Method for detecting target nucleic acids using RNA blocking molecules

Abstract

Embodiments of the present disclosure include compositions and methods for performing in situ hybridization reactions. In particular, the present disclosure provides RNA blocking molecules that improve detection of target RNA molecules (e.g., mRNA molecules, microRNA (miRNA) molecules, small non-coding RNA (sncRNA) molecules, PIWI-interacting RNA (piRNA) molecules, small interfering RNA (siRNA) molecules, and / or antisense oligo (ASO) molecules) by reducing binding between target probes and non-target RNA molecules in a sample.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 374,998, filed September 8, 2022, which is incorporated herein by reference in its entirety for all purposes.

[0002] Embodiments of the present disclosure include compositions and methods for performing in situ hybridization reactions. In particular, the present disclosure provides RNA blocking molecules that improve detection of target RNA molecules (e.g., mRNA molecules, microRNA (miRNA) molecules, small non-coding RNA (sncRNA) molecules, PIWI-interacting RNA (piRNA) molecules, and / or small interfering RNA (siRNA) molecules) by reducing binding between target probes and non-target RNA molecules in a sample. [Background technology]

[0003] In situ hybridization (ISH) is a technique that enables precise detection of specific segments of nucleic acids within histological sections. The principle underlying ISH is that if nucleic acids are properly preserved in a histological specimen, they can be detected by applying a complementary nucleic acid strand to which a reporter molecule is attached. The target nucleic acid can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Different types of RNA exist within cells: messenger RNA (mRNA), which encodes the amino acid sequence of a polypeptide; transfer RNA (tRNA), which guides amino acids to ribosomes during translation; ribosomal RNA (rRNA), which constitutes the ribosome, the organelle that translates mRNA; and sncRNA, which is involved in RNA processing. Specifically, sncRNA, miRNA, siRNA, and piRNA are of great importance in research, diagnostics, and drug discovery. However, increasing the sensitivity of RNA detection, especially small RNA molecules, within cell or tissue samples is challenging.

[0004] For example, miRNAs (single-stranded, non-coding, small RNAs) contain only approximately 16–22 nucleotides. These RNAs are evolutionarily conserved and regulate gene expression by post-translational modification or by interfering with mRNA degradation. Unstable expression of a single miRNA can lead to abnormal expression of multiple genes. Due to their importance in regulating physiological and pathological processes, they are used as biomarkers for various diseases, including cancer, autoimmune diseases, and neurodegenerative disorders. RNAScope® is an in situ hybridization (ISH)-based technology that detects small RNAs, including miRNAs, siRNAs, and antisense oligonucleotides (ASOs), in cells and tissues. This technology uses ISH probes that bind to miRNAs and reveal the cellular and subcellular spatial expression patterns of small RNAs. Probes for RNAScope® are designed in silico, and up to 34% are predicted to have non-target binding. Therefore, enhanced detection methods are needed to reduce or eliminate non-target binding to provide more accurate diagnostic and therapeutic information. Summary of the Invention

[0005] Embodiments of the present disclosure include RNA blocking molecules comprising at least one RNA blocking domain comprising a non-probe-targeted region and a probe-targeted region. In some embodiments, the non-probe-targeted region and the probe-targeted region are adjacent.

[0006] In some embodiments, the non-probe-targeted region of the RNA blocking domain is complementary to a non-probe-targeted region of the target RNA molecule. In some embodiments, the probe-targeted region of the RNA blocking domain is complementary to a portion of the probe-targeted region of the target RNA molecule.

[0007] In some embodiments, the target RNA molecule is an mRNA molecule, a microRNA (miRNA) molecule, a small non-coding RNA (sncRNA) molecule, a PIWI-interacting RNA (piRNA) molecule, a small interfering RNA (siRNA) molecule, and / or an antisense oligo (ASO).

[0008] In some embodiments, the non-probe-targeted region of the target mRNA comprises a non-coding region, and the probe-targeted region of the target mRNA comprises a coding region. In some embodiments, the non-probe-targeted region of the target miRNA comprises a region that is not present in the corresponding mature miRNA molecule, and the probe-targeted region of the target miRNA comprises a region that is present in the corresponding mature miRNA molecule.

[0009] In some embodiments, the non-probe-targeted region of the target comprises a vector region, such as in adeno-associated viruses (AAV), lentiviral vectors, and other viral vectors, and the probe-targeted region of the target comprises an ASO.

[0010] In some embodiments, the non-probe-targeted region of the RNA blocking domain is between 2 and 50 nucleotides in length. In some embodiments, the probe-targeted region of the RNA blocking domain is between 2 and 20 nucleotides in length.

[0011] In some embodiments, the RNA blocking molecule further comprises a second RNA blocking domain comprising a second non-probe-targeted region and a second probe-targeted region, and in some embodiments, the second non-probe-targeted region and the second probe-targeted region are adjacent.

[0012] In some embodiments, the first and second RNA blocking domains comprise the same number of nucleotides. In some embodiments, the non-probe-targeted regions of the first and second RNA blocking domains comprise the same number of nucleotides. In some embodiments, the probe-targeted regions of the first and second RNA blocking domains comprise the same number of nucleotides.

[0013] In some embodiments, the first and second RNA blocking domains are connected by a linker region. In some embodiments, the linker region is 1 to 10 nucleotides in length. In some embodiments, the linker region is 2 to 8 nucleotides in length. In some embodiments, the linker region is 2 to 5 nucleotides in length. In some embodiments, the linker region comprises at least two types of nucleotides. In some embodiments, the linker region comprises one type of nucleotide. In some embodiments, the linker region consists of thymine nucleotides. In some embodiments, the linker region consists of 1 to 10 thymine nucleotide(s).

[0014] In some embodiments, the linker region comprises at least one nucleotide that is non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 1 to 10 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 20% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule.

[0015] In some embodiments, the total length of the RNA blocking molecule is between 15 and 100 nucleotides.

[0016] Embodiments of the present disclosure also include kits that include any of the RNA blocker molecules described herein.

[0017] In some embodiments, the kit further comprises at least one target probe that specifically hybridizes to a probe-targeted region of the target RNA molecule. In some embodiments, the kit further comprises one or more target probe sets, each target probe set comprising a pair of target probes that specifically hybridize to a probe-targeted region of the target RNA molecule.

[0018] In some embodiments, the kit comprises a reagent for permeabilizing cells, a cross-linking reagent, and / or a protease.

[0019] In some embodiments, the kit includes one or more components of a signal-generating complex, which components include: (i) at least one label and at least one labeled probe, wherein the at least one labeled probe is capable of hybridizing to at least one target probe; or (ii) at least one label, at least one labeled probe, and at least one amplification agent that hybridizes to the at least one labeled probe and that can hybridize to the at least one target probe; or (iii) at least one label, at least one labeled probe, at least one amplification agent hybridized to the at least one labeled probe, and at least one pre-amplification agent that hybridizes to the at least one amplification agent and that can hybridize to the target probe.

[0020] In some embodiments, the kit includes instructions for performing an in situ hybridization reaction.

[0021] Embodiments of the present disclosure also include compositions comprising any of the RNA blocking molecules described herein.

[0022] In some embodiments, the composition comprises a hybridization buffer, hi some embodiments, the composition comprises a reagent for permeabilizing cells, a cross-linking reagent, and / or a protease.

[0023] In some embodiments, the composition comprises a biological sample. In some embodiments, the biological sample is or is derived from a tissue sample. In some embodiments, the biological sample is or is derived from a blood sample. In some embodiments, the biological sample is or is derived from a cytological sample. In some embodiments, the biological sample is cultured cells or an exosome-containing sample.

[0024] In some embodiments, the composition further comprises at least one target probe that specifically hybridizes to a probe-targeted region of a target RNA molecule in the biological sample.

[0025] In some embodiments, the composition comprises one or more components of a signal-generating complex, in some embodiments, the components of the signal-generating complex comprise: (i) at least one label and at least one labeled probe, wherein the at least one labeled probe is capable of hybridizing to at least one target probe; or (ii) at least one label, at least one labeled probe, and at least one amplification agent that hybridizes to the at least one labeled probe and that can hybridize to the at least one target probe; or (iii) at least one label, at least one labeled probe, at least one amplification agent hybridized to the at least one labeled probe, and at least one pre-amplification agent that hybridizes to the at least one amplification agent and that can hybridize to the target probe.

[0026] Embodiments of the present disclosure also include methods of performing in situ hybridization reactions using any of the RNA blocking molecules described herein, any of the kits described herein, and / or any of the compositions described herein.

[0027] Embodiments of the present disclosure also include methods for improving signal efficiency in in situ hybridization reactions. According to these embodiments, the methods include contacting a biological sample containing a target RNA molecule with any of the RNA blocking molecules described herein, contacting the biological sample with at least one target probe that specifically hybridizes to a probe-targeted region of the target RNA molecule, and contacting the biological sample with a signal-generating complex to detect the target RNA molecule. In some embodiments of the methods, the signal efficiency for the target RNA molecule is improved compared to an in situ hybridization reaction that does not include contacting the biological sample with an RNA blocking molecule. In some embodiments of the methods, the improved signal efficiency includes reducing binding of the target probe to non-target RNA molecules in the sample.

[0028] In some embodiments, the target RNA molecule is an mRNA molecule, a microRNA (miRNA) molecule, a small non-coding RNA (sncRNA) molecule, a PIWI-interacting RNA (piRNA) molecule, and / or a small interfering RNA (siRNA) molecule.

[0029] In some embodiments of the method, the biological sample is or is derived from a tissue specimen, a blood sample or is derived from a blood sample, or a cell sample or is derived from a cell sample.

[0030] In some embodiments of the method, the signal-generating complex comprises: (i) at least one label and at least one labeled probe, wherein the at least one labeled probe is capable of hybridizing to at least one target probe; or (ii) at least one label, at least one labeled probe, and at least one amplification agent that hybridizes to the at least one labeled probe and that can hybridize to the at least one target probe; or (iii) at least one label, at least one labeled probe, at least one amplification agent hybridized to the at least one labeled probe, and at least one pre-amplification agent that hybridizes to the at least one amplification agent and that can hybridize to the target probe.

[0031] In some embodiments, the methods include treating the biological sample with a reagent for permeabilizing cells, a cross-linking reagent, and / or a protease. [Brief explanation of the drawings]

