Method for sequential detection of nucleic acids

Abstract

The present invention relates to a method for multiplexed detection of multiple target nucleic acids by contacting a sample with an acid reagent to remove bound nucleic acid detection systems, thereby allowing the same detection system to be used again to detect different target nucleic acids and providing a higher level of multiplexing. The present invention also relates to a kit comprising the acid reagent and, optionally, probes for the detection of the target nucleic acids.

Description

Detailed description of the invention

[0001] [Technical Field] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 938,138, filed on November 20, 2019, the entirety of which is incorporated herein by reference in full.

[0002] [Background technology] This invention relates, in general terms, to the detection of nucleic acids, and more specifically, to the multiplex detection of nucleic acids.

[0003] RNA in situ hybridization (ISH) is a widely used molecular biological technique for measuring and localizing specific RNA sequences, such as messenger RNA (mRNA), long non-coding RNA (lncRNA), and microRNA (miRNA) within cells, such as circulating tumor cells (CTCs) or tissue sections, while preserving the cellular and tissue context. Therefore, RNA ISH provides spatiotemporal visualization and quantification of gene expression within cells and tissues. It has a wide range of applications in research and diagnosis (Hu et al., Biomark.Res.2(1):1-13,doi:10.1186 / 2050-7771-2-3(2014), Ratan et al., Cureus 9(6):e1325.doi:10.7759 / cureus.1325(2017), Weier et al., Expert Rev.Mol.Diagn.2(2):109-119 (2002)). Fluorescent RNA ISH utilizes fluorescent dyes and a fluorescence microscope for RNA labeling and detection, respectively. Fluorescent RNA ISH typically provides limited multiplexing of 4-5 target sequences. This limited multiplexing capability is largely due to the small number of spectrally distinct fluorescent dyes that can be distinguished by the fluorescence microscope's optical system. In areas such as generating cell and tissue maps to understand complex biological systems, and especially in human health and disease, a higher level of multiplexing is highly desirable.

[0004] Several approaches have been introduced that utilize a sequential round of hybridization, imaging, label removal, and re-hybridization to distinct targets, theoretically providing multiple imaging of 4-5 targets within the same cell or tissue section (Shah et al., Neuron 92(2):342-357 (2016), Codeluppi et al., Nature Methods 15(11):932-935 (2018), Kishi et al., Nat. Methods 16:533-544 (2019)). However, in practice, the aforementioned sequential fluorescence ISH (FISH) methods can result in substantial loss of nucleic acid detection sensitivity, particularly RNA detection sensitivity, and cell morphology during sequential rounds of hybridization and detection.

[0005] The enzyme DNAse I is commonly used, for example, in target probe removal and signal amplification systems based on hybridization chain reactions (Shah et al., above, 2016). However, DNAse I digestion can damage nuclear architecture and cell morphology, thus interfering with subsequent image registration and analysis steps. The enzyme exonuclease I has also been reported to detach amplification systems based on concatemers of long DNA (Kishi et al., above, 2019). However, this method does not allow for direct measurement and control of enzyme activity, and the procedure requires relatively large amounts of enzyme, longer incubation times at higher temperatures, and extensive post-fixation and washing steps.

[0006] Therefore, there is a need for a simple, reliable, and effective methodology for a signal amplification system that removes target probes and minimizes impact on the integrity of cellular nucleic acids, such as RNA integrity, as well as a morphology for achieving multiple rounds of hybridization. This invention satisfies this need and also provides the relevant advantages.

[0007] [Summary of the Invention] The present invention provides a method for removing a probe bound to nucleic acids within a cell, the method comprising contacting a cell with an acid reagent, wherein the cell contains a first probe hybridized to a first target nucleic acid within the cell, and the acid reagent disrupts the hybridization between the first probe and the first target nucleic acid; and removing the first probe from the cell. Optionally, the steps may be repeated to provide a sequential round of multiplex detection of nucleic acids within the cell.

[0008] [Brief explanation of the drawing] [Figure 1] Multiplex assay (RNAscope® HiPlex A schematic diagram of the workflow (of the sey workflow) is shown. The N target sequence is hybridized to a target probe (shown as a double Z probe), and the signal is simultaneously amplified by an amplification system such as RNAscope®. In the shown embodiment, the first four targets are detected via four non-spectral overlapping fluorescent dye-conjugated oligonucleotides (labeled probes) and imaged using a conventional fluorescence microscope or scanner. The fluorophores are then cleaved from the labeled probes, and the next four targets are labeled and imaged using the same method. After detection of each of the four targets in L rounds, the images are registered using an image registration software algorithm to create a final composite image superimposed at single-cell resolution. [Figure 2A] Sequential removal of nucleic acid probes using an acid reagent to remove the target probe. A schematic diagram of hybridization is shown. A schematic diagram of acid treatment and removal of target(s) bound to target(s) nucleic acids(s) for sequential hybridization is shown. The N target probe is hybridized to the target nucleic acid, for example, in an in situ hybridization assay. The diagram shows the amplification of an arbitrary signal of the target probe hybridized to the target nucleic acid. Cells may be counterstained to facilitate cell visualization; for example, the nucleus may be stained with 4',6-diamidino-2-phenylindole (DAPI). The target probe and counterstained cells are visualized, for example, by imaging, thereby detecting and imaging the target nucleic acid. When using the RNAscope® assay, the RNAscope® Double Z probe and signal are amplified simultaneously by the RNAscope® amplification system. An acid treatment step is performed to remove the target(s) bound to each target(s). One or more additional sets of N target nucleic acids may be detected by repeating the entire Nplex workflow for one or more rounds. After all targets have been detected, the images are registered using an image registration software algorithm to create a final composite image superimposed at single-cell resolution. The total number of multiplex levels available in this method is K rounds, with N targets (multiple) × N plexings per round. Generally, N is greater than or equal to 1, and K is greater than or equal to 2 if an acid removal step is included. In the diagram shown in Figure 2A, when K=1, only one round of target probe hybridization and imaging is required, and therefore the acid removal step is not necessary. [Figure 2B] Sequential removal of nucleic acid probes using an acid reagent to remove the target probe. A schematic diagram of hybridization is shown. A schematic diagram of acid treatment and removal of the probe bound to the target nucleic acid for sequential hybridization is shown. The N target probe is hybridized to the target nucleic acid using an in situ hybridization assay, such as the RNAscope® HiPlex assay. For example, the N target probe is hybridized to the target sequence using the RNAscope® double Z probe, and the signal is simultaneously amplified, for example, by the RNAscope® amplification system. The diagram shows arbitrary signal amplification of the target probe hybridized to the target nucleic acid. Cells may be counterstained to facilitate cell visualization; for example, the nucleus may be stained with 4′,6-diamidino-2-phenylindole (DAPI). The N target nucleic acid is detected by labeling, e.g., fluorescent labeling, imaging, and cleavage of the detectable label, e.g., repeated rounds of cleavage of the fluorescent label. In the diagram, a subset of the N target (N target) is obtained by hybridizing the N target nucleic acid to the N target probe and repeatedly detecting it. サブセット1 ) is labeled and detected, the label is cleaved from the labeled nucleic acid subset (L-round labeled probe hybridization, L=1), and then a second subset of the N target (N target) is extracted. サブセット2The process involves labeling and detecting the N-target nucleic acids, then cleaving the labels from a subset of the labeled nucleic acids (L rounds of labeled probe hybridization, L=2), and so on, until all N-target nucleic acids are detected. After all N-target nucleic acids have been detected with the desired number of labels (L=desired number of labeling rounds), an acid treatment step is performed to remove the hybridized N-target probes (e.g., ZZ probe signal-generating complexes). One or more additional sets of N-target nucleic acids (e.g., N'-target nucleic acids, N''-target nucleic acids, etc.) can be detected by repeating the entire Nplex workflow for one or more rounds. After all targets have been detected, the images are registered using image registration software algorithms to create a final synthesis of superimposed images at single-cell resolution. The total level of multiplex available in this method is K rounds of N-target nucleic acids × N-plexing per round ("N-plexing" refers to the flow from "hybridization of N-target probes" to "acid removal of probes and amplifiers" or, in the final round, to the last "counterstaining and imaging" step). Generally, N=1 or greater, and if an acid removal step is included, K=2 or greater. In the diagram shown in Figure 2B, when K=1, only L-round labeled probe hybridization, imaging, and fluorophore cleavage are required, and therefore the acid removal step for the probe and amplifier is not necessary. [Figure 3A] Acid treatment for sequential rounds of target nucleic acid detection. Acid treatment is performed on new This demonstrates the effective removal of target probes and amplification complexes in fresh frozen mouse brain. It shows the detection of four highly expressed positive control genes—glyceraldehyde-3-phosphate dehydrogenase (Gapdh), phosphoglycerate kinase 1 (Pgk1), basic helix-loop helix family member E22 (Bhlhe22), and complexin 2 (Cplx2)—in mouse brain prepared as fresh frozen sections. Target probes (ZZ probes) for the four genes were hybridized together, and the signals were amplified together using the RNAscope® HiPlex amplification system. The four genes were detected in the first round of repeated detection using fluorescently labeled probes corresponding to the signal amplification systems assigned to these four target probes. Alexa 488, ATTO 550, ATTO 647N, and Alexa 750 fluorophores were used to detect Gapdh, Pgk1, Bhlhe22, and Cplx2, respectively, and the nuclei were stained blue with DAPI (4′,6-diamidino-2-phenylindoline) (upper panel). After signal detection, tissue sections were treated with an acidic solution (20% acetic acid, 6.4x SSC) for 5 minutes at room temperature (RT), and the acid treatment was repeated two more times. The sections were then used for a second round of hybridization and amplification without the addition of target probes. In the second round after acid treatment, little to no signal was detected (lower panel), thus indicating complete removal of the previously hybridized target probes and signal amplification components. [Figure 3B] Shows acid treatment for sequential rounds of target nucleic acid detection. This demonstrates minimal impact on cellular RNA and tissue morphology of frozen mouse brains. As shown in Figure 3A (top panel), four positive control genes (Gapdh, Pgk1, Bhlhe22, and Cplx2) were detected in mouse brains prepared as fresh frozen sections. After signal detection, sections were treated with acid solution as shown in Figure 3A, except that the acid treatment was repeated four times instead of twice. The treated sections were then used for a second round of hybridization and amplification to detect the same four genes. Comparing the signals detected in the second round of hybridization (bottom panel) with those detected in the first round of hybridization (top panel), two rounds of hybridization and signal amplification of the target probe yielded similar expression patterns, indicating minimal RNA loss from repeated acid treatment. [Figure 4] Good shape after two rounds of acid treatment and sequential hybridization. Figure 4 shows the state and signal detection. In Figure 4, the top panel shows the detection of four positive control genes, RNA polymerase II subunit A (Polr2A), peptidyl prolyl isomerase B (Ppib), ubiquitin C (Ubc), and hypoxanthine phosphoribosyltransferase 1 (Hprt1), in fresh frozen mouse brain sections during the first round of target probe hybridization (k=1) and the third round of repeated detection (l=3), substantially performed as outlined in the workflow in Figure 2B. Twelve target probes (RNAscope® HiPlex 12 plex mouse positive controls) were simultaneously hybridized and amplified using the RNAscope® Hiplex assay. The four genes were detected in the first round using Alexa 488, ATTO 550, ATTO 647N, and Alexa 750 fluorophores, and the fluorophores were cleaved after imaging. The following four genes were detected in a second round of detection using the same four fluorophores, and the fluorophores were cleaved after imaging. A third round of repeated detection is shown in the upper panel of Figure 4. Alexa 488, ATTO 550, ATTO 647N, and Alexa 750 fluorophores were used to detect Polr2a, Ppib, Ubc, and Hprt1, respectively, and the nuclei were stained with blue DAPI. In Figure 4, the lower panel shows the detection of four distinct low-expression targets in the striatal region of the mouse brain: 5-hydroxytryptamine receptor 7 (Htr7), protocadherin 8 (Pcdh8), solute carrier family 32 member 1 (Slc32a1), and tyrosine hydroxylase (Th) in a third round of target probe hybridization and amplification (k=3) and a first round of repeated detection (l=1). The acid treatment, target hybridization, and amplification steps were performed after rounds 1 and 2 of target hybridization, as shown in Figure 3A. A second round of target probe hybridization and amplification was performed after the acid treatment. The target probe was omitted, and a probe diluent was used instead.A third round of target probe hybridization and amplification was performed after the second acid treatment using 12 different target probes. Four of the 12 target probes were initially detected in the first round of detection, as shown in the lower panel. [Figure 5] A-C show the aforementioned detection method for nucleic acid targets using signal-generating complexes (SGCs). A schematic diagram is shown. PPA stands for pre-pre-amplifier; PA stands for pre-amplifier; AMP stands for amplifier; LP stands for labeled probe. [Figure 6A] Schematic diagram of orthogonal labeling of target nucleic acids. RNAscope® App Figure 6A shows orthogonal labeling of target nucleic acids based on Sei. Figure 6A also shows three exemplary target nucleic acid labeling using each signal-generating complex (SGC). It shows the binding of target probe pair 1 (TP1a and TP1b) to target nucleic acid 1. It shows that the pre-amplifier (PA1) is bound to the target probe pair (TP1a and TP1b). It shows that multiple amplifiers (AMP1) are bound to PA1. It shows multiple labeled probes (LP1) bound to the amplifiers. Similar configurations are shown for targets 2 and 3, each having SGC components (target probe, pre-amplifier, amplifier, labeled probe) specific to each target. [Figure 6B] A schematic diagram of the orthogonal labeling of the target nucleic acid is shown. It shows a modification of the configuration shown in Figure 6A. . Two exemplary target nucleic acid labelings using each signal-generating complex (SGC) are shown in Figure 6B. The binding of target probe pair 1 (TP1a and TP1b) to target nucleic acid 1 is shown. The pre-preamplifier (PPA1) is shown to be bound to the target probe pair (TP1a and TP1b). Multiple preamplifiers (PA1) are shown to be bound to PPA1. Multiple amplifiers (AMP1) are shown to be bound to PA1. For simplicity, the amplifiers are shown to be bound to one preamplifier, but it should be understood that the amplifiers can be bound to all amplifiers. Multiple labeled probes (LP1) bound to the amplifiers are shown. A similar configuration for target 2 is shown, having SGC components (target probe, pre-preamplifier, preamplifier, amplifier, labeled probe) specific to each of the targets. [Figure 6C] Schematic diagram of orthogonal labeling of target nucleic acids. Basescope (trademark) Assembly Figure 6C shows orthogonal labeling of target nucleic acids based on (i). Figure 6C also shows two exemplary target nucleic acid labelings using each signal-generating complex (SGC). Binding of target probe pair 1 (TP1a and TP1b) to target nucleic acid 1 is shown. It is shown that a pair of pre-pre-amplifiers (PPA1a and PPA1b) are bound to their respective target probe pairs (TP1a and TP1b). It is shown that a pre-amplifier (PA1) is bound to the pre-amplifier pair (PPA1a and PPA1b). It is shown that multiple amplifiers (AMP1) are bound to PA1. For simplicity, the amplifier is shown as being bound to one pre-amplifier, but it should be understood that the amplifier can be bound to all amplifiers. Multiple labeled probes (LP1) bound to the amplifiers are shown. A similar configuration for target 2, which has SGC components (target probe, pre-pre-amplifier, pre-amplifier, amplifier, labeled probe) specific to each of the targets, is shown.

[0009] [Modes for carrying out the invention] The present invention relates, for example, to a method for sequential multiplex analysis of nucleic acids by in situ hybridization. The method of the present invention enables the detection of multiple target nucleic acids within the same sample and within the same cell.

[0010] This specification describes a chemical method for rapidly and effectively removing oligonucleotide probes and branched-chain DNA-like signal amplification systems that have little or no effect on cellular nucleic acids or cell morphology. This is generally achieved by using an acid-containing solution applied to a cell or tissue sample for a short period of time at room temperature, with little or no washing steps in between. The method of the present invention enables nucleic acid detection using a higher level of multiplexing, achieving detection of more nucleic acids in a sample and even within the same cell than the aforementioned nucleic acid detection assays.

[0011] One method of nucleic acid detection utilizes RNA ISH technology called RNAscope®, which uses specially designed oligonucleotide probes, sometimes referred to as "double Z" or ZZ probes, in combination with a branched-chain DNA-like signal amplification system to reliably detect single-molecule sensitive RNA as small as 1 kilobase 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)). Such probe design significantly improves the specificity of signal amplification, as signal amplification can only occur if both probes in each pair bind to their intended targets. RNAscope® technology can simultaneously distinguish up to four or five RNA targets using fluorescence detection.

[0012] Another recently described nucleic acid detection method (see U.S. Provisional Application No. 62 / 806,574, filed February 15, 2019) uses L(L=1, 2, 3, ...) rounds of repeated fluorescent labeling of I(I=2, 3, 4, ...) targets, followed by imaging and cleavage of the label as fluorophore cleavage, as shown in Figure 1. The iterative detection method provides simultaneous visualization of distinct target sequences (N) of LxI from single-round hybridization and amplification steps. In Figure 1, "N" indicates the total number of targets to be detected, and "I" above indicates the total number of targets per repeat in each L round. Generally, the number of targets (I) in each iterative round of labeling is the maximum number that can be clearly labeled and detected in a single round, as disclosed herein, such as 2 or more, 3 or more, 4 or more, etc. A single target nucleic acid (I=1) can be detected in a round if desired. In some embodiments, the number of targets in I may differ in each round. For example, in a detection of three rounds where I is 4, 4, and 1 in each round, a total of 9 targets will be detected. Therefore, the number of targets is N = sum(i1, i2, i3...i L ) where i1, i2, and i3 are the number of targets detected in each iteration round, and they may be the same or different, and L is the total number of rounds. If the numerical value "i" is the same in each round, then N = L x I.

[0013] To provide the ability to perform repeatable detection steps, the label, such as a fluorescent dye, is cleavable, for example, chemically cleavable, allowing detection in subsequent rounds using the same label as the first round. Within a round, a fluorescent dye conjugate, which can be spectrally separated using conventional fluorescence microscopy, is used. The fluorescent dye is then cleaved. One exemplary cleavage agent is the reducing agent tris(2-carboxyethyl)phosphine (TCEP). After cleavage of the detectable label, such as a fluorescent dye, subsequent rounds of target detection and imaging are performed. The assay strategy eliminates the need to detach the labeled probe from the previous round (which could disrupt existing signal amplification complexes associated with the remaining targets) and also preserves tissue and RNA integrity for maximized detection.

[0014] Similar to RNAscope® probes, each target probe contains a target binding segment (target binding site) that binds to a specific sequence within the target nucleic acid. The probe also contains a “tail” sequence that binds to a signal amplification molecule, as described herein. Two probes bind as a pair to adjacent sites in the target nucleic acid sequence. Only when both probes bind to their respective target sites can a complete binding site for the signal amplification molecule be formed (e.g., pre-amplifier in Figure 6A or pre-pre-amplifier in Figures 6B and 6C), enabling successful signal amplification and detection. Probes for different target sequences detected in the same round are designed to have independent and distinct binding sequences for the corresponding orthogonal amplification molecules. Such sequences can be easily designed using appropriate algorithms to achieve probe hybridization and parallel signal amplification of multiple targets. Targets are detected in multiple rounds using detectable labels, such as fluorophores, that are spectrally distinct within each round.

[0015] This detection strategy was adapted to an RNA ISH technique called RNAscope®. The result is an assay called RNAscope® HiPlex, which simultaneously provides specific and amplified detection of multiple target nucleic acid sequences at single-cell resolution. This is achieved by simultaneous targeted hybridization and amplification of all target sequences, followed by repeated detection of generally 3–5 nucleic acid targets in successive rounds. The number of target sequences that can be detected in this assay is limited by the number of orthogonal RNAscope® signal amplification systems used.

[0016] Assays and detection strategies are described herein that provide specific detection of two or more target nucleic acid sequences in a repeating manner at single-cell resolution, using a detection system, e.g., an RNAscope® signal amplification system, and a fluorescent dye having a common spectral profile. One way to increase the multiplex level without requiring an additional orthogonal RNAscope® signal amplification system is to completely remove the target probes and their associated signal amplification systems after all targets have been detected and imaged. The same detection system can then be reused for subsequent rounds of target probe hybridization and signal amplification.

[0017] The present invention relates to a method for enabling highly sensitive and specific detection of nucleic acid sequences in cells. The method of the present invention has numerous practical applications in research and diagnostics (Hu et al., Biomark.Res.2(1):1-13,doi:10.1186 / 2050-7771-2-3(2014), Ratan et al., Cureus 9(6):e1325.doi:10.7759 / cureus.1325(2017), Weier et al., Expert Rev.Mol.Diagn.2(2):109-119(2002)). The methods of the present invention can be used, for example, for spatial tissue mapping in highly complexed tissues such as the nervous system and tumor microenvironment, identification of known and new cell types, identification of cellular states, detection of modified gene expression in diseased cells and tissues, localization of modified gene expression in specific cell types, analysis of tumor heterogeneity, detection of biomarkers for cancer diagnosis and prognosis or other disease conditions, detection of biomarkers for companion diagnostics, and detection and identification of pathogens (e.g., bacteria, viruses, fungi, microbial parasites).

