Method for detecting target nucleic acids using RNA blocking molecules

Abstract

Embodiments of the present disclosure include compositions and methods for conducting in situ hybridization reactions. In particular, the disclosure provides RNA blocking molecules that enhance the detection of target RNA molecules (e.g., mRNA molecules, micro RNA (miRNA) molecules, small non-coding RNA (sncRNA) molecules, PIWI interacting RNA (piRNA) molecules, and / or small interfering RNA (siRNA) molecules) by reducing the binding of a target probe to non-target RNA molecules in a sample.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 374,998, filed on September 8, 2022, which is incorporated herein by reference in its entirety and for all purposes. Technical Field

[0003] Embodiments of the present disclosure include compositions and methods for performing in situ hybridization reactions. Specifically, the present disclosure provides RNA blocking molecules that enhance the detection of target RNA molecules (e.g., mRNA molecules, microRNA (miRNA) molecules, small noncoding RNA (sncRNA) molecules, PIWI interacting RNA (piRNA) molecules and / or small interfering RNA (siRNA) molecules) by reducing the binding of target probes to non-target RNA molecules in a sample. Background Art

[0004] In situ hybridization (ISH) is a technique that allows precise detection of specific segments of nucleic acids within histological sections. The fundamental basis of ISH is that if nucleic acids are properly preserved in histological specimens, they can be detected by applying complementary nucleic acid strands attached to reporter molecules. The target nucleic acid can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). There are different types of RNA in cells, including messenger RNA (mRNA), which encodes the amino acid sequence of a polypeptide; transfer RNA (tRNA), which brings amino acids to the ribosome during translation; ribosomal RNA (rRNA), which constitutes the ribosome, the organelle that translates mRNA; and sncRNA, which is involved in various RNA processing. Specifically, sncRNA, miRNA, siRNA, piRNA are of great significance in research, diagnosis, and drug development; however, the detection sensitivity of RNA (especially small RNA) in cell or tissue samples is challenging.

[0005] For example, miRNA (single-stranded, non-coding, small RNA) contains only about 16-22 nucleotides. These RNAs are evolutionarily conserved and regulate gene expression by disrupting post-translational modification or degradation of mRNA. Abnormal expression of a single miRNA may lead to the misexpression of multiple genes. Due to their importance in regulating physiological and pathological processes, they are used as biomarkers for various diseases, including cancer, autoimmune disorders and neurodegenerative diseases. It is an in situ hybridization (ISH)-based technique that can detect small RNAs, including miRNAs, siRNAs, and antisense oligonucleotides (ASOs) in cells and tissues. The technique uses ISH probes that bind to miRNAs and reveal the cellular and subcellular spatial expression patterns of small RNAs. The probes are designed in silico, and up to 34% are expected to exhibit off-target binding. Therefore, enhanced detection methods are needed to reduce or eliminate off-target binding to provide more accurate diagnostic and therapeutic information. Summary of the invention

[0006] Embodiments of the present disclosure include RNA blocking molecules comprising at least one RNA blocking domain comprising a non-probe targeting region and a probe targeting region. In some embodiments, the non-probe targeting region and the probe targeting region are continuous.

[0007] In some embodiments, the non-probe targeting region of the RNA blocking domain is complementary to the non-probe targeting region of the target RNA molecule. In some embodiments, the probe targeting region of the RNA blocking domain is complementary to the probe targeting region of the target RNA molecule.

[0008] In some embodiments, the target RNA molecule is an mRNA molecule, a microRNA (miRNA) molecule, a small noncoding RNA (sncRNA) molecule, a PIWI-interacting RNA (piRNA) molecule, a small interfering RNA (siRNA) molecule, and / or an antisense oligonucleotide (ASO).

[0009] In some embodiments, the non-probe targeting region of the target mRNA includes a non-coding region, and wherein the probe targeting region of the target mRNA includes a coding region. In some embodiments, the non-probe targeting region of the target miRNA includes a region that is not present in the corresponding mature miRNA molecule, and wherein the probe targeting region of the target miRNA includes a region that is present in the corresponding mature miRNA molecule.

[0010] In some embodiments, the non-probe-targeted region of the target comprises a region of a vector, such as adeno-associated virus (AAV), lentivirus, and other viral vectors, and wherein the probe-targeted region of the target comprises an ASO.

[0011] In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 2 to 50 nucleotides. In some embodiments, the length of the probe targeting region of the RNA blocking domain is from 2 to 20 nucleotides.

[0012] In some embodiments, the RNA blocking molecule further comprises a second RNA blocking domain, the second RNA blocking domain comprising a second non-probe targeting region and a second probe targeting region. In some embodiments, the second non-probe targeting region and the second probe targeting region are continuous.

[0013] In some embodiments, the first RNA blocking domain and the second RNA blocking domain comprise the same number of nucleotides. In some embodiments, the non-probe targeting region of the first RNA blocking domain and the second RNA blocking domain comprise the same number of nucleotides. In some embodiments, the probe targeting region of the first RNA blocking domain and the second RNA blocking domain comprises the same number of nucleotides.

[0014] In some embodiments, the first RNA blocking domain and the second RNA blocking domain are connected by a joint region. In some embodiments, the length of the joint region is from 1 to 10 nucleotides. In some embodiments, the length of the joint region is from 2 to 8 nucleotides. In some embodiments, the length of the joint region is from 2 to 5 nucleotides. In some embodiments, the joint region comprises at least two types of nucleotides. In some embodiments, the joint region comprises one type of nucleotide. In some embodiments, the joint region consists of thymine nucleotides. In some embodiments, the joint region consists of 1 to 10 thymine nucleotides.

[0015] In some embodiments, the linker region comprises at least one nucleotide that is not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises from 1 to 10 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 20% to about 80% of the nucleotides that make up the linker region are not complementary to a portion of the probe targeting region of the target RNA molecule.

[0016] In some embodiments, the total length of the RNA blocking molecule is from 15 to 100 nucleotides.

[0017] Embodiments of the present disclosure also include kits comprising any of the RNA blocking molecules described herein.

[0018] In some embodiments, the kit further comprises at least one target probe that specifically hybridizes with the probe targeting region of the target RNA molecule. In some embodiments, the kit further comprises one or more target probe groups, and each target probe group comprises a pair of target probes that specifically hybridize with the probe targeting region of the target RNA molecule.

[0019] In some embodiments, the kit comprises reagents for permeabilizing cells, cross-linking reagents, and / or proteases.

[0020] In some embodiments, the kit comprises one or more components of a signal-generating complex. In some embodiments, the components of the signal-generating complex include: (i) at least one label and at least one label probe, wherein the at least one label probe is capable of hybridizing with the at least one target probe; or (ii) at least one label, at least one label probe, and at least one amplifier that hybridizes with the at least one label probe and is capable of hybridizing with the at least one target probe; or (iii) at least one label, at least one label probe, at least one amplifier that hybridizes with the at least one label probe, and at least one preamplifier that hybridizes with the at least one amplifier and is capable of hybridizing with the probe.

[0021] In some embodiments, the kit comprises instructions for performing an in situ hybridization reaction.

[0022] Embodiments of the present disclosure also include compositions comprising any of the RNA blocking molecules described herein.

[0023] In some embodiments, the composition comprises a hybridization buffer. In some embodiments, the composition comprises an agent for permeabilizing cells, a cross-linking agent, and / or a protease.

[0024] In some embodiments, the composition comprises a biological sample. In some embodiments, the biological sample is a tissue specimen or is derived from a tissue specimen. In some embodiments, the biological sample is a blood sample or is derived from a blood sample. In some embodiments, the biological sample is a cytological sample or is derived from a cytological sample. In some embodiments, the biological sample is a cultured cell or a sample containing exosomes.

[0025] In some embodiments, the composition comprises at least one target probe that specifically hybridizes to a probe-targeted region of a target RNA molecule in a biological sample.

[0026] 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 include: (i) at least one label and at least one label probe, wherein the at least one label probe is capable of hybridizing with the at least one target probe; or (ii) at least one label, at least one label probe, and at least one amplifier that hybridizes with the at least one label probe and is capable of hybridizing with the at least one target probe; or (iii) at least one label, at least one label probe, at least one amplifier that hybridizes with the at least one label probe, and at least one preamplifier that hybridizes with the at least one amplifier and is capable of hybridizing with the probe.

[0027] Embodiments of the present disclosure also include methods of performing in situ hybridization reactions using any RNA blocking molecule described herein, any kit described herein, and / or any composition described herein.

[0028] Embodiments of the present disclosure also include methods for enhancing 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 RNA blocking molecule described herein; contacting the biological sample with at least one target probe that specifically hybridizes with the probe targeting 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 method, the signal efficiency for the target RNA molecule is enhanced as compared to an in situ hybridization reaction, and the in situ hybridization reaction does not include contacting the biological sample with an RNA blocking molecule. In some embodiments of the method, enhancing signal efficiency includes reducing the binding of the target probe to non-target RNA molecules in the sample.

[0029] In some embodiments of the method, the target RNA molecule is an mRNA molecule, a microRNA (miRNA) molecule, a small noncoding RNA (sncRNA) molecule, a PIWI-interacting RNA (piRNA) molecule, and / or a small interfering RNA (siRNA) molecule.

[0030] In some embodiments of the methods, the biological sample is a tissue specimen or is derived from a tissue specimen, a blood sample or is derived from a blood sample, or a cytology sample or is derived from a cytology sample.

[0031] In some embodiments of the method, the components of the signal-generating complex include: (i) at least one label and at least one label probe, wherein the at least one label probe is capable of hybridizing with the at least one target probe; or (ii) at least one label, at least one label probe, and at least one amplifier that hybridizes with the at least one label probe and is capable of hybridizing with the at least one target probe; or (iii) at least one label, at least one label probe, at least one amplifier that hybridizes with the at least one label probe, and at least one pre-amplifier that hybridizes with the at least one amplifier and is capable of hybridizing with the probe.

[0032] In some embodiments, the method comprises treating the biological sample with a cell permeabilizing agent, a cross-linking agent, and / or a protease. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figures 1A to 1C : Representative RNA detection blocking strategies tested according to various embodiments of the present disclosure ( Figure 1ATo test the efficiency of the RNA blocking molecules disclosed herein in reducing off-target binding of the target probe, oligonucleotides were designed as surrogate off-target molecules for the low-expression gene PPIB. Four different blocking strategies / designs were tested to determine the reduction of PPIB signal ( Figure 1A Oligonucleotides with complementary sequences on both sides of the probe design region connected by a T linker (the "double competitive blocking 2" strategy) were most effective in blocking the signal from PPIB ( Figure 1B ). Similar oligonucleotides were designed and tested for the highly expressed gene UBC. RNA blocking molecules designed using the same strategy ("double competitive blocking 2" strategy) effectively reduced the signal from the UBC probe ( Figure 1C ).

[0034] FIG. 2A to FIG. 2B Representative results show that the RNA blocking molecules of the present disclosure against pri-miR-21 and pre-miR-205 do not affect miRNA-21 ( Figure 2A ) and miRNA-205( Figure 2B ) detection.