[0032] [Figure 1A] Representative RNA detection blocking strategies tested according to various embodiments of the present disclosure (FIG. 1A). To test the efficiency of the disclosed RNA blocking molecules for reducing non-target binding of target probes, oligos were designed using PPIB, a low-expression gene, as a surrogate non-target molecule. Four different blocking strategies / designs were tested to identify a reduction in signal from PPIB (FIG. 1A). Oligos with complementary sequences on both sides of the probe design region connected with a T-linker (the "double competitive blocking 2" strategy) were most efficient at blocking signal from PPIB (FIG. 1B). Similar oligos were designed and tested against the highly expressed gene UBC. RNA blocking molecules designed using the same strategy (the "double competitive blocking 2" strategy) efficiently reduced signal from the UBC probe (FIG. 1C). [Figure 1B]Representative RNA detection blocking strategies tested according to various embodiments of the present disclosure (FIG. 1A). To test the efficiency of the disclosed RNA blocking molecules for reducing non-target binding of target probes, oligos were designed using PPIB, a low-expression gene, as a surrogate non-target molecule. Four different blocking strategies / designs were tested to identify a reduction in signal from PPIB (FIG. 1A). Oligos with complementary sequences on both sides of the probe design region connected with a T-linker (the "double competitive blocking 2" strategy) were most efficient at blocking signal from PPIB (FIG. 1B). Similar oligos were designed and tested against the highly expressed gene UBC. RNA blocking molecules designed using the same strategy (the "double competitive blocking 2" strategy) efficiently reduced signal from the UBC probe (FIG. 1C). [Figure 1C] Representative RNA detection blocking strategies tested according to various embodiments of the present disclosure (FIG. 1A). To test the efficiency of the disclosed RNA blocking molecules for reducing non-target binding of target probes, oligos were designed using PPIB, a low-expression gene, as a surrogate non-target molecule. Four different blocking strategies / designs were tested to identify a reduction in signal from PPIB (FIG. 1A). Oligos with complementary sequences on both sides of the probe design region connected with a T-linker (the "double competitive blocking 2" strategy) were most efficient at blocking signal from PPIB (FIG. 1B). Similar oligos were designed and tested against the highly expressed gene UBC. RNA blocking molecules designed using the same strategy (the "double competitive blocking 2" strategy) efficiently reduced signal from the UBC probe (FIG. 1C). [Figure 2A] Representative results showing that the RNA blocking molecules of the present disclosure targeting pri-miR-21 and pre-miR-205 do not affect the detection of miRNA-21 (Figure 2A) and miRNA-205 (Figure 2B), respectively. [Figure 2B] Representative results showing that the RNA blocking molecules of the present disclosure targeting pri-miR-21 and pre-miR-205 do not affect the detection of miRNA-21 (Figure 2A) and miRNA-205 (Figure 2B), respectively. [Figure 3]Representative results demonstrating that RNA blocking molecules of the present disclosure effectively reduce the signal from pre- and pri-miRNA21 (Figure 3). [Figure 4A] Representative results showing that the efficiency of the disclosed RNA blockers varies with the length of the overlapping region (probe-targeted region), with longer overlap lengths being somewhat more efficient for both probes targeting UBC (Figure 4A) and pri-miR-21 (Figures 4B and 4C). [Figure 4B] Representative results showing that the efficiency of the disclosed RNA blockers varies with the length of the overlapping region (probe-targeted region), with longer overlap lengths being somewhat more efficient for both probes targeting UBC (Figure 4A) and pri-miR-21 (Figures 4B and 4C). [Figure 4C] Representative results showing that the efficiency of the disclosed RNA blockers varies with the length of the overlapping region (probe-targeted region), with longer overlap lengths being somewhat more efficient for both probes targeting UBC (Figure 4A) and pri-miR-21 (Figures 4B and 4C). [Figure 5A] Representative results show that the efficiency of the disclosed RNA blockers varies with varying linker length (the linker connecting two RNA blocking molecules). Shorter linker lengths (e.g., T-linker lengths) are somewhat more efficient (Figure 5A), with linkers of approximately 1 to 3 nucleotides being the most efficient (Figures 5B and 5C). The results also show that T-linkers are somewhat more efficient than linkers composed of other nucleotides (Figure 5D), and that overall RNA blocker concentrations of approximately 20 nM to approximately 30 nM are most efficient (Figure 5E). In summary, one representative optimized RNA blocker design is provided (Figure 5F). [Figure 5B]Representative results show that the efficiency of the disclosed RNA blockers varies with varying linker length (the linker connecting two RNA blocking molecules). Shorter linker lengths (e.g., T-linker lengths) are somewhat more efficient (Figure 5A), with linkers of approximately 1 to 3 nucleotides being the most efficient (Figures 5B and 5C). The results also show that T-linkers are somewhat more efficient than linkers composed of other nucleotides (Figure 5D), and that overall RNA blocker concentrations of approximately 20 nM to approximately 30 nM are most efficient (Figure 5E). In summary, one representative optimized RNA blocker design is provided (Figure 5F). [Figure 5C] Representative results show that the efficiency of the disclosed RNA blockers varies with varying linker length (the linker connecting two RNA blocking molecules). Shorter linker lengths (e.g., T-linker lengths) are somewhat more efficient (Figure 5A), with linkers of approximately 1 to 3 nucleotides being the most efficient (Figures 5B and 5C). The results also show that T-linkers are somewhat more efficient than linkers composed of other nucleotides (Figure 5D), and that overall RNA blocker concentrations of approximately 20 nM to approximately 30 nM are most efficient (Figure 5E). In summary, one representative optimized RNA blocker design is provided (Figure 5F). [Figure 5D] Representative results show that the efficiency of the disclosed RNA blockers varies with varying linker length (the linker connecting two RNA blocking molecules). Shorter linker lengths (e.g., T-linker lengths) are somewhat more efficient (Figure 5A), with linkers of approximately 1 to 3 nucleotides being the most efficient (Figures 5B and 5C). The results also show that T-linkers are somewhat more efficient than linkers composed of other nucleotides (Figure 5D), and that overall RNA blocker concentrations of approximately 20 nM to approximately 30 nM are most efficient (Figure 5E). In summary, one representative optimized RNA blocker design is provided (Figure 5F). [Figure 5E]Representative results show that the efficiency of the disclosed RNA blockers varies with varying linker length (the linker connecting two RNA blocking molecules). Shorter linker lengths (e.g., T-linker lengths) are somewhat more efficient (Figure 5A), with linkers of approximately 1 to 3 nucleotides being the most efficient (Figures 5B and 5C). The results also show that T-linkers are somewhat more efficient than linkers composed of other nucleotides (Figure 5D), and that overall RNA blocker concentrations of approximately 20 nM to approximately 30 nM are most efficient (Figure 5E). In summary, one representative optimized RNA blocker design is provided (Figure 5F). [Figure 5F] Representative results show that the efficiency of the disclosed RNA blockers varies with varying linker length (the linker connecting two RNA blocking molecules). Shorter linker lengths (e.g., T-linker lengths) are somewhat more efficient (Figure 5A), with linkers of approximately 1 to 3 nucleotides being the most efficient (Figures 5B and 5C). The results also show that T-linkers are somewhat more efficient than linkers composed of other nucleotides (Figure 5D), and that overall RNA blocker concentrations of approximately 20 nM to approximately 30 nM are most efficient (Figure 5E). In summary, one representative optimized RNA blocker design is provided (Figure 5F). DETAILED DESCRIPTION OF THE INVENTION

[0033] Embodiments of the present disclosure include compositions and methods for performing in situ hybridization (ISH) reactions. In particular, the present disclosure provides RNA blocking molecules that improve detection of target RNA molecules (e.g., mRNA molecules, microRNA (miRNA) molecules, small non-coding RNA (sncRNA) molecules, PIWI-interacting RNA (piRNA) molecules, and / or small interfering RNA (siRNA) molecules) by reducing binding of target probes to non-target RNA molecules in a sample. In accordance with these embodiments, the present disclosure provides methods for improving the signal efficiency of ISH reactions using RNA blocking molecules, as further described herein.

[0034] definition As used herein, the terms "fixation" or "fixing," when referring to fixing a biological sample in an in situ hybridization process, refer to a procedure that protects the biological sample from decay, e.g., by autolysis or putrefaction. This procedure halts any ongoing biochemical reactions and may also improve the mechanical strength or stability of the treated tissue.

[0035] As used herein, the term "one or more" means, 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 higher number as desired for a particular application.

[0036] As used herein, the term "detecting" generally refers to any form of measurement, including determining whether a component is present or not. This term includes quantitative and / or qualitative determinations.

[0037] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to polymers of any length composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides) or synthetically produced compounds that can hybridize in a sequence-specific manner with naturally occurring nucleic acids, e.g., compounds that can participate in Watson-Crick base pairing interactions, similar to the sequence-specific hybridization of two naturally occurring nucleic acids. The term "bases" (or "bases"), when used herein in the context of a polynucleotide sequence, is synonymous with "nucleotides" (or "nucleotides"), i.e., the monomeric subunits of a polynucleotide. The terms "nucleoside" and "nucleotide" are intended to encompass not only moieties containing the known purine and pyrimidine bases, but also moieties containing modified or other heterocyclic bases. Such modifications include methylated purines, methylated pyrimidines, acylated purines, acylated pyrimidines, alkylated riboses, or other heterocycles. Furthermore, the terms "nucleoside" and "nucleotide" encompass not only moieties containing traditional ribose and deoxyribose sugars, but also moieties containing other sugars. Modified nucleosides or nucleotides also include modifications to the sugar moiety, such as replacing one or more of the hydroxyl groups with halogen atoms or aliphatic groups, or functionalizing with ethers, amines, etc. "Analog" refers to a molecule that is a mimetic, a derivative, has a similar structure, or has structural features recognized in the literature as other similar terms, including, for example, polynucleotides incorporating non-natural nucleotides, nucleotide mimics (such as 2'-modified nucleosides), peptide nucleic acids, oligomeric nucleoside phosphonates, and any polynucleotide to which a substituent (such as a protecting group or linking moiety) has been added.

[0038] As used herein, the terms "non-coding," "non-coding region," or "non-coding sequence" with respect to an RNA molecule generally refer to an RNA sequence / region that is not translated into an amino acid sequence and does not encode a protein, whereas a "coding" sequence / region is translated into an amino acid sequence. Generally, non-coding regions of an mRNA molecule include introns, promoters, enhancers, 5'UTR, 3'UTR, etc. Additionally, other types of RNA molecules comprise (or consist of) non-coding sequences / regions, including, but not limited to, microRNAs (miRNAs), small non-coding RNAs (sncRNAs), PIWI-interacting RNAs (piRNAs), and small interfering RNAs (siRNAs).

[0039] As used herein, the term "small noncoding RNA" or "sncRNA" generally refers to a large family of RNA molecules (e.g., 18-200 nt in length) that regulate cellular functions. Small ncRNAs are involved in nearly every developmental and pathological process in mammals. While the precise function of many ncRNAs remains unknown, numerous studies have revealed the direct involvement of various small ncRNAs in regulating gene expression at the level of post-transcriptional mRNA processing and ribosome biogenesis. Mammalian cells express several classes of small ncRNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), PIWI-interacting RNAs (piRNAs), and tRNA-derived small RNAs (tRFs).

[0040] As used herein, the term "small interfering RNA" or "siRNA" generally refers to a small interfering RNA duplex (generally 18-30 base pairs) that induces the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21-mers with a central 19-bp duplex region and symmetric two-base 3'-overhangs at the termini; however, it has recently been demonstrated that chemically synthesized RNA duplexes of 25-30 bases in length can be up to 100-fold more potent than 21-mers in the same location. The observed increase in potency obtained using longer RNAs in inducing RNAi has been suggested to be due to the provision of substrate (27-mer) instead of product (21-mer) to Dicer, which is thought to improve the rate or efficiency of RISC entry of siRNA duplexes.

[0041] As used herein, the terms "microRNA," "miRNA," and "miR" generally refer to a collection of non-coding, single-stranded RNA molecules, approximately 19-28 nucleotides in length, that regulate gene expression. miRNAs are found in a wide range of organisms, including viruses, and have been shown to play roles in development, homeostasis, and disease pathogenesis. Initially, pre-miRNAs exist as long, imperfect, double-stranded stem-loop RNAs, which are further processed by Dicer into siRNA-like duplexes containing similarly sized fragments known as the mature guide strand (miRNA) and passenger strand (miRNA). miRNAs and miRNAs can be derived from the opposing arms of pri-miRNAs and pre-miRNAs. miRNA sequences can be found in libraries of cloned miRNAs, but typically occur less frequently than miRNAs. While initially present as a duplex with miRNA, miRNAs are ultimately incorporated as single-stranded RNAs into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Various proteins can form RISC, resulting in variations in specificity for miRNA / miRNA duplexes, binding sites on target genes, miRNA activity (repression or activation), and which strand of the miRNA / miRNA duplex is loaded into RISC. When the miRNA strand of a miRNA:miRNA duplex is loaded into RISC, the miRNA is removed and degraded. The strand of the miRNA:miRNA duplex with the more loosely paired 5' end is loaded into RISC. When both ends of the miRNA:miRNA have nearly equal 5' pairing, both the miRNA and the miRNA can have gene silencing activity. RISC identifies target nucleic acids based on the high level of complementarity between the miRNA and mRNA, particularly nucleotides 2-8 of the miRNA (called the "seed sequence").

[0042] As used herein, the term "PIWI-interacting RNA" or "piRNA" generally refers to a class of small noncoding RNAs whose primary function in mammals is the silencing of germline-resident retrotransposons. PIWI-interacting RNAs (piRNAs) are 26-31 nucleotide single-stranded ncRNAs that interact with the germline-specific Argonaute family of proteins, the P-element-induced wimpiness (PIWI) proteins. piRNAs exhibit highly diverse sequences compared to any other known cellular RNA family and constitute one of the largest classes of known ncRNAs. piRNAs have been shown to be involved in both posttranscriptional and epigenetic silencing of retrotransposons and other genetic elements in the germline, particularly during spermatogenesis. They are 5' monophosphorylated and 3' 2'-O-methyl modified, features that are proposed to enhance piRNA stability.

[0043] 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 generate a predetermined 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), G pairs with C), although small regions of mismatched, inserted, or deleted sequences (e.g., less than about 3 bases) may exist.

[0044] As used herein, the term "sample" refers to a substance or mixture of substances containing one or more components of interest. The term "sample" encompasses "biological samples," which refer to samples obtained from a biological subject, such as samples derived from biological tissues or fluids obtained, reached, or harvested in vivo or in situ. Biological samples also include samples derived from regions of a biological subject containing precancerous cells, cancerous cells, precancerous tissue, or cancerous tissue. 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 lysates, cell cultures, cell lines, tissues, oral tissues, gastrointestinal tissues, organs, organelles, biological fluids, blood samples, urine samples, skin samples, and the like. Preferred biological samples include, but are not limited to, whole blood, partially purified blood, PBMCs, tissue biopsies, and the like.