[0018] The present invention extends the probe design principles at the core of RNAscope® technology and branched DNA-like signal amplification (Wang et al., supra, 2012) to the sensitive and specific detection of multiple nucleic acid targets (more than one target nucleic acid detected in a repetitive pattern) within the same cell and / or tissue section. The same iterative assay design strategy can be used with the Basescope™ signal amplification system, which can be applied to detect short sequences (Baker et al., Nat. Commun. 8(1):1998, doi:10.1038 / s41467-017-02295-5 (2017)). The method can also be applied to the DNA detection of multiple targets. The methods of the present invention can also be applied to other signal amplification methods known in the art, such as hybridization chain reaction (HCR) (Choi et al., Development 145(12), pii: dev165753, doi:10.1242 / dev.165753 (2018)). The most recent version of HCR (HCR v3.0) uses a paired probe design similar to RNAscope® (Choi et al., supra, 2018). Other signal amplification systems to which the methods of the present invention can be applied include, but are not limited to, rolling circle amplification (Larsson et al., Nature Methods 7(5):395-397 (2010)), clampFISH (Rouhanifard et al., BioRxiv, 222794 (doi.org / 10.1101 / 222794) (2018)), and SABER (Kishi et al., supra, 2019).

[0019] Using the methods of the present invention, multiple gene targets in single cells and / or tissue sections can be labeled. This can be used with both fluorescence-based detection and imaging mass cytometry (Schulz et al., Cell Syst. 6(1):25-36 (2018)) for the fluorescent detection of multiple biomarkers in the spatial context of the tissue microenvironment. The methods can be used for research and diagnostic applications.

[0020] The method of the present invention using acid treatment can be commonly used to remove various target probes and, optionally, an amplification system when used, and to provide sequential rounds of hybridization to increase the multiplex level. The method of the present invention is applicable to oligonucleotide-based signal amplification methods such as hybridization chain reaction (Choi et al., supra, 2018), rolling circle amplification (Larsson et al., supra, 2010), clampFISH (Rouhanifard et al., supra, 2018), and SABER (Kishi et al., supra, 2019).

[0021] The method of the present invention can be used in combination with the RNAscope® HiPlex assay to further increase the number of gene targets detected in single cells / tissue sections. The method of the present invention can also be used in conjunction with any other DNA oligonucleotide-based ISH detection that requires sequential hybridization to remove existing probes and amplifiers. The method can be used, for example, to fluorescently detect multiple biomarkers in the spatial context of the tissue microenvironment.

[0022] In one embodiment, the RNAscope® HiPlex assay is used to detect one or more sets of N (e.g., 12) targets using K rounds of target probe hybridization and signal amplification, and repetitive detection using spectrally distinct fluorophores of I for each imaging round (see Figure 2B). In the embodiment shown in Figure 2B, the K sequential rounds (outer loop) are used to detect one or more sets of N targets, and the L repeat rounds (inner loop) are used to detect a subset of N targets in each K round. After detecting the first N targets, the target probe and signal amplification system are removed from the tissue / cells using an acidic solution containing a mixture of acids and salts, such as acetic acid. Acid treatment is reliable, fast, and highly effective, and causes minimal damage to cellular RNA and tissue morphology (see Example I and Figures 3A and 3B). The acid-treated tissue sample is then ready to be labeled with a new set of target probes, for example, using the RNAscope® HiPlex assay. This process can be repeated K times to detect a total of N × K targets within the same cell or tissue. As shown in Example II and Figure 4, two sets of targets were detected in sequential rounds after acid treatment of fresh frozen mouse brain tissue.

[0023] In Figures 1, 5, and 6, the target probe is shown in a "Z" configuration, as described, for example, in U.S. Patent No. 7,709,198, U.S. Publications 2008 / 0038725 and 2009 / 0081688, and WO2007 / 001986 and WO2007 / 002006. The Z configuration shown in Figures 1, 5, and 6 has a target binding site 5' relative to the pre-amplifier (Figures 5A and 6A) or pre-pre-amplifier (Figures 5B, 5C, 6B, and 6C) binding site of the target probe. It should be understood that such configurations are merely illustrative, as shown in Figures 1, 5, and 6, and the orientation may be reversed, i.e., the target binding site may be 3' relative to the pre-amplifier or pre-pre-amplifier binding site. It should be understood that the target probe pair can be independently oriented in any way; that is, one member of the target probe pair may have a target binding site 5' or 3' for a pre-amplifier or pre-pre-amplifier binding site, and may pair with a second probe having a binding site 5' or 3' for a pre-amplifier or pre-pre-amplifier.

[0024] As used herein, the term “labeled probe” refers to an entity that binds directly or indirectly, generally indirectly, to a target molecule, enabling the detection of the target. A labeled probe (or “LP”) typically comprises a nucleic acid-binding moiety, which is a single-stranded polynucleotide or oligonucleotide, containing one or more labels that directly or indirectly provide a detectable signal. The labels may be covalently bound to the polynucleotide, or the polynucleotide may be configured to bind to the labels. For example, a biotinylated polynucleotide may bind to a streptavidin-associated label. A labeled probe may, for example, directly hybridize to a target nucleic acid. Generally, a labeled probe may hybridize to a nucleic acid that is subsequently hybridized to the target nucleic acid, or to one or more other nucleic acids that are hybridized to the target nucleic acid. Thus, a labeled probe may include a polynucleotide sequence of the target nucleic acid, in particular a polynucleotide sequence that is complementary to it in part. Alternatively, a labeled probe may include at least one polynucleotide sequence that is complementary to a polynucleotide sequence in an amplifier, pre-amplifier, pre-pre-amplifier, signal-generating complex (SGC), etc., as described herein. Generally, in embodiments of the present invention, the labeled probe is bound to an amplifier. As used herein, an enzyme-labeled labeled probe refers to a labeled probe comprising a nucleic acid-binding moiety, such as an oligonucleotide, and an enzyme bound to the nucleic acid-binding moiety. As disclosed herein, the binding of the enzyme to the nucleic acid-binding moiety may be by covalent bond or by high-affinity binding interaction, such as biotin / avidin or other similar high-affinity binding molecules.

[0025] As used herein, “target probe” is a polynucleotide that hybridizes to a target nucleic acid and can capture or bind a labeled probe or signal-generating complex (SGC) component, such as an amplifier, pre-amplifier, or pre-pre-amplifier, to that target nucleic acid. The target probe can hybridize directly to the target probe, or it can hybridize to one or more nucleic acids that subsequently hybridize to a labeled probe. For example, the target probe can hybridize to an amplifier, pre-amplifier, or pre-pre-amplifier within an SGC. Thus, the target probe comprises a first polynucleotide sequence complementary to the polynucleotide sequence of the target nucleic acid, and a second polynucleotide sequence complementary to the polynucleotide sequence such as the labeled probe, amplifier, pre-amplifier, or pre-pre-amplifier. Generally, in embodiments of the present invention, the target probe binds to a pre-amplifier as shown in Figures 5A and 6A, or to a pre-pre-amplifier as shown in Figures 5B, 5C, 6B, and 6C. Target probes are generally single-stranded so that their complementary sequences can be used for hybridization with the corresponding target nucleic acid, labeled probe, amplifier, pre-amplifier, or pre-pre-amplifier. In embodiments of the present invention, target probes are provided as pairs.

[0026] As used herein, “amplifier” is a molecule, typically a polynucleotide, that can hybridize to multiple labeled probes. Typically, an amplifier hybridizes to multiple identical labeled probes. An amplifier may also hybridize to a target nucleic acid, to at least one target probe of a target probe pair, to both target probes of a target probe pair, or to nucleic acids bound to a target probe, such as an amplifier, a preamplifier, or a pre-preamplifier. For example, an amplifier may hybridize to at least one target probe and multiple labeled probes, or to a preamplifier and multiple labeled probes. Generally, in embodiments of the present invention, an amplifier may hybridize to a preamplifier. An amplifier may be, for example, a linear, forked, comb-like, or branched nucleic acid. As described herein for all polynucleotides, an amplifier may include modified nucleotides and / or non-standard internucleotide bonds, as well as standard deoxyribonucleotides, ribonucleotides, and / or phosphodiester bonds. Suitable amplifiers are described, for example, in U.S. Patents 5,635,352, 5,124,246, 5,710,264, 5,849,481, and 7,709,198, as well as U.S. Publications 2008 / 0038725 and 2009 / 0081688, each of which is incorporated by reference. Generally, in embodiments of the present invention, the amplifier is coupled to a pre-amplifier and a labeled probe (see Figures 5 and 6).

[0027] As used herein, “pre-amplifier” is a molecule, typically a polynucleotide, that functions as an intermediate binding component between one or more target probes and one or more amplifiers. Typically, the pre-amplifier hybridizes simultaneously with one or more target probes and multiple amplifiers. Exemplary pre-amplifiers are described, for example, in U.S. Patents 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. Generally, in embodiments of the present invention, the pre-amplifier binds to both members of a target probe pair (see Figures 5A and 6A), to a pre-pre-amplifier that can bind to a target probe pair (Figures 5B and 6B), or to both members of a pre-pre-amplifier pair that can bind to a target probe pair (see Figures 5C and 6C). The pre-amplifier also binds to the amplifier (see Figures 5 and 6).

[0028] As used herein, “pre-preamplifier” is a molecule, typically a polynucleotide, that functions as an intermediate binding component between one or more target probes and one or more preamplifiers. Typically, a pre-preamplifier hybridizes simultaneously with one or more target probes and multiple preamplifiers. Exemplary pre-preamplifiers are described, for example, in 2017 / 0101672, incorporated by reference. Generally, in embodiments of the present invention, a pre-preamplifier binds to a target probe pair (see Figures 5B and 6B), or to a member of a target probe pair (see Figures 5C and 6C), and to a preamplifier.

[0029] As used herein, the term “plural” is understood to mean two or more. Therefore, plural means, for example, 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, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 or more, 43 or more, 44 or more, 45 or more , 46 or above, 47 or above, 48 or above, 49 or above, 50 or above, 55 or above, 60 or above, 65 or above, 70 or above, 75 or above, 80 or above, 85 or above, 90 or above, 95 or above, 100 or above, 110 or above, 120 or above, 130 or above, 140 or above, 150 or above, 160 or above, 170 or above, 180 or above, 190 or above, 200 or above, 300 or above, 400 or above, 500 or above, 600 or above, 700 or above, 800 or above, 900 or above, or 1000 or above, or even larger numbers if desired for a particular use.

[0030] As described herein, the present invention relates to the multiplex detection of target nucleic acids, and the method provides detection of a greater number of target nucleic acids than previously described in situ hybridization methods. The method can be combined with further multiplexing by repeated rounds of detection of the probe acid removal, followed by hybridization of the target probe and detection, and an orthogonal amplification system can be used to clearly detect multiple target nucleic acids in repeated rounds of detection.

[0031] Figure 2A shows a schematic diagram of an embodiment of the present invention using acid treatment and removal of target(s) bound to target(s) nucleic acids(s) for sequential hybridization. The N-target probe is hybridized to the target nucleic acid, for example, in an in situ hybridization assay. The diagram shows the amplification of an arbitrary signal of the target probe hybridized to the target nucleic acid. Cells may be counterstained to facilitate cell visualization; for example, the nucleus may be stained with 4′,6-diamidino-2-phenylindole (DAPI). The target probe and counterstained cells are visualized, for example, by imaging, thereby detecting and imaging the target nucleic acid. When using an RNAscope® assay, the RNAscope® Double Z probe and signal are amplified simultaneously by an RNAscope® amplification system. An acid treatment step is performed to remove the target(s) bound to each target(s). One or more additional sets of N-target nucleic acids may be detected by repeating the entire Nplex workflow for one or more rounds. After all targets have been detected, the images are registered using an image registration software algorithm to create a final composite image superimposed at single-cell resolution. The total number of multiplex levels available in this method is K rounds, with N targets (multiple) × N plexings per round. Generally, N is greater than or equal to 1, and K is greater than or equal to 2 if an acid removal step is included. In the diagram shown in Figure 2A, if K=1, only one round of target probe hybridization and imaging is sufficient, and therefore the acid removal step is not required.

[0032] In one embodiment, the present invention provides a method for disrupting the binding of a probe bound to a nucleic acid in a cell, the method comprising contacting a cell with an acid reagent, the cell containing a first probe hybridized to a first target nucleic acid in the cell, and the acid reagent disrupting the hybridization between the first probe and the first target nucleic acid.

[0033] In one embodiment, contact of cells with an acid reagent is repeated one or more times. In one embodiment, the method further includes removing the first probe from the cells.

[0034] In one embodiment, the method further comprises the step of contacting cells with a second probe, the second probe hybridizing to a second target nucleic acid in the cell, the second target nucleic acid being identical to or different from the first target nucleic acid. In one embodiment, the method further comprises the step of contacting cells with an acid reagent, the acid reagent disrupting the hybridization between the second probe and the second target nucleic acid. In one embodiment, contacting cells with the acid reagent is repeated one or more times. In one embodiment, the method further comprises the step of removing the second probe from the cells.

[0035] In one embodiment, the present invention provides a method for disrupting the binding of a probe bound to a nucleic acid in a cell, the method comprising contacting a cell with an acid reagent, the cell comprising one or more first probes hybridized to one or more first target nucleic acids in the cell, and the acid reagent disrupting the hybridization between the one or more first probes and the one or more first target nucleic acids.

[0036] In one embodiment, contact of cells with an acid reagent is repeated one or more times. In one embodiment, the method further includes removing one or more first probes from the cells. In one embodiment, the cells contain two or more first probes hybridized to two or more first target nucleic acids. In one embodiment, each of the first target nucleic acids is labeled by hybridization to the first probe, and the label on each first target nucleic acid is distinguishable from the labels on other first target nucleic acids hybridized to the first probe.

[0037] In one embodiment, the method further comprises the step of contacting a cell with one or more second probes, the one or more second probes hybridizing to one or more second target nucleic acids in the cell, the one or more second target nucleic acids being identical to or different from one or more first target nucleic acids. In one embodiment, the cell contains two or more second probes hybridized to two or more second target nucleic acids. In one embodiment, each of the second target nucleic acids is labeled by hybridization to the second probe, and the label on each second target nucleic acid is distinguishable from the labels on other second target nucleic acids hybridized to the second probe.

[0038] In one embodiment, the method further comprises the step of contacting cells with an acid reagent, the acid reagent disrupting hybridization between a second probe and one or more second target nucleic acids. In one embodiment, contacting cells with the acid reagent is repeated one or more times. In one embodiment, the method further comprises the step of removing the second probe from the cells.

[0039] This invention is based on the discovery that acid reagents can be applied to samples containing cells, which contain nucleic acids hybridized to one or more probes, such as probes used to detect nucleic acids, thereby causing the acid reagent to disrupt the hybridization between the nucleic acid and the probe. It was previously not recognized that acid reagents could be used to remove probes bound to target nucleic acids within cells, while still preserving the integrity of the nucleic acids within the cells and the morphology of the cells, and allowing for repeated rounds of nucleic acid hybridization and detection within the cells. Nucleic acid integrity refers to the ability of the nucleic acid to be detected by hybridization to a detectable probe. As used herein, “acid reagent” is a solution comprising an acid and, optionally, a salt, which results in the disruption of hybridization between a nucleic acid probe and a target nucleic acid, thereby disrupting the binding between probes bound to the target nucleic acid, and, if used, disrupting the binding between signal amplification molecule within the cells, thereby allowing for the removal of probes and, if used, any constructed signal amplification complexes from cells, wherein treatment of cells with an acid reagent preserves the morphology of the cells and the integrity of the nucleic acids within the cells, so that one or more additional rounds of probe hybridization to target nucleic acids can be applied to the cells. The present invention provides compositions comprising the acid reagent of the present invention, as disclosed herein.

[0040] As described herein, the method of the present invention provides multiplex detection of a target nucleic acid by using an acid reagent to disrupt the binding of a probe bound to the target nucleic acid, thereby enabling the same detection system to be used for sequential round detection. As described herein, a nucleic acid-bound probe generally refers to a probe having at least several components that are nucleic acids, thereby providing probe binding to the target nucleic acid by nucleic acid hybridization, as is well known in the art. It should be understood that the method of the present invention, which uses an acid reagent to disrupt the binding of a probe to a target nucleic acid, can be applied to any probe bound to a target nucleic acid by hybridization. It should be further understood that a probe bound to a target nucleic acid, whose binding between the probe and the target nucleic acid can be disrupted using an acid reagent, may be a probe that is a single nucleic acid directly bound to the target nucleic acid, or a probe that is a complex of multiple nucleic acid components. Thus, the binding of a probe to a target nucleic acid that is disrupted by the acid reagent of the present invention may be the disruption of a probe directly bound to the target nucleic acid and / or any component of a probe complex bound to the target nucleic acid. Examples of probes, which are complexes of multiple nucleic acid components, include the signal-generating complexes (SGCs) disclosed herein, as well as other types of probes and probe systems that can be used to detect target nucleic acids (e.g., hybridization chain reaction (HCR) (Choi et al., Development 145(12), pii:dev165753, doi:10.1242 / dev.165753 (2018), rolling circle amplification (Larsson et al., Nature Methods 7(5):395-397 (2010), clampFISH (Rouhanifard et al., BioRxiv, 222794 (doi.org / 10.1101 / 222794) (2018), and SABER (Kishi et al., Nat. Methods 16:533-544 (2019)).Therefore, any desired probe or probe system bound to a target nucleic acid can be destroyed using the method of the present invention, which involves disrupting the binding of a probe to a target nucleic acid using an acid reagent. After acid treatment, the same probe or probe system can be used again to detect the same or a different target nucleic acid.

[0041] The acid reagent of the present invention contains an acid. Examples of suitable acids for use in the acid reagent include, but are not limited to, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, oxalic acid, malonic acid, succinic acid, malic acid, tartaric acid, and citric acid. The acid reagent generally contains an acid concentration of about 5-40%. In one embodiment, the acid reagent contains 20-30% acid. For example, an acid reagent may contain 5-40% acid, e.g., 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% acid (volume / vol%). In certain embodiments, the acid reagent contains 5-40% acetic acid, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% acetic acid (volume / vol%), or concentrations in between.

[0042] Optionally, the acid reagent may also include salts. In one embodiment, the acid reagent comprises, in addition to the acid, saline sodium citrate (SSC), where 20x SSC corresponds to 3.0 M NaCl and 0.3 M sodium citrate at pH 7.0 (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001) and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). In one embodiment, the acid reagent contains 1x to 13x SSC. For example, acid reagents are 1x, 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x , 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, 4.9x, 5x, 5.1x, 5.2x, 5.3x, 5.4x, 5.5x, 5.6x, 5.7x, 5 .8x, 5.9x, 6x, 6.1x, 6.2x, 6.3x, 6.4x, 6.5x, 6.6x, 6.7x, 6.8x, 6.9x, 7x, 7.1x, 7.2x, 7.3x, 7.4x, 7.5x, 7.6x, 7.7x, 7.8x, 7.9x, 8x, 8.1x, 8.2x, 8.3x, 8.4x, 8.5x, 8.6x, 8.7x, 8.8x, 8.9x, 9x, 9.1x , 9.2x, 9.3x, 9.4x, 9.5x, 9.6x, 9.7x, 9.8x, 9.9x, 10x, 10.1x, 10.2x, 10.3x, 10.4x, 10.5x, 10.6x It may contain SSCs at concentrations of 10.7 times, 10.8 times, 10.9 times, 11 times, 11.1 times, 11.2 times, 11.3 times, 11.4 times, 11.5 times, 11.6 times, 11.7 times, 11.8 times, 11.9 times, 12 times, 12.1 times, 12.2 times, 12.3 times, 12.4 times, 12.5 times, 12.6 times, 12.7 times, 12.8 times, 12.9 times, 13 times, etc., or concentrations in between.

[0043] In another embodiment, the acid reagent, in addition to the acid, contains sodium chloride, sodium phosphate, ethylenediaminetetraacetic acid (EDTA) (SSPE), with a pH of 7.4, and 20 times SSPE, 3.0 M sodium chloride, 0.2 M sodium hydrogen phosphate (NaH2PO4), 0.02 M corresponds to ethylenediaminetetraacetic acid (EDTA) (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001)). In one embodiment, the acid reagent contains 1x to 13x SSPE. For example, the acid reagent may be 1x, 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x , 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, 4.9x, 5x, 5.1x, 5.2x, 5.3x, 5.4x, 5.5x, 5.6x, 5.7x, 5 .8x, 5.9x, 6x, 6.1x, 6.2x, 6.3x, 6.4x, 6.5x, 6.6x, 6.7x, 6.8x, 6.9x, 7x, 7.1x, 7.2x, 7.3x, 7.4x, 7.5x, 7.6x, 7.7x, 7.8x, 7.9x, 8x, 8.1x, 8.2x, 8.3x, 8.4x, 8.5x, 8.6x, 8.7x, 8.8x, 8.9x, 9x, 9.1x , 9.2x, 9.3x, 9.4x, 9.5x, 9.6x, 9.7x, 9.8x, 9.9x, 10x, 10.1x, 10.2x, 10.3x, 10.4x, 10.5x, 10.6x It may contain SSPE at concentrations of 10.7 times, 10.8 times, 10.9 times, 11 times, 11.1 times, 11.2 times, 11.3 times, 11.4 times, 11.5 times, 11.6 times, 11.7 times, 11.8 times, 11.9 times, 12 times, 12.1 times, 12.2 times, 12.3 times, 12.4 times, 12.5 times, 12.6 times, 12.7 times, 12.8 times, 12.9 times, 13 times, etc., or concentrations in between.