[0035] Figure 3 : Representative results show that the RNA blocking molecules disclosed herein effectively reduce the signals from precursor miRNA21 and initial miRNA21 ( Figure 3 ).

[0036] FIG. 4A to FIG. 4C : Representative results show that for targeting UBC ( Figure 4A ) and pri-miR-21( Figure 4B and Figure 4C ), the efficiency of the RNA blocker disclosed herein varies with the length of the overlapping region (probe targeting region), and the efficiency of the probe with a longer overlapping length is slightly higher.

[0037] FIG. 5A to FIG. 5F : Representative results show that the efficiency of the RNA blockers of the present disclosure varies with the length of the linker (the linker connects the two RNA blocking molecules). Shorter linker lengths (e.g., T linker lengths) are slightly more effective ( Figure 5A ), among which the linker with a length of about 1-3 nucleotides has the highest efficiency ( Figure 5B and 5C The results also showed that T linkers are more effective than linkers composed of other nucleotides ( Figure 5D ), and the overall RNA blocker concentration is most effective at about 20 nM to about 30 nM ( Figure 5E In summary, a representative optimized RNA blocker design is provided ( Fig. 5F ). DETAILED DESCRIPTION

[0038] Embodiments of the present disclosure include compositions and methods for performing in situ hybridization (ISH) reactions. Specifically, the present disclosure provides RNA blocking molecules that enhance 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 the binding of target probes to non-target RNA molecules in samples. According to these embodiments, the present disclosure provides methods for enhancing the signal efficiency of ISH reactions using RNA blocking molecules further described herein.

[0039] definition

[0040] As used herein, the term "fixation" or "fixing" when referring to a fixed biological sample during in situ hybridization refers to a procedure that preserves the biological sample to prevent decay due to, for example, autolysis or putrefaction. It terminates any ongoing biochemical reactions and may also increase the mechanical strength or stability of the treated tissue.

[0041] 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 more, if required for a particular purpose.

[0042] As used herein, the term "detecting" generally refers to any form of measurement, and includes determining whether an element is present or not. The term includes quantitative and / or qualitative determination.

[0043] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe polymers of any length or synthetically produced compounds composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides) that can hybridize with naturally occurring nucleic acids in a sequence-specific manner similar to two naturally occurring nucleic acids, for example, can participate in Watson-Crick base pairing interactions. As used herein, in the context of polynucleotide sequences, the terms "multiple bases" (or "bases") are synonymous with "multiple nucleotides" (or "nucleotides"), i.e., monomeric subunits of polynucleotides. The terms "nucleoside" and "nucleotide" are intended to include those containing not only known purine and pyrimidine bases, but also other modified heterocyclic bases. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated ribose or other heterocycles. In addition, the terms "nucleoside" and "nucleotide" include those containing not only conventional ribose and deoxyribose, but also other sugars. Modified nucleosides or nucleotides also include modifications located on the sugar moiety, for example, where one or more of the hydroxyl groups are substituted with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, etc. "Analogs" refer to molecules with structural features that are considered in the literature to be mimetics, derivatives, having similar structures or other similar terms, and include, for example, polynucleotides incorporating non-natural nucleotides, nucleotide mimetics (such as 2'-modified nucleosides), peptide nucleic acids, oligonucleoside phosphonates, and any polynucleotides to which substituents (such as protecting groups or linking moieties) have been added.

[0044] As used herein, the term "non-coding", "non-coding region" or "non-coding sequence" with respect to RNA molecules generally refers to an RNA sequence / region that is not translated into an amino acid sequence and does not encode a protein, which corresponds to a "coding" sequence / region that is translated into an amino acid sequence. In general, the non-coding region of an mRNA molecule includes introns, promoters, enhancers, 5'UTR, 3'UTR, etc. In addition, other types of RNA molecules include non-coding sequences / regions (or are composed of non-coding sequences / regions), including but not limited to microRNA (miRNA), small non-coding RNA (sncRNA), PIWI interacting RNA (piRNA) and small interfering RNA (siRNA).

[0045] As used herein, the term "small noncoding RNA" or "sncRNA" generally refers to a large class of RNA molecules (e.g., 18 to 200 nt in length) that regulate cellular function. Small ncRNAs are involved in almost all developmental and pathological processes in mammals. Although the exact functions of many ncRNAs remain unclear, a large number of studies have revealed that various small ncRNAs are directly involved in gene expression regulation at the level of post-transcriptional mRNA processing and ribosome biogenesis. Mammalian cells express several types 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).

[0046] As used herein, the term "small interfering RNA" or "siRNA" generally refers to a small inhibitory RNA duplex (generally between 18-30 base pairs) that induces an RNA interference (RNAi) pathway. Typically, siRNA is chemically synthesized as a 21-mer with a 19bp duplex region at the center and a symmetrical 2-base 3'-overhang at the end, but it has recently been reported that the effectiveness of chemically synthesized RNA duplexes of 25-30 bases in length can be increased by 100 times compared to 21-mers at the same position. The measured increased effectiveness obtained when using longer RNA to trigger RNAi is believed to be that Dicer provides a substrate (27mer) rather than a product (21mer), and this increases the rate or efficiency of the siRNA duplex entering RISC.

[0047] As used herein, the terms "microRNA", "miRNA" and "miR" generally refer to a group of non-coding single-stranded RNA molecules of about 19-28 nucleotides in length that regulate gene expression. MiRNA is present in a variety of organisms including viruses, and has been shown to play a role in development, homeostasis and disease etiology. Initially, pre-miRNA is presented as a long imperfect double-stranded stem-loop RNA, which is further processed by Dicer into a siRNA-like duplex, comprising a mature guide strand (miRNA) and a fragment (miRNA) of similar size called a passenger strand. MiRNA and miRNA may be derived from the relative arms of pri-miRNA and pre-miRNA. MiRNA A sequences can be found in cloned miRNA libraries, but are generally less frequent than miRNA. Although initially presented as a double-stranded form with miRNA, miRNA will eventually be integrated into a ribonucleoprotein complex called RNA-induced silencing complex (RISC) in the form of single-stranded RNA. Various proteins can form RISC, which leads to variability in the specificity of the miRNA / miRNA duplex, the binding sites of target genes, the activity of the miRNA (inhibition or activation), and which strand of the miRNA / miRNA duplex is loaded into RISC. When the miRNA strand of the miRNA A:miRNA duplex is loaded into RISC, the miRNA is removed and degraded. The strand of the miRNA:miRNA duplex loaded into RISC is the strand with a less tight 5′ end pairing. When both ends of the miRNA:miRNA have approximately equal 5′ pairing, both the miRNA and the miRNA may have gene silencing activity. RISC recognizes target nucleic acids based on the high degree of complementarity between miRNA and mRNA, especially 2-8 nucleotides of the miRNA (called the “seed sequence”).

[0048] 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 to silence germline-resident retrotransposons. PIWI-interacting RNAs (piRNAs) are single-stranded ncRNAs of 26-31 nucleotides that interact with P-element-induced weak testis (PIWI) proteins, a germline-specific Argonaute family. Compared to any other known cellular RNA family, piRNAs display a very diverse set of nucleotide sequences, which also contain the largest class of ncRNAs known. piRNAs have been shown to be associated with the silencing of retrotransposons, both at the post-transcriptional and epigenetic levels, as well as other genetic elements in the germline, particularly during spermatogenesis. They are 5' monophosphorylated and 2'-O-methyl modified at the 3' end, properties that are thought to increase the stability of piRNAs.

[0049] The term "complementary" refers to specific binding between polynucleotides based on polynucleotide sequences. As used herein, a first polynucleotide and a second polynucleotide are complementary if they bind to each other in a hybridization assay under stringent conditions, e.g., if they produce a given or detectable level of signal in a hybridization assay. If the portions of the polynucleotides follow conventional base pairing rules, e.g., A pairs with T (or U) and G pairs with C, they are complementary to each other, but there may be small regions (e.g., less than about 3 bases) with mismatches, insertions, or deletions of sequence.

[0050] The term "sample" as used herein refers to a material or mixture of materials containing one or more components of interest. The term "sample" includes a "biological sample", which refers to a sample obtained from a biological subject, including a sample of biological tissue or fluid origin obtained, reached or collected in vivo or in situ. Biological samples also include samples from areas containing precancerous or cancerous cells or tissues from biological subjects. Such samples can be, but are not limited to, organs, tissues, cells and exosomes isolated from mammals. 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, etc. Preferred biological samples include, but are not limited to, whole blood, partially purified blood, PBMCs, tissue biopsies, etc.

[0051] As used herein, the term "probe" refers to a capture agent for a specific target mRNA sequence. Therefore, each probe of a probe group has a respective target mRNA sequence. In some embodiments, the probe provided herein is a "nucleic acid probe" or an "oligonucleotide probe", which refers to a nucleic acid that can be bound to a target nucleic acid (such as an mRNA biomarker provided herein) with a complementary sequence by forming a hydrogen bond through complementary base pairing. As used herein, the probe may include a natural base (e.g., A, G, C or T) or a modified base (7-deazaguanosine, inosine, etc.). In addition, the bases in the probe may be connected by a bond other than a phosphodiester bond, as long as it does not interfere with hybridization. The probe may be directly or indirectly labeled with a label, such as a chromophore, a luminophore or a chromophore. By determining the presence or absence of the probe, one can detect the presence or absence of a target mRNA biomarker of interest.

[0052] As used herein, the term "endogenous" refers to substances originating from within an organism. As used herein, the term "exogenous" refers to substances originating from outside an organism.

[0053] As used in the disclosure and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0054] It should be understood that whenever an embodiment is described herein using the term "comprising", other similar embodiments described with "consisting of" and / or "consisting essentially of" are also provided. It should also be understood that whenever an embodiment is described herein using the phrase "consisting essentially of", other similar embodiments described with "consisting of" are also provided.

[0055] As used in such terms “between A and B” or “between AB”, the term “between” means a range that includes both A and B.

[0056] As used herein in phrases such as “A and / or B”, the term “and / or” is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used in phrases such as “A, B, and / or C”, the term “and / or” is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0057] RNA blocking molecules

[0058] Embodiments of the present disclosure include an RNA blocking molecule or multiple RNA blocking molecules, which include at least one RNA blocking domain comprising a non-probe targeting region and a probe targeting region. The non-probe targeting region of the RNA blocking domain is generally a region that does not overlap with the target region designed to bind to the target probe (see, e.g., Fig. 5F In contrast, the probe targeting region of the RNA blocking domain is generally a region that overlaps with the target region to which the target probe is designed to bind (see, e.g., Fig. 5F ). In some embodiments, the non-probe targeting region and the probe targeting region are continuous. According to these embodiments, the RNA blocking molecules of the present disclosure can bind to non-target molecules and reduce non-specific binding of target probes, thereby enhancing signal efficiency.