[0045] The term "probe" as used herein refers to a capture substance directed to a specific target mRNA sequence. Thus, each probe in a set of probes has its own target mRNA sequence. In some embodiments, the probes provided herein are "nucleic acid probes" or "oligonucleotide probes," which refer to nucleic acids that can bind to target nucleic acids with complementary sequences, such as the mRNA biomarkers provided herein, typically through complementary base pairing via the formation of hydrogen bonds. As used herein, a probe may contain natural bases (e.g., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, bases within a probe may be linked by bonds other than phosphodiester bonds, as long as they do not interfere with hybridization. Probes can be directly or indirectly labeled with tags, such as chromophores, lumiphores, or chromophores. The presence or absence of a target mRNA biomarker of interest can be detected by assaying for the presence or absence of the probe.

[0046] As used herein, the term "endogenous" refers to a substance that originates from within a living organism. As used herein, the term "exogenous" refers to a substance that originates outside of a living organism.

[0047] As used in this disclosure and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0048] When embodiments are described herein in conjunction with the term "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. Also, when embodiments are described herein in conjunction with the phrase "consisting essentially of," it is understood that other similar embodiments described in terms of "consisting of" are also provided.

[0049] The term "between" as used in expressions such as "between A and B" or "between AB" indicates a range that includes A and B.

[0050] As used herein, the term "and / or," as in phrases such as "A and / or B," is intended to include A and B, A or B, A only, and B only. Similarly, the term "and / or," as in phrases such as "A, B and / or C," is intended to encompass each of the following specific examples: 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 only; B only; and C only.

[0051] RNA blocking molecules Embodiments of the present disclosure include an RNA blocking molecule, or multiple RNA blocking molecules, comprising at least one RNA blocking domain including a non-probe-targeting region and a probe-targeting region. The non-probe-targeting region of an RNA blocking domain is generally a region that does not overlap with the region of the target designed to be bound by the target probe (see, e.g., FIG. 5F). Conversely, the probe-targeting region of an RNA blocking domain is generally a region that does not overlap with the region of the target designed to be bound by the target probe (see, e.g., FIG. 5F). In some embodiments, the non-probe-targeting region and the probe-targeting region are adjacent. According to these embodiments, the RNA blocking molecule of the present disclosure can bind to non-target molecules, reducing non-specific binding of the target probe and thereby improving signal efficiency.

[0052] In some embodiments, the non-probe-targeted region of the RNA blocking domain is complementary to the non-probe-targeted region of the target RNA molecule. In some embodiments, the non-probe-targeted region of the RNA blocking domain is at least 95% complementary to the non-probe-targeted region of the target RNA molecule. In some embodiments, the non-probe-targeted region of the RNA blocking domain is at least 90% complementary to the non-probe-targeted region of the target RNA molecule. In some embodiments, the non-probe-targeted region of the RNA blocking domain is at least 85% complementary to the non-probe-targeted region of the target RNA molecule. In some embodiments, the non-probe-targeted region of the RNA blocking domain is at least 80% complementary to the non-probe-targeted region of the target RNA molecule.

[0053] In some embodiments, the probe targeting region of the RNA blocking domain is complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the probe targeting region of the RNA blocking domain is at least 95% complementary to the probe targeting region of the target RNA molecule. In some embodiments, the probe targeting region of the RNA blocking domain is at least 90% complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the probe targeting region of the RNA blocking domain is at least 85% complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the probe targeting region of the RNA blocking domain is at least 80% complementary to a portion of the probe targeting region of the target RNA molecule.

[0054] In some embodiments, the RNA blocking molecules of the present disclosure are particularly useful for improving the detection of target RNA molecules, including target RNA molecules of smaller size. In some embodiments, the target RNA molecules include, but are not limited to, mRNA molecules, microRNA (miRNA) molecules, small non-coding RNA (sncRNA) molecules, PIWI-interacting RNA (piRNA) molecules, and / or small interfering RNA (siRNA) molecules. In some embodiments, the RNA blocking molecules of the present disclosure can also be used to improve the detection of DNA molecules, such as antisense oligonucleotides (ASOs).

[0055] In some embodiments, the RNA blocking molecules of the present disclosure improve the detection of one or more target messenger RNA (mRNA) molecules in a sample (e.g., a cell or tissue sample), such as detecting the target messenger (mRNA) using in situ hybridization (ISH). According to these embodiments, the non-probe-targeted region of the target mRNA comprises a non-coding region, and the probe-targeted region of the target mRNA comprises a coding region. In another embodiment, the RNA blocking molecules of the present disclosure improve the detection of one or more target microRNA (miRNA or miR) molecules in a sample (e.g., a cell or tissue sample), such as detecting the target (miRNA) using in situ hybridization (ISH). According to these embodiments, the non-probe-targeted region of the target miRNA comprises a region that is not present in the corresponding mature miRNA molecule (i.e., pr-microRNA or pre-microRNA), and the probe-targeted region of the target miRNA comprises a region that is present in the corresponding mature miRNA molecule.

[0056] In some embodiments, the non-probe-targeted region of the RNA blocking domain is 2-50 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 2-45 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 2-40 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 2-35 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 2-30 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 2-25 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 2-20 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 2-15 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 2-10 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 5-50 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 10-50 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 15-50 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 20-50 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 25-50 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 30-50 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 35-50 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 40-50 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 45-50 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 10-40 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 10-30 nucleotides in length.In some embodiments, the non-probe-targeted region of the RNA blocking domain is 15-35 nucleotides in length. In some embodiments, the non-probe-targeted region of the RNA blocking domain is 20-40 nucleotides in length.

[0057] In some embodiments, the probe targeting region of the RNA blocking domain is 2-20 nucleotides in length. In some embodiments, the probe targeting region of the RNA blocking domain is 5-20 nucleotides in length. In some embodiments, the probe targeting region of the RNA blocking domain is 10-20 nucleotides in length. In some embodiments, the probe targeting region of the RNA blocking domain is 15-20 nucleotides in length. In some embodiments, the probe targeting region of the RNA blocking domain is 2-15 nucleotides in length. In some embodiments, the probe targeting region of the RNA blocking domain is 2-10 nucleotides in length. In some embodiments, the probe targeting region of the RNA blocking domain is 2-5 nucleotides in length. In some embodiments, the probe targeting region of the RNA blocking domain is 10-15 nucleotides in length.

[0058] In some embodiments, the RNA blocking molecule of the present disclosure further comprises a second RNA blocking domain. According to these embodiments, the second RNA blocking domain of the RNA blocking molecule comprises a second non-probe-targeting region and a second probe-targeting region, as further described herein. In some embodiments, the second non-probe-targeting region and the second probe-targeting region are adjacent. In some embodiments, the first and second RNA blocking domains comprise the same number of nucleotides. In some embodiments, the first and second RNA blocking domains comprise a different number of nucleotides. In some embodiments, the non-probe-targeting regions of the first and second RNA blocking domains comprise the same number of nucleotides. In some embodiments, the non-probe-targeting regions of the first and second RNA blocking domains comprise a different number of nucleotides. In some embodiments, the probe-targeting regions of the first and second RNA blocking domains comprise the same number of nucleotides. In some embodiments, the probe-targeting regions of the first and second RNA blocking domains comprise a different number of nucleotides.

[0059] In some embodiments, the first and second RNA blocking domains are connected by a linker region. In some embodiments, the linker region is 1 to 10 nucleotides in length. In some embodiments, the linker region is 2 to 8 nucleotides in length. In some embodiments, the linker region is 2 to 5 nucleotides in length. In some embodiments, the linker region comprises at least two types of nucleotides. In some embodiments, the linker region comprises one type of nucleotide. In some embodiments, the linker region consists of thymine nucleotides. In some embodiments, the linker region consists of 1 to 10 thymine nucleotide(s).

[0060] In some embodiments, the linker region comprises at least one nucleotide that is non-complementary to a portion of the probe targeting region of the target RNA molecule. For example, if the linker region comprises a thymine nucleotide, at least one nucleotide in the probe targeting region of the target RNA molecule comprises a cytosine or guanine. In some embodiments, the linker region comprises 1 to 10 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 2 to 10 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 3 to 10 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 4 to 10 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 5 to 10 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 6 to 10 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 7 to 10 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 8 to 10 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 9 to 10 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 1 to 9 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 1 to 8 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 1 to 7 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 1 to 6 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule.In some embodiments, the linker region comprises 1 to 5 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 1 to 4 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 1 to 3 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 1 to 2 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 2 to 8 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 4 to 6 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 2 to 3 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises 5 to 7 nucleotides that are non-complementary to a portion of the probe targeting region of the target RNA molecule.

[0061] In some embodiments, about 20% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 25% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 30% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 35% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 40% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 45% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 50% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 55% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 60% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 65% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 70% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 75% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 20% to about 75% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 20% to about 70% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule.In some embodiments, about 20% to about 65% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 20% to about 60% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 20% to about 55% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 20% to about 50% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 20% to about 45% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 20% to about 40% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 20% to about 35% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 20% to about 30% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 20% to about 25% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 30% to about 70% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 40% to about 60% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, about 50% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of the target RNA molecule.

[0062] In some embodiments, the total length of the RNA blocking molecule is between 15 and 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is between 20 and 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is between 25 and 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is between 30 and 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is between 35 and 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is between 40 and 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is between 45 and 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is between 50 and 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is between 55 and 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is between 60 and 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is between 65 and 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is between 70 and 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 75-100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 80-100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 85-100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 90-100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 95-100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-95 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-90 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-85 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-80 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-75 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-70 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-65 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-60 nucleotides.In some embodiments, the total length of the RNA blocking molecule is 15-55 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-50 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-45 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-40 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-35 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-30 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-25 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 15-20 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 20-80 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 30-70 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 40-60 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 50-90 nucleotides. In some embodiments, the total length of the RNA blocking molecule is 30-60 nucleotides.

[0063] According to these embodiments, the present disclosure provides compositions and methods for performing an in situ hybridization (ISH) reaction to detect a target nucleic acid (e.g., RNA) in a sample using the RNA blocking molecules described herein. In some embodiments, the in situ hybridization detects a target nucleic acid comprising fewer than 100 nucleotides. In some embodiments, the target nucleic acid comprises 15-100 nucleotides. In some embodiments, the target nucleic acid comprises 15-80 nucleotides. In some embodiments, the target nucleic acid comprises 15-60 nucleotides. In some embodiments, the target nucleic acid comprises 15-50 nucleotides. In some embodiments, the target nucleic acid comprises 15-40 nucleotides. In some embodiments, the target nucleic acid comprises fewer than 90 nucleotides. In some embodiments, the target nucleic acid comprises fewer than 80 nucleotides. In some embodiments, the target nucleic acid comprises fewer than 70 nucleotides. In some embodiments, the target nucleic acid comprises fewer than 60 nucleotides. In some embodiments, the target nucleic acid comprises fewer than 50 nucleotides. In some embodiments, the target nucleic acid comprises fewer than 40 nucleotides. In some embodiments, the target nucleic acid comprises fewer than 30 nucleotides. In some embodiments, the target nucleic acid comprises fewer than 20 nucleotides. In some embodiments, the target nucleic acid contains fewer than 16 nucleotides. In some embodiments, the in situ hybridization is for detecting DNA. In some embodiments, the in situ hybridization is for detecting RNA. The methods provided herein can also be used to detect longer nucleic acids, for example, nucleic acids containing 100, 200, 300, 500, or 1,000 or more nucleotides.

[0064] In some embodiments, the methods provided herein comprise detecting small RNA species in a sample. In one embodiment, the RNA to be detected is less than 100 nucleotides. In one embodiment, the RNA to be detected is less than 50 nucleotides. In one embodiment, the RNA to be detected is less than 40 nucleotides. In one embodiment, the RNA to be detected is 10-40 nucleotides. In one embodiment, the RNA to be detected is 15-40 nucleotides. In one embodiment, the RNA to be detected is 30-40 nucleotides. In some embodiments, the methods provided herein detect naturally occurring small nucleic acids. Naturally occurring small nucleic acids perform vital and diverse cellular functions, from transcription and RNA processing to translation. A common characteristic of these RNAs is their size, ranging from 15-40 nucleotides in length. In another embodiment, the methods provided herein detect synthetic small nucleic acids.

[0065] In one embodiment, the method comprises detecting sncRNA. In one embodiment, the method comprises detecting miRNA. In one embodiment, the method comprises detecting siRNA. In one embodiment, the method comprises detecting piRNA. In one embodiment, the method comprises detecting endogenous RNA. In another embodiment, the method comprises detecting exogenous RNA. sncRNAs have been shown to have the ability to efficiently regulate gene expression in clinically relevant model systems as beneficial therapeutics for the medical treatment of disease (Watts et al., Journal of Pathology, 226(2), 365-379, 2012; Schoch et al., Neuron Review, 94, 1056-1070, 2017). miRNAs are naturally occurring small (approximately 22 nucleotide) regulatory RNAs present in all multicellular organisms, unicellular algae, and some viruses (Molnar et al., Nature, 447(7148), 2007; Bartel, Cell, 173, 20-51, 2018). To date, over 15,000 miRNAs from animals, plants, and viruses have been cataloged (www.mirbase.org), and many are expressed in tissue-, cell-type-, and cell-state-specific manners. Dysregulation of miRNA expression can lead to serious conditions, such as neurological disorders, infertility, immunodeficiency, or cancer.