[0044] In another embodiment, the acid reagent contains, in addition to the acid, 10–500 mM sodium phosphate at pH 7.8, or optionally in a pH range of 7–8. For example, the acid reagent may be 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 2 It may contain sodium phosphate at concentrations of 80 mM, 290 mM, 300 mM, 310 mM, 320 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 380 mM, 390 mM, 400 mM, 410 mM, 420 mM, 430 mM, 440 mM, 450 mM, 460 mM, 470 mM, 480 mM, 490 mM, 500 mM, etc., or concentrations in between.

[0045] In another embodiment, the acid reagent contains 10 mM to 6 M sodium chloride (NaCl) in addition to the acid. For example, the acid reagents may be 10 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, 1 M, 1.1 M, 1.2 M, 1.3 M It may contain sodium chloride in concentrations of 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, 5M, 5.1M, 5.2M, 5.3M, 5.4M, 5.5M, 5.6M, 5.7M, 5.8M, 5.9M, 6M, etc., or concentrations in between.

[0046] In some embodiments, the acid reagent contains 5–40% acid and 1–12.8 times SSC. It should be understood that the acid reagent may independently contain 5–40% acid and 1–12.8 times SSC in any of the acid and SSC concentration combinations disclosed herein. In some embodiments, the acid reagent contains 20–30% acid and 3.2–12.8 times SSC, or independently, increases in acid and SSC between those two. In some embodiments, the acid is acetic acid. In some embodiments, the acid is formic acid. In some embodiments, the acid reagent contains 20% acid and 3.2 times SSC. In another embodiment, the acid reagent contains 20% acid and 6.4 times SSC. In another specific embodiment, the acid reagent contains 20% acid and 12.8 times SSC. In another specific embodiment, the acid reagent contains 30% acid and 3.2 times SSC. In another specific embodiment, the acid reagent contains 30% acid and 6.4 times SSC. In another specific embodiment, the acid reagent contains 30% acid and 12.8 times SSC. In a specific embodiment, the acid reagent contains 20% acetic acid and 3.2 times SSC. In another specific embodiment, the acid reagent contains 20% acetic acid and 6.4 times SSC. In another specific embodiment, the acid reagent contains 20% acetic acid and 12.8 times SSC. In another specific embodiment, the acid reagent contains 30% acetic acid and 3.2 times SSC. In another specific embodiment, the acid reagent contains 30% acetic acid and 6.4 times SSC. In another specific embodiment, the acid reagent contains 30% acetic acid and 12.8 times SSC.

[0047] In some embodiments, the method of the present invention relating to applying an acid reagent to cells to cause disruption of hybridization between probes hybridized to target nucleic acids within cells is carried out at room temperature. In other embodiments, the acid reagent may be applied to cells at a temperature just below or above room temperature. Thus, the method of the present invention for applying an acid reagent to cells to cause disruption between probes hybridized to target nucleic acids within cells may be carried out at a temperature, for example, about 4°C to about 40°C. For example, the method can be carried out at temperatures of 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C, or increasing temperatures in between.

[0048] The present invention relates to a method of applying an acid reagent to cells to cause disruption of hybridization between probes hybridized to target nucleic acids within the cells. The method is performed for a set period of time and optionally repeated. The acid reagent is generally allowed to come into contact with the cells for 1 to 30 minutes or longer. For example, the acid reagent is allowed to come into contact with the cells for 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes or longer, or an increasing number in between. In some embodiments, the acid reagent treatment is repeated 1 to 10 times. For example, the acid reagent treatment is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 times (i.e., up to 10 times). In another example, the acid reagent treatment is performed 1, 2, 3, 4, 5, or 6 times (i.e., up to 5 times). In some embodiments, the acid reagent is brought into sequential contact with cells without removing the acid reagent (e.g., by aspirating the acid reagent from the cells) or without washing the cells (e.g., by washing the cells between applications of the acid reagent). Optionally, the acid reagent may be removed from contact with cells, for example, by aspirating the acid reagent from the cells or by washing the cells with a suitable buffer. Suitable washing buffers include, but are not limited to, buffers routinely used in in situ hybridization assays.

[0049] It should be understood that, as disclosed herein, the conditions for removing probes bound to target nucleic acids within cells can be readily determined, as disclosed herein, depending on the components and concentrations of the acid reagent, the incubation time of the acid reagent with the cells, and the number of times the incubation is repeated. The effectiveness of probe removal from cells can be readily determined by analyzing the cells using the same method used to detect the target nucleic acid and confirming whether residual probes are detectable (see Examples I and II). If residual probes are still present, the acid reagent treatment should simply be repeated until the previously detected probes are no longer detectable or are detected at sufficiently low levels, allowing for the detectable labeling of target nucleic acids with the same label in subsequent rounds of labeling. Similarly, the number of times cells can be treated with an acid reagent while preserving the cellular morphology and integrity of the nucleic acid within the cell, in order to enable subsequent detection of the nucleic acid, can be easily determined by repeatedly treating cells with a given acid reagent and determining whether the target nucleic acid can still be detected under a given set of conditions, for example, by determining the ability to detect a positive control nucleic acid within the cell, or by determining whether a similar cellular morphology can be detected within the cell after the cell has been treated with the acid reagent one or more times (see Examples I and II). Once a set of conditions for incubation time and number of repetitions for applying a given acid reagent is determined, the conditions can be applied to other cell samples. As disclosed herein, a range of acid reagents and conditions have been tested and shown to be effective in disrupting hybridization between the probe and the target nucleic acid while preserving cellular morphology and nucleic acid integrity, so that a new round of detection of the target nucleic acid can be applied (see Example III).

[0050] In some embodiments, a single target nucleic acid is detected in each round. In such cases, the sample is contacted with a set of target probes that can specifically hybridize to the target nucleic acid, rather than contacting the sample with a set of target probes for multiple target nucleic acids. In other embodiments, multiple targets are detected in a single round, as disclosed herein.

[0051] Accordingly, the present invention relates to the use of unique labels for multiple nucleic acid targets and repeated detection of subsets of target nucleic acids to achieve higher multiplex detection of target nucleic acids, followed by acid reagent treatment to remove bound probes to achieve higher multiplex detection of target nucleic acids. The effectiveness of the present invention's method for performing multiplex detection of target nucleic acids in repeated rounds is demonstrated in the present invention, as described in the examples.

[0052] As shown in Figure 1, in one embodiment, the method of the present invention uses simultaneous hybridization of target probes to multiple target nucleic acids and an amplification system to detect target nucleic acids. However, rather than detecting all target nucleic acids at once, the labeling and detection of target nucleic acids are performed in iterative rounds, in which the first round detects only the target nucleic acid subset to which the target probe is bound. This is schematically shown in Figure 1, where targets 1-4 are detected in the first round. By using cleavable labels, after imaging the detectable labels bound to the target nucleic acids (targets 1-4 in Figure 1) in the first round, the labels are removed from the sample ("fluorophore cleavage" in Figure 1). Once the labels bound to the target nucleic acids in the first round are cleaved, the detection in the second round is applied by adding a second group of labels that generally detect different subsets of the target nucleic acids (shown as targets 5-8 in Figure 1). By cleaving the labels from the target nucleic acids in the first round, the same detectable labels (shown as fluorophores in Figure 1) as in the first round can be used in the second round. Such a cycle of cleaving a label bound to the target nucleic acid and adding a new label to detect a new subset of the target nucleic acid allows for the detection of even more nucleic acid targets within the same sample and the same cells than previously described methods. Further layers of the multiplex can be achieved by acid treatment of the sample to remove the target probe and amplification system (e.g., SGC), as shown in Figure 2B, so that a new round of the method shown in Figure 1 can be repeated.

[0053] Figure 2B shows a schematic diagram of acid treatment and removal of probes bound to target nucleic acids for sequential hybridization. The N-target probe is hybridized to the target nucleic acid using an in situ hybridization assay, such as the RNAscope® HiPlex assay. The N-target probe is hybridized to the target sequence (e.g., RNAscope® Double Z probe) and the signal is amplified simultaneously (e.g., RNAscope® amplification system). The diagram in Figure 2B shows arbitrary signal amplification of the target probe hybridized to the target nucleic acid. Cells may be counterstained to facilitate cell visualization; for example, the nucleus may be stained with 4′,6-diamidino-2-phenylindole (DAPI). The N-target nucleic acid is detected by labeling, e.g., fluorescent labeling, imaging, and cleavage of the detectable label, e.g., repeated rounds of fluorescent label cleavage. In the diagram, a subset of the N target (N target) is detected by hybridizing the N-target nucleic acid to the N-target probe and repeatedly detecting it. サブセット1 ) is labeled and detected, the label is cleaved from the labeled nucleic acid subset (L-round labeled probe hybridization, L=1), and then a second subset of the N target (N target) is extracted. サブセット2The process involves labeling and detecting the N-target nucleic acids, then cleaving the labels from a subset of the labeled nucleic acids (L rounds of labeled probe hybridization, L=2), and so on, until all N-target nucleic acids are detected. After all N-target nucleic acids have been detected for the desired number of labels (L=rounds of desired labels), an acid treatment step is performed to remove the hybridized N-target probes (e.g., removal of signal-generating complexes (SGCs) as described herein). One or more additional sets of N-target nucleic acids (e.g., N'-target nucleic acids, N''-target nucleic acids, etc.) may be detected by repeating the entire Nplex workflow for one or more rounds. After all targets have been detected, the images are registered using an image registration software algorithm to create a final synthesis of superimposed images at single-cell resolution. The total level of multiplex available in this method is K rounds of N-target nucleic acids × N-plexing per round ("N-plexing" refers to the flow from "hybridization of N-target probes" to "acid removal of probes and amplifiers" or, in the final round, to the last "counterstaining and imaging" step). Generally, if N=1 or greater and includes an acid removal step, K=2 or greater. In the diagram shown in Figure 2B, when K=1, only L-round labeled probe hybridization, imaging, and fluorophore cleavage are required, and therefore the acid removal step of the probe and amplifier is not necessary. Furthermore, in the final round of detection of N-target nucleic acids in which no additional N-target nucleic acids are detected (i.e., K=N 最終 It should be understood that, as "N" is the total number of K rounds, no acid treatment step is required. Therefore, in the schematic diagram shown in Figure 2B, after all desired target nucleic acids have been detected and imaged, image registration is performed for analysis of the target nucleic acids without requiring any further labeling, cleavage, and / or acid treatment steps.

[0054] Generally, when using distinct and distinguishable labels for multiplex detection of target nucleic acids, there is a limit to the number of distinct labels that can be distinguished simultaneously. For example, in the case of fluorescent labels, in order to detect multiple labels simultaneously, the spectra of multiple emissions from the fluorophores must be separated so that the fluorescence microscope can distinguish the fluorophores simultaneously. The need to separate the spectra of emissions from the fluorophores limits the number of fluorophores that can be visualized simultaneously. This invention overcomes this limitation by repeatedly detecting the labels so that the target nucleic acids can be detected in sequential rounds using the same fluorophores.

[0055] Figure 6 shows the orthogonality of the detection systems that may be used in the method of the present invention. Figure 6A shows one embodiment using three exemplary target nucleic acids and their respective orthogonal detection systems (also referred to in the present invention as signal-generating complexes (SGCs)). As shown in Figure 6A, each target nucleic acid is hybridized to a specific target probe pair (TP1a and TP1b, TP2a and TP2b, TP3a ​​and TP3b), then to its respective specific pre-amplifier (PA1, PA2, PA3), then to its respective specific multiple amplifiers (AMP1, AMP2, AMP3), and then to its respective specific multiple labeled probes (LP1, LP2, LP3). Figure 6B shows another embodiment using two exemplary target nucleic acids and their respective orthogonal detection systems. As shown in Figure 6B, each target nucleic acid is hybridized to a specific target probe pair (TP1a and TP1b, TP2a and TP2b), then to its respective specific pre-pre-amplifier (PPA1, PPA2), then to its respective specific multiple pre-amplifiers (PA1, PA2), then to its respective specific multiple amplifiers (AMP1, AMP2), and finally to its respective specific multiple labeled probes (LP1, LP2). Figure 6C shows another embodiment using two exemplary target nucleic acids and their respective orthogonal detection systems. As shown in Figure 6C, each target nucleic acid hybridizes to a specific target probe pair (TP1a and TP1b, TP2a and TP2b), then to its respective specific pre-preamplifier pair (PPA1a and PPA1b, PPA2a and PPA2b), then both pre-preamplifiers hybridize to their respective specific preamplifiers (PA1 and PA2), then to their respective specific amplifiers (AMP1 and AMP2), and finally to their respective specific labeled probes (LP1 and LP2). For simplicity, multiple amplifiers are shown bound to one of the preamplifiers, but it should be understood that an amplifier can bind to any of the preamplifiers.As shown in Figure 6, each nucleic acid target has a specific detection system, and the binding of the components of this system is mediated by a unique binding site that binds to one specific complex but not to others. Such unique binding sites for hybridization of SGC components to specific target nucleic acids can be achieved by designing the binding site (nucleic acid sequence) to obtain the desired specificity, as is well known in the art and as described herein. This orthogonal detection system, in which each target is uniquely labeled, makes it possible to detect multiple target nucleic acids within the same sample.

[0056] In some embodiments described herein, the method utilizes an orthogonal amplification system to uniquely label target nucleic acids so that multiple target nucleic acids can be analyzed within the same sample and even within the same cell. The present invention utilizes the construction of a signal-generating complex (SGC) that is specific to a particular target nucleic acid to enable unique identification of each target nucleic acid. In one embodiment, the sample is contacted with a set of target probes, including a pair of target probes that can specifically hybridize to a target nucleic acid. The sample is also contacted with a set of pre-amplifiers that are specific to each set of target probes, as well as a set of pre-amplifiers that can hybridize to the target probe pair that hybridizes to each respective target nucleic acid. Such an embodiment is schematically shown in Figure 6A. The sample is also contacted with an amplifier, which includes a subset of amplifiers specific to each pre-amplifier that is specific to the target probe pair that is specific to the target nucleic acid. That is, each target nucleic acid has an assembly consisting of a unique component of the SGC that distinguishes between target nucleic acids, i.e., a target probe pair, a pre-amplifier, and an amplifier. In additional embodiments, a pre-pre-amplifier may be coupled to the target probe pair as an additional amplification layer between the target probe pair and the pre-amplifier (see Figures 5B and 6B).

[0057] In another embodiment, the sample is contacted with a set of target probes, each containing a pair of target probes that can specifically hybridize to a target nucleic acid. The sample is also contacted with a set of pre- and pre-amplifiers that can hybridize to the target probe pairs that hybridize to each respective target nucleic acid, along with a pair of pre- and pre-amplifiers specific to each set of target probes. Such an embodiment is schematically shown in Figure 6C. The sample is also contacted with a set of pre-amplifiers, each containing a pre-amplifier that can specifically bind to both pairs of pre- and pre-amplifiers that are specific to the target probe pair that is specific to the target. The sample is also contacted with an amplifier, which comprises a subset of amplifiers specific to each pre-amplifier that is specific to the pre- and pre-amplifier pair that is specific to the target nucleic acid pair. That is, each target nucleic acid has an assembly of SGC-specific components, i.e., target probe pairs, pre- and pre-amplifiers, pre-amplifiers, and amplifiers, which perform discrimination between target nucleic acids.

[0058] To detect target nucleic acids, a set of labeled probes is brought into contact with the sample. Instead of bringing the sample into contact with a set of labeled probes that can detect all target nucleic acids, the sample is brought into contact with a set of labeled probes that can detect a subset of target nucleic acids. Thus, target nucleic acids are detected in iterative detection rounds rather than detecting all target nucleic acids at once. Within a single round, the labeled probes specific to each target nucleic acid are distinguishable from one another so that all target nucleic acids associated with the applied labeled probe in the first round can be detected simultaneously.

[0059] The number of target nucleic acids that can be detected simultaneously in a single round depends on the type of label used in the labeling probe and the method by which the label can be distinguished. For example, when using fluorescent labels, the fluorophores used in a single round must be distinguishable, and therefore spectral separation of the fluorophore emission must be performed. The number of fluorophores that can be distinguished simultaneously is up to 10, depending on the detection system and the availability of filters and / or software that can be used to distinguish fluorophores with overlapping emission, and spectral separation is considered to exist if they can be distinguished, as is well known in the art. Imaging systems for detecting multiple fluorescent labels are well known in the art (e.g., Vectra Polaris, Perkin Elmer, Waltham MA).

[0060] In one embodiment, the method of the present invention is applied to the detection of one or more target nucleic acids per round, for example, in each round, one, two, three, four, five, six, seven, eight, nine, ten, or more target nucleic acids are detected. As disclosed herein, those skilled in the art can select suitable separate labels and a suitable number of rounds of detection so that a desired number of target nucleic acids can be detected in the sample.

[0061] In some embodiments, the label on the label probe is cleavable so that the distinguishable features of the label used in the first round of detection can be utilized in subsequent rounds. Therefore, when the first round of detection is performed to detect a subset of target nucleic acids, the label bound to the target nucleic acids is cleaved to remove the label from the first subset of target nucleic acids. Exemplary cleavable conjugates of labels to label probes are described herein. Once the label is cleaved, a second round of detection is performed by contacting the sample with a second set of label probes specific to target nucleic acids that are generally different from the target nucleic acids detected in the first round. Since the label from the first round has been cleaved from each target nucleic acid, the same distinguishable label may be used in the label probe for the second round of detection. Further multiplexing may be achieved by acid treatment of the sample to remove the binding probe and amplification system (e.g., SGC) so that the above steps can be repeated to detect different sets of target nucleic acids, as described herein. It should be understood that while the same label may be used in iterative detection rounds of subsets of target nucleic acids, it is not necessary to use the same label in subsequent detection rounds, as long as the label used in the same round is distinguishable.

[0062] Once the detection of a subset of the target nucleic acid in the second round has been performed, one or more additional rounds of cleavage, labeling, and detection may be optionally applied to the sample. For example, after the detection of a second subset of the target nucleic acid, the label may be cleaved from the SGC assembled on the second subset of the target nucleic acid, and a third round of labeling and detection may be performed to detect a third subset of the target nucleic acid, distinct from the first and second subsets. Such iterative rounds of detection of the target nucleic acid can be performed for the purpose of detecting multiple target nucleic acids in a desired number by utilizing the fact that the detection is repeated a desired number of times. Further multiplexing may be achieved by acid treatment of the sample to remove the binding probe and amplification system (e.g., SGC) so that the above steps can be repeated to detect the same or different sets of the target nucleic acid in order to detect a desired number of target nucleic acids, as described herein. It should be understood that in the final iterative detection round, the label probe does not need to contain a cleavable label, as no further detection rounds will be performed. Therefore, in the final iterative detection round, the use of a labeled probe containing a cleavable label is optional; that is, the labeled probe may or may not contain a cleavable label. Generally, detection rounds are performed so that a different target nucleic acid is detected in each round. However, it should be understood that subsequent rounds of detection may include the detection of one or more target nucleic acids (including up to all nucleic acids in previous rounds) that overlap with previous rounds, so that the same or overlapping target nucleic acids are detected in one or more sequential rounds. Thus, using the detection of the same or overlapping target nucleic acids in sequential rounds, a transient barcode can be generated for each target nucleic acid when the same target nucleic acid is detected in a given color sequence in each round. Such a method has been previously described, for example, as sequential barcode fluorescence in situ hybridization (seqFISH) (see Shah et al., Neuron 92(2):342-357 (2016)).Therefore, the present invention, which uses an acid reagent to remove probes bound to target nucleic acids within cells, can be applied to methods such as seqFISH to provide an efficient method for performing multiple rounds of hybridization to generate barcodes for a higher level of multiplexing.

[0063] In one embodiment, the present invention provides a method for multiplex detection of multiple target nucleic acids in a cell, the method comprising (A) contacting a sample comprising cells containing multiple target nucleic acids with a set of probes specific to one or more target nucleic acids, wherein the probes for the target nucleic acids include (a) a set of target probes, the set of target probes comprising one or more pairs of target probes that specifically hybridize to the target nucleic acids, and (b) a set of preamplifiers, the set of preamplifiers comprising multiple preamplifiers, the preamplifiers comprising binding sites for pairs of target probes and multiple binding sites for the amplifiers. (c) a set of pre-amplifiers, (d) a set of amplifiers, each set comprising multiple amplifiers, each containing a binding site for the pre-amplifier and multiple binding sites for the labeled probes, and (b) a set of labeled probes, each of which contains a label and a binding site for the amplifiers, and (c) contacting the sample with an acid reagent to break the binding of the probes to the target nucleic acids (see Figures 2A and 6A).

[0064] In one embodiment, the present invention provides a method for multiplex detection of multiple target nucleic acids in a cell, the method comprising (A) contacting a sample comprising cells containing multiple target nucleic acids with a set of probes specific to one or more target nucleic acids, wherein the probes for the target nucleic acids include (a) a set of target probes, the set of target probes comprising one or more pairs of target probes that specifically hybridize to the target nucleic acids; (b) a set of pre-preamplifiers, the set of pre-preamplifiers comprising one or more pre-preamplifiers, each pre-preamplifier comprising a binding site for one or more pairs of target probes; and (c) a set of preamplifiers, the set of preamplifiers comprising multiple preamplifiers (b) contacting a set of preamplifiers comprising an agent, wherein the preamplifier includes a binding site for the preamplifier and a plurality of binding sites for the amplification agent; (d) a set of amplification agents, wherein the set of amplification agents comprises a plurality of amplification agents, wherein the amplification agents include a binding site for the preamplifier and a plurality of binding sites for the labeled probe; and (e) a set of labeled probes, wherein each labeled probe in the set of labeled probes includes a label and a binding site for the amplification agent; (b) detecting a detectable label bound to each target nucleic acid; and (c) contacting the sample with an acid reagent to disrupt the binding of the probe bound to the target nucleic acid (see, for example, Figures 2A and 6B).