[0059] In some embodiments, the non-probe targeting region of the RNA blocking domain is complementary to the non-probe targeting region of the target RNA molecule. In some embodiments, the non-probe targeting region of the RNA blocking domain is at least 95% complementary to the non-probe targeting region of the target RNA molecule. In some embodiments, the non-probe targeting region of the RNA blocking domain is at least 90% complementary to the non-probe targeting region of the target RNA molecule. In some embodiments, the non-probe targeting region of the RNA blocking domain is at least 85% complementary to the non-probe targeting region of the target RNA molecule. In some embodiments, the non-probe targeting region of the RNA blocking domain is at least 80% complementary to the non-probe targeting region of the target RNA molecule.

[0060] In some embodiments, the probe targeting region of the RNA blocking domain is 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 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 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 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 the probe targeting region of the target RNA molecule.

[0061] In some embodiments, the RNA blocking molecules of the present disclosure are particularly useful for enhancing the detection of target RNA molecules (including target RNA molecules with smaller sizes). 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 interaction 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 enhance the detection of DNA molecules, such as antisense oligonucleotides (ASOs).

[0062] In some embodiments, the RNA blocking molecules of the present invention enhance the detection of one or more target messenger RNA (mRNA) molecules in a sample (e.g., a cell or tissue sample), such as using in situ hybridization (ISH) to detect the target messenger mRNA. According to these embodiments, the non-probe targeting region of the target mRNA includes a non-coding region, and the probe targeting region of the target mRNA includes a coding region. In other embodiments, the RNA blocking molecules of the present invention enhance the detection of one or more target messenger RNA (mRNA) molecules in a sample (such as a cell or tissue sample), such as using in situ hybridization (ISH) to detect the target messenger mRNA. According to these embodiments, the non-probe targeting region of the target miRNA includes a region that is not present in the corresponding mature miRNA molecule, and the probe targeting region of the target miRNA includes a region that exists in the corresponding mature miRNA molecule.

[0063] In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 2 to 50 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 2 to 45 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 2 to 40 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 2 to 35 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 2 to 30 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 2 to 25 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 2 to 20 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 2 to 15 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 2 to 10 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 5 to 50 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 10 to 50 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 15 to 50 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 20 to 50 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 25 to 50 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 30 to 50 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 35 to 50 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 40 to 50 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 45 to 50 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 10 to 40 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 10 to 30 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 15 to 35 nucleotides. In some embodiments, the length of the non-probe targeting region of the RNA blocking domain is from 20 to 40 nucleotides.

[0064] In some embodiments, the length of the probe targeting region of the RNA blocking domain is from 2 to 20 nucleotides. In some embodiments, the length of the probe targeting region of the RNA blocking domain is from 5 to 20 nucleotides. In some embodiments, the length of the probe targeting region of the RNA blocking domain is from 10 to 20 nucleotides. In some embodiments, the length of the probe targeting region of the RNA blocking domain is from 15 to 20 nucleotides. In some embodiments, the length of the probe targeting region of the RNA blocking domain is from 2 to 15 nucleotides. In some embodiments, the length of the probe targeting region of the RNA blocking domain is from 2 to 10 nucleotides. In some embodiments, the length of the probe targeting region of the RNA blocking domain is from 2 to 5 nucleotides. In some embodiments, the length of the probe targeting region of the RNA blocking domain is from 10 to 15 nucleotides.

[0065] In some embodiments, the RNA blocking molecule of the present disclosure further comprises a second RNA blocking domain. According to these embodiments, as further described herein, the second RNA blocking domain of the RNA blocking molecule includes a second non-probe targeting region and a second probe targeting region. In some embodiments, the second non-probe targeting region and the second probe targeting region are continuous. In some embodiments, the first RNA blocking domain and the second RNA blocking domain comprise the same number of nucleotides. In some embodiments, the first RNA blocking domain and the second RNA blocking domain comprise different numbers of nucleotides. In some embodiments, the non-probe targeting region of the first RNA blocking domain and the second RNA blocking domain comprises the same number of nucleotides. In some embodiments, the non-probe targeting region of the first RNA blocking domain and the second RNA blocking domain comprises different numbers of nucleotides. In some embodiments, the probe targeting region of the first RNA blocking domain and the second RNA blocking domain comprises the same number of nucleotides. In some embodiments, the probe targeting region of the first RNA blocking domain and the second RNA blocking domain comprises different numbers of nucleotides.

[0066] In some embodiments, the first RNA blocking domain and the second RNA blocking domain are connected by a joint region. In some embodiments, the length of the joint region is from 1 to 10 nucleotides. In some embodiments, the length of the joint region is from 2 to 8 nucleotides. In some embodiments, the length of the joint region is from 2 to 5 nucleotides. In some embodiments, the joint region comprises at least two types of nucleotides. In some embodiments, the joint region comprises one type of nucleotide. In some embodiments, the joint region consists of thymine nucleotides. In some embodiments, the joint region consists of 1 to 10 thymine nucleotides.

[0067] In some embodiments, the joint region includes at least one nucleotide that is not complementary to a part of the probe targeting region of the target RNA molecule. For example, if the joint region includes thymine nucleotides, at least one nucleotide in the probe targeting region of the target RNA molecule includes cytosine or guanine. In some embodiments, the joint region includes from 1 to 10 nucleotides that are not complementary to a part of the probe targeting region of the target RNA molecule. In some embodiments, the joint region includes from 2 to 10 nucleotides that are not complementary to a part of the probe targeting region of the target RNA molecule. In some embodiments, the joint region includes from 3 to 10 nucleotides that are not complementary to a part of the probe targeting region of the target RNA molecule. In some embodiments, the joint region includes from 4 to 10 nucleotides that are not complementary to a part of the probe targeting region of the target RNA molecule. In some embodiments, the joint region includes from 5 to 10 nucleotides that are not complementary to a part of the probe targeting region of the target RNA molecule. In some embodiments, the joint region includes from 6 to 10 nucleotides that are not complementary to a part of the probe targeting region of the target RNA molecule. In some embodiments, the joint region comprises from 7 to 10 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the joint region comprises from 8 to 10 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the joint region comprises from 9 to 10 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the joint region comprises from 1 to 9 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the joint region comprises from 1 to 8 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the joint region comprises from 1 to 7 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the joint region comprises from 1 to 6 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the joint region comprises from 1 to 5 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises from 1 to 4 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises from 1 to 3 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises from 1 to 2 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises from 2 to 8 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule.In some embodiments, the linker region comprises from 4 to 6 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises from 2 to 3 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, the linker region comprises from 5 to 7 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule.

[0068] In some embodiments, from about 20% to about 80% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 25% to about 80% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 30% to about 80% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 35% to about 80% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 40% to about 80% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 45% to about 80% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 50% to about 80% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 55% to about 80% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 60% to about 80% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 65% to about 80% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 70% to about 80% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 75% to about 80% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 20% to about 75% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 20% to about 70% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 20% to about 65% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 20% to about 60% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 20% to about 55% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 20% to about 50% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule.In some embodiments, from about 20% to about 45% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 20% to about 40% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 20% to about 35% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 20% to about 30% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 20% to about 25% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 30% to about 70% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 40% to about 60% of the nucleotides constituting the joint region are not complementary to a portion of the probe targeting region of the target RNA molecule. In some embodiments, from about 50% to about 80% of the nucleotides making up the linker region are not complementary to a portion of the probe targeting region of the target RNA molecule.

[0069] In some embodiments, the total length of the RNA blocking molecule is from 15 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 20 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 25 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 30 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 35 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 40 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 45 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 50 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 55 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 60 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 65 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 70 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 75 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 80 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 85 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 90 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 95 to 100 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 95 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 90 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 85 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 80 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 75 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 70 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 65 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 60 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 55 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 50 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 45 nucleotides.In some embodiments, the total length of the RNA blocking molecule is from 15 to 40 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 35 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 30 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 25 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 15 to 20 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 20 to 80 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 30 to 70 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 40 to 60 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 50 to 90 nucleotides. In some embodiments, the total length of the RNA blocking molecule is from 30 to 60 nucleotides.

[0070] According to these embodiments, the present disclosure provides compositions and methods for performing in situ hybridization (ISH) reactions to detect target nucleic acids (e.g., RNA) in samples using RNA blocking molecules as described herein. In some embodiments, the in situ hybridization detection comprises a target nucleic acid of less 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 less than 90 nucleotides. In some embodiments, the target nucleic acid comprises less than 80 nucleotides. In some embodiments, the target nucleic acid comprises less than 70 nucleotides. In some embodiments, the target nucleic acid comprises less than 60 nucleotides. In some embodiments, the target nucleic acid comprises less than 50 nucleotides. In some embodiments, the target nucleic acid comprises less than 40 nucleotides. In some embodiments, the target nucleic acid comprises less than 30 nucleotides. In some embodiments, the target nucleic acid comprises less than 20 nucleotides. In some embodiments, the target nucleic acid comprises less than 16 nucleotides. In some embodiments, the in situ hybridization is used to detect DNA. In some embodiments, the in situ hybridization is used to detect RNA. The method provided herein can also be used to detect longer nucleic acids, for example, nucleic acids comprising more than 100, 200, 300, 500, 1000 or more nucleotides.

[0071] In some embodiments, the method provided herein includes a small RNA material in a detection sample. In one embodiment, the RNA detected is less than 100 nucleotides. In one embodiment, the RNA detected is less than 50 nucleotides. In one embodiment, the RNA detected is less than 40 nucleotides. In one embodiment, the RNA detected is between 10 and 40 nucleotides. In one embodiment, the RNA detected is between 15 and 40 nucleotides. In one embodiment, the RNA detected is between 30 and 40 nucleotides. In some embodiments, the method provided herein detects naturally occurring small nucleic acids. Naturally occurring small nucleic acids play a vital and different cellular function from transcription and RNA processing to translation. The common feature of these RNAs is that their size is in the range of 15 to 40 nucleotide lengths. In other embodiments, the method provided herein detects synthetic small nucleic acids.

[0072] In one embodiment, the method includes detecting sncRNA. In one embodiment, the method includes detecting miRNA. In one embodiment, the method includes detecting siRNA. In one embodiment, the method includes detecting piRNA. In one embodiment, the method includes detecting endogenous RNA. In another embodiment, the method includes detecting exogenous RNA. sncRNA has become a valuable treatment method for disease intervention, which can efficiently regulate gene expression in clinically relevant model systems (Watts et al., Journal of Pathology, 226 (2), 365-379, 2012; Schoch et al., Neuron Review, 94, 1056-1070, 2017). miRNA is a naturally occurring small (~22 nucleotides) regulatory RNA 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, more than 15,000 miRNAs from animals, plants and viruses have been registered (www.mirbase.org), many of which are expressed in a tissue-, cell-type- and cell-state-specific manner. Dysregulation of miRNA expression may lead to serious pathologies such as neurological disorders, infertility, immune disorders or cancer.

