodiments, the epitope retrieval step comprises heating the sample. In some embodiments, the pretreatment step comprises a permeabilization step to retain the nucleic acid targets in the cell and to permit the anchor probes, linker oligonucleotides, signal-generating complex components etc. to enter the cell. In some embodiments, the permeabilization step comprises a digestion with a protease. Detergents (e.g., Triton X-100 or SDS) and Proteinase K can also 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 target mRNA. Optimal concentrations and durations of treatment can be experimentally determined as is known in the art. A cell washing step can follow, to remove the dissolved materials produced in the pretreatment step. When the sample is in a formalin-fixed paraffin embedded tissue, a de-paraffinization step can be used to remove paraffin and rehydratethe 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.

[0133] 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 protein targets or other nucleic acid targets. In some embodiments, the methods further comprise contacting the biological sample with one of more protein or nucleic acid detection agents. In some embodiments, the methods comprise one or more immunohistochemical or immunocytochemical assays of the biological sample for protein targets and / or other nucleic acids targets.

[0134] In some embodiments, the methods provided herein can detect binding or a target srON with a target nucleic acid with spatial and temporal resolution. In specific embodiments, the methods can be used to determine the presence or absence of binding of the srON to the target nucleic acid. In some embodiments, the methods can be used to determine the spatial or temporal presence or absence of binding of the srON to the target nucleic acid (e.g., in a subject or a disease model). In some embodiments, the methods facilitate determination of efficiency of binding of the srON to the target over time. In specific embodiments, the methods can be used to calculate stability of a half-life of the srON.

[0135] In specific embodiments, the method can be used for determining the efficiency of binding of the srON to the target following delivery into a subject or disease model. For example, the quantity of free srON can be determined using known assays, see for example, WO2024054571. Target nucleic acid concentrations (e.g., mRNAs) can also be assayed using known methods. Using the quantities of bound srON, free srON, and target nucleic acids concentrations, total srON and binding efficiencies can be determined.

[0136] In specific embodiments, the method can be used for detecting the presence of the srONs following the delivery of the srONs to a subject. In specific embodiments, the method can be used for detecting the presence of the srONs following the delivery of the srONs into disease models. In specific embodiments, the method can be used for monitoring the binding following the delivery of the srON into a subject or disease models.

[0137] In some embodiments, the methods provided herein are to determine or monitor the effectiveness of a nucleic acid-based therapies. In one embodiment, the methods provided hereinare to monitor and / or determine the effectiveness of gene silencing or downregulating oligonuclcotidc-bascd therapy. In one embodiment, the methods provided herein arc to monitor and / or determine the effectiveness of nucleic acid mediated editing (e.g., gene editing).

[0138] In specific embodiments, the methods can be used to determine the dosing of the nucleic acid-based therapies to see a desired effect. In one embodiment, the methods provided herein can be used to determine the dosing of the gene silencing or downregulating oligonucleotide-based therapy to see a desired effect. In one embodiment, the methods provided herein can be used to determine the dosing of nucleic acid mediated editing (e.g., gene editing) to see the desired effect. d. Image Processing

[0139] In some embodiments, the methods include an image processing method, such as the methods described in International Patent Application PCT / US22 / 24975, which is herein incorporated by reference. The method is implemented at least in part with a computer having corresponding instructions stored on a memory (i.e., 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.

[0140] 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 (i.e., "‘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 (i.e., 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.

[0141] 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).

[0142] The method further includes modifying the background image to create an adjusted background image (c.g., transformed, intensity-adjusted blank image) based on at least one image 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.

[0143] 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. e. Kits

[0144] Also provided herein is a kit for performing the methods described herein.

[0145] In some embodiments, provided herein is a kit detecting, quantifying, localizing, and / or determining the efficiency of one or more small regulatory oligonucleotides (srONs) binding to a target nucleic acid, comprising: at least one anchor probe and a signal-generating complex, as described herein. In some embodiments, the kit further comprises a signal blocking probe, as described herein. In some embodiments, the kit further comprises one or more linker oligonucleotides, as described herein.