[0066] According to these embodiments, the methods and compositions of the present disclosure comprise performing an in situ hybridization reaction to detect and / or quantify RNA in a biological sample. In one embodiment, the biological sample is or is derived from a tissue sample. In one embodiment, the biological sample is or is derived from a blood sample. In one embodiment, the biological sample is or is derived from a cytological sample. In one embodiment, the biological sample is cultured cells. In another embodiment, the biological sample is an exosome.

[0067] Tissue samples include, for example, tissue biopsy samples. Blood samples include, for example, blood samples collected for diagnostic purposes. In the case of blood samples, the blood can be analyzed directly, as in a blood smear, or the blood can be processed, for example, by lysing red blood cells, isolating PBMCs or white blood cells, isolating target cells, etc., so that the cells in the sample analyzed by the methods of the present disclosure are present in or extracted from the blood sample. Similarly, tissue samples can be processed, for example, by cutting the tissue sample into small pieces and treating it physically or enzymatically to break down the tissue into individual cells or cell clusters. Additionally, cytological samples can be treated to isolate cells or break down cell clusters, if desired. Thus, tissue, blood, and cytological samples can be obtained and processed using methods in the art. The methods of the present disclosure can be used in diagnostic applications to determine the presence or absence of diseased cells based on the presence or absence of nucleic acid targets that are biomarkers indicative of a disease state.

[0068] Based on the present disclosure, those skilled in the art will understand that any number of suitable samples can be used to detect target nucleic acids using the methods and compositions provided herein. The samples used in the methods provided herein are generally biological or tissue samples. Such samples can be obtained from a living subject and include samples derived from biological tissues or fluids collected from an individual or any other source of biological material (e.g., biopsy, autopsy, or diagnostic material). Biological samples also include samples obtained from areas of a living subject that contain or are suspected of containing precancerous cells, cancerous cells, precancerous tissue, or cancerous tissue, such as tissue biopsies (including fine needle aspirates, blood samples, or cytological specimens). Such samples can be, but are not limited to, organs, tissues, tissue fragments, cells, and / or exosomes isolated from an organism, such as a mammal. Exemplary biological samples include, but are not limited to, cell cultures (including primary cell cultures), cell lines, tissues, organs, organelles, biological fluids, and the like. Additional biological samples include, but are not limited to, skin samples, tissue biopsies (including fine needle aspirates), cytological samples, stool, bodily fluids (including blood and / or serum samples, saliva, semen), etc. Such samples can be used for diagnostic purposes in human or veterinary medicine.

[0069] Obtaining cytological samples for analysis by the methods provided herein is well known in the art (see, for example, 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).

[0070] For example, methods for processing samples (including tissue biopsies and cytological samples) for analysis of cervical tissue are well known in the art (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, Sellors 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).

[0071] In some embodiments, the sample is or is derived from a tissue sample. In some embodiments, the tissue sample is formalin-fixed, paraffin-embedded (FFPE). In some embodiments, the tissue sample is fresh-frozen. In some embodiments, the tissue sample is prepared with a fixative other than formalin. In some embodiments, the fixative other than formalin is selected from the group consisting of ethanol, methanol, Bouin's fixative, B5, and IBF. In other embodiments, the sample is or is derived from a blood sample. In other embodiments, the sample is or is derived from a cytological sample. In some embodiments, the method comprises treating the biological sample with a reagent for permeabilizing cells, a cross-linking reagent, and / or a protease.

[0072] Method for detecting target nucleic acid Embodiments of the present disclosure also include methods and compositions for performing in situ hybridization reactions using any of the RNA blocking molecules described herein, any of the kits described herein, and / or any of the compositions described herein. Embodiments of the present disclosure also include methods for improving signal efficiency in in situ hybridization reactions. According to these embodiments, the method includes contacting a biological sample containing a target RNA molecule with any of the RNA blocking molecules described herein. In some embodiments, the method includes contacting the biological sample with at least one target probe that specifically hybridizes to a probe-targeted region of the target RNA molecule. In some embodiments, the method includes contacting the biological sample with a signal-generating complex to detect the target RNA molecule. In some embodiments of the method, the signal efficiency for the target RNA molecule is improved compared to an in situ hybridization reaction that does not include contacting the biological sample with an RNA blocking molecule. In some embodiments of the method, the improved signal efficiency includes reducing binding of the target probe to non-target RNA molecules in the sample.

[0073] As further described herein, in some embodiments, the target RNA molecule is an mRNA molecule, a microRNA (miRNA) molecule, a small non-coding RNA (sncRNA) molecule, a PIWI-interacting RNA (piRNA) molecule, and / or a small interfering RNA (siRNA) molecule. As further described herein, in some embodiments of the method, the biological sample is or is derived from a tissue specimen, a blood sample, or is derived from a blood sample, or is or is derived from a cell sample.

[0074] In some embodiments of the method, the signal generating complex (SGC) comprises at least one label and at least one labeled probe, wherein the at least one labeled probe is capable of hybridizing to at least one target probe. In some embodiments of the method, the SGC comprises at least one label, at least one labeled probe, and at least one amplification agent hybridized to the at least one labeled probe and capable of hybridizing to the at least one target probe. In some embodiments of the method, the SGC comprises at least one label, at least one labeled probe, and an amplification agent hybridized to the at least one labeled probe, and at least one pre-amplification agent hybridized to the at least one amplification agent and capable of hybridizing to the target probe.

[0075] In some embodiments, the methods provided herein detect relatively short nucleic acids. For example, in some embodiments, in situ hybridization detects target nucleic acids containing fewer than 100 nucleotides. In some embodiments, the target nucleic acid contains 15-100 nucleotides. In some embodiments, the target nucleic acid contains 15-80 nucleotides. In some embodiments, the target nucleic acid contains 15-60 nucleotides. In some embodiments, the target nucleic acid contains 15-50 nucleotides. In some embodiments, the target nucleic acid contains 15-40 nucleotides. In some embodiments, the target nucleic acid contains fewer than 90 nucleotides. In some embodiments, the target nucleic acid contains fewer than 80 nucleotides. In some embodiments, the target nucleic acid contains fewer than 70 nucleotides. In some embodiments, the target nucleic acid contains fewer than 60 nucleotides. In some embodiments, the target nucleic acid contains fewer than 50 nucleotides. In some embodiments, the target nucleic acid contains fewer than 40 nucleotides. In some embodiments, the target nucleic acid contains fewer than 30 nucleotides. In some embodiments, the target nucleic acid contains fewer than 20 nucleotides. In some embodiments, the target nucleic acid contains fewer than 16 nucleotides.

[0076] In some embodiments, the in situ hybridization is for detecting small RNA species. In one embodiment, the RNA to be detected has fewer than 100 nucleotides. In one embodiment, the RNA to be detected has fewer than 50 nucleotides. In one embodiment, the RNA to be detected has fewer than 40 nucleotides. In one embodiment, the RNA to be detected has 10-40 nucleotides. In one embodiment, the RNA to be detected has 15-40 nucleotides. In one embodiment, the RNA to be detected has 30-40 nucleotides. In one embodiment, the method is for detecting sncRNA. In one embodiment, the method is for detecting miRNA. In one embodiment, the method is for detecting siRNA. In one embodiment, the method is for detecting piRNA. In one embodiment, the method is for detecting ASO. In one embodiment, the method is for detecting endogenous RNA. In one embodiment, the method is for detecting exogenous RNA.

[0077] In some embodiments, the in situ hybridization provided herein includes providing at least one set of one or more target probe(s) capable of hybridizing to the target nucleic acid; providing a signal-generating complex capable of hybridizing to the set of one or more target probe(s), wherein the signal-generating complex comprises a nucleic acid component capable of hybridizing to the set of one or more target probe(s) and a labeled probe; hybridizing the target nucleic acid to the set of one or more target probe(s); and capturing the signal-generating complex on the set of one or more target probe(s), thereby capturing the signal-generating complex on the target nucleic acid. In some embodiments, each set of one or more target probe(s) comprises a single probe. In other embodiments, each set of one or more target probe(s) comprises two probes. In still other embodiments, each set of one or more target probe(s) comprises three or more probes. In some embodiments, when each set of target probes comprises a single target probe, a signal-generating complex is formed when the single target probe binds to the target nucleic acid. In other embodiments, when each set of target probes comprises two target probes, a signal-generating complex is formed when both members of the target probe pair bind to the target nucleic acid.

[0078] In certain embodiments, the RNA ISH used herein is RNAscope®, which is described in more detail in, for example, U.S. Patent Nos. 7,709,198, 8,604,182, and 8,951,726. Specifically, RNAscope® describes the use of specially designed oligonucleotide probes in combination with branched DNA-like signal-generating complexes to reliably detect RNAs as small as 1 kilobase with single-molecule sensitivity under standard bright-field microscopy (Anderson et al., J. Cell. Biochem. 117(10):2201-2208(2016); Wang et al., J. Mol. Diagn. 14(1):22-29(2012)).

[0079] In some embodiments, each target probe comprises a target (T) section and a label (L) section, wherein the T section is a nucleic acid sequence complementary to a section on the target nucleic acid and the L section is a nucleic acid sequence complementary to a section on a nucleic acid component of the signal generation complex, wherein the T section of one or more target probe(s) is complementary to a non-overlapping region of the target nucleic acid and the L section of one or more target probe(s) is complementary to a non-overlapping region of the nucleic acid component of the signal generation complex.

[0080] In some embodiments, one set of one or more target probe(s) is used to detect the target nucleic acid. In other embodiments, two or more sets of one or more target probe(s) are used to detect the target nucleic acid. In some embodiments, two sets of one or more target probe(s) are used to detect the target nucleic acid. In some embodiments, three sets of one or more target probe(s) are used to detect the target nucleic acid. In some embodiments, four sets of one or more target probe(s) are used to detect the target nucleic acid. In some embodiments, five sets of one or more target probe(s) are used to detect the target nucleic acid. In some embodiments, six sets of one or more target probe(s) are used to detect the target nucleic acid. In some embodiments, seven sets of one or more target probe(s) are used to detect the target nucleic acid. In some embodiments, eight sets of one or more target probe(s) are used to detect the target nucleic acid. In some embodiments, nine sets of one or more target probe(s) are used to detect the target nucleic acid. In some embodiments, ten sets of one or more target probe(s) are used to detect the target nucleic acid. In some embodiments, eleven or more sets of one or more target probe(s) are used to detect the target nucleic acid. In some embodiments, 16 or more sets of one or more target probe(s) are used to detect the target nucleic acid. In some embodiments, 21 or more sets of one or more target probe(s) are used to detect the target nucleic acid. In some embodiments, 31 or more sets of one or more target probe(s) are used to detect the target nucleic acid. In some embodiments, the methods provided herein are for detecting a plurality of nucleic acid targets. In some embodiments, all of the plurality of nucleic acid targets comprise fewer than 100 nucleotides. In other embodiments, some of the nucleic acid targets comprise fewer than 100 nucleotides, while other targets comprise more than 100 nucleotides.

[0081] As used herein, "target probe" generally refers to a polynucleotide that can hybridize to a target nucleic acid to capture or bind a labeled probe or a component of a signal generating complex (SGC) to that target nucleic acid. The target probe can hybridize directly to the labeled probe or to one or more nucleic acids that sequentially hybridize to the labeled probe; for example, the target probe can hybridize to an amplifier, preamplifier, or pre-preamplifier in an SGC. That is, the target probe comprises a first polynucleotide sequence that is complementary to the polynucleotide sequence of the target nucleic acid and a second polynucleotide sequence that is complementary to the polynucleotide sequence of the labeled probe, amplifier, preamplifier, or pre-preamplifier. Target probes are generally single-stranded, such that the complementary sequence is available for hybridization to the corresponding target nucleic acid, labeled probe, amplifier, preamplifier, or pre-preamplifier. In some embodiments, target probes are provided in pairs.

[0082] As used herein, the term "labeled probe" refers to an entity that binds directly or indirectly, generally indirectly, to a target molecule, making the target detectable. A labeled probe (or "LP") comprises a nucleic acid-binding moiety, typically a single-stranded polynucleotide or oligonucleotide, containing one or more labels that provide a directly or indirectly 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 be attached to a streptavidin-linked label. A labeled probe can, for example, hybridize directly to a target nucleic acid. Generally, a labeled probe can hybridize to a nucleic acid that is sequentially hybridized to a target nucleic acid, or to one or more other nucleic acids that are hybridized to the target nucleic acid. That is, a labeled probe can comprise a polynucleotide sequence that is complementary to the polynucleotide sequence of a target nucleic acid, particularly a portion of the target nucleic acid. Alternatively, a labeled probe can comprise at least one polynucleotide sequence that is complementary to the polynucleotide sequence of an amplifier, preamplifier, or pre-preamplifier in an SGC.