[0073] According to these embodiments, the methods and compositions of the present disclosure include performing an in situ hybridization reaction to detect and / or quantify RNA in a biological sample. In one embodiment, the biological sample is a tissue specimen or is derived from a tissue specimen. In one embodiment, the biological sample is a blood sample or is derived from a blood sample. In one embodiment, the biological sample is a cytological sample or is derived from a cytological sample. In one embodiment, the biological sample is a cultured cell. In another embodiment, the biological sample is an exosome.

[0074] Tissue specimens 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, for example, in a blood smear, or the blood can be processed, for example, lysing red blood cells, separating PBMC or white blood cells, separating target cells, etc., so that the cells in the sample analyzed by the disclosed method are in the blood sample or derived from the blood sample. Similarly, tissue specimens can be processed, for example, mincing and physically or enzymatically treating tissue specimens to break the tissue into single cells or cell clusters. In addition, if necessary, cytological samples can be processed to separate cells or destroy cell clusters. Therefore, tissue, blood and cytological samples can be obtained and processed using methods in the art. The method disclosed herein can be used for diagnostic applications to identify the presence or absence of pathological cells based on the presence or absence of nucleic acid targets as biomarkers indicating pathology.

[0075] Those skilled in the art will appreciate based on the present disclosure that any of a number of suitable samples can be used to detect target nucleic acids using the methods and compositions provided herein. Samples for the methods provided herein are typically biological samples or tissue samples. Such samples can be obtained from biological subjects, including samples of biological tissue or fluid sources, which are collected from individuals or some other sources of biological materials, such as biopsies, autopsies, or forensic materials. Biological samples also include samples from biological subject areas containing or suspected of containing precancerous or cancerous cells or tissues, 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 fractions, cells, and / or exosomes isolated from organisms such as mammals. 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, feces, body fluids (including blood and / or serum samples), saliva, semen, and the like. These samples can be used for medical or veterinary diagnostic purposes.

[0076] The collection of cytology samples for analysis by the methods provided herein is known in the art (see, e.g., Dey, "Cytology Sample Procurement, Fixation and Processing," in Basic and Advanced Laboratory Techniques in Histopathology and Cytology, pp. 121-132, Springer, Singapore (2018); "Non-Gynecological Cytology Practice Guideline," by the American Society of Cytopathology (adopted by the ASC Executive Committee on March 2, 2004)).

[0077] For example, methods for processing samples for cervical tissue analysis, including tissue biopsies and cytology samples, are known in the art (see, e.g., Cecil Textbook of Medicine, Bennett and Plum, eds., 20th ed., WB Saunders, Philadelphia (1996); Colposcopy and Treatment of Cervical Intraepithelial Neoplasia: A Beginner's Manual, 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 for Cytopathology (ASC) Executive Committee on November 10, 2000)).

[0078] In some embodiments, the sample is a tissue specimen or is derived from a tissue specimen. In some embodiments, the tissue specimen is formalin fixed paraffin embedded (FFPE). In some embodiments, the tissue specimen is fresh frozen. In some embodiments, the tissue specimen 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 fixative, B5 and IBF. In other embodiments, the sample is a blood sample or is derived from a blood sample. In other embodiments, the sample is a cytological sample or is derived from a cytological sample. In some embodiments, the method includes treating the biological sample with a reagent, a cross-linking reagent and / or a protease that permeabilizes the cell.

[0079] Method for detecting target nucleic acid

[0080] Embodiments of the present disclosure also include methods and compositions for in situ hybridization reactions using any RNA blocking molecules described herein, any kits described herein, and / or any compositions described herein. Embodiments of the present disclosure also include methods for enhancing signal efficiency in in situ hybridization reactions. According to these embodiments, the method includes contacting a biological sample comprising a target RNA molecule with any RNA blocking molecule described herein. In some embodiments, the method includes contacting the biological sample with at least one target probe that specifically hybridizes with the probe targeting 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 enhanced as compared with the in situ hybridization reaction, and the in situ hybridization reaction does not include contacting the biological sample with an RNA blocking molecule. In some embodiments of the method, enhancing signal efficiency includes reducing the binding of the target probe to non-target RNA molecules in the sample.

[0081] In some embodiments of the method, as further described herein, the target RNA molecule is an mRNA molecule, a microRNA (miRNA) molecule, a small non-coding RNA (sncRNA) molecule, a PIWI interaction RNA (piRNA) molecule and / or a small interfering RNA (siRNA) molecule. In some embodiments of the method, as further described herein, the biological sample is a tissue specimen or is derived from a tissue specimen, a blood sample or is derived from a blood sample, or a cytological sample or is derived from a cytological sample.

[0082] In some embodiments of the method, the signal generating complex (SGC) includes at least one label and at least one label probe, wherein the at least one label probe is capable of hybridizing with the at least one target probe. In some embodiments of the method, the SGC includes at least one label, at least one label probe, and at least one amplifier, the amplifier hybridizing with the at least one label probe and capable of hybridizing with the at least one target probe. In some embodiments of the method, the SGC includes at least one label, at least one label probe, at least one amplifier hybridizing with the at least one label probe, and at least one preamplifier hybridizing with the at least one amplifier and capable of hybridizing with the target probe.

[0083] In some embodiments, the method provided herein detects relatively short nucleic acids. For example, in some embodiments, the in situ hybridization detects a target nucleic acid comprising less 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 less than 90 nucleotides. In some embodiments, the target nucleic acid comprises less than 80 nucleotides. In some embodiments, the target nucleic acid comprises less than 70 nucleotides. In some embodiments, the target nucleic acid comprises less than 60 nucleotides. In some embodiments, the target nucleic acid comprises less than 50 nucleotides. In some embodiments, the target nucleic acid comprises less than 40 nucleotides. In some embodiments, the target nucleic acid comprises less than 30 nucleotides. In some embodiments, the target nucleic acid comprises less than 20 nucleotides. In some embodiments, the target nucleic acid comprises less than 16 nucleotides.

[0084] In some embodiments, the in situ hybridization is used to detect small RNA substances. In one embodiment, the detected RNA is less than 100 nucleotides. In one embodiment, the detected RNA is less than 50 nucleotides. In one embodiment, the detected RNA is less than 40 nucleotides. In one embodiment, the detected RNA is between 10 and 40 nucleotides. In one embodiment, the detected RNA is between 15 and 40 nucleotides. In one embodiment, the detected RNA is between 30 and 40 nucleotides. In one embodiment, the method is used to detect sncRNA. In one embodiment, the method is used to detect miRNA. In one embodiment, the method is used to detect siRNA. In one embodiment, the method is used to detect piRNA. In one embodiment, the method is used to detect ASO. In one embodiment, the method is used to detect endogenous RNA. In one embodiment, the method is used to detect exogenous RNA.

[0085] In some embodiments, the in situ hybridization provided herein includes providing at least one group of one or more target probes capable of hybridizing with the target nucleic acid; providing a signal-generating complex capable of hybridizing with one or more target probes of the group, the signal-generating complex comprising a nucleic acid component capable of hybridizing with one or more target probes and a label probe of the group; hybridizing the target nucleic acid with one or more target probes of the group; and capturing the signal-generating complex to one or more target probes of the group, thereby capturing the signal-generating complex to the target nucleic acid. In some embodiments, each group of one or more target probes comprises a single probe. In other embodiments, each group of one or more target probes comprises two probes. In some embodiments, each group of one or more target probes comprises more than two probes. In some embodiments, when each group of target probes comprises a single target probe, when a single target probe is bound to a target nucleic acid, a signal-generating complex is formed. In other embodiments, when each group of target probes comprises two target probes, the signal-generating complex is formed when both members of the target probe pair are bound to the target nucleic acid.

[0086] In some specific embodiments, the RNA ISH used herein is It is described in more detail in, for example, U.S. Pat. Nos. 7,709,198, 8,604,182, and 8,951,726. Specifically, The use of specially designed oligonucleotide probes in combination with branched DNA-like signal-generating complexes has been described 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)).

[0087] In some embodiments, each target probe comprises a target (T) segment and a label (L) segment, wherein the T segment is a nucleic acid sequence complementary to a segment on the target nucleic acid, and the L segment is a nucleic acid sequence complementary to a segment on the nucleic acid component of the signal-generating complex, and wherein the T segment of one or more target probes is complementary to a non-overlapping region of the target nucleic acid, and the L segment of one or more target probes is complementary to a non-overlapping region of the nucleic acid component of the signal-generating complex.

[0088] In some embodiments, one or more target probes are used to detect target nucleic acids. In other embodiments, two or more target probes are used to detect target nucleic acids. In some embodiments, two or more target probes are used to detect target nucleic acids. In some embodiments, three or more target probes are used to detect target nucleic acids. In some embodiments, four or more target probes are used to detect target nucleic acids. In some embodiments, five or more target probes are used to detect target nucleic acids. In some embodiments, six or more target probes are used to detect target nucleic acids. In some embodiments, seven or more target probes are used to detect target nucleic acids. In some embodiments, eight or more target probes are used to detect target nucleic acids. In some embodiments, nine or more target probes are used to detect target nucleic acids. In some embodiments, ten or more target probes are used to detect target nucleic acids. In some embodiments, more than 10 groups of one or more target probes are used to detect target nucleic acids. In some embodiments, more than 15 groups of one or more target probes are used to detect target nucleic acids. In some embodiments, more than 20 groups of one or more target probes are used to detect target nucleic acids. In some embodiments, more than 30 groups of one or more target probes are used to detect target nucleic acids. In some embodiments, the methods provided herein are used to detect multiple nucleic acid targets. In some embodiments, all of the plurality of nucleic acid targets comprise less than 100 nucleotides. In other embodiments, some of the nucleic acid targets comprise less than 100 nucleotides, while other targets comprise greater than 100 nucleotides.

[0089] As used herein, "target probe" generally refers to a polynucleotide capable of hybridizing with a target nucleic acid and capturing or binding a label probe or a signal-generating complex (SGC) component to the target nucleic acid. The target probe may be directly hybridized with the label probe, or it may be hybridized with one or more nucleic acids, which in turn hybridize with the label probe; for example, the target probe may hybridize with an amplifier, a preamplifier, or a pre-preamplifier in the SGC. Therefore, the target probe includes a first polynucleotide sequence complementary to the polynucleotide sequence of the target nucleic acid and a second polynucleotide sequence complementary to the polynucleotide sequence of the label probe, amplifier, preamplifier, pre-preamplifier, etc. The target probe is typically single-stranded so that the complementary sequence can be used to hybridize with the corresponding target nucleic acid, label probe, amplifier, preamplifier, or pre-preamplifier. In some embodiments, the target probe is provided as a pair.