[0146] 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.

[0147] In a specific embodiment, the kit provided herein comprises agents for performing RNAscope™ as described in more detail in, e.g., US Patent Nos. 7,709,198, 8,604,182, and 8,951,726. In a specific embodiment, the kit provided herein comprises agents for performing BaseScope™ as described in more detail in, e.g., U.S. Patent No. 11,078,528.

[0148] 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. Inone 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 (sec 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 et al., Histochem. 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 Bums 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).

[0149] 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 INaCL. 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 aldchydc-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- 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.

[0150] 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.

[0151] 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.).

[0152] Kits provided herein can include labels or inserts, which can include information for which the kit component may be used for. Labels or inserts can include instructions for carrying out any of the methods disclosed herein.EXAMPLES

[0153] 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 dataassociated with the teachings of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (c.g., amounts, percentages, etc.), but some experimental error and deviation may be present.Materials and Methods

[0154] srON-target Binding Assay - Mouse Tissue Array as in FIGS. 2-9 Antisense oligos (ASOs) were designed to target the coding sequence (CDS) of mouse Lamp5 and Malatl transcripts. Both chromogenic and fluorescent assay were performed using a BOND RX Automated Stainer. Briefly, FFPE mouse tissue sections were deparaffinized and pretreated. Slides were incubated with ASOs targeting Lamp5 / Malatl transcript for Ih at 37°C. Blocking probes were incubated for Ih at 37°C. Anchoring probes and blocking probes were then mixed and incubated for 2h at 42°C before 1-hour linker oligos hybridization at 42°C. For chromogenic assay, RNAscope amplification was performed following manufacturer’s protocol. Slides were counterstained by Hematoxylin and imaged on a Nanozoomer whole slide scanner (Hamamatsu Photonics, Japan). For fluorescent assay, RNAscope signal amplification was conducted using manufacturer’s protocol, and the fluorescent signal was developed using Opal 650 reagent (Akoya Biosciences, MA, USA) at 1:1000 dilution for 30min at room temperature. Slides were counterstained by DAPI and imaged by either a PhenoImager HT 2.0 (Akoya Biosciences, MA, USA) or a Zeiss Axio Imager 2 (Zeiss, Germany). All oligos were applied at 20nM unless mentioned otherwise.

[0155] srON-target Binding Assay - Cell Culture as in FIG. 13 An occupancy-only ASO was designed to the 3’UTR of the target gene. HCT116 cells were cultured, treated with the ASO at concentrations of 3nM, lOnM, and 30nM, harvested, and processed into FFPE sections. Untreated HCT116 cells served as negative control. The srON-target binding assay was performed using a BOND RX Automated Stainer. FFPE sections of HCT116 cell pellets were deparaffinized, and pretreatment steps were canned out as per ACD’s protocol. Blocking probe was hybridized to the target gene for 2h at 37°C prior to the anchor probe hybridization 2h at 42°C. RNAscope signal amplification was conducted following manufacturer’s protocol, and the fluorescent signal was developed using iFluor® 546 Styramide (AAT Bioquest, CA, USA) at 1:1000 dilution for 15min at room temperature. Slides were counterstained with DAPI and imaged using a SLIDEVIEW VS 200 Universal Whole Slide Imaging Scanner.Example 1

[0156] An antisense oligonucleotide (ASO) targeting Malatl mRNA was designed and applied to FFPE mouse tissues. Following sequential incubation with the anchor probes, linker oligonucleotide, and signal-generating complex, positive signals were detected in two different regions of mouse brain tissue (FIGS. 2A and 2C). Since the methods do not directly target ASO sequences, the positive signals come from the target-bound ASOs but not the free-floating ones. Control tissues that were not incubated with the ASO showed low to no unspecific signals (FIGS. 2B and 2D). Similar results were seen in lung (FIGS. 3C-3D), liver (FIGS. 3E-3F), and intestine (FIGS. 3G-3H).