[0065] In one embodiment, the present invention provides a method for multiplex detection of multiple target nucleic acids in a cell, the method comprising (A) contacting a sample comprising cells containing multiple target nucleic acids with a set of probes specific to one or more target nucleic acids, wherein the probes for the target nucleic acids include (a) a set of target probes, the set of target probes comprising one or more pairs of target probes that specifically hybridize to the target nucleic acids, (b) a set of pre-preamplifiers, the set of pre-preamplifiers comprising one or more pairs of pre-preamplifiers, each pre-preamplifier in the pair comprising a binding site for one of the target probes of the pair of target probes, and (c) a set of preamplifiers, the set of preamplifiers (d) a set of pre-amplifiers comprising a plurality of pre-amplifiers, each pre-amplifier comprising a binding site for a pre-pre-amplifier pair and a plurality of binding sites for an amplifier; (e) a set of amplifiers comprising a plurality of amplifiers, each amplifier comprising a binding site for a pre-amplifier and a plurality of binding sites for a labeled probe; and (b) a set of labeled probes, each labeled probe comprising a label and a binding site for an amplifier; and (c) contacting the sample with an acid reagent to break the binding of the probe to the target nucleic acid (see, for example, Figures 2A and 6C).

[0066] In one embodiment, contact of cells with an acid reagent is repeated one or more times. In one embodiment, the method further includes repeating steps (A) and (B) one or more times, or repeating steps (A), (B), and (C).

[0067] In one embodiment, the present invention provides a method for detecting multiple target nucleic acids, the method comprising: (A) contacting a sample comprising cells containing multiple nucleic acids with a set of multiple target probes, wherein each set of target probes comprises a pair of target probes that specifically hybridize with the target nucleic acids; (B) contacting the sample with a set of pre-amplifiers, wherein the set of pre-amplifiers comprises multiple pre-amplifiers, each pre-amplifier being specific to each set of target probes, each pre-amplifier comprising a binding site for one pair of target probes from the set of target probes and multiple binding sites for the amplifier; (C) contacting the sample with a set of amplifiers, wherein the set of amplifiers comprises multiple subsets of amplifiers specific to each pre-amplifier, each subset of amplifiers comprising multiple amplifiers, each amplifier of the subset of amplifiers comprising a binding site for one of the pre-amplifiers specific to the set of target probes and multiple binding sites for the labeled probe; and (D) contacting the sample with a first set of labeled probes, wherein the labeled probes (E) detecting the labeled probes of the first set of labeled probes bound to the target nucleic acid, thereby detecting the first subset of the target nucleic acid, (F) cleaving the label from the first set of labeled probes bound to the first subset of the target nucleic acid, and (G) contacting the sample with a second set of labeled probes, wherein the second set of labeled probes comprises a plurality of second subsets of labeled probes, and each subset of labeled probes includes a label and binding site for one of the amplification agents, the label in each first subset of labeled probes is distinguishable between the first subsets of labeled probes, the label is cleavable, and the first set of labeled probes specifically labels a first subset of the target nucleic acid hybridized with a plurality of target probes, (E) detecting the labeled probes of the first set of labeled probes bound to the target nucleic acid, thereby detecting the first subset of the target nucleic acid, (F) cleaving the label from the first set of labeled probes bound to the first subset of the target nucleic acid, and (G) contacting the sample with a second set of labeled probes, wherein the second set of labeled probes comprises a plurality of second subsets of labeled probes, and each subset of labeled probes isThe label probe is specific to one of the amplification agents in a subset of amplification agents, and the second subset of the label probe is specific to an amplification agent in a subset of amplification agents different from that of the first subset of the label probe, each subset of the label probe includes multiple label probes, each label probe in the subset of the label probe includes a label and binding site for one of the amplification agents in the subset of amplification agents, the labels in each second subset of the label probe are distinguishable between the second subsets of the label probe, the labels are optionally cleavable, and the second set of label probes is the target nucleus The method includes (H) specifically labeling and contacting a second subset of the target nucleic acid hybridized with a set of multiple target probes different from a first subset of the acid, and detecting the second subset of the target nucleic acid by detecting the second set of labeled probes bound to the target nucleic acid, thereby detecting multiple target nucleic acids, and (I) contacting the sample with the acid reagent to break the binding of the probes bound to the target nucleic acid (see, for example, Figures 2B and 6A (L=2, and the label is cleaved from SGC)).

[0068] In one embodiment of such a method, the method is to (J) cleave the label from a second set of labeled probes bound to a second subset of a target nucleic acid before step (I), and (K) contact the sample with a third set of labeled probes, wherein the third set of labeled probes comprises a plurality of third subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of amplifiers, the third subset of labeled probes is specific to an amplifier of a subset of amplifiers different from the first and second subsets of labeled probes, each subset of labeled probes comprises a plurality of labeled probes, each labeled probe of the subset of labeled probes is amplified (L) Includes a labeling and binding site for one of the amplification agents from a subset of the agent, wherein the label in each third subset of the labeling probe is distinguishable among the third subsets of the labeling probe, the label is optionally cleavable, and the third set of labeling probes specifically labels a third subset of the target nucleic acid hybridized with multiple sets of target probes different from the first and second subsets of the target nucleic acid; and includes detecting the labeling probe of the third set of labeling probes bound to the target nucleic acid, thereby detecting the third subset of the target nucleic acid (see, for example, Figures 2B and 6A (where L=3 and the label is cleaved from the SGC)).

[0069] In one embodiment, the method includes repeating steps (J) to (L) one or more times (see, for example, Figures 2B and 6A (where L = 4+ and the label is cut from the SGC)).

[0070] Optionally, in the method of the present invention, contact between cells and an acid reagent is repeated one or more times.

[0071] In one embodiment, the method further includes repeating steps (A) to (I) or steps (A) to (H), (J) to (L), and (I) one or more times (see, for example, Figures 2B and 6A (where K=2+ and the label is cut from SGC)). In one embodiment of such a method, the method further includes repeating steps (A) to (H) or steps (A) to (H) and (J) to (L).

[0072] In one embodiment, the present invention provides a method for detecting multiple target nucleic acids, the method comprising: (A) contacting a sample comprising cells containing multiple nucleic acids with a set of multiple target probes, each set of target probes comprising a pair of target probes that specifically hybridize with the target nucleic acids; and (B) contacting the sample with a set of pre-preamplifiers, each set of pre-preamplifiers comprising multiple pre-preamplifiers, each pre-preamplifier comprising a pre-preamplifier specific to each set of target probes, and each pre-preamplifier being specific to the target probes (C) Contacting the sample with a set of preamplifiers, wherein the set of preamplifiers comprises a plurality of subsets of preamplifiers specific to each preamplifier, each subset of preamplifiers comprises a plurality of preamplifiers, and the preamplifiers of the subset of preamplifiers comprises a binding site for one of the preamplifiers specific to the set of target probes and a plurality of binding sites for the amplification agent, and (D) Contacting the sample with the set of amplification agents (E) Contacting the sample with a first set of labeled probes, wherein the set of amplifiers comprises a plurality of subsets of amplifiers specific to each subset of pre-amplifiers, each subset of amplifiers comprises a plurality of amplifiers, and the amplifiers of the subset of amplifiers comprises a binding site for one of the pre-amplifiers from the subset of pre-amplifiers and a plurality of binding sites for the labeled probes; and (E) Contacting the sample with a first set of labeled probes, wherein the first set of labeled probes comprises a plurality of first subsets of labeled probes, each subset of labeled probes is specific to one of the amplifiers from the subset of amplifiers, each subset of labeled probes comprises a plurality of target probes, each of the labeled probes comprises a label and a binding site for one of the amplifiers from the subset of amplifiers, the label in each first subset of labeled probes is distinguishable between the first subsets of labeled probes, the label is cleavable, and the first set of labeled probes specifically labels a first subset of target nucleic acids hybridized to a plurality of target probes.(F) detecting a first set of labeled probes bound to a target nucleic acid, thereby detecting a first subset of the target nucleic acid; (G) cleaving the label from the first set of labeled probes bound to the first subset of the target nucleic acid; and (H) contacting the sample with a second set of labeled probes, wherein the second set of labeled probes comprises a plurality of second subsets of labeled probes, each subset of labeled probes is specific to one of the amplification agents in the subset of amplification agents, and the second subset of labeled probes is specific to an amplification agent in a subset of amplification agents different from that of the first subset of labeled probes, each subset of labeled probes comprises a plurality of labeled probes, and each labeled probe in the subset of labeled probes is specific to one of the amplification agents in the subset of amplification agents. The method includes (I) detecting the labeled probes of the second set of labeled probes bound to the target nucleic acid, thereby detecting the second subset of the target nucleic acid, wherein the label in each second subset of the labeled probes is distinguishable between the second subsets of the labeled probes, the label is optionally cleavable, and the second set of labeled probes specifically labels and contacts a second subset of the target nucleic acid that has been hybridized with multiple sets of target probes different from the first subset of the target nucleic acid; and (J) contacting the sample with an acid reagent to break the binding of the probes bound to the target nucleic acid (see, for example, Figures 2B and 6B (L=2 and the label is cleaved from SGC)).

[0073] In one embodiment, the method is to (K) cleave the label from a second set of labeled probes bound to a second subset of the target nucleic acid before step (J), and (L) contact the sample with a third set of labeled probes, wherein the third set of labeled probes comprises a plurality of third subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of amplifiers, and the third subset of labeled probes is specific to an amplifier of a subset of amplifiers different from the first and second subsets of labeled probes, each subset of labeled probes comprises a plurality of labeled probes, and each labeled probe of the subset of labeled probes is specific to a subset of amplifiers (M) Includes labeling and binding sites for one of the amplification agents in the set, wherein the labeling in each third subset of the labeling probes is distinguishable among the third subsets of the labeling probes, the labeling is optionally cleavable, and the third set of labeling probes specifically labels a third subset of the target nucleic acid hybridized with multiple sets of target probes different from the first and second subsets of the target nucleic acid; and (M) detects the labeling probes of the third set of labeling probes bound to the target nucleic acid, thereby detecting the third subset of the target nucleic acid (see, for example, Figures 2B and 6B (L=3 and the labeling is cleaved from the SGC)).

[0074] In one embodiment, the method includes repeating steps (K) to (M) one or more times (see, for example, Figures 2B and 6B (where L=4+ and the label is cut from the SGC)).

[0075] In one embodiment, contact between cells and an acid reagent is repeated one or more times.

[0076] In one embodiment, the method further includes repeating steps (A) to (J) or steps (A) to (I), (K) to (M), and (J) one or more times (see, for example, Figures 2B and 6B (where K = 2+ and the label is cut from SGC)). In one embodiment of such a method, the method further includes repeating steps (A) to (I) or steps (A) to (I) and (K) to (M).

[0077] In one embodiment, the present invention provides a method for detecting multiple nucleic acids, the method comprising: (A) contacting a sample comprising cells containing multiple nucleic acids with a set of multiple target probes, each set of target probes comprising a pair of target probes that specifically hybridize with a target nucleic acid; and (B) contacting the sample with a set of pre-preamplifiers, each set of pre-preamplifiers comprising a set of multiple pairs of pre-preamplifiers, each set of pre-preamplifiers comprising a pair of pre-preamplifiers specific to each pair of target probes in the set of target probes. - Contacting a sample with a set of pre-amplifiers, wherein each pre-pre-amplifier in a pre-amplifier pair includes a binding site for one of the target probes in a set of target probes, and the pre-pre-amplifier includes multiple binding sites for the pre-amplifier; and (C) Contacting a sample with a set of pre-amplifiers, wherein the set of pre-amplifiers includes multiple pre-amplifiers, each of which includes a pre-amplifier specific to each pair of pre-pre-amplifiers, and each pre-amplifier includes a binding site for one of the pre-pre-amplifier pairs in the set of pre-pre-amplifiers and multiple binding sites for the amplifier. (D) Contacting the sample with a set of amplifiers, wherein the set of amplifiers comprises a plurality of subsets of amplifiers specific to each pair of pre-pre-amplifiers, and the amplifiers in the subset of amplifiers comprises a binding site to one of the pre-pre-amplifiers specific to each pair of pre-pre-amplifiers and a plurality of binding sites to the labeling probe; and (E) Contacting the sample with a first set of labeling probes, wherein the first set of labeling probes comprises a plurality of first subsets of labeling probes, and each subset of labeling probes comprises a subset of amplifiers The labeling probe is specific to one of the amplification agents in the set, each subset of the labeling probe contains multiple target probes, each of the labeling probe subsets contains a label and binding site for one of the amplification agents in the subset of the amplification agents, the label in each first subset of the labeling probe is distinguishable among the first subsets of the labeling probe, the label is cleavable, and the first set of labeling probes specifically labels the first subset of the target nucleic acid hybridized with the set of multiple target probes, by contact,(F) detecting a first set of labeled probes bound to a target nucleic acid, thereby detecting a first subset of the target nucleic acid; (G) cleaving the label from the first set of labeled probes bound to the first subset of the target nucleic acid; and (H) contacting the sample with a second set of labeled probes, wherein the second set of labeled probes comprises a plurality of second subsets of labeled probes, each subset of labeled probes is specific to one of the amplification agents in the subset of amplification agents, and the second subset of labeled probes is specific to an amplification agent in a subset of amplification agents different from that of the first subset of labeled probes, each subset of labeled probes comprises a plurality of labeled probes, and each labeled probe in the subset of labeled probes is specific to one of the amplification agents in the subset of amplification agents. The method includes (I) detecting the labeled probes of the second set of labeled probes bound to the target nucleic acid, thereby detecting the second subset of the target nucleic acid, wherein the labels in each second subset of the labeled probes are distinguishable between the second subsets of the labeled probes, the labels are optionally cleavable, and the second set of labeled probes specifically labels and contacts a second subset of the target nucleic acid that has been hybridized with multiple sets of target probes different from the first subset of the target nucleic acid; and (J) contacting the sample with an acid reagent to break the binding of the probes bound to the target nucleic acid (see, for example, Figures 2B and 6C (L=2 and the labels are cleaved from SGC)).

[0078] In one embodiment, the method is to (K) cleave the label from a second set of labeled probes bound to a second subset of the target nucleic acid before step (J), and (L) contact the sample with a third set of labeled probes, wherein the third set of labeled probes comprises a plurality of third subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of amplifiers, and the third subset of labeled probes is specific to an amplifier of a subset of amplifiers different from the first and second subsets of labeled probes, each subset of labeled probes comprises a plurality of labeled probes, and each labeled probe of the subset of labeled probes is specific to a subset of amplifiers (M) Includes a labeling and binding site for one of the amplification agents in the set, wherein the label in each third subset of the labeling probes is distinguishable among the third subsets of the labeling probes, the label is optionally cleavable, and the third set of labeling probes specifically labels a third subset of the target nucleic acid hybridized with multiple sets of target probes different from the first and second subsets of the target nucleic acid; and includes detecting the labeling probes of the third set of labeling probes bound to the target nucleic acid, thereby detecting the third subset of the target nucleic acid (see, for example, Figures 2B and 6C (L=3 and the label is cleaved from SGC)).

[0079] In one embodiment, the method includes repeating steps (K) to (M) one or more times (see, for example, Figures 2B and 6C (where L=4 and the label is cut from the SGC)).

[0080] In one embodiment, contact between cells and an acid reagent is repeated one or more times.

[0081] In one embodiment, the method further includes repeating steps (A) to (J) or steps (A) to (I), (K) to (M), and (J) one or more times (see, for example, Figures 2B and 6C (where K = 2+ and the label is cut from SGC)). In one embodiment of such a method, the method further includes repeating steps (A) to (I) or steps (A) to (I) and (K) to (M).

[0082] In one embodiment, the present invention provides a method for detecting multiple target nucleic acids, the method comprising: (A) contacting a sample comprising cells containing multiple nucleic acids with a set of multiple target probes, wherein each set of target probes comprises a pair of target probes that specifically hybridize to the target nucleic acids; (B) contacting the sample with a set of pre-amplifiers, wherein the set of pre-amplifiers comprises multiple pre-amplifiers, each pre-amplifier being specific to each set of target probes, each pre-amplifier comprising a binding site for one pair of target probes from the set of target probes and multiple binding sites for the amplifier; (C) contacting the sample with a set of amplifiers, wherein the set of amplifiers comprises multiple subsets of amplifiers specific to each pre-amplifier, each subset of amplifiers comprising multiple amplifiers, each amplifier of the subset of amplifiers comprising a binding site for one of the pre-amplifiers specific to the set of target probes and multiple binding sites for the labeled probe; and (D) contacting the sample with a first set of labeled probes. (E) a first set of labeled probes comprises a plurality of first subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of amplifiers, each subset of labeled probes comprises a plurality of target probes, each labeled probe in each subset of labeled probes comprises a labeling and binding site for one of the subsets of amplifiers, the labeling in each first subset of labeled probes is distinguishable among the first subsets of labeled probes, the melting temperature between the labeled probes and the amplifier is lower than the melting temperature between the target probes, pre-amplifier, and amplifier, the first set of labeled probes specifically labels a first subset of the target nucleic acid hybridized to a plurality of target probes, (E) detecting the labeled probes of the first set of labeled probes bound to the target nucleic acid, thereby detecting a first subset of the target nucleic acid, and (F) incubating the sample at a temperature above the melting temperature between the labeled probes and the amplifier, and below the melting temperature between the target probes, pre-amplifier, and amplifier.(G) removing the label from a first set of labeled probes bound to a first subset of the target nucleic acid, and (G) contacting the sample with a second set of labeled probes, wherein the second set of labeled probes comprises a plurality of second subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of amplifiers, and the second subset of labeled probes is specific to an amplifier of a subset of amplifiers different from that of the first subset of labeled probes, each subset of labeled probes comprises a plurality of labeled probes, each labeled probe of the subset of labeled probes comprises a label and binding site for one of the subsets of amplifiers, the label in each second subset of labeled probes is distinguishable between the second subsets of labeled probes, and optionally, the melting temperature between the labeled probe and the amplifier However, the process includes (H) contacting a second set of labeled probes that is lower than the melting temperature between the target probe, pre-amplifier, and amplifier, so that the second set of labeled probes specifically labels a second subset of the target nucleic acid that has hybridized with a set of multiple target probes different from the first subset of the target nucleic acid; (H) detecting the labeled probes of the second set of labeled probes bound to the target nucleic acid, thereby detecting the second subset of the target nucleic acid, so that multiple target nucleic acids are detected; and (I) contacting the sample with an acid reagent to break the binding of the probes bound to the target nucleic acid (see, for example, Figures 2B and 6A (where L=2, and the labeled probes are removed from the SGC using a temperature above the melting temperature between the labeled probes and the amplifier, and below the melting temperature between the other components of the SGC, rather than cleaving the label from the SGC)).

[0083] In one embodiment, the method is to (J) incubate the sample at a temperature above the melting temperature between the labeled probe and the amplifier, and below the melting temperature between the target probe, pre-amplifier, and amplifier, thereby removing the label from the second set of labeled probes bound to a second subset of the target nucleic acid, and (K) contact the sample with a third set of labeled probes, wherein the third set of labeled probes comprises a plurality of third subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of amplifiers, and the third subset of labeled probes is specific to an amplifier of a subset of amplifiers different from the first and second subsets of labeled probes, each subset of labeled probes comprises a plurality of labeled probes, and each labeled probe of the subset of labeled probes is specific to the label and (L) Includes contact with a third set of labeled probes that has been hybridized with a set of target probes that is different from the first and second subsets of the target nucleic acid, and (L) detects the labeled probe of the third set of labeled probes bound to the target nucleic acid, thereby detecting the third subset of the target nucleic acid (see, for example, Figures 2B and 6A (where L=3 and the labeled probe is removed from the SGC using a temperature above the melting temperature between the labeled probe and the amplifier and below the melting temperature between the other components of the SGC, rather than cleaving the label from the SGC)).

[0084] In one embodiment, the method includes repeating steps (J) to (L) one or more times (see, for example, Figures 2B and 6A (where L = 4+, and rather than cleaving the label from the SGC, the labeled probe is removed from the SGC using a temperature above the melting temperature between the labeled probe and the amplifying agent, and below the melting temperature between the other components of the SGC)).

[0085] In one embodiment, contact between cells and an acid reagent is repeated one or more times.

[0086] In one embodiment, the method further includes repeating steps (A) to (I) or steps (A) to (H), (J) to (L), and (J) one or more times (see, for example, Figures 2B and 6A (where K=2+, and rather than cleaving the label from the SGC, the labeled probe is removed from the SGC using a temperature above the melting temperature between the labeled probe and the amplifying agent and below the melting temperature between the other components of the SGC)). In one embodiment of such a method, the method further includes repeating steps (A) to (H) or steps (A) to (H) and (J) to (L).