[0090] As used herein, the term "label probe" refers to an entity that is directly or indirectly, usually indirectly bound to a target molecule and enables the target to be detected. The label probe (or "LP") contains a nucleic acid binding portion that is usually a single-stranded polynucleotide or oligonucleotide, which contains one or more labels that directly or indirectly provide a detectable signal. The label can be covalently linked to the polynucleotide, or the polynucleotide can be configured to be combined with the label. For example, a biotinylated polynucleotide can be combined with a label that is combined with streptavidin. The label probe can, for example, directly hybridize with a target nucleic acid. In general, the label probe can hybridize with a nucleic acid, which in turn hybridizes with a target nucleic acid or with one or more other nucleic acids that hybridize with a target nucleic acid. Therefore, the label probe can include a polynucleotide sequence with a target nucleic acid, particularly a polynucleotide sequence that is complementary to a portion. Alternatively, the label probe can include at least one polynucleotide sequence complementary to a polynucleotide sequence in an amplifier, a preamplifier, or a pre-preamplifier in an SGC.

[0091] In some embodiments, the SGC provided herein includes additional annotations, such as amplifiers, preamplifiers and / or pre-preamplifiers. As used herein, "amplifiers" are molecules capable of hybridizing with multiple label probes, typically polynucleotides. Typically, amplifiers hybridize with multiple identical label probes. Amplifiers can also hybridize with target nucleic acids, hybridize with at least one target probe in a pair of target probes, hybridize with two target probes in a pair of target probes, or hybridize with nucleic acids that bind target probes, such as amplifiers, preamplifiers or pre-preamplifiers. For example, amplifiers can hybridize with at least one target probe and multiple label probes, or hybridize with preamplifiers and multiple label probes. Amplifiers can be, for example, linear, forked, comb-shaped or branched nucleic acids. As described herein for all polynucleotides, amplifiers can include modified nucleotides and / or non-standard nucleotide connections as well as standard deoxyribonucleotides, ribonucleotides and / or phosphodiester bonds. Suitable amplicons 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. Publications 2008 / 0038725 and 2009 / 0081688, each of which is incorporated by reference.

[0092] As used herein, a "preamplifier" is a molecule, typically a polynucleotide, that acts as an intermediate binding component between one or more target probes and one or more amplifiers. Typically, the preamplifier hybridizes with one or more target probes and multiple amplifiers simultaneously. Exemplary preamplifiers are described, for example, in U.S. Patent Nos. 5,635,352, 5,681,697, and 7,709,198 and U.S. Publications 2008 / 0038725, 2009 / 0081688, and 2017 / 0101672, each of which is incorporated by reference.

[0093] As used herein, a "pre-preamplifier" is a molecule, typically a polynucleotide, that acts as an intermediate binding component between one or more target probes and one or more preamplifiers. Typically, the pre-preamplifier hybridizes with one or more target probes and multiple preamplifiers simultaneously. Exemplary pre-preamplifiers are described in, for example, U.S. Patent No. 11,078,528, which is incorporated herein by reference.

[0094] Labels are commonly used to detect target nucleic acids in RNA in situ hybridization. As used herein, a "label" is a portion that makes a molecule easily detectable. Common labels include fluorescence, luminescence, light scattering and / or colorimetric labels. Suitable labels include enzymes, fluorescent and chromogenic moieties, and radionuclides, substrates, cofactors, inhibitors, chemiluminescent moieties, magnetic particles, rare earth metals, metal isotopes, etc. In a specific embodiment, the label is an enzyme. Exemplary enzyme labels include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, glucose oxidase, etc., and various proteases. Other labels include, but are not limited to, fluorophores, dinitrophenyl (DNP), etc. Labels are known to those skilled in the art, such as described 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 are commercially available and can be used in the methods and assays of the present disclosure, including detectable enzyme / substrate combinations (Pierce, Rockford IL; Santa Cruz Biotechnology, Dallas TX; Life Technologies, Carlsbad CA). In specific 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.

[0095] Any enzyme or non-enzyme labeling of many enzymes or non-enzyme labels can be used, as long as the enzyme activity or non-enzyme labeling can be detected respectively. The enzyme thus produces a detectable signal, which can be used to detect the target nucleic acid. Particularly useful detectable signals are chromogenic or fluorescent signals. Therefore, particularly useful enzymes used as labels include enzymes that can obtain chromogenic or fluorescent substrates. Such chromogenic or fluorescent substrates can be converted into easily detectable chromogenic or fluorescent products by enzymatic reactions, which can be easily detected and / or quantified using a microscope or spectroscopy. Such enzymes are known to those skilled in the art, including but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, etc. (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Other enzymes with known chromogenic or fluorescent substrates include various peptidases, in which chromogenic or fluorescent peptide substrates can be used to detect proteolytic cleavage reactions. The use of chromogenic and fluorogenic substrates is also known in bacterial diagnostics, including but not limited to the use of α-galactosidase and β-galactosidase, β-glucuronidase, 6-phospho-β-D-galactoside 6-phosphogalactohydrolase, β-glucosidase, α-glucosidase, amylase, neuraminidase, esterase, lipase, etc. (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.

[0096] Various chromogenic or fluorogenic substrates for generating a detectable signal are known to those skilled in the art and are commercially available. Exemplary substrates that can be used to generate a detectable signal include, but are not limited to, 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; 5-amino-1-hydroxy-2-nitro-1-nitro ...2-nitro-1-nitro-2-nitro-2-nitro-1-nitro-2-nitro-2-nitro-1 -bromo-4-chloro-3-indolyl-1-phosphate (BCIP), nitroblue tetrazolium (NBT), Fast Red (Fast Red TR / AS-MX) and p-nitrophenyl phosphate (PNPP); 1-methyl-3-indolyl-β-D-galactopyranoside and 2-methoxy-4-(2-nitrovinyl)phenyl β-D-galactopyranoside for β-galactosidase; 2-methoxy-4-(2-nitrovinyl)phenyl β-D-glucopyranoside for β-glucosidase, etc. 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 for horseradish peroxidase TM and Quintolet; 4-methylumbelliferyl β-D-galactopyranoside, fluorescein di(β-D-galactopyranoside) and naphthyl fluorescein di(β-D-galactopyranoside) for β-galactosidase; 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 for generating detectable signals are also described, for example, in U.S. Publication 2012 / 0100540. Various detectable enzyme substrates, including chromogenic or fluorogenic substrates, are known and commercially available (Pierce, Rockford IL; Santa Cruz Biotechnology, Dallas TX; Invitrogen, Carlsbad CA; 42 Life Science; Biocare). Typically, the substrate is converted to a product that forms a precipitate, which is deposited at the site of the target nucleic acid. Other exemplary substrates include, but are not limited to, HRP-Green (42Li feScience), Betazoid DAB, Cardassian DAB, Romulin AEC, Bajoran Purple, Vina Green, Deep Space Black TM 、Warp Red TM, Vulcan Fast Red, and Ferangi Blue from Biocare (Concord CA; biocare.net / products / detection / chromogens).

[0097] Exemplary rare earth metals and metal isotopes suitable as detectable labels include, but are not limited to, lanthanide (III) isotopes such as 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Nd, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 155Gd, 156Gd, 158Gd, 159Tb, 160Gd, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 166Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, and 176Yb. Metal isotopes can be measured, for example, using time-of-flight mass spectrometry (TOF-MS) (eg, Fluidigm Helios and Hyperion systems, fluidigm.com / systems; South San Francisco, CA).

[0098] Biotin-avidin (or biotin-streptavidin) is a well-known signal amplification system, which is based on the fact that the two molecules have a particularly high affinity for each other and one avidin / streptavidin molecule can bind four biotin molecules. Antibodies are widely used for signal amplification in immunohistochemistry and ISH. Tyramide signal amplification (TSA) is based on the deposition of a large number of haptenized tyramide molecules due to peroxidase activity. Tyramide is a phenolic compound. In the presence of a small amount of hydrogen peroxide, fixed horseradish peroxidase (HRP) converts the labeled substrate into a short-lived, highly reactive intermediate. The activated substrate molecule then reacts very quickly and covalently binds to the electron-rich part of the protein such as tyrosine at or near the peroxidase binding site. In this way, many hapten molecules conjugated to tyramide can be introduced in situ at the hybridization site. Subsequently, the deposited tyramide-hapten molecules can be visualized directly or indirectly. Such detection systems are described in more detail in, for example, U.S. Patent No. 8,658,361.

[0099] Embodiments described herein can utilize enzymes to produce detectable signals using suitable chromogenic or fluorescent substrates. It is understood that, alternatively, the label probe can have a detectable label that is directly coupled to the nucleic acid portion of the label probe. Exemplary detectable labels are known to those skilled in the art, including but not limited to chromogenic or fluorescent labels (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Exemplary fluorophores that can be used as labels include, but are not limited to, rhodamine derivatives, such as tetramethylrhodamine, rhodamine B, rhodamine 6G, sulforhodamine B, Texas Red (sulforhodamine 101), rhodamine 110, and their derivatives, such as tetramethylrhodamine-5-(or 6), lissamine rhodamine B, etc.; 7-nitrobenzene-2-oxa-1,3-diazole (NBD); fluorescein and its derivatives; naphthalene, 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), etc.; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY TM ) and its derivatives (Molecular Probes; Eugene, OR); pyrene and sulfonated pyrene, such as Cascade Blue TM and its derivatives, including 8-methoxypyrene-1,3,6-trisulfonic acid, etc.; pyridyl oxazole derivatives and dabachoxy derivatives (Molecular Probes); fluorescent yellow (3,6-disulfonate-4-amino-naphthalene diimide) and its derivatives; CyDye TMFluorescent 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, ATTO542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO101, 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 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, nitroaromatic compounds such as nitrophenyl, diazo dyes, dabsyl (4-dimethylaminoazobenzene-4'-sulfonyl), etc.

[0100] As disclosed herein, the method provided herein can be used for the parallel detection of multiple target nucleic acids.When using a fluorophore as a mark, the fluorophore selected for detecting multiple target nucleic acids is distinguishable, and can be detected in parallel in a fluorescence microscope when detecting target nucleic acids in parallel.Such a fluorophore is selected to separate the emission spectrum so that the different labels of target nucleic acids can be detected in parallel.The method for selecting a suitable distinguishable fluorophore for the disclosed method is known in the art (see, for example, Johnson and Spence, "Molecular Probes Handbook, a Guide to Fluorescent Probes and Labeling Technologies", 11th edition, Life Technologies (2010)).

[0101] The chromogenic, fluorescent or metal detectable signal associated with the corresponding target nucleic acid can be visualized using methods such as microscopy, cytometry (e.g., mass cytometry, cytometry based on time of flight (CyTOF), flow cytometry), or spectroscopy. In general, if different labels are used in the same assay, a chromogenic substrate or a fluorescent substrate, or a chromogenic or fluorescent label, or a rare earth metal isotope will be used for a specific assay so that a single type of instrument can be used to detect nucleic acid targets in the same sample.