[0157] An antisense oligonucleotide (ASO) targeting Lamp5 mRNA was designed and applied to FFPE mouse brain. Following sequential incubation with the anchor probes, linker oligonucleotide, and signal-generating complex, positive signals were detected in different regions of mouse brain tissues (FIG. 4A). Signals were particularly enriched in the hindbrain and certain layers of cortex, which was consistent with the distribution pattern characterized previously by other studies (FIGS 4B and 4D). Control tissues that were not incubated with the ASO showed low to no unspecific signals (FIGS 4C and 4E).Example 2

[0158] An alternative to a single linker probe is to use dual linker oligonucleotides with two or more anchor probes. Such a configuration increases specificity of linkers oligonucleotides as strong and stable hybridization of the signal-generating complex oligonucleotide can only interact with the linker oligonucleotide when both anchor probes are hybridized to the correct location about the target binding site. As shown in FIG. 5, dual linker oligonucleotides largely reduced background staining to levels comparable to background in negative controls in mouse brain and lung. As shown in FIG. 6, when utilized with FFPE mouse brain sections, the dual linker oligonucleotides largely reduced the background staining while maintaining high signal-to-noise ratio.

[0159] Additionally, the signal blocking probe was modified to include multiple locked nucleic acids in the signal-generating blocking region. Additionally, the signal blocking probe was incubated with the target nucleic acid prior to the anchor probes or the linker oligonucleotide. As shown in FIG. 7, the inclusion of the locked nucleic acids reduced background staining in FFPE mouse brain sections. Without being bound by theory, the locked nucleic acids may be increasingthe binding affinity between the signal blocking probe and the linker oligonucleotides, preventing or reducing competitive binding between the signal-generating complex oligonucleotides and the signal blocking probe for the linker oligonucleotide. The signal-to-noise ratio was further improved when signal blocking probes containing the locked nucleic acids were pre-incubated. The pre-incubation may facilitate more complete blocking of the target binding site on the target nucleic acid.

[0160] The use of dual linker oligonucleotides, signal blocking probes containing locked nucleic acids, and pre-incubation of the signal blocking probes improved the signal-to-noise ratio. FIGS. 9A-9D show the dose dependency of the ASO on signal intensity.

[0161] 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

CLAIMS1. A small regulatory oligonucleotide (srON) detection system comprising: at least one anchor probe, wherein each anchor probe comprises: a target nucleic acid binding region complementary to one or more regions of a target nucleic acid flanking a target srON binding site on the target nucleic acid; and a signal-generating binding region complementary to a section of one or more linker oligonucleotides or a nucleic acid component of a signal-generating complex.

2. The srON detection system of claim 1, wherein the system comprises a single anchor probe.

3. The srON detection system of claim 1, wherein the system comprises a first anchor probe and a second anchor probe.

4. The srON detection system of claim 3, wherein the first anchor probe and the second anchor probe are complementary to adjacent regions upstream or downstream of the target srON binding site.

5. The srON detection system of claim 3, wherein the first anchor probe is complementary to a region upstream of the target srON binding site and the second anchor probe is complementary to a region downstream of the target srON binding site.

6. The srON detection system of any one of claims 1-5, wherein the signal-generating binding region of each of the at least one anchor probe is individually 5’ or 3’ of the target nucleic acid binding region.

7. The srON detection system of any one of claims 1-6, wherein the signal-generating binding region is 10 to 40 nucleotides in length.

8. The srON detection system of any one of claims 1-7, wherein the target nucleic acid binding region is 10 to 50 nucleotides in length.

9. The srON detection system of any one of claims 1-8, wherein each anchor probe further comprises a non-targeting region separating the signal-generating binding region from the target nucleic acid binding region.

10. The srON detection system of any of claims 1-9, wherein the system further comprises one or more linker oligonucleotides.