[0087] In one embodiment, the present invention provides a method for detecting multiple target nucleic acids, the method comprising: (A) contacting a sample comprising cells containing multiple nucleic acids with a set of multiple target probes, wherein each set of target probes comprises a pair of target probes that specifically hybridize with the target nucleic acids; and (B) contacting the sample with a set of pre-preamplifiers, wherein the set of pre-preamplifiers comprises multiple pre-preamplifiers, each pre-preamplifier comprising a pre-preamplifier specific to each set of target probes, and each pre-preamplifier being a target (C) Contacting a sample with a set of preamplifiers, wherein the set of preamplifiers comprises a plurality of subsets of preamplifiers specific to each preamplifier, each subset of preamplifiers comprises a plurality of preamplifiers, and the preamplifiers of the subset of preamplifiers comprises a binding site for one of the preamplifiers specific to the set of target probes and a plurality of binding sites for the amplifier, and (D) Contacting a sample (E) Contacting the sample with a set of amplifiers, wherein the set of amplifiers comprises a plurality of subsets of amplifiers specific to each subset of pre-amplifiers, each subset of amplifiers comprises a plurality of amplifiers, and the amplifiers of the subset of amplifiers comprises a binding site for one of the pre-amplifiers from the subset of pre-amplifiers and a plurality of binding sites for the labeling probes, and (E) Contacting the sample with a first set of labeling probes, wherein the first set of labeling probes comprises a plurality of first subsets of labeling probes, each subset of labeling probes is specific to one of the amplifiers from the subset of amplifiers, each subset of labeling probes comprises a plurality of target probes, the labeling probes in each subset of labeling probes comprises labeling and binding sites for one of the amplifiers from the subset of amplifiers, the labeling in each first subset of labeling probes is distinguishable among the first subsets of labeling probes, the melting temperature between the labeling probes and the amplifiers is lower than the melting temperature between the target probes, pre-pre-amplifiers, pre-amplifiers, and amplifiers, and the first set of labeling probes(F) Contacting a first subset of a hybridized target nucleic acid with a set of multiple target probes; (G) Detecting the labeled probes of the first set of labeled probes bound to the target nucleic acid, thereby detecting the first subset of the target nucleic acid; (H) Incubating the sample at a temperature above the melting temperature between the labeled probes and the amplifier, and below the melting temperature between the target probes, pre-pre-amplifier, pre-amplifier, and amplifier, thereby removing the label from the first set of labeled probes bound to the first subset of the target nucleic acid; and (H) Contacting the sample with a second set of labeled probes, wherein the second set of labeled probes comprises multiple second subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of amplifiers, and the second subset of labeled probes is specific to an amplifier of a subset of amplifiers different from that of the first subset of labeled probes, each subset of labeled probes comprises multiple labeled probes, and each labeled probe of the subset of labeled probes is specific to a subset of amplifiers (I) The labeling probe includes a labeling and binding site for one amplification agent, wherein the labeling in each second subset of the labeling probe is distinguishable between the second subsets of the labeling probe, optionally the melting temperature between the labeling probe and the amplification agent is lower than the melting temperature between the target probe, pre-pre-amplifier, pre-amplifier, and amplification agent, and the second set of labeling probes specifically labels a second subset of the target nucleic acid that has been hybridized to a set of multiple target probes different from the first subset of the target nucleic acid, and (I) the labeling probe bound to the target nucleic acid (J) detecting a second set of labeled probes on the lobes, thereby detecting a second subset of the target nucleic acid, wherein multiple target nucleic acids are detected, and (J) contacting the sample with an acid reagent to break the binding of the probes bound to the target nucleic acids (see, for example, Figures 2B and 6B (where L=2, and the labeled probe is removed from the SGC using a temperature above the melting temperature between the labeled probe and the amplifier, and below the melting temperature between the other components of the SGC, rather than cleaving the label from the SGC)).

[0088] In one embodiment, the method is to (K) incubate the sample at a temperature above the melting temperature between the labeled probe and the amplifier, and below the melting temperature between the target probe, pre-pre-amplifier, pre-amplifier and amplifier, thereby removing the label from the second set of labeled probes bound to a second subset of the target nucleic acid, and (L) contact the sample with a third set of labeled probes, wherein the third set of labeled probes comprises a plurality of third subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of amplifiers, and the third subset of labeled probes is specific to an amplifier of a subset of amplifiers different from the first and second subsets of labeled probes, each subset of labeled probes comprises a plurality of labeled probes, and each labeled probe of the subset of labeled probes is specific to the label and (M) Contacting the third set of labeled probes, which includes a binding site, wherein the labeling in each third subset of the labeled probes is distinguishable among the third subsets of the labeled probes, and optionally, the melting temperature between the labeled probes and the amplifier is lower than the melting temperature between the target probe, pre-pre-amplifier, pre-amplifier, and amplifier, and the third set of labeled probes specifically labels a third subset of the target nucleic acid hybridized to multiple sets of target probes different from the first and second subsets of the target nucleic acid; and (M) detecting the labeled probes of the third set of labeled probes bound to the target nucleic acid, thereby detecting the third subset of the target nucleic acid (see, for example, Figures 2B and 6B (where L=3 and the labeled probes are removed from the SGC using a temperature above the melting temperature between the labeled probes and the amplifier, and below the melting temperature between the other components of the SGC, rather than cleaving the label from the SGC).

[0089] In one embodiment, the method includes repeating steps (K) to (M) one or more times (see, for example, Figures 2B and 6B (where L = 4+, and rather than cleaving the label from the SGC, the labeled probe is removed from the SGC using a temperature above the melting temperature between the labeled probe and the amplifier, and below the melting temperature between the other components of the SGC)).

[0090] In one embodiment, contact between cells and an acid reagent is repeated one or more times.

[0091] In one embodiment, the method further includes repeating steps (A) to (J) or steps (A) to (I), (K) to (M), and (J) one or more times (see, for example, Figures 2B and 6B (where K = 2+, and the labeled probe is removed from the SGC using a temperature above the melting temperature between the labeled probe and the amplifying agent, and below the melting temperature between the other components of the SGC, rather than cleaving the label from the SGC)). In one embodiment of such a method, the method further includes repeating steps (A) to (I) or steps (A) to (I) and (K) to (M).

[0092] In one embodiment, the present invention provides a method for detecting multiple nucleic acids, the method comprising: (A) contacting a sample comprising cells containing multiple nucleic acids with a set of multiple target probes, each set of target probes comprising a pair of target probes that specifically hybridize to a target nucleic acid; and (B) contacting the sample with a set of pre-preamplifiers, each set of pre-preamplifiers comprising a set of multiple pairs of pre-preamplifiers, each set of pre-preamplifiers comprising a pair of pre-preamplifiers specific to each pair of target probes in the set of target probes. (c) Contacting a sample with a set of pre-amplifiers, wherein each pre-amplifier in a pre-amplifier pair includes a binding site for one of the target probes in a set of target probes, and the pre-amplifier includes multiple binding sites for the pre-amplifier; and (c) Contacting a sample with a set of pre-amplifiers, wherein the set of pre-amplifiers includes multiple pre-amplifiers, each of which includes a pre-amplifier specific to each pair of pre-amplifiers, and each pre-amplifier includes a binding site for one of the pre-amplifier pairs in the set of pre-amplifiers and multiple binding sites for the amplifier. (D) Contacting the sample with a set of amplifiers, wherein the set of amplifiers comprises a plurality of subsets of amplifiers specific to each pre-pre-amplifier pair, and the amplifiers of the subsets of amplifiers comprises binding sites to one of the pre-pre-amplifiers specific to the pre-pre-amplifier pair and a plurality of binding sites to the labeling probe, and (E) Contacting the sample with a first set of labeling probes, wherein the first set of labeling probes comprises a plurality of first subsets of labeling probes, and each subset of labeling probes The label probe is specific to one of the amplification agents in a subset of amplification agents, each subset of the label probes includes multiple target probes, each label probe in each subset of the label probes includes a label and binding site for one of the amplification agents in a subset of amplification agents, the label in each first subset of the label probes is distinguishable among the first subsets of the label probes, the melting temperature between the label probe and the amplification agent is lower than the melting temperature between the target probe, pre-pre-amplifier, pre-amplifier, and amplification agent, and the first set of label probes is(F) Contacting a first subset of a hybridized target nucleic acid with a set of multiple target probes; (G) Detecting the labeled probes of the first set of labeled probes bound to the target nucleic acid, thereby detecting the first subset of the target nucleic acid; (H) Incubating the sample at a temperature above the melting temperature between the labeled probes and the amplifier, and below the melting temperature between the target probes, pre-pre-amplifier, pre-amplifier, and amplifier, thereby removing the label from the first set of labeled probes bound to the first subset of the target nucleic acid; and (H) Contacting the sample with a second set of labeled probes, wherein the second set of labeled probes comprises multiple second subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of amplifiers, and the second subset of labeled probes is specific to an amplifier of a subset of amplifiers different from that of the first subset of labeled probes, each subset of labeled probes comprises multiple labeled probes, and each labeled probe of the subset of labeled probes is specific to a subset of amplifiers (I) The labeling probe includes a labeling and binding site for one amplification agent, wherein the labeling in each second subset of the labeling probe is distinguishable between the second subsets of the labeling probe, optionally the melting temperature between the labeling probe and the amplification agent is lower than the melting temperature between the target probe, pre-pre-amplifier, pre-amplifier, and amplification agent, and the second set of labeling probes specifically labels a second subset of the target nucleic acid that has been hybridized to a set of multiple target probes different from the first subset of the target nucleic acid, and (I) the labeling probe bound to the target nucleic acid (J) Detection of a second set of labeled probes on the lobes, thereby detecting a second subset of the target nucleic acid, wherein multiple target nucleic acids are detected, and (J) contacting the sample with an acid reagent, thereby breaking the binding of the probes bound to the target nucleic acids (see, for example, Figures 2B and 6C (where L=2, and the labeled probe is removed from the SGC using a temperature above the melting temperature between the labeled probe and the amplifier, and below the melting temperature between the other components of the SGC, rather than cleaving the label from the SGC)).

[0093] In one embodiment, the method is to (K) incubate the sample at a temperature above the melting temperature between the labeled probe and the amplifier, and below the melting temperature between the target probe, pre-pre-amplifier, pre-amplifier and amplifier, thereby removing the label from the second set of labeled probes bound to a second subset of the target nucleic acid, and (L) contact the sample with a third set of labeled probes, wherein the third set of labeled probes comprises a plurality of third subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of amplifiers, and the third subset of labeled probes is specific to an amplifier of a subset of amplifiers different from the first and second subsets of labeled probes, each subset of labeled probes comprises a plurality of labeled probes, and each labeled probe of the subset of labeled probes is specific to the label and (M) Contacting the third set of labeled probes, which includes a binding site, wherein the labeling in each third subset of the labeled probes is distinguishable among the third subsets of the labeled probes, and optionally, the melting temperature between the labeled probes and the amplifier is lower than the melting temperature between the target probe, pre-pre-amplifier, pre-amplifier, and amplifier, and the third set of labeled probes specifically labels a third subset of the target nucleic acid hybridized to multiple sets of target probes different from the first and second subsets of the target nucleic acid; and (M) detecting the labeled probes of the third set of labeled probes bound to the target nucleic acid, thereby detecting the third subset of the target nucleic acid (see, for example, Figures 2B and 6C (where L=3 and the labeled probes are removed from the SGC using a temperature above the melting temperature between the labeled probes and the amplifier, and below the melting temperature between the other components of the SGC, rather than cleaving the label from the SGC).

[0094] In one embodiment, the method includes repeating steps (K) to (M) one or more times (see, for example, Figures 2B and 6C (where L = 4+, and rather than cleaving the label from the SGC, the labeled probe is removed from the SGC using a temperature above the melting temperature between the labeled probe and the amplifier, and below the melting temperature between the other components of the SGC)).

[0095] In one embodiment, contact between cells and an acid reagent is repeated one or more times.

[0096] In one embodiment, the method further comprises repeating steps (A) to (J) or steps (A) to (I), (K) to (M), and (J) one or more times (see, for example, Figures 2B and 6C (where K = 2+, and the labeled probe is removed from the SGC using a temperature above the melting temperature between the labeled probe and the amplifying agent, and below the melting temperature between the other components of the SGC, rather than cleaving the label from the SGC)). In one embodiment of such a method, the method further comprises repeating steps (A) to (I) or steps (A) to (I) and (K) to (M).

[0097] In some embodiments of the present invention's method using a set of target probes, each set of target probes comprises two or more pairs of target probes that specifically hybridize to the same target nucleic acid.

[0098] In some embodiments of the method of the present invention, the acid reagent comprises 5-40% or 20-30% of an acid, or other concentrations disclosed herein. In one embodiment, the acid is selected from the group consisting of acetic acid, formic acid, propionic acid, butyric acid, valeric acid, oxalic acid, malonic acid, succinic acid, malic acid, tartaric acid, and citric acid.

[0099] In some embodiments, the acid reagent contains a salt. In one embodiment, the acid reagent contains SSC. In one embodiment, the acid reagent contains 1 to 13 times SSC or 3.2 to 12.8 times SSC.

[0100] In some embodiments of the method of the present invention, the target nucleic acid is independently DNA or RNA. In one embodiment, the target nucleic acid, which is RNA, is independently selected from the group consisting of messenger RNA (mRNA), microRNA (miRNA), ribosomal RNA (rRNA), mitochondrial RNA, and non-coding RNA.

[0101] In some embodiments of the method of the present invention, the sample is a tissue specimen or derived from a tissue specimen. In some embodiments of the method of the present invention, the sample is a blood specimen or derived from a blood specimen. In some embodiments of the method of the present invention, the sample is a cytological specimen or derived from a cytological specimen.

[0102] The method of the present invention is applied to the multiplex detection of target nucleic acids. As disclosed herein, the method of the present invention is carried out in repeated detection rounds of a subset of target nucleic acids. Also disclosed herein, the number of target nucleic acids that can be detected in a single round depends on the type of labeling probe used and its ability to distinguish between labeling probes specific to different target nucleic acids when detected simultaneously. A higher level of multiplexing is achieved by repeated rounds of detection and acid treatment. For example, in the exemplary embodiment shown in Figure 1, four target nucleic acids are detected in one round of labeling and detection, eight target nucleic acids are detected in a second round of labeling and detection, and twelve target nucleic acids are detected in a third round of labeling and detection. By including an acid treatment step, an additional set of target nucleic acids can be detected, and in this particular embodiment, up to 12 additional target nucleic acids can be detected for a total of 24 target nucleic acids in the same sample. Those skilled in the art can easily determine the desired number of target nucleic acids to be detected by the assay of the present invention. In some embodiments, two rounds of labeling and detection are used in the method of the present invention. In some embodiments, three rounds of labeling and detection are used. In some embodiments, four, five, six, seven, eight, nine, or more rounds of labeling and detection can be used, provided that there are enough SGCs to independently label and detect each target nucleic acid, and the SGCs remain sufficiently bound to the target nucleic acid throughout the assay conditions for detecting the target nucleic acid. Those skilled in the art can easily determine the number of labeling and detection rounds that can be applied in the method of the present invention, and that the target nucleic acid can be detected in each round.

[0103] Since subsequent rounds of detection are applied to the sample, the subsequent detection of the target nucleic acid can be registered with the detection of the target nucleic acid in the previously detected round(s), thereby allowing for the determination of the expression of all target nucleic acids detected within the same sample and even within the same cell (see Figures 1, 2A, and 2B). The registration of target nucleic acid detection rounds can be achieved using image analysis software that overlays images of target nucleic acids detected in different rounds. Such registration algorithms for the alignment and overlay of multiple images are well known in the art, for example, the Scale-Invariant Feature Transform (SIFT) algorithm (Lowe, "Distinctive Image Features from Scale-Invariant Keypoints," Internat. J. Computer Vision 60(2):91-110 (2004)). Essentially, these algorithms compare an input image with a reference image to generate a transformation matrix to accommodate translation and rotation. For example, ImageJ has a tool that can automate this task (imagej.net / Registration) (Schneider et al., Nature Methods 9(7):671-675(2012), Schindelin et al., Mol.Reprod.Dev.82(7-8):518-529(2015)).

[0104] Additional multiplexing can be achieved by utilizing an SGC assembly containing the same SSG but with different sets of target probes for different target nucleic acids. In such cases, after detecting the first set of target nucleic acids with the SGC complex detected in iterative rounds labeling a subset of target nucleic acids, the SGC complex can be removed from the target nucleic acid using conditions appropriate to denature the hybridization of the SGC to the target nucleic acid. Once the SGC complex is removed from the sample, the same pre-amplifier / amplifier / labeling probe, or the same pre-pre-amplifier / pre-amplifier / amplifier / labeling probe, can be used with a set of target probes designed to be specific to different sets of target nucleic acids detected in the first round of SGC detection. In this way, detection of additional target nucleic acids can be achieved. As described herein, further multiplexing can be achieved by acid-treating the sample to remove the bound target probe and SGC, and then, optionally, reusing the same SGC, but targeting different target nucleic acids and labeling different target nucleic acids in subsequent rounds.

[0105] In yet another embodiment, the method of the present invention can be applied to the simultaneous detection of double-stranded and single-stranded nucleic acids, for example, the detection of DNA and RNA in the same sample. In such cases, the probe can be designed to detect single-stranded nucleic acids such as RNA (see, for example, U.S. Patent No. 7,709,198, U.S. Publications 2008 / 0038725, 2009 / 0081688, and 2017 / 0101672) and double-stranded nucleic acids, so that both double-stranded and single-stranded nucleic acids (such as DNA and RNA) can be detected in the same sample.

[0106] In some embodiments, each set of target probes specific to a target nucleic acid includes two or more pairs of target probes that specifically hybridize to the same target nucleic acid. In such cases, the pairs of target probes in the target nucleic acid-specific set bind to different non-overlapping sequences of the target nucleic acid. When using a set of target probes having two or more pairs of target probes that can specifically hybridize to the same target nucleic acid, the molecules that bind to those target probe pairs, i.e., pre-amplifiers (see Figures 5A and 6A) or pre-pre-amplifiers (see Figures 5B, 5C, 6B, and 6C), are generally the same in the target probe pairs within the same set of target probes. Therefore, target probe pairs that bind to the same target nucleic acid can be designed to include the same binding site for the above-mentioned molecules in the SGC that bind to those target probe pairs, i.e., pre-amplifiers or pre-pre-amplifiers. By using multiple target probe pairs to detect a target nucleic acid, a more advanced signal can be obtained on the same target nucleic acid, associated with an assembly of multiple SGCs. In some embodiments, the number of target probe pairs used to bind to the same target nucleic acid is in the range of 1 to 10 pairs, 1 to 20 pairs, 1 to 30 pairs, 1 to 40 pairs, 1 to 50 pairs, 1 to 60 pairs, 1 to 70 pairs, 1 to 80 pairs, 1 to 90 pairs, 1 to 100 pairs, 1 to 110 pairs, 1 to 120 pairs, 1 to 130 pairs, 1 to 140 pairs, 1 to 150 pairs, 1 to 160 pairs, 1 to 170 pairs, 1 to 180 pairs, 1 to 190 pairs, 1 to 200 pairs, or any integer number of pairs between these ranges, for example, 1 pair, 2 pairs, 3 pairs, 4 pairs, 5 pairs, 6 pairs, 7 pairs, 8 pairs, 9 pairs, 10 pairs , 11 vs 12 vs 13 vs 14 vs 15 vs 16 vs 17 vs 18 vs 19 vs 20 vs 21 vs 22 vs 23 vs 24 vs 25 vs 26 vs 27 vs 28 vs 29 vs 30 vs 31 vs 32 vs 33 vs 34 vs 35 vs 36 vs 37 vs 38 vs 39 vs 40 vs 41 vs 4 2 vs, 43 vs, 44 vs, 45 vs, 46 vs, 47 vs, 48 ​​vs, 49 vs, 50 vs, 51 vs, 52 vs, 53 vs, 54 vs, 55 vs, 56 vs, 57 vs, 58 vs, 59 vs, 60 vs, 61 vs, 62 vs, 63 vs, 64 vs, 65 vs, 66 vs, 67 vs, 68 vs, 69 vs, 70 vs, 71 vs, 72 vs, 73 vs,74 vs 75 vs 76 vs 77 vs 78 vs 79 vs 80 vs 81 vs 82 vs 83 vs 84 vs 85 vs 86 vs 87 vs 88 vs 89 vs 90 vs 91 vs 92 vs 93 vs 94 vs 95 vs 96 vs 97 vs 98 vs 99 vs 100 vs 101 vs 102 vs 103 vs 104 vs 105 vs 106 vs 107 vs 108 vs 109 vs , 110 vs 111 vs 112 vs 113 vs 114 vs 115 vs 116 vs 117 vs 118 vs 119 vs 120 vs 121 vs 122 vs 123 vs 124 vs 125 vs 126 vs 127 vs 128 vs 129 vs 130 vs 131 vs 132 vs 133 vs 134 vs 135 vs 136 vs 137 vs 138 vs 139 vs 140 141 vs, 142 vs, 143 vs, 144 vs, 145 vs, 146 vs, 147 vs, 148 vs, 149 vs, 150 vs, 151 vs, 152 vs, 153 vs, 154 vs, 155 vs, 156 vs, 157 vs, 158 vs, 159 vs, 160 vs, 161 vs, 162 vs, 163 vs, 164 vs, 165 vs, 166 vs, 167 vs, 168 vs, 169 vs, 170 vs, 1 These include 71, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, and so on.