[0083] In some embodiments, the SGCs provided herein include additional descriptions regarding such amplification agents, preamplification agents, and / or pre-preamplification agents. As used herein, an "amplification agent" is a molecule, typically a polynucleotide, that can hybridize to multiple labeled probes. Typically, an amplification agent hybridizes to multiple identical labeled probes. An amplification agent can also hybridize to a target nucleic acid, at least one target probe of a target probe pair, both target probes of a target probe pair, or a nucleic acid bound to a target probe (such as an amplification agent, preamplification agent, or pre-preamplification agent). For example, an amplification agent can hybridize to at least one target probe and multiple labeled probes, or a preamplification agent and multiple labeled probes. An amplification agent can be, for example, a linear, forked, comb-like, or branched nucleic acid. As described herein for all polynucleotides, an amplification agent can include modified nucleotides and / or non-standard internucleotide linkages, as well as standard deoxyribonucleotides, ribonucleotides, and / or phosphodiester linkages. Suitable amplification agents are described, for example, in U.S. Pat. Nos. 5,635,352, 5,124,246, 5,710,264, 5,849,481, and 7,709,198, and U.S. Publication Nos. 2008 / 0038725 and 2009 / 0081688, each of which is incorporated by reference.

[0084] As used herein, "preamplifier" refers to a molecule, typically a polynucleotide, that serves as an intermediate binding component between one or more target probes and one or more amplification agents. Typically, a preamplifier is simultaneously hybridized to one or more target probes and multiple amplification agents. Exemplary preamplifiers are described in, for example, U.S. Patent Nos. 5,635,352, 5,681,697 and 7,709,198, and U.S. Publication Nos. 2008 / 0038725, 2009 / 0081688 and 2017 / 0101672, each of which is incorporated by reference.

[0085] As used herein, a "pre-preamplifier" is a molecule, typically a polynucleotide, that serves as an intermediate binding component between one or more target probes and one or more preamplifiers. Typically, a pre-preamplifier simultaneously hybridizes to one or more target probes and multiple preamplifiers. Exemplary pre-preamplifiers are described, for example, in U.S. Pat. No. 11,078,528, which is incorporated by reference.

[0086] Labels are typically used to detect target nucleic acids in RNA in situ hybridization. As used herein, a "label" is a moiety that facilitates detection of a molecule. Common labels include fluorescent labels, luminescent labels, light scattering labels, and / or colorimetric labels. Suitable labels include enzymes, fluorescent and chromogenic moieties, as well as radionuclides, substrates, cofactors, inhibitors, chemiluminescent moieties, magnetic particles, rare earth metals, metal isotopes, and the like. In certain embodiments, the label is an enzyme. Exemplary enzymatic labels include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, glucose oxidase, and various proteases. Other labels include, but are not limited to, fluorophores and dinitrophenyl phosphate (DNP). 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. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Many labels, including detectable enzyme / substrate combinations (Pierce, Rockford IL; Santa Cruz Biotechnology, Dallas TX; Life Technologies, Carlsbad CA), are commercially available and can be used in the methods and assays of the present disclosure. In certain embodiments of the present disclosure, the enzyme can utilize a chromogenic or fluorogenic substrate to generate a detectable signal, as described herein. Exemplary labels are described herein.

[0087] Any number of enzymatic or non-enzymatic labels can be used, so long as they are detectable, respectively. The enzyme generates a detectable signal that can be used to detect the target nucleic acid. Particularly useful detectable signals are chromogenic or fluorescent signals. Therefore, particularly useful enzymes for use as labels include enzymes for which chromogenic or fluorescent substrates are available. Such chromogenic or fluorescent substrates are converted by an enzymatic reaction into a readily detectable chromogenic or fluorescent product, which can be readily detected and / or quantified using microscopic or spectroscopic methods. Such enzymes are well known to those skilled in the art and include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, and the like (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Other enzymes with well-known chromogenic or fluorescent substrates include various peptidases, whose chromogenic or fluorescent peptide substrates can be used to detect protein cleavage reactions. The use of chromogenic and fluorogenic substrates is also well known in bacterial diagnostics and includes, but is not limited to, the use of α-galactosidase, β-galactosidase, β-glucuronidase, 6-phospho-β-D-galactoside 6-phosphogalactohydrolase, β-glucosidase, α-glucosidase, amylase, neuraminidase, esterase, and lipase (Manafi et al., Microbiol. Rev. 55:335-348 (1991)), and such enzymes with known chromogenic or fluorogenic substrates can be readily adapted for use in the methods provided herein.

[0088] Various chromogenic or fluorogenic substrates for producing a detectable signal are well known to those skilled in the art and are commercially available. Exemplary substrates that can be used to produce a detectable signal include 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), chloronaphthol (4-CN) (4-chloro-1-naphthol), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine dihydrochloride (OPD), and 3-amino-9-ethylcarbazole (AEC) for horseradish peroxidase, and 5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP), nitroblue tetrazolium (NBT), Fast Red (Fast Red), and the like for alkaline phosphatase. β-D-glucosidase. Exemplary fluorescent substrates include, but are not limited to, 4-(trifluoromethyl)umbelliferyl phosphate for alkaline phosphatase, 4-methylumbelliferyl phosphate bis(2-amino-2-methyl-1,3-propanediol), 4-methylumbelliferyl phosphate bis(cyclohexylammonium) and 4-methylumbelliferyl phosphate for phosphatase, QuantaBlu™ and Quintolet™ for horseradish peroxidase, and β-galactosidase. Examples of suitable galactosidase inhibitors include, but are not limited to, 4-methylumbelliferyl β-D-galactopyranoside, fluorescein di(β-D-galactopyranoside) and naphthofluorescein di(β-D-galactopyranoside) for β-glucosidase, 3-acetylumbelliferyl β-D-glucopyranoside and 4-methylumbelliferyl-β-D-glucopyranoside for β-glucosidase, and 4-methylumbelliferyl-α-D-galactopyranoside for α-galactosidase.Exemplary enzymes and substrates that generate detectable signals are also described, for example, in U.S. Publication No. 2012 / 0100540. A variety of detectable enzyme substrates, including chromogenic or fluorogenic substrates, are well known and commercially available (Pierce, Rockford, IL; Santa Cruz Biotechnology, Dallas, TX; Invitrogen, Carlsbad, CA; 42 Life Science, Biocare). Generally, the substrate is converted to a product that forms a precipitate that deposits at the site of the target nucleic acid. 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).

[0089] Exemplary rare earth metals and metal isotopes suitable as detectable labels include, but are not limited to, lanthanide(III) isotopes such as Pr, Nd, Nd, Nd, Nd, Nd, Sm, Nd, Sm, Nd, Eu, Sm, Eu, Sm, Gd, Gd, Gd, Tb, Gd, Dy, Dy, Dy, Dy, Ho, Er, Er, Tm, Er, Yb, Yb, Yb, Lu, and Yb. Metal isotopes can be detected, for example, using time-of-flight mass spectrometry (TOF-MS) (eg, Fluidigm's Helios and Hyperion systems, fluidigm.com / systems; South San Francisco, Calif.).

[0090] Biotin-avidin (or biotin-streptavidin) is a well-known signal amplification system based on the observation that the two molecules have a very high affinity for each other and that one avidin / streptavidin molecule can bind four biotin molecules. Antibodies are widely used for signal amplification in immunohistochemistry and ISH. Tyramide signal amplification (TSA) is based on the deposition of numerous haptenized tyramide molecules by peroxidase activity. Tyramine is a phenolic compound. In the presence of small amounts of hydrogen peroxide, immobilized horseradish peroxidase (HRP) converts a labeled substrate into a highly reactive, short-lived intermediate. The activated substrate molecule then reacts very rapidly with and covalently binds to electron-rich protein moieties (e.g., tyrosine) at or near the peroxidase binding site. In this manner, many hapten molecules conjugated to tyramide can be introduced in situ at the hybridization site. The deposited tyramide-hapten molecules can then be visualized directly or indirectly. Such detection systems are described in further detail, for example, in U.S. Patent No. 8,658,361.

[0091] In the embodiments described herein, enzymes can be used to generate a detectable signal using an appropriate chromogenic or fluorogenic substrate. Alternatively, it is understood that labeled probes can have a detectable label directly attached to the nucleic acid portion of the labeled probe. Exemplary detectable labels are well known to those of skill in the art and include, but are not limited to, chromogenic or fluorescent labels (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Exemplary fluorophores useful as labels include rhodamine derivatives, such as 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, etc.; 7-nitrobenz-2-oxa-1,3-diazole (NBD); fluorescein and its derivatives; naphthalenes, such as dansyl (5-dimethylaminonaphthalene-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 Fluorescent Dyes (Molecular Probes); 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY™) and its derivatives (Molecular Probes; Eugene, OR); pyrene and sulfonated pyrenes, such as Cascade Blue™ and its derivatives, such as 8-methoxypyrene-1,3,6-trisulfonic acid; pyridyloxazole derivatives and dapoxyl derivatives (Molecular Probes); Lucifer Yellow (3,6-disulfonate-4-amino-naphthalimide) and its derivatives; 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 Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 643, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740, Cyan 500 NHS-Ester (ATTO-TECH, Siegen, Germany), etc. Exemplary chromophores include, but are not limited to, phenolphthalein, malachite green, aromatic nitro compounds (such as nitrophenyl), diazo dyes, dabcyl (4-dimethylaminoazobenzene-4'-sulfonyl), etc.;

[0092] As disclosed herein, the methods provided herein can be used for the simultaneous detection of multiple target nucleic acids. When fluorophores are used as labels, the fluorophores used for the detection of multiple target nucleic acids are selected so that, in the case of simultaneous detection of target nucleic acids, each fluorophore is distinguishable and the fluorophores can be simultaneously detected by a fluorescence microscope. Such fluorophores are selected so that the emission lines are spectrally separated so that the distinct labels of the target nucleic acids can be simultaneously detected. Methods for selecting distinguishable fluorophores suitable for use in the methods of the present disclosure are well known in the art (see, for example, Johnson and Spence, "Molecular Probes Handbook, a Guide to Fluorescent Probes and Labeling Technologies," 11th ed., Life Technologies (2010)).

[0093] Methods such as microscopy, cytometry (e.g., mass cytometry, time-of-flight cytometry (CyTOF), flow cytometry), or spectroscopy can be used to visualize the detectable chromogenic, fluorescent, or metallic signals associated with each target nucleic acid. Generally, when different labels are used in the same assay, such that a single instrument can be used to detect nucleic acid targets in the same sample, either chromogenic or fluorogenic substrates, or chromogenic or fluorescent labels, or rare earth metal isotopes are used, depending on the particular assay.

[0094] As disclosed herein, the label can be designed to be 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, to use the same label in a second or subsequent round of labeling and detecting a target nucleic acid. Generally, the label is conjugated to the label probe by a cleavable chemical linker. Methods for conjugating a label to a label probe so that the label is cleavable are well known to those skilled in the art (see, for example, Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996); Daniel et al., BioTechniques 24(3):484-489 (1998)). One specific system for labeling oligonucleotides is the FastTag™ system (Daniel et al., supra, 1998, Vector Laboratories, Burlinghame CA). Various cleavable moieties can be included in the linker to allow the label to be cleaved from the label probe. Such cleavable moieties include groups that can be cleaved chemically, photochemically, or enzymatically. Cleavable chemical linkers can include cleavable chemical moieties such as disulfides that can be cleaved by reduction, glycols or diols that can be cleaved by periodate, diazo bonds that can be cleaved by dithionite, esters that can be cleaved by hydroxylamine, sulfones that can be cleaved by base, and the like (see Hermanson, 1996, supra). One particularly useful cleavable linker is a linker containing a disulfide bond that can be cleaved by reducing the disulfide bond. In another embodiment, the linker can include a site for enzymatic cleavage. For example, the linker can include a proteolytic cleavage site. Typically, such cleavage sites are for sequence-specific proteases. Examples of such proteases include human rhinovirus 3C protease (cleavage site: LEVLFQ / GP), enterokinase (cleavage site: DDDDK / ), factor X protease (cleavage site: DDDDK / ), and factor X protease (cleavage site: DDDDK / ). aExamples of cleavable moieties include, but are not limited to, thrombin (cleavage site: LVPR / GS), tobacco etch virus protease (cleavage site: ENLYFQ / G), and thrombin (cleavage site: LVPR / GS) (see, e.g., Oxford Genetics, Oxford, UK). Other cleavable moieties 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)).