11. The srON detection system of claim 10, wherein the system comprises a single linker oligonucleotide.

12. The srON detection system of claim 11, wherein the linker oligonucleotide is 50 to 120 nucleotides in length.

13. The srON detection system of claim 10, wherein the system comprises a first linker oligonucleotide and a second linker oligonucleotide.

14. The srON detection system of 13, wherein each of the first and second linker oligonucleotides are 25 to 60 nucleotides in length.

15. The srON detection system of any one of claims 1-14, further comprising a signal-generating complex comprising a nucleic acid component capable of hybridizing to the one or more linker oligonucleotides or the signal-generating binding region of the at least one anchor probe.

16. The srON detection system of claim 15, wherein the signal-generating complex comprises: a pre-amplifier complementary to a section of the one or more linker oligonucleotides or the signal-generating binding region of the at least one anchor probe; one or more amplifiers complementary to a portion of the pre-amplifier; and a plurality of label probes each complementary to a portion of the one or more amplifiers and each comprising a detectable label.

17. The srON detection system of claim 16, wherein the detectable label comprises a fluorescent moiety or a chromogenic moiety.

18. The srON detection system of claim 16 or 17, wherein the detectable label comprises an enzyme or an enzyme substrate.

19. The srON detection system of any of claims 16-18, wherein the detectable label comprises a cleavable label.

20. The srON detection system of any one of claims 1-19, further comprising a signal blocking probe configured to hybridize with: the linker oligonucleotide or the signal-generating binding region of the at least one anchor probe; and the target srON binding site in the absence of a srON bound to the target nucleic acid.

21. The srON detection system of claim 20, wherein the signal blocking probe comprises: a srON region complementary to the target srON binding site; and at least one signal-generating blocking region, wherein each signal-generating blocking region is complementary to a region of the linker oligonucleotide configured to hybridize to the signal-generating complex or the signal-generating binding region of the at least one anchor probe, wherein the srON region and the at least one signal-generating blocking region are separated by one or more nucleotides.

22. The srON detection system of claim 21, wherein the signal blocking probe comprises two signal-generating blocking regions flanking the srON region.

23. The srON detection system of claim 22, wherein each of the two signal-generating blocking regions are separated from the srON region by one or more nucleotides.

24. The srON detection system of any one of claims 21-23, wherein the at least one signalgenerating blocking region comprises one or more locked nucleic acid (LNA) nucleotides.

25. The srON detection system of any one of claims 1-24, further comprising the target nucleic acid.

26. The srON detection system of claim 25, wherein the target nucleic acid comprises or is suspected of comprising the target srON binding site.

27. The srON detection system of any one of claims 1-26, further comprising a target srON.

28. The srON detection system of claim 27, wherein the target srON is an RNA molecule.

29. The srON detection system of claim 28, wherein the target srON is a microRNA (miRNA) molecule, a small non-coding RNA (sncRNA) molecule, a PlWI-interacting RNA (piRNA) molecule, a small interfering RNA (siRNA) molecule, or an antisense oligonucleotide (ASO).

30. A method for detecting, quantifying, localizing, and / or determining the efficiency of one or more small regulatory oligonucleotides (srONs) binding to a target nucleic acid comprising:(i) contacting a sample comprising a target srON and a target nucleic acid with at least one anchor probe complementary to one or more regions of the target nucleic acid flanking a target srON binding site;(ii) contacting the sample with: a) one or more linker oligonucleotides complementary to a region in each of the least one anchor probes and a signal-generating complex comprising a nucleic acid component capable of hybridizing to the one or more linker oligonucleotides in the presence of a srON bound to the target nucleic acid, or b) a signal-generating complex comprising a nucleic acid component capable of hybridizing to the at least one anchor probe oligonucleotides in the presence of a srON bound to the target nucleic acid; and(iii) detecting a signal from the signal-generating complex.

31. The method of claim 30, wherein: step (i) and step (ii) are performed simultaneously; or step (i) is performed before step (ii).