[0107] The method of the present invention can be used to achieve the detection of a desired target nucleic acid. In one embodiment, a target nucleic acid is detected using multiple target probe pairs. In such cases, the target probe pairs are designed to bind to two or more regions of the target nucleic acid so as to enable the assembly of multiple SGCs to the target nucleic acid. It should be understood that when multiple target probe pairs are used to bind to the same target nucleic acid, the target binding sites of one target probe pair do not overlap with the target binding sites of another target probe pair.

[0108] In one embodiment of the present invention, the target nucleic acid detected by the method of the present invention may be any nucleic acid present in the cell sample, including, but not limited to, messenger RNA (mRNA), microRNA (miRNA), ribosomal RNA (rRNA), mitochondrial RNA, non-coding RNA, or RNA including DNA. In certain embodiments, the nucleic acid is RNA. It should be understood that in the method of the present invention for multiplex detection of nucleic acids, the target nucleic acid may independently be DNA or RNA. In other words, the detected target nucleic acids may, but not necessarily, be the same type of nucleic acid. Therefore, the target nucleic acids detected in the assay of the present invention may be DNA and RNA. When the target nucleic acid is RNA, it should be understood that the target nucleic acid may independently be selected from the group consisting of messenger RNA (mRNA), microRNA (miRNA), ribosomal RNA (rRNA), mitochondrial RNA, and non-coding RNA. Therefore, the target nucleic acid may independently be DNA or any type of RNA.

[0109] As described herein, the methods of the present invention generally relate to the in situ detection of target nucleic acids. Methods for the in situ detection of nucleic acids are well known to those skilled in the art (see, for example, US2008 / 0038725, US2009 / 0081688, Hicks et al., J.Mol.Histol.35:595-601(2004)). As used herein, “in situ hybridization” or “ISH” refers to a type of hybridization in which a specific nucleic acid, such as DNA or RNA, is localized in a sample, particularly a tissue or cell part or section (in situ), by binding to a directly or indirectly labeled complementary DNA or RNA strand, such as a probe. The types of probes may be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded complementary RNA (sscRNA), messenger RNA (mRNA), microRNA (miRNA), ribosomal RNA, mitochondrial RNA, and / or synthetic oligonucleotides. The term "fluorescent in situ hybridization" or "FISH" refers to a type of ISH that utilizes fluorescent labeling. The term "chromogenic in situ hybridization" or "CISH" refers to a type of ISH that uses chromogenic labeling. The ISH, FISH, and CISH methods are well known to those skilled in the art (see, for example, Stoler, Clinics in Laboratory Medicine 10(1):215-236 (1990); In situ hybridization. A practical approach, Wilkinson, ed., IRL Press, Oxford (1992); Schwarzacher and Heslop-Harrison, Practical in situ hybridization, BIOS Scientific Publishers Ltd, Oxford (2000)).

[0110] In the present invention's method for in situ detection of intracellular nucleic acid targets, including but not limited to in situ hybridization or flow cytometry, cells are optionally fixed and / or permeabilized before hybridization of the target probe. Fixing and permeabilizing the cells encourages the nucleic acid target to remain within the cell, allowing the target probe, labeled probe, amplifier, pre-amplifier, pre-pre-amplifier, etc., to enter the cell and reach the target nucleic acid molecule. The cells are optionally washed to remove any material not captured by the nucleic acid target. After any of the various steps, for example, after hybridization of the target probe to the nucleic acid target, or after hybridization of pre-pre-amplifier, pre-amplifier, amplifier, and / or labeled probe to the target probe, the cells may be washed to remove unbound target probes. Methods for fixing and permeabilizing cells for in situ detection of nucleic acids, as well as methods for hybridizing, washing, and detecting target nucleic acids, are also well known in the art (e.g., US2008 / 0038725, US2009 / 0081688, Hicks et al., J.Mol.Histol.35:595-601(2004), Stoler, Clinics in Laboratory Medicine 10(1):215-236(1990), In situ hybridization. A practical approach, Wilkinson, ed., IRL Press, Oxford(1992), Schwarzacher and Heslop-Harrison, Practical in situ hybridization, BIOS Scientific Publishers Ltd, Oxford(2000), Shapiro, Practical Flow Cytometry 3rd ed., Wiley-Liss, New York(1995), Ormerod, Flow See Cytometry, 2nd ed., Springer (1999). Examples of fixatives, but not limited to these, include aldehydes (formaldehyde, glutaraldehyde, etc.), acetone, and alcohols (methanol, ethanol, etc.).Examples of permeabilizing agents include, but are not limited to, alcohols (methanol, ethanol, etc.), acids (glacial acetic acid, etc.), detergents (Triton, NP-40, Tween® 20, etc.), saponins, digitonin, Leucoperm® (BioRad, Hercules, CA), and enzymes (e.g., lysozyme, lipase, protease, and peptidase). Permeabilization can also be carried out by mechanical disruption, as in tissue sections.

[0111] For in situ detection of double-stranded nucleic acids, the sample is generally treated to denature the double-stranded nucleic acids in the sample, making the target probe reachable so that the target probe can bind to one strand of the target double-stranded nucleic acid by hybridization. Conditions for denature of double-stranded nucleic acids are well known in the art and include denaturation by heat and chemicals, such as bases (NaOH), formamide, and dimethyl sulfoxide (Wang et al., Environ. Health Toxicol. 29:e2014007 (doi:10.5620 / eht.2014.29.e2014007) 2014, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001), Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). For example, double-stranded nucleic acids such as DNA can be denatured using NaOH, LiOH, KOH, or other high-pH buffers (pH above 11). In addition, thermal denaturation methods and chemical denaturation methods can be used in combination.

[0112] Such in situ detection methods can be used for tissue specimens immobilized on glass slides, single cells in suspensions such as peripheral blood mononuclear cells (PBMCs) isolated from blood samples, etc. Examples of tissue specimens include tissue biopsy specimens. Examples of blood samples include blood samples collected for diagnostic purposes. In the case of blood samples, the blood can be analyzed directly, as in blood smears, or the blood can be processed, for example, by lysing red blood cells, isolating PBMCs or white blood cells, or isolating target cells, so that the cells to be analyzed by the method of the present invention are present in or extracted from the blood sample. Similarly, tissue specimens can be processed, for example, by finely cutting the tissue specimen and processing it physically or enzymatically to destroy the tissue into individual cells or cell clusters. Furthermore, if desired, cytological specimens can be processed to isolate cells or destroy cell clusters. Thus, tissue, blood, and cytological specimens can be obtained and processed using methods well known in the art. The method of the present invention can be used for diagnostic purposes to identify the presence or absence of diseased cells based on the presence or absence of nucleic acid targets, which are biomarkers indicating a pathological state.

[0113] Those skilled in the art will understand that any number of suitable samples can be used to detect target nucleic acids using the method of the present invention. Samples for use in the method of the present invention are generally biological or tissue samples. Such samples can be obtained from a living subject and include samples derived from biological tissue or bodily fluids taken from an individual or some other source of biological material (such as a live specimen, autopsy specimen, or forensic material). Biological samples also include samples from areas of a living subject that contain or are suspected of containing precancerous cells, cancer cells, precancerous tissue, or cancerous tissue, such as tissue biopsies (including fine-needle aspirations, blood samples, or cytological specimens). Such samples may be organs, tissues, tissue fragments, and / or cells 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, and bodily fluids. Additional biological samples include, but are not limited to, skin samples, tissue biopsies (including fine-needle aspirations), cytological samples, stool, and bodily fluids (including blood and / or serum samples, saliva, and semen). Such samples can be used for diagnostic purposes in medicine or veterinary medicine. Samples can be obtained from other sources, such as food, soil, object surfaces, and other substances from which detection of target nucleic acids is desired. Thus, the method of the present invention can be used to detect one or more pathogens (such as viruses, bacteria, fungi, and single-celled organisms like parasites) from biological samples taken from individuals or other sources.

[0114] The method of collecting cytological samples for analysis using the present invention is well known in the art (see, for example, Dey, “Cytology Sample Procurement, Fixation and Processing” in Basic and Advanced Laboratory Techniques in Histopathology and Cytology pp.121-132, Springer, Singapore (2018), and “Non-Gynecological Cytology Practice Guideline” American Society of Cytopathology, Adopted by the ASC executive board March 2, 2004). Methods for processing samples (including biopsy specimens and cytological specimens) for analysis of cervical tissue are well known in the art (e.g., Cecil Textbook of Medicine, Bennett and Plum, eds., 20th ed., WB Saunders, Philadelphia (1996), Colposcopy and Treatment of Cervical Intraepithelial Neoplasia: A Beginner's Manual, Sellors and Sankaranarayanan, eds., International Agency for Research on Cancer, Lyon, France (2003), Kalaf and Cooper, J. Clin. Pathol. 60:449-455 (2007), Brown and Trimble, Best Pract. Res. Clin. Obstet. Gynaecol. 26:233-242 (2012), Waxman et al.) See also al., Obstet. Gynecol. 120:1465-1471 (2012), Cervical Cytology Practice Guidelines TOC, Approved by the American Society of Cytopathology (ASC) Executive Board, November 10, 2000).In one embodiment, the cytological sample is a cervical sample, such as a Pap smear. In another embodiment, the sample is a fine-needle aspiration.

[0115] In certain embodiments of the present invention, the sample is a tissue specimen or derived from a tissue specimen. In another particular embodiment of the present invention, the sample is a blood specimen or derived from a blood specimen. In yet another particular embodiment of the present invention, the sample is a cytological specimen or derived from a cytological specimen.

[0116] The present invention is based on constructing a complex between target nucleic acids in order to label them with a detectable label. Such a complex may be referred to as a signal-generating complex (SGC, see, e.g., US20170101672). Such a complex, i.e., an SGC, is achieved by constructing a layer of molecules that allows for the binding of multiple labels to the target nucleic acid.

[0117] The method of the present invention may utilize a signal-generating complex (SGC), which comprises multiple molecules rather than a single molecule. Such SGCs are particularly useful for amplifying a detectable signal and providing more sensitive detection of a target nucleic acid. Such methods for amplifying a signal are described, for example, in U.S. Patents 5,635,352, 5,124,246, 5,710,264, 5,849,481, and 7,709,198, as well as U.S. Publications 2008 / 0038725 and 2009 / 0081688, and WO2007 / 001986 and WO2012 / 054795, each of which is incorporated herein by reference. The generation of SGCs is based on the principle of the RNAscope® assay (see U.S. Patents No. 7,709,198, 8,658,361, and 9,315,854, U.S. Publications 2008 / 0038725, 2009 / 0081688, and 2016 / 0201117, and WO2007 / 001986 and WO2012 / 054795, each of which is incorporated herein by reference).

[0118] The basic signal-generating complex (SGC) is shown in Figure 5A (incorporated herein by reference; see also US2009 / 0081688). The pair of target probes shown in Figure 5 as the "Z" pair hybridizes to a complementary molecular sequence labeled "Target". Each target probe contains an additional sequence complementary to a pre-amplifier molecule (PA, shown in green), which must hybridize simultaneously to both members of the target probe pair for stable binding. The pre-amplifier molecule consists of two domains: one domain with a region to hybridize to each target probe, and another domain containing a series of nucleotide sequence repeats complementary to the sequences on the amplifier molecule (Amp, shown in black). The presence of multiple repeats in this sequence allows multiple amplifier molecules to hybridize to a single pre-amplifier, thereby increasing the overall signal amplification. Each amplification molecule consists of two domains: one domain having a region for hybridization to a pre-amplifier, and another domain containing a series of nucleotide sequence repeats complementary to the sequence on the labeled probe (LP, shown in yellow). This allows multiple labeled probes to hybridize to each amplification molecule, thereby further increasing the overall signal amplification. Each labeled probe contains two components. One component consists of a nucleotide sequence complementary to the repeat sequence on the amplification molecule to hybridize the labeled probe. This nucleotide sequence is linked to a second component, which may be a signal product containing, as described herein, a fluorescent or chromogenic label for direct visualization, a directly detectable metal isotope, or an enzyme or other chemical that can facilitate a chemical reaction that produces fluorescence, color, or other detectable signals. In Figure 5A, the labeled probes are depicted as lines representing the nucleic acid component and asterisks representing the signal-generating component. Collectively, the assembly from the target probe to the labeled probe is called a signal-generating complex (SGC).

[0119] Figure 5B shows SGC enhanced by the addition of an amplification molecular layer, in this case a pre-pre-amplifier molecule (PPA, shown in red). In one domain, the PPA binds to both target probes, and in the other domain, it binds to multiple pre-amplifiers (PAs).

[0120] Figure 5C shows different SGC structures that utilize synergistic hybridization at the pre-amplifier level (see US2017 / 0101672, incorporated herein by reference). Similar to the SGCs formed in Figures 5A and 5B, the target probe pair hybridizes to the target molecular sequence. Each target probe contains an additional sequence complementary to a unique pre-pre-amplifier molecule (PPA-1, shown in purple; PPA-2, shown in red). The use of two independent molecules establishes the foundation upon which synergistic hybridization may be required. Each pre-pre-amplifier molecule consists of two domains: one domain having a region to hybridize to one of the target probes, and a domain sequence containing a series of nucleotide sequence repeats, each containing a sequence complementary to the sequence in the pre-amplifier molecule (PA, shown in green), as well as a spacer sequence that facilitates the binding efficiency of PPA and PA. To ensure stable binding to the growing SGC, each PA must hybridize to both PPA molecules simultaneously. Each pre-amplifier molecule consists of two domains: one domain containing sequences complementary to both pre-pre-amplifiers to enable hybridization, and another domain containing a series of nucleotide sequence repeats complementary to the sequences on the amplifier molecule (AMP, shown in black). Multiple repeats of the amplifier hybridization sequence allow multiple amplifier molecules to hybridize to each pre-amplifier, further increasing signal amplification. For simplification of the diagram, the amplifier molecules are shown hybridizing to one pre-amplifier molecule, but it is understood that amplifiers can bind to each pre-amplifier. Each amplifier molecule contains a series of nucleotide sequence repeats complementary to the sequence in the labeled probe (LP, shown in yellow), thereby allowing several labeled probes to hybridize to each amplifier molecule. Each labeled probe contains a signal-generating element for detecting the signal.

[0121] As described above, regardless of whether the configurations shown in Figures 5A, 5B, 6A, or 6B, or Figures 5C and 6C, are used, the components of the SGC are designed so that binding to both target probes is required to construct the SGC. In the configurations of Figures 5A, 5B, 6A, or 6B, the pre-amplifier (or pre-pre-amplifier in Figures 5B and 6B) must bind to both members of the target probe pair to achieve stable binding. This is achieved by designing the binding sites of the target probe and the pre-amplifier (or pre-pre-amplifier) ​​such that the melting temperature (Tm) of the binding of both target probes to the pre-amplifier (or pre-pre-amplifier) ​​is higher than the Tm of the binding of one target probe to the pre-amplifier (or pre-pre-amplifier), and that the state in which one target probe is bound is unstable under assay conditions. This design has been previously described, for example, in U.S. Patent No. 7,709,198, U.S. Publications 2008 / 0038725 and 2009 / 0081688, WO2007 / 001986, WO2007 / 002006, Wang et al., 2012, and Anderson et al., 2016. By constructing the components of the SGC in this manner, background noise is reduced because, upon binding of both target probes to the target nucleic acid and pre-amplifier, the SGC is assembled, thereby minimizing the number of SGCs assembled as false positives.

[0122] In the configurations of Figures 5C and 6C, the requirement that SGCs should only be formed when both members of the target probe pair are bound to the target nucleic acid is met by requiring that the pre-amplifier binds to both pre-pre-amplifiers that are sequentially bound to both members of the target probe pair. This requirement is met by designing the binding sites of the pre-pre-amplifiers and pre-amplifiers such that the melting temperature (Tm) of the binding of both pre-pre-amplifiers to the pre-amplifier is higher than the melting temperature of either pre-pre-amplifier alone, and that the state in which one of the pre-pre-amplifiers is bound to the pre-amplifier is unstable under assay conditions. This design has been previously described, for example, in US20170101672, WO2017 / 066211, and Baker et al., 2017. Unless the pre-amplifier binds to both pre-pre-amplifiers, the amplifier and labeled probe cannot assemble into SGCs bound to the target nucleic acid, thereby minimizing the number of false-positive SGCs assembled and reducing background noise.

[0123] As disclosed herein, the method may be based on constructing a signal-generating complex (SGC) bound to a target nucleic acid in order to detect the presence of the target nucleic acid within a cell. The components for constructing an SGC generally include nucleic acids, so that the components of the SGC are bound to the target nucleic acid by utilizing a nucleic acid hybridization reaction. Methods for selecting appropriate regions and designing specific and selective reagents that bind to the target nucleic acid, specifically oligonucleotides or probes that bind specifically and selectively to the target nucleic acid, or other components of the SGC, are well known to those skilled in the art (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). Target probes are designed to hybridize specifically to the target nucleic acid. Desired specificity can be achieved using appropriately selected target nucleic acid regions and binders (such as oligonucleotides or probes) of appropriate length, and such selection methods are well known to those skilled in the art. Therefore, those skilled in the art can readily grasp and determine suitable reagents (such as oligonucleotides or probes) that can be used to target one specific target nucleic acid to another, or to provide binding to the components of an SGC. Similar specificity can be achieved in target-specific SGCs by using appropriate selection of unique sequences such that given components of a target-specific SGC (e.g., target probe, pre- and pre-amplifiers, pre-amplifiers, amplifiers, labeling probes) bind to their respective components, causing the SGC to bind to a specific target (see Figure 6).

[0124] As described herein, embodiments of the present invention include the use of target probe pairs. When target probe pairs are coupled to the same pre-amplifier (Figures 5A and 6A) or pre-pre-amplifier (Figures 5B and 5B), probe configurations sometimes referred to as "Z" configurations may be used. Such configurations and their advantages for increasing sensitivity and reducing background are described, for example, in U.S. Patent No. 7,709,198, U.S. Publications 2008 / 0038725 and 2009 / 0081688, and WO2007 / 001986 and WO2007 / 002006, each of which is incorporated herein by reference. U.S. Patent No. 7,709,198, and U.S. Publications 2008 / 0038725 and 2009 / 0081688 also provide further details regarding the selection of target probe characteristics (including length, orientation, hybridization conditions, etc.), such as target probe pairs. A person skilled in the art can easily identify preferred configurations based on this specification and the teachings in, for example, U.S. Patent No. 7,709,198, U.S. Publications 2008 / 0038725, 2009 / 0081688, WO2007 / 001986, and WO2007 / 002006.

[0125] As described herein, the target binding site of a target probe in a target probe pair can be any desired orientation and combination. For example, one target binding site of a member of a target probe pair can be on the 5' or 3' side of the pre-amplifier or pre-pre-amplifier binding site, and the other member of the target probe pair can independently have an orientation having the target binding site on the 5' or 3' side of the pre-amplifier or pre-pre-amplifier binding site.

[0126] In another embodiment, the SGC used to detect the presence of a target nucleic acid is based on the synergistic hybridization of one or more components of the SGC (see US20170101672 and WO2017 / 066211, each incorporated herein by reference). Such synergistic hybridization is also referred herein to as BaseScope®. In the effect of synergistic hybridization, the binding between two components of the SGC is mediated by two binding sites, and the melting temperature when the two sites are bound simultaneously is higher than the melting temperature when one site is bound alone (see US20170101672 and WO2017 / 066211). The effect of synergistic hybridization can be enhanced by a set configuration of target probes as described in US20170101672 and WO2017 / 066211.

[0127] The method and related compositions of the present invention utilize synergistic hybridization to improve specificity and reduce background noise in in situ detection of nucleic acid targets (which can be noisy due to the complex physiological and chemical environment and the presence of an overwhelming number of non-target molecules). Using such a synergistic hybridization method, the labeled probe is bound only when the SGC is bound to the target nucleic acid. As described in US20170101672 and WO2017 / 066211, and shown in Figure 1, the method can be readily modified to provide a desired signal-to-noise ratio by increasing the number of synergistic hybridizations in one or more components of the SGC.

[0128] In another embodiment, synergistic hybridization can be applied to various components of SGCs. For example, the binding between components of an SGC may be a stable reaction, as described herein, or the binding may be configured to require synergistic hybridization, as also described herein. In such cases, the binding component to which synergistic hybridization is envisioned is designed to include two segments that bind to another component.

[0129] In other words, a method for detecting a target nucleic acid can utilize synergistic hybridization in the binding reaction of one or all of the components in a detection system that yield SGCs that specifically bind to the target nucleic acid. The number and types of components to which synergistic hybridization is applied can be selected based on the desired assay conditions, the type of sample to be assayed, the desired assay sensitivity, etc. The sensitivity and specificity of the assay can be improved by using one or a combination of synergistic hybridization binding reactions. In embodiments of the present invention, synergistic hybridization can be between pre- and pre-amplifiers and pre-amplifiers, between pre-amplifiers and amplifiers, between amplifiers and labeled probes, or a combination thereof (see, for example, US20170101672 and WO2017 / 066211).

[0130] As disclosed herein, the components are generally directly bonded to one another. In the case of nucleic acid-containing components, the binding reaction is generally by hybridization. In the case of hybridization reactions, the binding between components is direct. If desired, intermediate components may be included so that the binding between one component and another is indirect, for example, the intermediate components include complementary binding sites for bridging the two other components.