[0095] Cleavable labels can be removed by applying an agent such as a chemical agent or light to cleave the label and dissociate it from the labeled probe. As discussed above, useful cleavage agents for chemical cleavage include, but are not limited to, reducing agents, periodate, dithionite, hydroxylamine, and bases (see Hermanson, 1996, supra). One useful method for cleaving a linker containing a disulfide bond is to use 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 the agent that cleaves the label from the labeled probe.

[0096] In some embodiments, the methods for detecting a target nucleic acid in a cell provided herein include a pretreatment step prior to hybridization of a target probe(s). In some embodiments, the pretreatment step includes a blocking step in which specific blocking agent(s) are applied to block specific endogenous components of the cell, thereby reducing the background of the assay. As further described herein, the blocking step may include contacting the biological sample with a composition comprising a plurality of RNA blocking molecules described herein. According to these methods, the use of the disclosed RNA blocking molecules improves signal efficiency, resulting in improved detection of a target nucleic acid using a target probe or a series of target probes compared to when RNA blocking molecules are not used.

[0097] In some embodiments, various other blocking agents can be used in addition to the RNA blocking molecules of the present disclosure. For example, when horseradish peroxidase (HRP) is used as the detection enzyme in a subsequent step, hydrogen peroxide is the blocking agent. Hydrogen peroxide is added to inactivate endogenous HRP activity in the sample, thereby reducing the background of the assay. In certain embodiments, this blocking step is added as the first pretreatment step, immediately after deparaffinization. In some embodiments, the pretreatment step includes an epitope antigen retrieval step, in which specific epitope antigen retrieval buffer(s) can be added to expose the target nucleic acid. In some embodiments, the epitope antigen retrieval step includes heating the sample. In some embodiments, the epitope antigen retrieval step includes heating the sample to 50°C to 100°C. In one embodiment, the epitope antigen retrieval step includes heating the sample to approximately 88°C. In some embodiments, the pretreatment step includes a permeabilization step that retains nucleic acid targets in the cells and allows target probe(s), signal-generating complexes, and the like to enter the cells. In some embodiments, the permeabilization step includes digestion with a protease. Detergents (e.g., Triton X-100 or SDS) and Proteinase K can also be used to increase the permeability of fixed cells. Typically, detergent treatment using Triton X-100 or SDS is frequently used to extract lipids and permeabilize cell membranes. Proteinase K is a nonspecific protease that is active over a wide pH range and is not easily inactivated. Proteinase K is used to digest proteins surrounding target mRNA. The optimal concentration and duration of treatment can be determined empirically, as is well known in the art. A cell washing step can then be performed to remove lysed materials generated in any of the pretreatment steps. In some embodiments, the sample is in formalin-fixed, paraffin-embedded tissue, and a deparaffinization step is required to remove the paraffin.

[0098] In some embodiments, the methods for detecting a target nucleic acid in a cell provided herein include a post-fixation step at a specific time. In one embodiment, the post-fixation step is (i) after the first fixation step and (ii) before applying at least one set of one or more target probe(s) capable of hybridizing to the target nucleic acid. In one embodiment, the post-fixation step is (i) after the first fixation step and (ii) before the pretreatment step described in the immediately preceding paragraph. In one embodiment, the post-fixation step is (i) after the first fixation step and (ii) before the blocking step described in the immediately preceding paragraph. In one embodiment, the post-fixation step is (i) after the first fixation step and (ii) before the epitope antigen retrieval step described in the immediately preceding paragraph. In one embodiment, the post-fixation step is (i) after the first fixation step and (ii) before the permeabilization step described in the immediately preceding paragraph. In one embodiment, the post-fixation step is (i) after the deparaffinization step and (ii) before applying at least one set of one or more target probe(s) capable of hybridizing to the target nucleic acid. In one embodiment, the post-fixation step is (i) after the deparaffinization step and (ii) before the pretreatment step described in the immediately preceding paragraph. In one embodiment, the post-fixation step is (i) after the deparaffinization step and (ii) before the blocking step described in the immediately preceding paragraph. In one embodiment, the post-fixation step is (i) after the deparaffinization step and (ii) before the epitope antigen retrieval step described in the immediately preceding paragraph. In one embodiment, the post-fixation step is (i) after the deparaffinization step and (ii) before the permeabilization step described in the immediately preceding paragraph.

[0099] The methods provided herein have several applications in research and diagnostics (Hanna et al., Frontiers in Genetics, 10,1-6, 2019; Watts et al., Journal of Pathology, 226(2), 365-379, 2012). The methods provided herein may improve understanding of small nucleic acids, such as sncRNAs, miRNAs, siRNAs, piRNAs, and ASOs, in their natural context and the gene regulatory networks to which these small nucleic acids are linked, implicating various health and disease processes.

[0100] In some embodiments, the methods provided herein can detect small RNAs with temporal and spatial resolution. In one embodiment, the methods provided herein can be used to distinguish between tissues and cell types. In one embodiment, the methods provided herein can be used to distinguish between different stages of development. In one embodiment, the methods provided herein can be used to characterize adult tissues.

[0101] In some embodiments, the methods provided herein can be used to detect the expression of altered small RNAs or the presence of small RNAs associated with pathogens. In one embodiment, the methods provided herein can be used to diagnose a disease or disorder. In one embodiment, the methods provided herein can be used to diagnose a pathogen.

[0102] In some embodiments, the methods provided herein are for monitoring the effectiveness of small RNA-based therapy. In one embodiment, the methods provided herein are for monitoring the effectiveness of siRNA-based therapy. In one embodiment, the methods provided herein are for monitoring the effectiveness of ASO-based therapy. In some embodiments, the methods provided herein are for determining the effectiveness of small RNA-based therapy. In one embodiment, the methods provided herein are for determining the effectiveness of siRNA-based therapy. In one embodiment, the methods provided herein are for determining the effectiveness of ASO-based therapy.

[0103] In certain embodiments, the method can be used to detect the presence of siRNA after delivery of siRNA to a disease model.In certain embodiments, the method can be used to localize siRNA after delivery of siRNA to a disease model.In certain embodiments, the method can be used to quantify siRNA after delivery of siRNA to a disease model.In certain embodiments, the method can be used to quantify the RNA that siRNA targets after delivery of siRNA to a disease model.

[0104] In certain embodiments, the method can be used to detect the presence of ASO after delivery of ASO to a disease model.In certain embodiments, the method can be used to localize ASO after delivery of ASO to a disease model.In certain embodiments, the method can be used to quantify ASO after delivery of ASO to a disease model.In certain embodiments, the method can be used to quantify RNA targeted by ASO after delivery of ASO to a disease model.

[0105] Kits for in situ detection of target nucleic acids Embodiments of the present disclosure also include kits containing any of the RNA blocking molecules described herein. The kits can also include any of the components described herein for performing an in situ hybridization reaction. In some embodiments, the kits include at least one target probe that specifically hybridizes to a probe-targeted region of a target RNA molecule. In some embodiments, the kits further include one or more target probe sets, each target probe set including a pair of target probes that specifically hybridize to a probe-targeted region of a target RNA molecule. In some embodiments, the kits include a reagent for permeabilizing cells, a cross-linking reagent, and / or a protease. In some embodiments, the kits include instructions for performing an in situ hybridization reaction.

[0106] In some embodiments, the kit includes one or more components of a signal-generating complex, which components include: (i) at least one label and at least one labeled probe, wherein the at least one labeled probe is capable of hybridizing to at least one target probe; or (ii) at least one label, at least one labeled probe, and at least one amplification agent that hybridizes to the at least one labeled probe and that can hybridize to the at least one target probe; or (iii) at least one label, at least one labeled probe, at least one amplification agent hybridized to the at least one labeled probe, and at least one pre-amplification agent that hybridizes to the at least one amplification agent and that can hybridize to the target probe.

[0107] Embodiments of the present disclosure also include compositions comprising any of the RNA blocking molecules described herein. In some embodiments, the composition comprises a hybridization buffer. In some embodiments, the composition comprises a reagent for permeabilizing cells, a cross-linking reagent, and / or a protease. In some embodiments, the composition comprises a biological sample. In some embodiments, the biological sample is or is derived from a tissue sample. In some embodiments, the biological sample is or is derived from a blood sample. In some embodiments, the biological sample is or is derived from a cytological sample. In some embodiments, the biological sample is cultured cells or an exosome-containing sample. In some embodiments, the composition further comprises at least one target probe that specifically hybridizes to a probe-targeted region of a target RNA molecule in the biological sample.

[0108] In some embodiments, the composition comprises one or more components of a signal-generating complex, in some embodiments, the components of the signal-generating complex comprise: (i) at least one label and at least one labeled probe, wherein the at least one labeled probe is capable of hybridizing to at least one target probe; or (ii) at least one label, at least one labeled probe, and at least one amplification agent that hybridizes to the at least one labeled probe and that can hybridize to the at least one target probe; or (iii) at least one label, at least one labeled probe, at least one amplification agent hybridized to the at least one labeled probe, and at least one pre-amplification agent that hybridizes to the at least one amplification agent and that can hybridize to the target probe.

[0109] In some embodiments, the kit includes an agent for fixing a biological sample. In some embodiments, the kit includes fixative(s) suitable for preserving nucleic acids. In one embodiment, the fixative is FineFix (see Kothmaier et al., Arch. Pathol. Lab. Med. 135:744-752, 2011). In one embodiment, the fixative is Glyo-fix (see Lykidis et al., Nucleic Acids Res. 35:e85, 2007). In one embodiment, the fixative is Histochoice (see Vince et al., 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 PAXgene Tissue System (see Nietner et al., Int. J. Pathol. 461:259-269, 2012). In one embodiment, the fixative is RCL2 (see van Essen et al., Clin. Pathol. 63:1090-1094, 2010). In one embodiment, the fixative is Streck's Tissue Fixative (see Burns et al., Histochem. Cytochem. 57:257-264, 2009). In one embodiment, the fixative is UMFIX (see Nadji et al., Appl. Immunohistochem. 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 Cytochem.:Official J. Histochem. Soc. 58:237-246, 2010).

[0110] In some embodiments, the kits provided herein comprise 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 INaO4. 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 sulfate. In one embodiment, the aldehyde-containing fixative in the kit is Helly fixative, which is a solution of formaldehyde, potassium dichromate, sodium sulfate, and mercuric chloride. In one embodiment, the aldehyde-containing fixative in the kit is Hollande 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 solution, which is a solution of formaldehyde, ethanol, picric acid, and glacial acetic acid. In one embodiment, the aldehyde-containing fixative in the kit is alcohol-formalin, which is a solution of formaldehyde, ethanol, and calcium acetate.In one embodiment, the aldehyde-containing fixative in the kit is formol-alcohol acetate, which is a solution of formaldehyde, glacial acetic acid, and ethanol.In one embodiment, the aldehyde-containing fixative in the kit is a mixture of fixatives, where at least one fixative of the mixture is formaldehyde or glutaraldehyde. In one embodiment, the aldehyde-containing fixatives in the kit are non-simultaneously but sequentially used fixatives, where at least one fixative is formaldehyde or glutaraldehyde.

[0111] In some embodiments, the aldehyde-containing fixative in the kits provided herein contains about 5% to about 50% formaldehyde. In other embodiments, the aldehyde-containing fixative contains about 10% to about 40% formaldehyde. In still other embodiments, the aldehyde-containing fixative contains about 12% to about 37% formaldehyde.

[0112] In some embodiments, the aldehyde-containing fixative in the kits provided herein contains various concentrations of formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 5% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 6% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 7% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 8% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 9% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 10% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 11% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 12% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 13% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 14% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 15% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 16% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 17% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 18% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 19% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 20% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 30% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 35% formaldehyde, hi one embodiment, the aldehyde-containing fixative comprises about 40% formaldehyde, hi one embodiment, the aldehyde-containing fixative comprises about 50% formaldehyde.

[0113] In some embodiments, the kit further comprises a tool for obtaining a biological sample from a subject. In certain embodiments, the biological sample is or is derived from a tissue sample. In certain embodiments, the biological sample is or is derived from a blood sample. In certain embodiments, the biological sample is or is derived from a cytological sample.

[0114] In some embodiments, the target nucleic acid is DNA. In some embodiments, the target nucleic acid is RNA. In some embodiments, the target nucleic acid is long-chain RNA. In some embodiments, the target nucleic acid is short-chain RNA. In some embodiments, the target nucleic acid is RNA containing fewer than 100 nucleotides. In other embodiments, the target nucleic acid is RNA containing fewer than 50 nucleotides. In other embodiments, the target nucleic acid is RNA containing 15-40 nucleotides. In some embodiments, the target nucleic acid is sncRNA. In other embodiments, the target nucleic acid is miRNA, siRNA, piRNA, or ASO. In still other embodiments, the target nucleic acid is endogenous RNA or exogenous RNA.