[0102] As disclosed herein, the label can be designed so that the label is 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, so that the same label can be used in a subsequent round of target nucleic acid labeling and detection. Typically, the label is conjugated to the label probe via a cleavable chemical linker. Methods for conjugating a label to a label probe so that the label is cleavable are 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)). A specific system for labeling oligonucleotides is FastTag TM System (Daniel et al., supra, 1998; Vector Laboratories, Burlinghame CA). Various cleavable moieties may be included in the linker so that the label can be cleaved from the label probe. Such cleavable moieties include groups that can be cleaved chemically, photochemically or enzymatically. Cleavable chemical linkers may include cleavable chemical moieties such as disulfides that can be cleaved by reduction, diols 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, etc. (see Hermanson, supra, 1996). A particularly useful cleavable linker is a linker containing a disulfide bond that can be cleaved by reducing the disulfide bond. In other embodiments, the linker may include a site for being cleaved by an enzyme. For example, the linker may contain a proteolytic cleavage site. Typically, such cleavage sites are used for sequence-specific proteases. Such proteases include, but are not limited to, human rhinovirus 3C protease (cleavage site LEVLFQ / GP), enterokinase (cleavage site DDDDK / ), factor X a(cleavage site IEGR / ), tobacco etch virus protease (cleavage site ENLYFQ / G) and thrombin (cleavage site LVPR / GS) (see, e.g., Oxford Genetics, Oxford, UK). Another cleavable portion 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)).

[0103] The cleavable label can be removed by applying an agent such as a chemical agent or light to cleave the label and release the label from the label probe. As discussed above, useful cleavage agents for chemical cleavage include, but are not limited to, reducing agents, periodates, dithionites, hydroxylamines, bases, etc. (see Hermanson, supra, 1996). A useful method for cleaving a disulfide-containing joint 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 an agent for cleaving a label from a label probe.

[0104] In some embodiments, the method for detecting a target nucleic acid in a cell provided herein includes a pretreatment step before hybridization of one or more target probes. In some embodiments, the pretreatment step includes a blocking step, wherein one or more blocking agents are applied to block certain endogenous components of the cell, thereby reducing the assay background. 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 RNA blocking molecules disclosed herein can enhance signal efficiency, thereby improving the detection of target nucleic acid using a target probe or a target probe group compared to not using RNA blocking molecules.

[0105] In some embodiments, in addition to the RNA blocking molecules disclosed herein, various other blocking agents can also be used. For example, when horseradish peroxidase (HRP) is used as a detection enzyme in a later step, hydrogen peroxide is a blocking agent. Hydrogen peroxide is added to inactivate endogenous HRP activity in the sample, thereby reducing the assay background. In a specific embodiment, this blocking step is added as the first step of pretreatment performed immediately after dewaxing. In some embodiments, the pretreatment step includes an epitope repair step, wherein one or more epitope repair buffers may be added to unmask the target nucleic acid. In some embodiments, the epitope repair step includes heating the sample. In some embodiments, the epitope repair step includes heating the sample to 50°C to 100°C. In one embodiment, the epitope repair step includes heating the sample to about 88°C. In some embodiments, the pretreatment step includes a permeabilization step to retain the nucleic acid target in the cell and allow one or more target probes, signal-generating complexes, etc. to enter the cell. 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. Detergent treatments, usually performed using Triton X-100 or SDS, are often used to permeate membranes by extracting lipids. Proteinase K is a non-specific protease that is active and not easily inactivated in a wide pH range. It is used to digest proteins around the target mRNA. The optimal concentration and duration of treatment can be determined experimentally as known in the art. Cell washing steps can be performed subsequently to remove dissolved substances produced in any of the pretreatment steps. In some embodiments, the sample is located in formalin-fixed paraffin-embedded tissue, and a dewaxing step is required when removing paraffin.

[0106] In some embodiments, the method for detecting a target nucleic acid in a cell provided herein includes a post-fixation step at a specific moment. In one embodiment, the post-fixation step is (i) after the first fixing step; and (ii) before applying at least one set of one or more target probes capable of hybridizing with the target nucleic acid. In one embodiment, the post-fixation step is (i) after the first fixing 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 fixing 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 fixing step; and (ii) before the epitope repair step described in the immediately preceding paragraph. In one embodiment, the post-fixation step is (i) after the first fixing step; and (ii) before the permeabilization step described in the immediately preceding paragraph. In one embodiment, the post-fixation step is (i) after the dewaxing step; and (ii) before applying at least one set of one or more target probes capable of hybridizing with the target nucleic acid. In one embodiment, the post-fixation step is (i) after the dewaxing step; and (ii) before the pretreatment step described in the immediately preceding paragraph. In one embodiment, the post-fixation step is (i) after the dewaxing step; and (ii) before the blocking step described in the immediately preceding paragraph. In one embodiment, the post-fixation step is (i) after the dewaxing step; and (ii) before the epitope retrieval step described in the immediately preceding paragraph. In one embodiment, the post-fixation step is (i) after the dewaxing step; and (ii) before the permeabilization step described in the immediately preceding paragraph.

[0107] The methods provided herein have multiple applications in research and diagnosis (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 can improve our understanding of small nucleic acids (including sncRNA, miRNA, siRNA, piRNA and ASO) in their native environment and their related gene regulatory networks involved in various health stages and disease stages.

[0108] In some embodiments, the method provided herein can detect small RNA with spatial and temporal resolution. In one embodiment, the method provided herein can be used to identify tissues and cell types. In one embodiment, the method provided herein can be used to identify different developmental stages. In one embodiment, the method provided herein can be used to characterize adult tissues.

[0109] In some embodiments, the method provided herein can be used to detect the presence of the small RNA expression of the change or the small RNA associated with the pathogen. In one embodiment, the method provided herein can be used to diagnose a disease or illness. In one embodiment, the method provided herein can be used to diagnose a pathogen.

[0110] In some embodiments, the method provided herein is used to monitor the effectiveness of a therapy based on small RNA. In one embodiment, the method provided herein is used to monitor the effectiveness of a therapy based on siRNA. In one embodiment, the method provided herein is used to monitor the effectiveness of a therapy based on ASO. In some embodiments, the method provided herein is used to determine the effectiveness of a therapy based on small RNA. In one embodiment, the method provided herein is used to determine the effectiveness of a therapy based on siRNA. In one embodiment, the method provided herein is used to determine the effectiveness of a therapy based on ASO.

[0111] In a specific embodiment, the method can be used to detect the presence of the siRNA after the siRNA is delivered to a disease model. In a specific embodiment, the method can be used to locate the siRNA after the siRNA is delivered to a disease model. In a specific embodiment, the method can be used to quantify the siRNA after the siRNA is delivered to a disease model. In a specific embodiment, the method can be used to quantify the RNA that is the target of the siRNA after the siRNA is delivered to a disease model.

[0112] In specific embodiments, the methods can be used to detect the presence of the ASO after delivery of the ASO to a disease model. In specific embodiments, the methods can be used to localize the ASO after delivery of the ASO to a disease model. In specific embodiments, the methods can be used to quantify the ASO after delivery of the ASO to a disease model. In specific embodiments, the methods can be used to quantify the RNA that is the target of the ASO after delivery of the ASO to a disease model.

[0113] Kit for in situ detection of target nucleic acids

[0114] Embodiments of the present disclosure also include a kit, which includes any RNA blocking molecule described herein. The kit may also include any component described herein for performing in situ hybridization reactions. In some embodiments, the kit includes at least one target probe that specifically hybridizes with the probe targeting region of the target RNA molecule. In some embodiments, the kit also includes one or more target probe groups, and each target probe group includes a pair of target probes that specifically hybridize with the probe targeting region of the target RNA molecule. In some embodiments, the kit includes reagents, cross-linking agents and / or proteases for permeabilizing cells. In some embodiments, the kit includes instructions for performing in situ hybridization reactions.

[0115] In some embodiments, the kit comprises one or more components of a signal-generating complex. In some embodiments, the components of the signal-generating complex include: (i) at least one label and at least one label probe, wherein the at least one label probe is capable of hybridizing with the at least one target probe; or (ii) at least one label, at least one label probe, and at least one amplifier that hybridizes with the at least one label probe and is capable of hybridizing with the at least one target probe; or (iii) at least one label, at least one label probe, at least one amplifier that hybridizes with the at least one label probe, and at least one preamplifier that hybridizes with the at least one amplifier and is capable of hybridizing with the probe.

[0116] Embodiments of the present disclosure also include compositions comprising any RNA blocking molecules described herein. In some embodiments, the composition comprises a hybridization buffer. In some embodiments, the composition comprises reagents, cross-linking reagents and / or proteases for permeabilizing cells. In some embodiments, the composition comprises a biological sample. In some embodiments, the biological sample is a tissue specimen or is derived from a tissue specimen. In some embodiments, the biological sample is a blood sample or is derived from a blood sample. In some embodiments, the biological sample is a cytological sample or is derived from a cytological sample. In some embodiments, the biological sample is a cultured cell or a sample containing exosomes. In some embodiments, the composition comprises at least one target probe that specifically hybridizes with a probe targeting region of a target RNA molecule in a biological sample.

[0117] 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 include: (i) at least one label and at least one label probe, wherein the at least one label probe is capable of hybridizing with the at least one target probe; or (ii) at least one label, at least one label probe, and at least one amplifier that hybridizes with the at least one label probe and is capable of hybridizing with the at least one target probe; or (iii) at least one label, at least one label probe, at least one amplifier that hybridizes with the at least one label probe, and at least one preamplifier that hybridizes with the at least one amplifier and is capable of hybridizing with the probe.

[0118] In some embodiments, the kit includes an agent for fixing a biological sample. In some embodiments, the kit includes one or more fixatives 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 tissue fixative (see Burns et al., Histochem. Cytochem. 57: 257-264, 2009). In one embodiment, the fixative is UMFIX (see Nadji et al., Appl. Immunohistochem. Mol. Morphol. 13: 277-282, 2005). In one embodiment, the fixative is Z7 (see Lykidis et al., Nucleic Acids Res. 35: e85, 2007). In one embodiment, the fixative is ZBF (see Paavilainen et al., Histochem. Cytochem.: Official J. Histochem. Soc. 58: 237-246, 2010).

[0119] In some embodiments, the kit provided herein comprises an aldehyde-containing fixative. In one embodiment, the aldehyde-containing fixative in the kit is formaldehyde. In one embodiment, the aldehyde-containing fixative in the kit is glutaraldehyde. In one embodiment, the aldehyde-containing fixative in the kit is Bouin's fixative, which is a solution of picric acid, formaldehyde and acetic acid. In one embodiment, the aldehyde-containing fixative in the kit is a mixture of formaldehyde and glutaraldehyde. In one embodiment, the aldehyde-containing fixative in the kit is FAA, which is a solution of ethanol, acetic acid and formaldehyde. In one embodiment, the aldehyde-containing fixative in the kit is periodate-lysine-paraformaldehyde (PLP), which is a solution of paraformaldehyde, L-lysine and 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 calcium formaldehyde, which is a solution of formaldehyde and calcium chloride. In one embodiment, the aldehyde-containing fixative in the kit is formaldehyde 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's 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's fixative, which is a solution of formaldehyde, copper acetate, picric acid and acetic acid. In one embodiment, the aldehyde-containing fixative in the kit is Gendre's solution, which is a solution of formaldehyde, ethanol, picric acid and 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 formaldehyde acetic acid alcohol, 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, wherein at least one of the fixatives in the mixture is formaldehyde or glutaraldehyde. In one embodiment, the aldehyde-containing fixative in the kit is a fixative used not simultaneously but consecutively, wherein at least one of the fixatives is formaldehyde or glutaraldehyde.