[0131] As described herein, the composition of various components can be selected to yield the desired stable or synergistic hybridization binding reaction (see, for example, US20170101672). It should be understood that, whether a binding reaction is exemplified as a stable reaction or, as in synergistic hybridization, as an unstable reaction, either binding reaction can be modified as desired, as long as the target nucleic acid is detected. Furthermore, it should be understood that the composition can be changed and selected depending on the assay and hybridization conditions used. Generally, when it is desirable for the binding reaction to be stable, the complementary nucleic acid sequence segments between the constituent components are generally in the range of 16 to 30 nucleotides, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more. When a relatively unstable binding reaction is desirable, such as when using a synergistic hybridization binding reaction, the complementary nucleic acid sequence segments between the constituent components are generally in the range of 5 to 18 nucleotides, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides.It should be understood that this nucleotide length may be slightly shorter or longer than the above, depending on the sequence (e.g., GC content) and conditions used in the assay, for stable or unstable hybridization. Furthermore, it should be understood that the length of the binding segment can be shortened by improving the binding strength at the modified base using modified nucleotides such as locked nucleic acid (LNA) or cross-linked nucleic acid (BNA), as disclosed herein. That is, it should be understood that the length of nucleic acid segments complementary to other nucleic acid segments can be further shortened if desired. Those skilled in the art can easily determine an appropriate probe design, including length, the presence of modified nucleotides, etc., so that the interaction between nucleic acid components is as desired.

[0132] When designing a binding site between two nucleic acid sequences containing complementary sequences, the complementary sequences are optionally selected based on the difference in melting temperature (dT). m ) can be designed to maximize this. This can be done by using melting temperature calculation algorithms known in the art (see, for example, Santa Lucia, Proc. Natl. Acad. Sci. USA 95:1460-1465 (1998)). In addition, artificially modified bases such as locked nucleic acid (LNA) or cross-linked nucleic acid (BNA), and natural 2'-O-methyl RNA are known to increase the binding strength between complements (Petersen and Wengel, Trends Biotechnol. 21:74-81 (2003), Majlessi et al., Nucl. Acids Res. 26:2224-2229 (1998)). These modified bases can be strategically introduced, as desired, into the binding sites between the components of SGC.

[0133] One approach is to utilize modified nucleotides (LNA, BNA, or 2'-O-methyl RNA). Each modified base can increase the melting temperature, thus substantially shortening the length of the binding region between two nucleic acid sequences (i.e., complementary sequences). The binding strength of the modified base to its complement is stronger, resulting in a larger difference in melting temperature (dT m ). Yet another embodiment is to use, for example, three modified bases (e.g., three LNA, BNA or 2'-O-methyl RNA bases, or a combination of two or three different modified bases) within the complementary sequence of the nucleic acid component of the signal generating complex (SGC) that is to hybridize, or between two nucleic acid components. Such components can be, for example, pairs of pre-amplifiers, pre-amplifiers, amplifiers, labeled probes, or target probes.

[0134] Modified bases such as LNA or BNA can be used in segments of selected components of the SGC, specifically, segments that mediate binding between nucleic acid components, thereby improving the binding strength between that base and its complementary base and enabling shortening of the length of its complementary segment (see, for example, Petersen and Wengel, Trends Biotechnol. 21:74-81 (2003), U.S. Patent No. 7,399,845). An artificially extended genetic information system (AEGIS; Yang et al., Nucl. Acids Res. 34(21):6095-6101 (2006)) can be incorporated into the binding site among the interacting components of the SGC. These artificial bases can improve the specificity of this interacting component, which in turn allows for a less stringent hybridization reaction and the generation of a higher level of signal.

[0135] With regard to target probe pairs, the target probe pair can be designed to bind to a directly adjacent segment of the target nucleic acid, or to a segment having one to a substantial number of bases between the target probe binding sites of the target probe pair. Generally, target probe pairs are designed to bind to target nucleic acids, and generally, between the binding sites on the target nucleic acid, there are base lengths of 0 to 500, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 bases, or any integer in between. In certain embodiments, the binding sites for target probe pairs are 0–100 nucleotides long, 0–200 nucleotides long, or 0–300 nucleotides long, or any integer nucleotide length in between. It should be understood that in a set of target probes, if two or more target probe pairs are used to bind to the same target nucleic acid, which is RNA or single-stranded DNA, and there is a gap in the binding sites between target probe pairs, the binding sites of different target probe pairs will not overlap. When detecting double-stranded nucleic acids such as DNA, there may be some overlap between different target probe pairs, as long as the target probe pairs can simultaneously bind to their respective binding sites on the double-stranded target nucleic acid.

[0136] SGC also includes multiple labeled probes (LPs). Each LP contains a detectable segment. The detectable component can be directly bound to the LP, or the LP can hybridize to another nucleic acid containing the detectable component, i.e., the label. As used herein, “label” is a portion that facilitates the detection of a molecule. Common labels in the context of the present invention include fluorescent labels, luminescent labels, light scattering labels, and / or colorimetric labels. Preferred labels include enzyme moieties, fluorescent moieties, color-producing moieties, radionuclides, substrates, cofactors, inhibitors, chemiluminescent moieties, magnetic particles, rare earth metals, metal isotopes, and the like. In certain embodiments of the present invention, the label is an enzyme. Exemplary enzyme labels include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, glucose oxidase, and various proteases. Other labels include, but are not limited to, fluorophores, dinitrophenyl (DNP), and the like. The labels are well known to those skilled in the art, for example, as described in Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996), and U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Many labels, including detectable enzyme / substrate combinations (Pierce, Rockford IL; Santa Cruz Biotechnology, Dallas TX; Life Technologies, Carlsbad CA), are commercially available and can be used in the methods and assays of the present invention. In certain embodiments of the present invention, the enzyme can generate a detectable signal using a chromogenic substrate or a fluorescent substrate, as described herein. Exemplary labels are described herein.

[0137] Any number of enzyme-active or non-enzymatic labels can be used, as long as each is detectable. Enzymes produce detectable signals that can be used to detect target nucleic acids. Particularly useful detectable signals are chromogenic or fluorescent signals. Therefore, enzymes particularly useful for use as labels include those for which chromogenic or fluorescent substrates are available. Such chromogenic or fluorescent substrates can be readily converted by enzymatic reactions into detectable chromogenic or fluorescent products, which can be readily detected and / or quantified using microscopy or spectroscopy. Such enzymes are well known to those skilled in the art and include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose oxidase (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Other enzymes with known chromogenic or fluorescent substrates include various peptidases, and their chromogenic or fluorescent peptide substrates can be used to detect protein cleavage reactions. The use of chromogenic and fluorescent substrates is also well known in bacterial diagnosis, and is not limited to these, but includes the use of α-galactosidase, β-galactosidase, β-glucuronidase, 6-phospho-β-D-galactosidase-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 fluorescent substrates can be readily adapted for use in the method of the present invention.

[0138] Various chromogenic or fluorescent substrates for generating detectable signals are well known to those skilled in the art and are commercially available. Exemplary substrates that can be used to generate detectable signals 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, and alkaline phosphatase. Examples of effective phosphates include 5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP), nitrobluetetrazolium (NBT), Fastred (Fastred TR / AS-MX), and p-nitrophenyl phosphate (PNPP) for β-galactosidase; 1-methyl-3-indolyl-β-D-galactopyranoside and 2-methoxy-4-(2-nitrovinyl)phenyl β-D-galactopyranoside for β-galactosidase; and 2-methoxy-4-(2-nitrovinyl)phenyl β-D-glucopyranoside for β-glucosidase. Examples of 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, and QuantaBlu® and QuantaRed® for horseradish peroxidase. Examples include 4-methylumbelliferyl-β-D-galactopyranoside, fluoresceindi(β-D-galactopyranoside), and naphthofluoresceindi-(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 that generate detectable signals are described, for example, in U.S. Publication 2012 / 0100540. A variety of detectable enzyme substrates, including chromogenic or fluorescent substrates, are well known and commercially available (Pierce, Rockford IL; Santa Cruz Biotechnology, Dallas TX; Invitrogen, Carlsbad CA; 42 Life Science; Biocare). Generally, substrates are converted into products that form precipitates deposited at the site of the target nucleic acid. Other exemplary substrates, but not limited to these, include HRP-Green (42 Life Science), Betazoid DAB, Cardassian DAB, Romulin AEC, Bajoran Purple, Vina Green, Deep Space Black®, Warp Red®, Vulcan Fast Red, and Ferangi Blue (from Biocare) (Concord CA; biocare.net / products / detection / chromogens).

[0139] Suitable examples of rare earth metals and metal isotopes 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. Metallic isotopes can be detected using, for example, time-of-flight mass spectrometry (TOF-MS) (e.g., Fluidigm Helios and Hyperion systems, fluidigm.com / systems, South San Francisco, CA).

[0140] Biotin-avidin (or biotin-streptavidin) is a well-known signal amplification system based on the very high affinity between the two molecules and the fact that one avidin / streptavidin molecule can bind to four biotin molecules. Antibodies are widely used for signal amplification in immunohistochemistry and ISH. Tyramide signal amplification (TSA) is based on the deposition of numerous haptenized tyramide molecules via peroxidase activity. Tyramine is a phenolic compound. Immobilized horseradish peroxidase (HRP), in the presence of a small amount of hydrogen peroxide, converts the labeled substrate into a highly reactive, short-lived intermediate. Subsequently, the activated substrate molecule reacts very rapidly with an electron-rich protein moiety (such as tyrosine) at or near the peroxidase binding site, covalently binding to that moiety. 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, for example, U.S. Publication 2012 / 0100540.

[0141] In the embodiments described herein, enzymes can be used to generate a detectable signal using a suitable chromogenic or fluorescent substrate. Alternatively, it should be understood that the labeled probe may have a detectable label directly bound to the nucleic acid portion of the labeled probe. Exemplary detectable labels, including but not limited to those well known to those skilled in the art, include chromogenic or fluorescent labels (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Examples of fluorophores useful as labels include, but are not limited to, rhodamine derivatives, e.g., tetramethylrhodamine, rhodamine B, rhodamine 6G, sulfolphodamine B, Texas Red (sulfodamine 101), rhodamine 110, and their derivatives (e.g., tetramethylrhodamine-5-(or 6), lysaminerhodamine B); 7-nitrobenz-2-oxa-1,3-diazole (NBD); fluorescein and its derivatives; naphthalene (e.g., dansyl(5-dimethylaminonaphthalene-1-sulfonyl)); coumarin derivatives (e.g., 7-amino-4-methylcoumarin-3-acetic acid (AMCA), 7-diethylamino-3-[(4'-(iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA)); Alexa fluor dye (Molecular (Probes, etc.); 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY®) and its derivatives (Molecular Probes, Eugene, OR); pyrene and sulfonated pyrene (Cascade Blue®) and its derivatives (including 8-methoxypyrene-1,3,6-trisulfonic acid, etc.); pyridyloxazole derivatives and dapoxyl derivatives (Molecular Probes); Lucifer Yellow (3,6-disulfonate-4-amino-naphthalimide) and its derivatives; CyDye® fluorescent dyes (Amersham / GE Healthcare Life Sciences, Piscataway, NJ);ATTO 390, DyLight 395XL, ATTO 425, ATTO 465, ATTO 488, ATTO 490LS, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 643, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO Examples include 700, ATTO 725, ATTO 740, and Cyan 500 NHS-Ester (ATTO-TECH, Siegen, Germany). Exemplary chromophores, though not limited to these, include phenolphthalein, malachite green, aromatic nitro compounds (such as nitrophenyl), diazo dyes, and dabucil (4-dimethylaminoazobenzene-4'-sulfonyl).

[0142] As disclosed herein, the method can utilize the simultaneous detection of multiple target nucleic acids. When fluorophores are used as labels, the fluorophores used for the detection of multiple target nucleic acids are selected so that each fluorophore is distinguishable in the case of simultaneous detection of target nucleic acids and so that the fluorophores can be detected simultaneously by fluorescence microscopy. Such fluorophores are selected so that the emission line spectra are separated so that distinct labels of target nucleic acids can be detected simultaneously. Methods for selecting distinguishable fluorophores suitable for use in the method of the present invention are well known in the art (see, for example, Johnson and Spence, “Molecular Probes Handbook, a Guide to Fluorescent Probes and Labeling Technologies, 11th ed., Life Technologies (2010)”).

[0143] Well-known methods such as microscopy, cytometry (e.g., mass cytometry, time-of-flight cytometry (CyTOF), flow cytometry), or spectroscopy can be used to visualize detectable chromogenic, fluorescent, or metallic signals associated with each target nucleic acid. Generally, to allow the use of a single instrument for detecting nucleic acid targets within the same sample, different labels are used in the same assay, or, depending on the specific assay, either a chromogenic substrate, a fluorescent substrate, a chromogenic label, a fluorescent label, or a rare-earth metal isotope is used.

[0144] As disclosed herein, labeled probes may be designed such that the label is optionally cleavable. As used herein, a cleavable label refers to a label that is bound to or conjugated to a labeled probe so that the label can be removed from the SGC for use in a subsequent round of labeling and detecting a target nucleic acid, for example. Generally, the label is conjugated to the labeled probe by a cleavable chemical linker. Methods for conjugating a label to a labeled probe so that the label is cleavable are well known to those skilled in the art (see, e.g., Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996), Daniel et al., BioTechniques 24(3):484-489 (1998)). One particular system for labeling oligonucleotides is the FastTag® system (Daniel et al., above, 1998; Vector Laboratories, Burlinghame CA). Various cleavable moieties may be included in the linker to allow the label to be cleaved from the label probe. Such cleavable moieties include groups that can be cleaved chemically, photochemically, or enzymatically. A cleavable chemical linker may include cleavable chemical moieties such as disulfides that can be cleaved by reduction, glycols or diols that can be cleaved by periodates, diazo bonds that can be cleaved by dithionites, esters that can be cleaved by hydroxylamines, and sulfones that can be cleaved by bases (see Hermanson, above, 1996). One particularly useful cleavable linker is one that includes a disulfide bond that can be cleaved by reduction of the disulfide bond. In other embodiments, the linker may include sites for enzymatic cleavage. For example, the linker may include protein cleavage sites. Generally, such cleavage sites are for sequence-specific proteases.Examples of such proteases, but not limited to these, include human rhinovirus 3C protease (cleavage site LEVLFQ / GP), enterokinase (cleavage site DDDDK / ), factor Xa (cleavage site IEGR / ), tobacco etch virus protease (cleavage site ENLYFQ / G), and thrombin (cleavage site LVPR / GS) (see, e.g., Oxford Genetics, Oxford, UK). Another cleavable region may be uracil-DNA (uracil-containing DNA) that can be cleaved by uracil-DNA glycosylase (UNG) (see, e.g., Sidorenko et al., FEBS Lett. 582(3):410-404 (2008)).

[0145] The present invention relates, in some embodiments, to the use of cleavable labels so that a label bound to a target nucleic acid can be removed from the target nucleic acid. The cleavable label can be removed by applying a chemical agent or light for the purpose of cleaving the label and dissociating it from the label probe. As discussed above, useful cleaving agents for chemical cleavage include, but are not limited to, reducing agents, periodic acid, dithionites, hydroxylamines, and bases (see Hermanson, above, 1996). One useful method for cleaving a linker containing a disulfide bond is the use of tris(2-carboxyethyl)phosphine (TCEP) (see Moffitt et al., Proc. Natl. Acad. Sci. USA 113:11046-11051 (2016)). In one embodiment, TCEP is used as an agent for cleaving the label from the label probe.

[0146] In another embodiment, instead of using a cleavable label, the label probe bound to the target nucleic acid can be selectively removed or washed away by exposing the SGC to a temperature higher than the Tm of the binding sequence between the label probe and the amplifier. Methods for selectively removing components of an SGC, such as removing the label probe bound to the amplifier in the SGC by selecting suitable temperatures and conditions for disrupting the binding between the label probe and the amplifier in the SGC, are well known in the art and are disclosed herein. When selectively removing the label probe from the SGC is used, the components of the SGC should be designed so that interactions of the SGC components other than the interaction between the label probe and the amplifier remain stable even under conditions that disrupt the binding of the label probe to the amplifier. It should be understood that, as with using a label probe containing a cleavable label, since no further detection rounds are performed in the final iterative detection round, the melting temperature between the label probe and the amplifier does not necessarily need to be lower than the melting temperature between the target probe, pre-pre-amplifier (if used), pre-amplifier, and amplifier. Therefore, in the final iterative detection round, it is optional to set the melting temperature between the labeled probe and the amplifier lower than the melting temperature between the target probe, pre-pre-amplifier (if used), pre-amplifier, and amplifier, resulting in the labeled probe and label remaining bound to the SGC in the final iterative detection round.

[0147] The present invention as described herein generally relates to the detection of multiple target nucleic acids in a sample. It should be understood that the methods of the present invention may also be applied to the detection of multiple target nucleic acids and, optionally, other molecules in the sample, particularly within the same cells as the target nucleic acids. For example, in addition to detecting multiple target nucleic acids, intracellularly expressed proteins may also be detected simultaneously using a principle similar to that described for detecting target nucleic acids. In this case, in one or more rounds of detection of multiple target nucleic acids, one or more intracellularly expressed proteins may be optionally detected, for example, by using a detectable label for detecting proteins. If proteins are detected in the initial target nucleic acid detection rounds, the proteins may be detected by a cleavable label, similar to the cleavable label used for detecting the target nucleic acids. If proteins are detected in the final detection round, the label does not need to be cleavable. For example, it is well known to those skilled in the art that intracellular proteins can be detected by detecting the binding of a protein-specific antibody using one of the well-known detection systems, including the system described herein, for the detection of target nucleic acids. The detection of target nucleic acids and proteins within the same cell has been described (see also Schulz et al., Cell Syst. 6(1):25-36(2018)).

[0148] It should be understood that the present invention can be carried out in any desired order, as long as the target nucleic acid is detected. That is, in the method of the present invention, as long as the target nucleic acid is detected, the steps of contacting any component with cells to assemble the SGC can be carried out in any desired order, sequentially, or simultaneously, or, as desired, some steps can be carried out sequentially while others are carried out simultaneously, as long as the target nucleic acid is detected. Furthermore, it should be understood that, as desired, the embodiments disclosed herein can be used independently or combined with other embodiments disclosed herein for the purpose of using various configurations, component sizes, assay conditions, assay sensitivities, etc.

[0149] It should be understood that the present invention can be carried out in any form that provides detection of a target nucleic acid. While the implementation of the present invention has generally been described herein using in situ hybridization, it should be understood that the present invention can be carried out for detection of target nucleic acids in other forms, as is well known in the art, particularly for detection of target nucleic acids within cells. One method that can be used to detect target nucleic acids within cells is flow cytometry, which is well known in the art (see, for example, Shapiro, Practical Flow Cytometry, 3rd ed., Wiley-Liss, New York (1995), and Ormerod, Flow Cytometry, 2nd ed., Springer (1999)). Thus, the methods, samples, and kits of the present invention can be used in the form of an in situ hybridization assay or in another form such as flow cytometry. The application of nucleic acid detection methods, including in situ hybridization, to flow cytometry has been previously described (e.g., Hanley et al., PLoS One, 8(2):e57002.doi:10.1371 / journal.pone.0057002 (2013), Baxter et al., Nature Protocols 12(10):2029-2049 (2017)).

[0150] In some cases, it is desirable to reduce the number of steps in an assay, for example, by reducing the number of hybridization and washing steps. One way to reduce the number of assay steps is to pre-assemble some or all of the components of the SGC before contacting the cells. This pre-assembly can be done by hybridizing some or all of the components of the SGC together before contacting the target nucleic acid.

[0151] The present invention further provides kits comprising acid reagents, as disclosed herein. The acid reagents result in the disruption of hybridization between the target probe and each target nucleic acid, while preserving cell morphology and nucleic acid integrity. The components of the kits of the present invention may optionally be contained in a container, and optionally instructions for using the kit may be provided. The instructions may describe steps for carrying out the method of the present invention, for example, as disclosed herein. Optionally, the kit may contain one or more components of the SGC described herein, and the kit may not contain target nucleic acids. Such a kit may include a pre-amplifier (PA), an amplifier (AMP), and a labeled probe (LP), as disclosed herein, and optionally a pre-pre-amplifier (PPA). Optionally, the kit may include a target probe (TP) targeting a specific target nucleic acid or multiple target nucleic acids.

[0152] In one embodiment, the present invention provides a kit comprising one or more probes specific to one or more nucleic acid targets and instructions for carrying out the methods of the present invention disclosed herein.

[0153] In one embodiment, the present invention provides a kit comprising an acid reagent for use in a method for disrupting the binding of a probe bound to a nucleic acid in a cell, the method comprising contacting a cell with the acid reagent, the cell comprising a first probe hybridized to a first target nucleic acid in the cell, and the acid reagent disrupting the hybridization between the first probe and the first target nucleic acid.

[0154] In one embodiment of such a kit, contact between cells and an acid reagent is repeated one or more times.

[0155] In one embodiment of such a kit, the method further includes removing a first probe from cells. In one embodiment of such a kit, the method further includes contacting cells with a second probe, the second probe hybridizing to a second target nucleic acid in the cells, the second target nucleic acid being identical or different from the first target nucleic acid. In one embodiment of such a kit, the method further includes contacting cells with an acid reagent, the acid reagent disrupting the hybridization between the second probe and the second target nucleic acid. In one embodiment, contacting cells with the acid reagent is repeated one or more times. In one embodiment of such a kit, the method further includes removing the second probe from cells.