[0115] In certain embodiments, kits provided herein contain agents for performing RNAscope®, e.g., as described in more detail in U.S. Patent Nos. 7,709,198, 8,604,182, and 8,951,726. In some embodiments, the kits contain at least one set of one or more target probe(s) capable of hybridizing to a target nucleic acid and a signal-generating complex capable of hybridizing to the set of one or more target probe(s), wherein the signal-generating complex comprises a labeled probe and a nucleic acid component capable of hybridizing to the set of one or more target probe(s). In some embodiments, the target probe(s) comprise a target (T) section and a label (L) section, wherein the T section is a nucleic acid sequence complementary to a section on the target nucleic acid and the L section is a nucleic acid sequence complementary to a section on a nucleic acid component of the signal generation complex, wherein the T section of one or more target probe(s) is complementary to a non-overlapping region of the target nucleic acid and the L section of one or more target probe(s) is complementary to a non-overlapping region of the nucleic acid component of the signal generation complex.

[0116] In some embodiments, the kit further comprises a signal generation complex as described above, which may include a labeled probe, an amplification agent, a preamplification agent, and / or a pre-preamplification agent. In some embodiments, the kit further comprises other agents or materials for performing RNA ISH, such as fixatives and agents for treating the sample in preparation for hybridization, and agents for washing the sample.

[0117] The kit may further include "packaging material," which refers to a physical structure that contains the components of the kit. The packaging material can maintain the sterility of the components and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0118] The kits provided herein can include a label or insert. The label or insert includes information about the condition, disorder, disease, or symptom for which the kit components may be used. The label or insert can include instructions for a clinician or subject to use one or more of the kit components in a method, treatment protocol, or treatment regimen. In some embodiments, the kits can be used to identify tissues and cell types. In some embodiments, the kits can be used to distinguish different stages of development. In some embodiments, the kits can be used to detect clinical biomarkers for cancer. In some embodiments, the kits can be used to diagnose diseases or disorders based on the expression of one or more denatured small RNAs or the presence of small RNAs associated with pathogens. In some embodiments, the kits can be used to characterize adult tissues. In some embodiments, the kits can be used to detect clinical biomarkers for pathogen diagnosis. In some embodiments, the kits can be used to detect and characterize small RNA-based therapeutics. In some embodiments, the kits can be used for initial validation of small RNA-based therapeutics. In some embodiments, the kits can be used to monitor the effectiveness of small RNA-based therapies. In some embodiments, the kits can be used to determine the effectiveness of small RNA-based therapies. In some embodiments, the kits can be used to detect, localize, and quantify the presence of siRNAs. In some embodiments, the kits can be used to detect, localize, and quantify the presence of ASO molecules. In some embodiments, the kits can be used to detect and identify small RNAs derived from pathogens.

[0119] Image Processing Embodiments of the present disclosure also include methods for improving detection of a target (e.g., a target nucleic acid). In some embodiments, the methods include image processing methods, such as those described in International Patent Application PCT / US22 / 24975, incorporated herein by reference. The methods are performed, at least in part, using a computer with corresponding instructions stored on a storage device (i.e., a non-transitory computer-readable medium). Final images from the methods, and in some embodiments, intermediate images, are stored in the storage device. In some embodiments, the storage device is accessible by a network. In some embodiments, user input or instructions can be received or accessed over the network.

[0120] The method includes imaging a sample with a target signal to create a probe image, and imaging a sample without a target signal to create a background image (i.e., a "blank image"). In some embodiments, the "blank image" is an image that includes an RNA blocking molecule of the present disclosure. In some embodiments, the "blank image" is an image that does not include an RNA blocking molecule of the present disclosure. In some embodiments, the imaging utilizes a fluorescent microscope connected to a computer via a network. In some embodiments, the target signal is obtained by subjecting the sample to a fluorescent in situ hybridization assay and / or an immunofluorescence assay. In some embodiments, the background image without the target signal is obtained by removing the target signal from the sample (i.e., by a cleavage process). In other embodiments, the background image without the target signal is obtained before the assay is performed. In some embodiments, the target signal comprises a fluorescent label attached to the target nucleic acid. In other embodiments, the target signal comprises a fluorescent label attached to the target peptide or target polypeptide.

[0121] The method can also include registering the probe image with a background image (e.g., with or without the RNA blocking molecule). Possible background fluorescence differences between the probe image and the background image result in spatial pattern shifts due to global sample movement between different rounds of image acquisition. To remove such differences, image registration techniques (e.g., phase correlation) are utilized. Robust image registration utilizes image feature detection and matching to correct for any global sample movement (i.e., translation and rotation).

[0122] The method further includes correcting the background image (e.g., with or without the RNA blocking molecule) based on at least one image metric to create an adjusted background image (e.g., a transformed, intensity-adjusted blank image). As further described herein, the at least one image metric is a ratio factor, a multiplication factor, a local maximum 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.

[0123] In some embodiments, the method further includes subtracting the adjusted background image (e.g., with or without the RNA blocking molecule) from the probe image to create a final image containing an enhanced target signal. In other words, a modified (i.e., transformed, adjusted, scaled, etc.) blank image is used in the subtraction step instead of the original blank image. In some embodiments, the enhanced target signal includes contrast enhancement. In some embodiments, the method includes displaying the final image on a display (e.g., a computer display). The final image can be saved to a storage device and accessible to a user, for example, over a network. In this way, the method provides improved signal detection in the presence of a background involving tissue autofluorescence.

[0124] In some embodiments, the image metric is a ratio factor that accounts for the background intensity difference between the blank image and the probe image. The intensity difference can occur when image acquisition settings are different or from photobleaching during fluorophore excitation. To correct for the background intensity difference, the method includes determining a ratio factor that compares the overall background intensity of the probe image versus the blank image. First, the pixel location of the probe is estimated. The probe location in the probe image is estimated using, for example, a White Top Hat algorithm (Gonzalez & Woods, 2008, Digital Image Processing), a bandpass filter (Shenoi, 2006, Introduction to Digital Signal Processing and Filter Design), or any combination of suitable methods. After determining the estimated location of the target signal in the probe image, the pixels at the estimated probe location are removed from the probe image and the blank image, resulting in an image of only the background pixels (i.e., a background-only image). In other words, the method includes removing the estimated location from the probe image to create a first background-only image, and removing the estimated location from the blank image (background image) to create a second background-only image.

[0125] Following removal of the estimated probe location from both images, the method includes determining a ratio factor. In other words, statistical metrics for the probe-removed blank image and the probe-removed probe image are evaluated and incorporated into the ratio factor. In some embodiments, the ratio factor is utilized to modify the background image to create an adjusted background image. In other words, modifying the background image to create an adjusted background image may, in some embodiments, include scaling the background image by the ratio factor.

[0126] In some embodiments, at least one image metric is a ratio factor of a first background-only image and a second background-only image. For example, in some embodiments, the ratio factor is a first intensity to a second intensity, where the first intensity is determined from the first background-only image and the second intensity is determined from the second background-only image. In some embodiments, the first and second intensities used in the ratio factor are statistical metrics for any portion (including all) of the intensity values ​​in the image, such as a statistical mean, median, or a combination of both.

[0127] In some embodiments, the first intensity is the average of a plurality of pixel intensity values ​​in the first background-only image, and the second intensity is the average of a plurality of pixel intensity values ​​in the second background-only image. In some embodiments, the average is the average of all pixel intensity values ​​in the image. In other embodiments, the first intensity is the median of a plurality of pixel intensity values ​​in the first background-only image, and the second intensity is the median of a plurality of pixel intensity values ​​in the second background-only image. In some embodiments, the median is the median of all pixel intensity values ​​in the image. In another embodiment, the first intensity is the average of approximately the middle 80% of all pixel intensity values ​​(i.e., excluding approximately the top 10% and the bottom 10%) of all pixel intensity values ​​in the first background-only image, and the second intensity is the average of approximately the middle 80% of all pixel intensity values ​​in the second background-only image.

[0128] In some embodiments, the image metric is a multiplication factor that accounts for possible local intensity differences between the blank image and the probe image. In particular, the method includes determining a multiplication factor. In some embodiments, the multiplication factor is in the range of about 1.0 to about 1.2. In other embodiments, the multiplication factor is in the range of about 1.0 to about 1.1. In some embodiments, the multiplication factor is utilized to modify the background image to create an adjusted background image. In other words, modifying the background image to create an adjusted background image, in some embodiments, can include scaling the background image by the multiplication factor.

[0129] In some embodiments, the image metric is a local maximum transform. In particular, the method includes transforming the blank image using the local maximum transform. Even after global image registration, local background pattern shifts may remain, for example, from image acquisition at different focal planes, poor sample adhesion to the support (e.g., a glass slide), and partial sample movement between imaging sessions. To address this issue, the local shifts are corrected accordingly. In the illustrated embodiment, for each pixel ("pixel of interest") in the blank image, a neighborhood of a predetermined radius surrounding the pixel of interest is searched. The search process finds the pixel with the maximum intensity, and this maximum intensity is assigned to the pixel of interest. This search procedure is performed for each pixel of interest, and a neighborhood of each pixel of interest in the original blank image is searched to form a transformed blank image. As described in more detail herein, the transformed blank image can be used in place of the original blank image in a subsequent subtraction process. In some embodiments, the predetermined radius ("match distance") is adjustable.

[0130] In some embodiments, the predetermined radius used in the local maximum transform is in the range of about 0 to about 5 pixels. In other words, the local maximum transform includes a search radius in the range of about 0 to 5 pixels. For example, when there is no significant local background pattern deviation, a predetermined radius of 0 pixels is utilized. In some embodiments, the search region is simplified to reduce computation time by using eight equally spaced angled lines (i.e., 45 degree intervals), each a single pixel wide, radiating from the pixel of interest.

[0131] In some embodiments, the image metric is a block matching transform. In particular, the method includes, in some embodiments, transforming the blank image using a block matching transform. In some embodiments, the block matching transform is used instead of a local maximum transform to solve the problem of local misalignment. In some embodiments, a block ("block of interest") is used with a predetermined block size (e.g., a 3 pixel by 3 pixel block). Each block of the blank image is compared with a block of the same size in the probe image at a nearby location (i.e., within a predetermined block search size). The search determines the neighboring block that is most similar to the block of interest. Using a similarity metric that measures the similarity of the blocks, the searched neighboring block with the highest similarity metric is determined as the target block. The block of interest is then moved to the corresponding position of the target block. In some embodiments, the similarity metric is the mean absolute difference, sum of absolute differences, mean squared difference, or sum of squared difference, where the difference is the pixel intensity difference between the two blocks being compared. Thus, a block matching transform is performed on each block of interest, searching for its corresponding neighbor in the probe image and shifting its position accordingly to form a transformed blank image, which in some embodiments is used in place of the original blank image in the subsequent subtraction step.

[0132] In some embodiments, the predetermined block size and the predetermined block search size are adjustable. In some embodiments, the predetermined block size used in the block matching transform is in the range of about 1 to about 10 pixels. In other words, the block matching transform includes block sizes in the range of about 1 to about 10 pixels. In some embodiments, the predetermined block search size used in the block matching transform is in the range of about 1 to about 10 pixels. In other words, the block matching transform includes block search sizes in the range of about 1 to about 10 pixels.

[0133] In some embodiments, the methods for improving target detection include any combination of the steps described herein, in various orders. In some embodiments, steps may be omitted. Furthermore, the order of steps may be reversed, changed, or performed simultaneously.

[0134] In at least one embodiment, electronic-based aspects of the method may be implemented (e.g., stored on a non-transitory computer-readable medium) in software that is executable by a computer having one or more processing units, e.g., a microprocessor and / or an application-specific integrated circuit ("ASIC"). Some embodiments may include hardware, software, and electronic components or modules. Thus, it should be noted that a number of hardware- and software-based devices, as well as a number of different structural components, may be utilized to implement the embodiments. [Example]

[0135] The following is a description of various methods and materials used in the research, presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and is not intended to limit the scope of what the inventors regard as their disclosure, nor is it intended to represent that the experiments described below have been performed or are all that may be performed. It should be understood that exemplary descriptions written in the present tense have not necessarily been performed, but rather, that these descriptions can be performed to generate data, etc. relevant to the teachings of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental error and variation should be accounted for.