[0120] In some embodiments, the aldehyde-containing fixative in the kit comprises about 5% to about 50% formaldehyde. In other embodiments, the aldehyde-containing fixative comprises about 10% to about 40% formaldehyde. In still other embodiments, the aldehyde-containing fixative comprises about 12% to about 37% formaldehyde.

[0121] In some embodiments, the aldehyde-containing fixative in the kit provided herein comprises formaldehyde of various concentrations. In one embodiment, the aldehyde-containing fixative comprises about 5% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 6% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 7% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 8% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 9% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 10% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 11% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises 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. In one embodiment, the aldehyde-containing fixative comprises about 40% formaldehyde. In one embodiment, the aldehyde-containing fixative comprises about 50% formaldehyde.

[0122] In some embodiments, the kit also includes a tool for obtaining a biological sample from a subject. In certain embodiments, the biological sample is a tissue specimen or is derived from a tissue specimen. In certain embodiments, the biological sample is a blood sample or is derived from a blood sample. In certain embodiments, the biological sample is a cytological sample or is derived from a cytological sample.

[0123] 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 RNA. In some embodiments, the target nucleic acid is short RNA. In some embodiments, the target nucleic acid is an RNA comprising less than 100 nucleotides. In other embodiments, the target nucleic acid is an RNA comprising less than 50 nucleotides. In other embodiments, the target nucleic acid is an RNA comprising 15 to 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 some other embodiments, the target nucleic acid is endogenous RNA or exogenous RNA.

[0124] In specific embodiments, the kits provided herein comprise Agents, such as described in more detail in, for example, U.S. Pat. Nos. 7,709,198, 8,604,182, and 8,951,726. In some embodiments, the kit includes at least one set of one or more target probes capable of hybridizing to a target nucleic acid; a signal-generating complex capable of hybridizing to the one or more target probes of the set, wherein the signal-generating complex comprises a label probe and a nucleic acid component capable of hybridizing to the one or more target probes of the set. In some embodiments, the one or more target probes comprise a target (T) segment and a label (L) segment, wherein the T segment is a nucleic acid sequence complementary to a segment on the target nucleic acid, and the L segment is a nucleic acid sequence complementary to a segment on the nucleic acid component of the signal-generating complex, and wherein the T segment of the one or more target probes is complementary to a non-overlapping region of the target nucleic acid, and the L segment of the one or more target probes is complementary to a non-overlapping region of the nucleic acid component of the signal-generating complex.

[0125] In some embodiments, the kit further comprises a signal-generating complex as described above, which may include a labeled probe, an amplifier, a preamplifier, and / or a pre-preamplifier. In some embodiments, the kit further comprises other agents or materials for performing RNA ISH, including a fixative and an agent for treating a sample for making hybridization, an agent for washing a sample, and the like.

[0126] The kit may also include "packaging material," which refers to a physical structure that holds 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.).

[0127] The kit provided herein may include a label or insert. The label or insert includes information about the condition, illness, disease or symptom for which the kit components can be used. The label or insert may include instructions for clinicians or subjects to use one or more kit components in the kit components in methods, treatment strategies (protocols) or treatment regimens. In some embodiments, the kit may be used to identify tissues and cell types. In some embodiments, the kit may be used to identify different developmental stages. In some embodiments, the kit may be used to detect clinical biomarkers for cancer. In some embodiments, the kit may be used to diagnose diseases or conditions based on the expression of one or more altered small RNAs or the presence of pathogen-related small RNAs. In some embodiments, the kit may be used to characterize adult tissues. In some embodiments, the kit may be used to detect clinical biomarkers for pathogen diagnosis. In some embodiments, the kit may be used to detect and characterize small RNA-based therapies. In some embodiments, the kit may be used to confirm the initial efficiency of small RNA-based therapies. In some embodiments, the kit may be used to continue monitoring the efficiency of small RNA-based therapies. In some embodiments, the kit may be used to determine the efficiency of small RNA-based therapies. In some embodiments, the kit can be used to detect the presence, location and quantification of siRNA. In some embodiments, the kit can be used to detect the presence, location and quantification of ASO molecules. In some embodiments, the kit can be used to detect and identify pathogen-derived small RNA.

[0128] Image Processing

[0129] Embodiments of the present disclosure also include methods for enhancing the detection of a target (e.g., a target nucleic acid). In some embodiments, the method includes an image processing method, such as the method described in International Patent Application PCT / US22 / 24975, which is incorporated herein by reference. The method is at least partially implemented with a computer having corresponding instructions stored on a memory (i.e., a non-transient computer-readable medium). The final image from the method and the intermediate image in some embodiments are stored in a memory. In some embodiments, the memory can be accessed by a network. In some embodiments, user input or instructions can be received or accessed through a network.

[0130] The method includes imaging the sample with a target signal to create a probe image, and imaging the sample with no target signal to create a background image (i.e., a "blank image"). In some embodiments, a "blank image" is an image comprising an RNA blocking molecule of the present disclosure. In some embodiments, a "blank image" is an image that does not comprise an RNA blocking molecule of the present disclosure. In some embodiments, imaging utilizes a fluorescent microscope coupled to a computer via a network. In some embodiments, the target signal is obtained by performing a fluorescent in situ hybridization assay and / or an immunofluorescence assay on the sample. In some embodiments, a background image without a target signal is obtained by removing the target signal from the sample (i.e., by a cutting process). In other embodiments, a background image without a target signal is obtained before the assay is performed. In some embodiments, the target signal includes a fluorescent marker that is bound to a target nucleic acid. In other embodiments, the target signal includes a fluorescent marker that is bound to a target peptide or polypeptide.

[0131] The method can also include registering the probe image and the background image (e.g., with or without RNA blocking molecules). Potential background fluorescence differences between the probe image and the background image produce spatial pattern mismatches that occur due to overall sample movement between different rounds of image acquisition. To eliminate such differences, image registration techniques (e.g., phase correlation) are utilized. Robust image registration utilizes detection and matching of image features to compensate for any global sample movement (i.e., translation and rotation).

[0132] The method also includes modifying the background image (e.g., with or without RNA blocking molecules) based on at least one image metric to create an adjusted background image (e.g., a transformed, intensity-adjusted blank image). As further explained herein, at least one image metric is a ratio factor, a multiplication factor, a local maximum transformation, 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.

[0133] In some embodiments, the method also includes subtracting an adjusted background image (e.g., with or without RNA blocking molecules) from the probe image to create a final image including an enhanced target signal. In other words, a modified (i.e., transformed, adjusted, scaled, etc.) blank image is used in the subtracting step, rather than the original blank image. In some embodiments, the enhanced target signal includes an enhanced contrast. 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 memory and can be accessed by a user, for example, via a network. Therefore, the method provides improved signal detection in the presence of a background with tissue autofluorescence.

[0134] In some embodiments, image metrics are ratio factors that illustrate the intensity difference in the background between a blank image and a probe image. When image acquisition settings are different or from photobleaching during fluorophore excitation, intensity differences may occur. In order to compensate for background intensity differences, the method includes determining a ratio factor that compares the overall background intensity of the probe image with the blank image. First, the pixel position of the probe is estimated. Any combination of, for example, a white hat algorithm (Gonzalez & Woods, 2008, Digital Image Processing), a bandpass filter (Shenoi, 2006, Introduction to Digital Signal Processing and Filter Design) or a suitable method is used to estimate the probe position in the probe image. After determining the estimated position of the target signal in the probe image, the pixels at the estimated probe position are excluded from both the probe image and the blank image, thereby generating an image with only background pixels (i.e., an image with only background). In other words, the method includes removing the estimated position from the probe image to create a first image with only background, and removing the estimated position from the blank image (background image) to create a second image with only background.

[0135] After removing the estimated probe position from the two images, the method includes determining a ratio factor. In other words, statistical metrics of both the blank image excluding the probe and the probe image excluding the probe are evaluated and incorporated into the ratio factor. In some embodiments, the ratio factor is used to modify the background image to create an adjusted background image. In other words, in some embodiments, modifying the background image to create an adjusted background image may include scaling the background image by the ratio factor.

[0136] 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 ratio of 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 intensity and the second intensity used in the ratio factor are statistical metrics, such as a statistical mean, median, or a combination of both of the intensity values ​​of any portion (including all) in the image.

[0137] In some embodiments, the first intensity is the average of multiple pixel intensity values ​​in the first background-only image, and the second intensity is the average of multiple 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 multiple pixel intensity values ​​in the first background-only image, and the second intensity is the median of multiple 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 the central approximately 80% (i.e., excluding approximately the top 10% and approximately the bottom 10%) of all pixel intensity values ​​in the first background-only image, and the second intensity is the average of the central approximately 80% of all pixel intensity values ​​in the second background-only image.

[0138] In some embodiments, the image metric is a multiplication factor that illustrates potential local intensity differences between the blank image and the probe image. Specifically, the method includes determining the 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 background image is modified to create an adjusted background image using the multiplication factor. In other words, in some embodiments, modifying the background image to create an adjusted background image may include scaling the background image by the multiplication factor.

[0139] In some embodiments, the image metric is a local maximum transformation. Specifically, the method includes transforming a blank image with a local maximum transformation. Even after global image registration, there may still be a local background pattern mismatch, which comes from, for example, image acquisition at different focal planes, or the sample is not firmly attached to a supporting material (e.g., a slide) and partially moves between imaging periods. In order to solve this problem, local mismatches are compensated accordingly. In the illustrated embodiment, for each pixel in the blank image ("pixel of interest"), the neighborhood of the predefined radius around the pixel of interest is searched. The search process will find the pixel of maximum intensity, and the maximum intensity is assigned to the pixel of interest. The search procedure is performed for each pixel of interest, and its neighborhood is searched in the original blank image to form a transformed blank image. As explained in more detail herein, in the subtraction step later, the transformed blank image can be used to replace the original blank image. In some embodiments, the predefined radius ("matching distance") is adjustable.

[0140] In some embodiments, the predefined 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 obvious local background pattern mismatch, a predefined radius of 0 pixels is used. In some embodiments, the search area is simplified to reduce calculation time by using eight lines with equal angles (i.e., 45 degrees apart), each line having a single pixel width, radiating from the pixel of interest.