32. The method of claim 30 or 31, wherein the method comprises contacting the sample with a single anchor probe.

33. The method of claim 30 or 31, wherein the method comprises contacting the sample with a first anchor probe and a second anchor probe.

34. The method of claim 33, wherein the first anchor probe and the second anchor probe are complementary to adjacent regions upstream or downstream of the target srON binding site or the first anchor probe is complementary to a region upstream of the target srON binding site and the second anchor probe is complementary to a region downstream of the target srON binding site.

35. The method of any one of claims 30-34, wherein each anchor probe comprises: a target nucleic acid binding region complementary to one or more regions of a target nucleic acid flanking a target srON binding site on the target nucleic acid; and a signal-generating binding region complementary to a section of one or more linker oligonucleotides or a nucleic acid component of a signal-generating complex.

36. The method of claim 35, wherein the signal-generating binding region of each of the at least one anchor probe is individually 5’ or 3’ of the target nucleic acid binding region.

37. The method of claim 35 or 36, wherein the signal-generating binding region is 10 to 40 nucleotides in length.

38. The method of any one of claims 35-37, wherein the target nucleic acid binding region is 10 to 50 nucleotides in length.

39. The method of any one of claims 3-38, wherein each anchor probe further comprises a nontargeting region separating the signal-generating binding region from the target nucleic acid binding region.

40. The method of any one of claims 30-39, wherein the one or more linker oligonucleotides are each 25 to 120 nucleotides in length.

41. The method of any one of claims 30-40, wherein the signal-generating complex comprises: a pre-amplifier complementary to a section of the one or more linker oligonucleotides or complementary to the signal-generating binding region of the at least one anchor probe; one or more amplifiers complementary to a portion of the pre-amplifier; and a plurality of label probes each complementary to a portion of the one or more amplifiers and each comprising a detectable label.

42. The method of claim 41, wherein the detectable label comprises a fluorescent moiety, a chromogenic moiety an enzyme, or an enzyme substrate.

43. The method of claim 41 or 42, wherein the detectable label comprises a cleavable label.

44. The method of any one of claims 30-43, further comprising contacting the sample with a signal blocking probe configured to hybridize with: the one or more linker oligonucleotides or the signal-generating binding region of the at least one anchor probe; and the target srON binding site, in the absence of a srON bound to the target nucleic acid before step (i) or with step (i) and / or step (ii).

45. The method of claim 44, wherein the signal blocking probe comprises: a srON region complementary to the target srON binding site; and at least one signal-generating blocking region complementary to a region of the linker oligonucleotide configured to hybridize to the signal-generating complex or the signal-generating binding region of the at least one anchor probe,wherein the srON region and the at least one signal-generating blocking region are separated by one or more nucleotides.

46. The method of claim 45, wherein the signal blocking probe comprises two signal-generating blocking regions flanking the srON region, each separated from the srON region by one or more nucleotides.

47. The method of any one of claims 44-46, wherein the at least one signal-generating blocking region comprises one or more locked nucleic acid (LNA) nucleotides.

48. The method of any one of claims 30-47, further comprising contacting the biological sample with one of more nucleic acid or protein detection agents.

49. The method of any one of claims 30-48, wherein the sample is a biological sample.

50. The method of claim 49, wherein the biological sample is a tissue specimen or is derived from a tissue specimen, a blood sample or is derived from a blood sample, a cytological sample or is derived from a cytological sample, or cultured cells.

51. The method of any one of claims 30-50, wherein the method further comprises contacting the sample with a permeabilizing and / or antigen retrieval reagent or condition.

52. The method of any one of claims 30-51, wherein the method further comprises contacting the sample with a target srON and / or a target nucleic acid.

53. A kit comprising one or more components from a system of any one of claims 1-29.

54. The kit of claim 53, wherein the kit further comprises one or more permeabilizing and / or antigen retrieval reagents.

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