[0136] Design and evaluation of RNA blocking molecules We designed and tested various strategies to reduce non-target binding of target probes to nonspecific nucleic acids in the target sample while simultaneously improving binding of the target probes to their corresponding target nucleic acids in the target sample. Figure 1A provides the four design strategies evaluated, including (i) competitive blockade, (ii) dual competitive blockade, (iii) dual competitive blockade 2, and (iv) toehold displacement. To test the efficiency of each RNA blockade design strategy for blocking or reducing non-target binding of the probes, we chose the low-expression gene PPIB (peptidyl prolyl isomerase B, accession number NM_000942.4) as a surrogate non-target binding site and designed oligos against it. Four different blocking strategies / designs were tested to identify a reduction in signal from PPIB (Figure 1B). As shown, each of the four design strategies tested resulted in some reduction of off-target binding of the target probe; however, oligos with complementary sequences on both sides of the probe design region connected with a T-linker (the "double competitive blockade 2" strategy) were most efficient at blocking the signal from PPIB (Figure 1B).

[0137] Similar oligos were designed and tested against the highly expressed gene UBC. RNA blocker molecules designed using the same strategy (the "double competitive blocker 2" strategy) efficiently reduced the signal from the UBC probe (Figure 1C). The RNA blocker molecules used in these experiments were 19 base pairs (bp) long and contained 5'-O-methyl modifications.

[0138] Effect of blockers on immature pre-miRNA21 and pri-miRNA21 but no effect of blockers on target signaling After determining an effective design for the blocker, we tested its effect on the ability to detect target miRNAs. Experiments were conducted to evaluate T-linker blockers targeting miRNA-21 and miRNA-205. As shown in Figures 2A and 2B, the results showed that RNA blockers targeting pri-miR-21 and pre-miR-205 did not affect the detection of miRNA-21 (Figure 2A) and miRNA-205 (Figure 2B), respectively.

[0139] Further experiments were performed to evaluate RNA blocking molecules using RNAscope® probes designed to bind both mature and long immature miRNAs (pre-miRNA and pri-miRNA). To test the efficacy of RNA blockers in blocking signals from longer targets, a model system was developed by transfecting HeLa cells with pre-miRNA and pri-miRNA. Pre-miR21 and pri-miR21 were transfected at 1 nM concentrations, and a decrease in signal was determined after the addition of blockers. The presence of blockers reduced the signal in overexpressing cell pellets (Figure 3). Experiments were also performed to determine the effect of blockers on mature miR-21 signal. HeLa cell pellets containing endogenous levels of miR21 showed no decrease in signal, indicating that blockers had no effect on probe binding efficiency.

[0140] Optimization of RNA blocking molecules The probes used in the miRNAscope® compositions and methods vary in length from approximately 16 to approximately 22 nucleotides. These compositions and methods allow optimization of various features of the disclosed RNA blockers, including, but not limited to, longer pre- and pri-miRNA overlap lengths (probe targeting regions), the linker length connecting the two blockers, and the minimum concentration required for highest efficiency. To develop a universal blocker design, we tested various overlap lengths for the targets UBC (ubiquitin C, accession number NM_021009) and miR21. Results showed that longer overlap lengths of approximately 14 to approximately 16 nucleotides had higher blocking efficiency, as indicated by lower signals in samples with added blocker compared to samples without blocker (Figures 4A-4C).

[0141] Experiments were also performed using linkers of various lengths while keeping the overlap length constant. Blockers with different linker lengths (e.g., T-linker lengths) were designed for UBC and miR21, with a total overlap length of 14 or 16 nucleotides. Blockers with shorter T-linker lengths demonstrated higher efficiency in blocking nonspecific signals compared to longer T-linker blockers (Figure 5A). Linker lengths of approximately 1 to 3 nucleotides were most efficient (Figures 5B and 5C). Results also showed that T-linkers were slightly more efficient than linkers composed of other nucleotides (Figure 5D), although this may depend on the complementarity between the nucleotides constituting the probe-targeted region of the target RNA and the nucleotides in the linker region.

[0142] Furthermore, various blocker concentrations were tested to determine the optimal concentration for more efficient blockers. Blockers against UBC and miR21 were tested at concentrations of 10, 20, 30, 40, and 50 nM (Figure 5E). Blockers at 20 nM and 30 nM showed the highest inhibitory efficiency. These data suggest that a typical optimal RNA blocker design includes an overlap region (i.e., probe-targeted region) of approximately 14 to 16 nucleotides, a linker length of approximately 2 to 3 nucleotides, and a concentration of approximately 20 nM to 30 nM (Figure 5F).

Claims

1. 1. An RNA blocking molecule comprising at least one RNA blocking domain comprising a non-probe-targeted region and a probe-targeted region, said regions being contiguous.

2. 2. The RNA blocking molecule of claim 1, wherein the non-probe-targeted region of the RNA blocking domain is complementary to a non-probe-targeted region of a target RNA molecule.

3. 2. The RNA blocking molecule of claim 1, wherein the probe-targeting region of the RNA blocking domain is complementary to a portion of the probe-targeting region of a target RNA molecule.

4. 4. The RNA blocking molecule of claim 2 or claim 3, wherein the target RNA molecule is an mRNA molecule, a microRNA (miRNA) molecule, a small non-coding RNA (sncRNA) molecule, a PIWI-interacting RNA (piRNA) molecule, a small interfering RNA (siRNA) molecule, and / or an antisense oligonucleotide (ASO).

5. The RNA blocking molecule of any one of claims 1 to 4, wherein the non-probe-targeted region of the target mRNA comprises a non-coding region and the probe-targeted region of the target mRNA comprises a coding region.

6. The RNA blocking molecule of any one of claims 1 to 4, wherein the non-probe-targeted region of the target miRNA comprises a region that is not present in the corresponding mature miRNA molecule, and the probe-targeted region of the target miRNA comprises a region that is present in the corresponding mature miRNA molecule.

7. The RNA blocking molecule of any one of claims 1 to 6, wherein the non-probe-targeted region of the RNA blocking domain is 2 to 50 nucleotides in length.

8. The RNA blocking molecule of any one of claims 1 to 7, wherein the probe-targeting region of the RNA blocking domain is 2 to 20 nucleotides in length.

9. 9. The RNA blocking molecule of any one of claims 1 to 8, further comprising a second RNA blocking domain comprising a second non-probe-targeted region and a second probe-targeted region, wherein both second regions are adjacent.

10. The RNA blocking molecule of any one of claims 1 to 9, wherein the first RNA blocking domain and the second RNA blocking domain are connected by a linker region.

11. The RNA blocking molecule of any one of claims 1 to 10, wherein the first RNA blocking domain and the second RNA blocking domain comprise the same number of nucleotides.

12. The RNA blocking molecule of any one of claims 1 to 11, wherein the non-probe-targeted regions of the first and second RNA blocking domains comprise the same number of nucleotides.

13. The RNA blocking molecule of any one of claims 1 to 12, wherein the probe targeting regions of the first and second RNA blocking domains comprise the same number of nucleotides.

14. The RNA blocking molecule of any one of claims 10 to 13, wherein the linker region is 1 to 10 nucleotides in length.

15. The RNA blocking molecule of any one of claims 10 to 14, wherein the linker region is 2 to 8 nucleotides in length.

16. The RNA blocking molecule of any one of claims 10 to 15, wherein the linker region is 2 to 5 nucleotides in length.

17. The RNA blocking molecule of any one of claims 10 to 16, wherein the linker region comprises at least two types of nucleotides.

18. The RNA blocking molecule of any one of claims 10 to 17, wherein the linker region comprises one type of nucleotide.

19. 19. The RNA blocking molecule of claim 18, wherein the linker region consists of thymine nucleotides.

20. 22. The RNA blocking molecule of claim 21, wherein the linker region consists of 1 to 10 thymine nucleotides.

21. The RNA blocking molecule of any one of claims 10 to 20, wherein the linker region comprises at least one nucleotide that is non-complementary to a portion of the probe-targeted region of the target RNA molecule.

22. The RNA blocking molecule of any one of claims 10 to 21, wherein the linker region comprises 1 to 10 nucleotides that are non-complementary to a portion of the probe-targeted region of the target RNA molecule.

23. 22. The RNA blocking molecule of any one of claims 10 to 21, wherein about 20% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe-targeted region of the target RNA molecule.

24. The RNA blocking molecule of any one of claims 10 to 23, wherein the total length of the RNA blocking molecule is 15 to 100 nucleotides.

25. A kit comprising any of the RNA blocking molecules of claims 1 to 24.

26. 26. The kit of claim 25, further comprising at least one target probe that specifically hybridizes to a probe-targeted region of a target RNA molecule.

27. 26. The kit of claim 25, wherein the kit further comprises one or more target probe sets, each target probe set comprising a pair of target probes that specifically hybridize to a probe-targeted region of a target RNA molecule.

28. The kit according to any one of claims 25 to 27, comprising a reagent for permeabilizing cells, a cross-linking reagent, and / or a protease.

29. A kit according to any one of claims 25 to 28, comprising one or more components of a signal generating complex.

30. said one or more components of the signal generating complex being: (i) at least one label and at least one labeled probe capable of hybridizing to said at least one target probe; or (ii) at least one label, at least one labeled probe, and at least one amplification agent capable of hybridizing to the at least one target probe hybridized to the at least one labeled probe; or (iii) at least one label, at least one labeled probe, at least one amplification agent hybridized to the at least one labeled probe, and at least one pre-amplification agent hybridized to the at least one amplification agent and capable of hybridizing to the target probe; 30. The kit of claim 29, comprising:

31. The kit of any one of claims 25 to 30, comprising instructions for carrying out an in situ hybridization reaction.

32. A composition comprising any of the RNA blocking molecules of claims 1-24.

33. 33. The composition of claim 32, comprising a hybridization buffer.

34. The composition of any one of claims 32 to 33, comprising a reagent for permeabilizing cells, a cross-linking reagent, and / or a protease.

35. The composition of any one of claims 32 to 34, comprising a biological sample.

36. 36. The composition of claim 35, wherein the biological sample is or is derived from a tissue sample.

37. 36. The composition of claim 35, wherein the biological sample is or is derived from a blood sample.

38. 36. The composition of claim 35, wherein the biological sample is or is derived from a cytological sample.

39. The composition of claim 35, wherein the biological sample is a cultured cell or an exosome-containing sample.

40. 40. The composition of any one of claims 33 to 39, comprising at least one target probe that specifically hybridizes to a probe-targeted region of a target RNA molecule in the biological sample.

41. 41. A composition according to any one of claims 33 to 40, comprising one or more components of a signal generating complex.

42. said one or more components of the signal generating complex being: (i) at least one label and at least one labeled probe capable of hybridizing to said at least one target probe; or (ii) at least one label, at least one labeled probe, and at least one amplification agent capable of hybridizing to the at least one target probe hybridized to the at least one labeled probe; or (iii) at least one label, at least one labeled probe, at least one amplification agent hybridized to the at least one labeled probe, and at least one pre-amplification agent hybridized to the at least one amplification agent and capable of hybridizing to the target probe; 42. The composition of claim 41, comprising:

43. A method for carrying out an in situ hybridization reaction using any of the RNA blocking molecules of any of claims 1 to 24, any of the kits of claims 25 to 31, or any of the compositions of any of claims 32 to 42.

44. 1. A method for improving signal efficiency in an in situ hybridization reaction, comprising: contacting a biological sample containing a target RNA molecule with any of the RNA blocking molecules of any one of claims 1 to 24; contacting the biological sample with at least one target probe that specifically hybridizes to a probe-targeted region of the target RNA molecule; and contacting the biological sample with a signal generating complex to detect the target RNA molecule.

45. 45. The method of claim 44, wherein the signal efficiency for the target RNA molecule is improved compared to an in situ hybridization reaction that does not include contacting the biological sample with the RNA blocking molecule.

46. 46. ​​The method of claim 44 or claim 45, wherein improving signal efficiency comprises reducing binding of the target probe to non-target RNA molecules in the sample.

47. 47. The method of any one of claims 44 to 46, wherein the target RNA molecule is an mRNA molecule, a microRNA (miRNA) molecule, a small non-coding RNA (sncRNA) molecule, a PIWI-interacting RNA (piRNA) molecule, a small interfering RNA (siRNA) molecule, and / or an antisense oligonucleotide (ASO).

48. 48. The method of any one of claims 44 to 47, wherein the biological sample is or is derived from a tissue specimen, a blood sample or is derived from a blood sample, or a cell sample or is derived from a cell sample.

49. The signal generating complex comprises: (i) at least one label and at least one labeled probe capable of hybridizing to said at least one target probe; or (ii) at least one label, at least one labeled probe, and at least one amplification agent capable of hybridizing to the at least one target probe hybridized to the at least one labeled probe; or (iii) at least one label, at least one labeled probe, at least one amplification agent hybridized to the at least one labeled probe, and at least one pre-amplification agent hybridized to the at least one amplification agent and capable of hybridizing to the target probe; The method of any one of claims 44 to 48, comprising:

50. 50. The method of any one of claims 44 to 49, wherein the method comprises treating the biological sample with a reagent for cell permeabilization, a cross-linking reagent, and / or a protease.

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