[0141] In some embodiments, the image metric is a block matching transform. Specifically, in some embodiments, the method includes the step of transforming the blank image with a block matching transform. In some embodiments, a block matching transform is used instead of a local maximum transform to solve the problem of local mismatch. In some embodiments, a block ("block of interest") is used with a predefined block size (e.g., a block of 3 pixels by 3 pixels). Each block in the blank image is compared with a block of the same size in a probe image at a nearby position (i.e., within a predefined block search size). The search determines the nearby block that is most similar to the block of interest. The similarity of the blocks is measured using a similarity metric, and the nearby block searched with the highest similarity metric is determined as a target block. Then, the block of interest is moved to the corresponding position of the target block. In some embodiments, the similarity metric is the mean absolute difference, the sum of absolute differences, the mean square error, or the sum of variances, wherein the difference is the pixel intensity difference between the two blocks being compared. Therefore, a block matching transform is performed on each block of interest, and its corresponding neighborhood in the probe image is searched and its position is moved accordingly to form a transformed blank image. In some embodiments, the transformed blank image is used to replace the original blank image in a subsequent subtraction step.

[0142] In some embodiments, the predefined block size and the predefined block search size are adjustable. In some embodiments, the predefined 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 a block size in the range of about 1 to 10 pixels. In some embodiments, the predefined 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 a block search size in the range of about 1 to 10 pixels.

[0143] In some embodiments, the method for enhancing the detection of a target comprises any combination of the steps described herein in various orders. In some embodiments, steps may be omitted. In addition, the order of the steps may be reversed, changed, or performed simultaneously.

[0144] In at least one embodiment, the electronic-based aspects of the method can be implemented in software (e.g., stored on a non-transitory computer-readable medium) executable by a computer having one or more processing units such as a microprocessor and / or an application-specific integrated circuit ("ASIC"). Some embodiments may include hardware, software, and electronic components or modules. Therefore, it should be noted that embodiments may be implemented using a plurality of hardware- and software-based devices and a plurality of different structural components.

[0145] Example

[0146] The following is a description of various methods and materials used in the study, and is presented with the purpose of providing a person 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 the present disclosure as the inventors believe, nor does it represent that the following experiments were conducted and are all experiments that can be conducted. It should be understood that the exemplary description written in the present tense does not need to be conducted, but rather the description can be conducted to generate data associated with the teachings of the present disclosure, etc. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should also be considered.

[0147] Design and evaluation of RNA blocking molecules

[0148] Various strategies were designed and tested to reduce off-target binding of target probes to nonspecific nucleic acids in the sample, while enhancing the binding of target probes to the corresponding target nucleic acids in the sample. Figure 1A Four evaluated design strategies are provided, including (i) competitive blocking; (ii) dual competitive blocking; (iii) dual competitive blocking 2; and (iv) toehold replacement. To test the efficiency of each RNA blocking design strategy to block or reduce probe off-target binding, oligonucleotides were designed against the low-expressed gene PPIB (peptidyl prolyl isomerase B; accession number NM_000942.4), which was selected as a surrogate off-target binding. Four different blocking strategies / designs were tested to determine the reduction of PPIB signal ( Figure 1B As shown, although the four design strategies tested all reduced off-target binding of the target probe to some extent; however, the oligonucleotides complementary to the sequences on both sides of the probe design region and connected by T linkers (the "double competitive blocking 2" strategy) were the most effective in blocking PPIB signals ( Figure 1B ).

[0149] Similar oligonucleotides were designed and tested for the highly expressed gene UBC. RNA blocking molecules designed using the same strategy (“double competitive blocking 2” strategy) effectively reduced the signal from the UBC probe ( Figure 1C ). The RNA blocking molecules used in these experiments were 19 bp in length and contained a 5'-O-methyl modification.

[0150] The inhibitors affect immature precursor miRNA21 and initial miRNA21 but have no effect on target signaling

[0151] After determining an effective design for the blocker, the effect of the blocker on the ability to detect the target miRNA was tested. Experiments were performed to evaluate the effect of T-linker blockers against miRNA-21 and miRNA-205. Figure 2A and Figure 2B As shown, the results showed that RNA blocking molecules targeting initial miR-21 and precursor miR-205 had no effect on miRNA-21 ( Figure 2A ) and miRNA-205( Figure 2B ) detection.

[0152] In addition, experiments were performed to evaluate RNA blocking molecules, in which The probes are designed to bind to both mature and long immature miRNAs (pre-miRNA and naïve miRNA). To test the efficiency of RNA blockers in blocking the signal from longer targets, a model system was developed by transfecting HeLa cells with pre-miRNA and naïve miRNA. Pre-miR21 and naïve miR21 were transfected at 1 nM concentration and the signal was reduced after the addition of the blocker. The presence of the blocker reduced the signal from the overexpressing cell pellet ( Figure 3 Experiments were also performed to determine the effect of the blockers on mature miR-21 signaling. HeLa cell pellets containing endogenous levels of miR21 showed that the blockers had no effect on probe binding efficiency, showing no reduction in signal.

[0153] Optimization of RNA blocking molecules

[0154] The probe lengths used in the compositions and methods range from about 16 nucleotides to about 22 nucleotides. According to these compositions and methods, various features of the RNA blockers of the present disclosure can be optimized, including but not limited to the overlap length of the longer precursor miRNA and the initial miRNA (the probe targeting region of the blocker), the length of the linker connecting the two blockers, and the minimum concentration required for optimal efficiency. In order to develop a universal blocker design, different overlap lengths were tested for UBC (ubiquitin C; Accession No. NM_021009) and miR21 targets. The results showed that longer overlap lengths of about 14 to about 16 nucleotides had higher blocking efficiency, as indicated by the lower signal of the samples with added blockers compared to the samples without blockers ( FIG. 4A to FIG. 4C ).

[0155] Experiments were also performed using different linker lengths while keeping the overlap length constant. Blockers with different linker lengths (e.g., T linker lengths) were designed for UBC and miR21, while the overlap length was 14 or 16 nucleotides in total. Blockers with shorter T linker lengths showed higher efficiency in blocking nonspecific signals compared to longer T linker blockers ( Figure 5A ), among which the linker with a length of about 1-3 nucleotides has the highest efficiency ( Figure 5B and Figure 5C The results also showed that T linkers are somewhat more effective than linkers composed of other nucleotides ( Figure 5D ), although this may depend on the nucleotides that make up the probe targeting region of the target RNA and their complementarity with the nucleotides in the linker region.

[0156] In addition, different concentrations of blockers were tested to determine the optimal concentration of blockers with higher efficiency. Blockers of UBC and miR21 were tested at 10, 20, 30, 40, and 50 nM concentrations ( Figure 5E ). Blockers at 20 and 30 nM showed the highest blocking efficiency. These data suggest that a representative optimal RNA blocker design includes an overlap region of about 14-16 nucleotides (i.e., the probe targeting region), with a linker length of about 2-3 nucleotides and a concentration of about 20 nM to about 30 nM ( Fig. 5F ).

Claims

1. An RNA blocking molecule comprising at least one RNA blocking domain comprising a non-probe targeting region and a probe targeting region, wherein the regions are contiguous.

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

3. 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. 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 targeting region of the target mRNA comprises a non-coding region, and wherein the probe targeting 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 targeting region of the target miRNA comprises a region not present in the corresponding mature miRNA molecule, and wherein the probe targeting region of the target miRNA comprises a region present in the corresponding mature miRNA molecule.

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

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

9. The RNA blocking molecule of any one of claims 1 to 8, further comprising a second RNA blocking domain, wherein the second RNA blocking domain comprises a second non-probe targeting region and a second probe targeting region, wherein the second region is continuous.

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 targeting region of the first RNA blocking domain and the second RNA blocking domain comprises 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 RNA blocking domain and the second RNA blocking domain comprise the same number of nucleotides.

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

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

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

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

20. 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 not complementary to a portion of the probe targeting region of the target RNA molecule.

22. The RNA blocking molecule of any one of claims 10 to 21, wherein the linker region comprises from 1 to 10 nucleotides that are not complementary to a portion of the probe targeting region of the target RNA molecule.

23. The RNA blocking molecule of any one of claims 10 to 21, wherein from about 20% to about 80% of the nucleotides comprising the linker region are non-complementary to a portion of the probe targeting region of a 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 from 15 to 100 nucleotides.

25. A kit comprising any RNA blocking molecule according to claims 1 to 24.

26. The kit of claim 25, wherein the kit further comprises at least one target probe that specifically hybridizes to a probe-targeting region of a target RNA molecule.

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

28. The kit of any one of claims 25 to 27, wherein the kit comprises reagents for permeabilizing cells, cross-linking reagents and / or proteases.

29. The kit of any one of claims 25 to 28, wherein the kit comprises one or more components of a signal-generating complex.

30. The kit of claim 29, wherein the one or more components of the signal-generating complex comprise: (i) at least one label and at least one label probe, wherein the at least one label probe is capable of hybridizing to the at least one target probe; or (ii) at least one label, at least one label probe, and at least one amplifier that hybridizes to the at least one label probe and is capable of hybridizing to the at least one target probe; or (iii) at least one label, at least one label probe, at least one amplifier hybridized to the at least one label probe, and at least one preamplifier hybridized to the at least one amplifier and capable of hybridizing to the target probe.

31. The kit of any one of claims 25 to 30, wherein the kit comprises instructions for performing an in situ hybridization reaction.

32. A composition comprising any RNA blocking molecule according to claims 1 to 24.

33. The composition of claim 32, wherein the composition comprises a hybridization buffer.

34. A composition as described in any one of claims 32 to 33, wherein the composition comprises an agent for permeabilizing cells, a cross-linking agent and / or a protease.

35. The composition of any one of claims 32 to 34, wherein the composition comprises a biological sample.

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

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

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

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

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

41. The composition of any one of claims 33 to 40, wherein the composition comprises one or more components of a signal-generating complex.

42. The composition of claim 41, wherein the one or more components of the signal-generating complex comprise: (i) at least one label and at least one label probe, wherein the at least one label probe is capable of hybridizing to the at least one target probe; or (ii) at least one label, at least one label probe, and at least one amplifier that hybridizes to the at least one label probe and is capable of hybridizing to the at least one target probe; or (iii) at least one label, at least one label probe, at least one amplifier hybridized to the at least one label probe, and at least one preamplifier hybridized to the at least one amplifier and capable of hybridizing to the target probe.

43. A method for performing an in situ hybridization reaction using any RNA blocking molecule according to any one of claims 1 to 24, any kit according to any one of claims 25 to 31, or any composition according to any one of claims 32-42.

44. A method for enhancing signal efficiency in an in situ hybridization reaction, the method comprising: contacting a biological sample comprising a target RNA molecule with any RNA blocking molecule as described in 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 The biological sample is contacted with the signal-generating complex to detect the target RNA molecule.

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

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

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

49. The method of any one of claims 44 to 48, wherein the signal-generating complex comprises: (i) at least one label and at least one label probe, wherein the at least one label probe is capable of hybridizing to the at least one target probe; or (ii) at least one label, at least one label probe, and at least one amplifier that hybridizes to the at least one label probe and is capable of hybridizing to the at least one target probe; or (iii) at least one label, at least one label probe, at least one amplifier hybridized to the at least one label probe, and at least one preamplifier hybridized to the at least one amplifier and capable of hybridizing to the target probe.

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

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