[0156] In one embodiment, the present invention provides a kit comprising an acid reagent for use in a method for disrupting the binding of probes bound to nucleic acids within cells, the method comprising contacting cells with the acid reagent, the cells comprising one or more first probes hybridized to one or more first target nucleic acids within the cells, and the acid reagent disrupting the hybridization between the one or more first probes and the one or more first target nucleic acids.

[0157] In one embodiment of such a kit, contact between cells and an acid reagent is repeated one or more times.

[0158] In one embodiment of such a kit, the method further comprises removing one or more first probes from cells. In one embodiment of such a kit, the cells contain two or more first probes hybridized to two or more first target nucleic acids. In one embodiment of such a kit, each of the first target nucleic acids is labeled by hybridization to the first probe, and the label on each first target nucleic acid is distinguishable from the labels on other first target nucleic acids hybridized to the first probe.

[0159] In one embodiment of such a kit, the method further comprises the step of contacting cells with one or more second probes, the one or more second probes hybridizing to one or more second target nucleic acids in the cells, the one or more second target nucleic acids being identical to or different from one or more first target nucleic acids. In one embodiment, the cells contain two or more second probes hybridized to two or more second target nucleic acids.

[0160] In one embodiment of such a kit, each of the second target nucleic acids is labeled by hybridization to a second probe, and the label on each second target nucleic acid is distinguishable from the labels on other second target nucleic acids hybridized to the second probe. In one embodiment of such a kit, the method further comprises the step of contacting cells with an acid reagent, the acid reagent disrupting the hybridization between the second probe and one or more second target nucleic acids. In one embodiment, contacting cells with the acid reagent is repeated one or more times. In one embodiment, the method further comprises the step of removing the second probe from the cells.

[0161] In one embodiment, the present invention provides a kit for in situ detection of a target nucleic acid, comprising: (A) a set of pre-amplifiers, the set of pre-amplifiers comprising a plurality of pre-amplifiers, each pre-amplifier comprising a binding site for a pre-pre-amplifier pair and a plurality of binding sites for an amplifier; (B) a set of amplifiers, the set of amplifiers comprising a plurality of amplifiers, each amplifier comprising a binding site for a pre-amplifier and a plurality of binding sites for a labeled probe; (C) a set of labeled probes, the set of labeled probes comprising a label and a binding site for an amplifier; and (D) an acid reagent, the acid reagent causing disruption of hybridization between the target probe and each target nucleic acid (see, for example, Figures 2A and 6A). In one embodiment, the kit comprises a set of target probes, the set of target probes comprising one or more pairs of target probes that specifically hybridize to the target nucleic acid.

[0162] In one embodiment, the present invention provides a kit for in situ detection of target nucleic acids, comprising: (A) a set of pre-preamplifiers, each set comprising one or more pre-preamplifiers, each pre-preamplifier comprising a binding site for one or more pairs of target probes; (B) a set of preamplifiers, each set comprising a plurality of preamplifiers, each preamplifier comprising a binding site for the pre-preamplifiers and a plurality of binding sites for the amplification agents; (C) a set of amplification agents, each set comprising a plurality of amplification agents, each amplification agent comprising a binding site for the preamplifiers and a plurality of binding sites for the labeled probes; (D) a set of labeled probes, each labeled probe in the set of labeled probes comprising a label and a binding site for the amplification agents; and (E) an acid reagent, the acid reagent causing disruption of hybridization between the target probes and their respective target nucleic acids (see, for example, Figures 2A and 6B). In one embodiment, the kit includes a set of target probes, the set of target probes including one or more pairs of target probes that specifically hybridize to a target nucleic acid.

[0163] In one embodiment, the present invention comprises: (A) a set of pre-preamplifiers, the set of pre-preamplifiers comprising one or more pairs of pre-preamplifiers, each pre-preamplifier in the pair comprising a binding site for one of the target probes of the target probe pair; (B) a set of preamplifiers, the set of preamplifiers comprising a plurality of preamplifiers, each preamplifier comprising a binding site for a pair of pre-preamplifiers and a plurality of binding sites for an amplifier; (C) a set of amplifiers, the set of amplifiers comprising a plurality of amplifiers, each amplifier comprising a binding site for a preamplifier and a plurality of binding sites for a labeled probe; (D) a set of labeled probes, the set of labeled probes comprising each labeled probe comprising a label and a binding site for an amplifier; and (E) an acid reagent, the acid reagent causing disruption of hybridization between the target probe and each target nucleic acid. A kit for situ detection is provided (see, for example, Figures 2A and 6C). In one embodiment, the kit includes a set of target probes, the set of target probes including one or more pairs of target probes that specifically hybridize to a target nucleic acid.

[0164] In one embodiment, the present invention relates to the in of target nucleic acids. The kit provides for situ detection, the kit comprising: (A) a set of pre-amplifiers, the set of pre-amplifiers comprising a plurality of pre-amplifiers, each of which is specific to each of one or more sets of target probes, and each pre-amplifier comprising a binding site for one pair of target probes from the set of target probes and a plurality of binding sites for the amplifier; (B) a set of amplifiers, the set of amplifiers comprising a plurality of subsets of amplifiers specific to each pre-amplifier, each subset of amplifiers comprising a plurality of amplifiers, each amplifier comprising a binding site for one of the pre-amplifiers specific to the set of target probes and a plurality of binding sites for the labeled probe; and (C) a first set of labeled probes, the first set of labeled probes comprising a plurality of first subsets of labeled probes, each subset of labeled probes being specific to one of the amplifiers from the subset of amplifiers, each subset of labeled probes comprising a plurality of target probes, and each of the subsets of labeled probes is (D) A first set of labeling probes, each comprising a label and binding site for one of the amplification agents from a subset of amplification agents, wherein the label in each first subset of the labeling probes is distinguishable among the first subsets of the labeling probes, the label is cleavable, and the first set of labeling probes can specifically label a first subset of the target nucleic acid; and (D) a second set of labeling probes, each of which comprises a plurality of second subsets of labeling probes, each subset of labeling probes is specific to one of the amplification agents from the subset of amplification agents, and the second subset of labeling probes is specific to an amplification agent of a subset of amplification agents different from that of the first subset of labeling probes, each subset of labeling probes comprises a plurality of labeling probes, each labeling probe in the subset of labeling probes comprises a label and binding site for one of the amplification agents from the subset of amplification agents, the label in each second subset of labeling probes is distinguishable among the second subsets of labeling probes, the label is cleavable, and the second set of labeling probes is(E) an acid reagent, wherein the acid reagent causes the hybridization between the target probe and the respective target nucleic acid to be disrupted (see, for example, Figures 2B and 6A).

[0165] In one embodiment, the kit further comprises a third set of labeled probes, the third set of labeled probes comprising a plurality of third subsets of labeled probes, each subset of labeled probes being specific to one of the subsets of amplifiers, the third subset of labeled probes being specific to an amplifier of a subset of amplifiers different from the first and second subsets of labeled probes, each subset of labeled probes comprising a plurality of labeled probes, each labeled probe of the subset of labeled probes comprising a label and binding site for one of the subsets of amplifiers, the label in each third subset of labeled probes being distinguishable among the third subsets of labeled probes, the label being cleavable, and the third set of labeled probes being able to specifically label a third subset of target nucleic acid different from the first and second subsets of target nucleic acid.

[0166] In one embodiment, the present invention provides a kit for in situ detection of a target nucleic acid, the kit comprising: (A) a set of pre-preamplifiers, the set of pre-preamplifiers comprising a plurality of pre-preamplifiers, the plurality of pre-preamplifiers comprising pre-preamplifiers specific to each of one or more sets of target probes, each pre-preamplifier comprising a binding site for one pair of target probes from the set of target probes and a plurality of binding sites for the preamplifier; and (B) a set of preamplifiers, the set of preamplifiers comprising a plurality of subsets of preamplifiers specific to each pre-preamplifier, (C) A set of preamplifiers, where each subset of the broadener includes a plurality of preamplifiers, and the preamplifier of the subset of preamplifiers includes a binding site to one of the preamplifiers specific to the target probe set and a plurality of binding sites to the amplifier, and (C) a set of amplifiers, where the set of amplifiers includes a plurality of subsets of amplifiers specific to each subset of preamplifiers, and each subset of amplifiers includes a plurality of amplifiers, and the amplifier of the subset of amplifiers includes a binding site to one of the preamplifiers in the subset of preamplifiers and a plurality of binding sites to the labeled probe. (D) A first set of labeled probes, wherein the first set of labeled probes comprises a set of amplification agents including binding sites, and each subset of labeled probes is specific to one of the subsets of amplification agents, each subset of labeled probes comprises a set of target probes, each subset of labeled probes comprises a label and binding site for one of the subsets of amplification agents, the label in each subset of labeled probes is distinguishable between the first subsets of labeled probes, the label is cleavable, and the first set of labeled probes can specifically label a first subset of target nucleic acids, and (E) a second set of labeled probes, wherein the second set of labeled probes comprises a set of second subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of amplification agents, and each subset of labeled probes is specific to an amplification agent of a subset of amplification agents different from that of the first subset of labeled probes.The second set of labeled probes comprises (F) an acid reagent, the acid reagent causing disruption of hybridization between the target probe and the respective target nucleic acid (see, for example, Figures 2B and 6B).

[0167] In one embodiment, the kit further comprises a third set of labeled probes, the third set of labeled probes comprising a plurality of third subsets of labeled probes, each subset of labeled probes being specific to one of the subsets of amplifiers, the third subset of labeled probes being specific to an amplifier of a subset of amplifiers different from the first and second subsets of labeled probes, each subset of labeled probes comprising a plurality of labeled probes, each labeled probe of the subset of labeled probes comprising a label and binding site for one of the subsets of amplifiers, the label in each third subset of labeled probes being distinguishable among the third subsets of labeled probes, the label being cleavable, and the third set of labeled probes being able to specifically label a third subset of target nucleic acid different from the first and second subsets of target nucleic acid.

[0168] In one embodiment, the present invention provides a kit for in situ detection of a target nucleic acid, the kit comprising (A) a set of pre-amplifiers, the set of pre-amplifiers comprising a plurality of pairs of pre-amplifiers, the set of pre-amplifiers comprising a pair of pre-amplifiers specific to each target probe of a pair of target probes in a set of one or more target probes, each pre-amplifier in the pair of pre-amplifiers comprising a binding site for one of the target probes of the pair of target probes in the set of target probes, and the set of pre-amplifiers comprising a plurality of binding sites for the pre-amplifiers, and (B) a set of pre-amplifiers (C) A set of pre-amplifiers comprising a plurality of pre-amplifiers, each of which includes a pre-amplifier specific to each pair of pre-pre-amplifiers, and each pre-amplifier comprising a binding site for one of the pre-pre-amplifier pairs in the set of pre-pre-amplifiers and a plurality of binding sites for the amplifier, and (C) a set of amplifiers comprising a plurality of subsets of amplifiers specific to each pre-amplifier specific to each pair of pre-pre-amplifiers, and the amplifiers of the subsets of amplifiers comprising a binding site for one of the pre-amplifiers specific to each pair of pre-pre-amplifiers and a binding site for the labeled probe (D) A first set of labeled probes, wherein the first set of labeled probes comprises a set of amplifiers including multiple binding sites, and each subset of labeled probes is specific to one of the subsets of amplifiers, each subset of labeled probes comprises a set of target probes, each of the subsets of labeled probes comprises a label and binding site for one of the subsets of amplifiers, the label in each first subset of labeled probes is distinguishable between the first subsets of labeled probes, the label is cleavable, and the first set of labeled probes can specifically label a first subset of target nucleic acids, and (E) a second set of labeled probes, wherein the second set of labeled probes comprises a set of second subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of amplifiers, and each subset of labeled probes is specific to an amplifier of a subset of amplifiers different from that of the first subset of labeled probes.The second set of labeled probes comprises (F) an acid reagent, the acid reagent causing the hybridization between the target probe and the respective target nucleic acid to be disrupted (see Figures 2B and 6C).

[0169] In one embodiment, the kit further comprises a third set of labeled probes, the third set of labeled probes comprising a plurality of third subsets of labeled probes, each subset of labeled probes being specific to one of the subsets of amplifiers, the third subset of labeled probes being specific to an amplifier of a subset of amplifiers different from the first and second subsets of labeled probes, each subset of labeled probes comprising a plurality of labeled probes, each labeled probe of the subset of labeled probes comprising a label and binding site for one of the subsets of amplifiers, the label in each third subset of labeled probes being distinguishable among the third subsets of labeled probes, the label being cleavable, and the third set of labeled probes being able to specifically label a third subset of target nucleic acid different from the first and second subsets of target nucleic acid.

[0170] In one embodiment, the kit of the present invention, which includes a severable label, includes a cutting agent for cutting the severable label from the label probe.

[0171] In one embodiment, the present invention relates to the in of target nucleic acids. The kit provides for situ detection, and the kit comprises: (A) a set of pre-amplifiers, each set of pre-amplifiers comprising a plurality of pre-amplifiers, each pre-amplifier being specific to each of one or more sets of target probes, each pre-amplifier comprising a binding site for one pair of target probes from the set of target probes and a plurality of binding sites for the amplifier; (B) a set of amplifiers, each set of amplifiers comprising a plurality of subsets of amplifiers specific to each pre-amplifier, each subset of amplifiers comprising a plurality of amplifiers, each amplifier comprising a binding site for one of the pre-amplifiers specific to the set of target probes and a plurality of binding sites for the labeled probe; and (C) a first set of labeled probes, each first set of labeled probes comprising a plurality of first subsets of labeled probes, each subset of labeled probes being specific to one of the amplifiers from the subset of amplifiers, each subset of labeled probes comprising a plurality of target probes, and each labeled probe in each subset of labeled probes being specific to the amplifier (D) A first set of labeled probes comprising a label and binding site for one of the amplification agents from a subset, wherein the label in each first subset of the labeled probes is distinguishable among the first subsets of the labeled probes, the melting temperature between the labeled probe and the amplification agent is lower than the melting temperature between the target probe, pre-amplifier and amplification agent, and the first set of labeled probes is capable of specifically labeling a first subset of the target nucleic acid, and (D) a second set of labeled probes comprising a plurality of second subsets of labeled probes, each subset of the labeled probes being specific to one of the amplification agents from the subset of amplification agents, each subset of the labeled probes being specific to an amplification agent of a subset of amplification agents different from the first subset of the labeled probes, each subset of the labeled probes comprising a plurality of labeled probes, each labeled probe of the subset of labeled probes comprising a label and binding site for one of the amplification agents from the subset of amplification agents, and the label in each second subset of the labeled probes being distinguishable among the second subsets of the labeled probes,(E) an acid reagent, wherein the melting temperature between the labeled probe and the amplifier is lower than the melting temperature between the target probe, pre-amplifier, and amplifier, and the second set of labeled probes can specifically label a second subset of the target nucleic acid that is different from the first subset of the target nucleic acid; and (E) an acid reagent, wherein the acid reagent causes disruption of hybridization between the target probe and the respective target nucleic acid (see, for example, Figures 2B and 6A).

[0172] In one embodiment, the kit further comprises a third set of labeled probes, the third set of labeled probes comprising a plurality of third subsets of labeled probes, each subset of labeled probes being specific to one of the subsets of amplifiers, the third subset of labeled probes being specific to an amplifier of a subset of amplifiers different from the first and second subsets of labeled probes, each subset of labeled probes comprising a plurality of labeled probes, each labeled probe of the subset of labeled probes comprising a labeling and binding site for one of the subsets of amplifiers, the labeling in each third subset of labeled probes being distinguishable among the third subsets of labeled probes, the melting temperature between the labeled probe and the amplifier being lower than the melting temperature between the target probe, pre-amplifier, and amplifier, and the third set of labeled probes being able to specifically label a third subset of the target nucleic acid different from the first and second subsets of the target nucleic acid.

[0173] In one embodiment, the present invention provides a kit for in situ detection of a target nucleic acid, the kit comprising: (A) a set of pre-amplifiers, the set of pre-amplifiers comprising a plurality of pairs of pre-amplifiers, the set of pre-amplifiers comprising a pair of pre-amplifiers specific to each of the pairs of target probes of one or more sets of target probes, each pre-amplifier in the pair of pre-amplifiers comprising a binding site for one of the target probes of the pairs of target probes of the set of target probes, the set of pre-amplifiers comprising a plurality of binding sites for the pre-amplifiers; and (B) a set of pre-amplifiers, the pre-amplifiers comprising a plurality of binding sites for the pre-amplifiers. (C) A set of preamplifiers comprising a set of preamplifiers, wherein the set of preamplifiers comprises a preamplifier specific to each pair of preamplifiers, and each preamplifier comprises a binding site for one of the preamplifier pairs in the set of preamplifiers and a multiple binding site for the amplifier, and (C) a set of amplifiers comprising a set of amplifiers comprising a set of amplifiers comprising a subset of amplifiers specific to each preamplifier specific to each pair of preamplifiers, and the amplifiers of the subset of amplifiers comprising a binding site for one of the preamplifiers specific to each pair of preamplifiers and a multiple binding site for the labeled probe (D) A first set of labeled probes, comprising a set of amplifiers including a binding site, and (E) a second set of labeled probes, wherein the second set of labeled probes comprises a plurality of first subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of amplifiers, each subset of labeled probes comprises a plurality of target probes, each of the subsets of labeled probes includes a label and binding site for one of the subsets of amplifiers, the label in each first subset of labeled probes is distinguishable among the first subsets of labeled probes, the melting temperature between the labeled probe and the amplifier is lower than the melting temperature between the target probe, pre-pre-amplifier, pre-amplifier, and amplifier, and the first set of labeled probes can specifically label a first subset of the target nucleic acid, and (E) a second set of labeled probes, comprising a plurality of second subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of amplifiers, and the second subset of labeled probes is(F) an acid reagent, the second set of labeling probes, which is specific to a subset of amplifiers different from a first subset of labeling probes, each subset of labeling probes comprises multiple labeling probes, each labeling probe in the subset of labeling probes comprises a labeling and binding site for one of the amplifiers in the subset of amplifiers, the labeling in each second subset of labeling probes is distinguishable between the second subsets of labeling probes, the labeling is cleavable, and the second set of labeling probes can specifically label a second subset of target nucleic acids different from a first subset of target nucleic acids; and (F) an acid reagent, the acid reagent causing disruption of hybridization between the target probes and the respective target nucleic acids (see, for example, Figures 2B and 6C).

[0174] In one embodiment, the kit further comprises a third set of labeled probes, the third set of labeled probes comprising a plurality of third subsets of labeled probes, each subset of labeled probes being specific to one of the subsets of amplifiers, the third subset of labeled probes being specific to an amplifier of a subset of amplifiers different from the first and second subsets of labeled probes, each subset of labeled probes comprising a plurality of labeled probes, each labeled probe of the subset of labeled probes comprising a labeling and binding site for one of the subsets of amplifiers, the labeling in each third subset of labeled probes being distinguishable among the third subsets of labeled probes, the melting temperature between the labeled probe and the amplifier being lower than the melting temperature between the target probe, the pre-pre-amplifier, the pre-amplifier, and the amplifier, and the third set of labeled probes being able to specifically label a third subset of the target nucleic acid different from the first and second subsets of the target nucleic acid.

[0175] In one embodiment, the present invention provides a kit for in situ detection of a target nucleic acid, the kit comprising: (A) a set of pre-preamplifiers, the set of pre-preamplifiers comprising a plurality of pre-preamplifiers, the plurality of pre-preamplifiers comprising pre-preamplifiers specific to each of one or more sets of target probes, each pre-preamplifier comprising a binding site for one pair of target probes from the set of target probes and a plurality of binding sites for the preamplifier; and (B) a set of preamplifiers, the set of preamplifiers comprising a plurality of subsets of preamplifiers specific to each pre-preamplifier, (C) A set of preamplifiers, each subset of the agent comprises a plurality of preamplifiers, and the preamplifiers of the subset of preamplifiers comprises a binding site for one of the preamplifiers specific to the target probe set and a plurality of binding sites for the amplifier, and (C) a set of amplifiers, each set of amplifiers comprises a plurality of subsets of amplifiers specific to each subset of preamplifiers, each subset of amplifiers comprises a plurality of amplifiers, and the amplifiers of the subset of amplifiers comprises a binding site for one of the preamplifiers in the subset of preamplifiers and a plurality of binding sites for the labeled probe. (D) A first set of labeled probes, wherein the first set of labeled probes comprises a plurality of first subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of labeled probes, each subset of labeled probes comprises a plurality of target probes, each labeled probe in each subset of labeled probes comprises a label and binding site for one of the subsets of labeled probes, the label in each first subset of labeled probes is distinguishable among the first subsets of labeled probes, the melting temperature between the labeled probe and the labeled probe is lower than the melting temperature between the target probe, pre-pre-amplifier, pre-amplifier, and amplifier, and the first set of labeled probes can specifically label a first subset of the target nucleic acid, and (E) a second set of labeled probes, wherein the second set of labeled probes comprises a plurality of second subsets of labeled probes, each subset of labeled probes is specific to one of the subsets of labeled probes, and the second subset of labeled probes isThe second set of labeled probes is specific to a subset of amplifiers different from the first subset of labeled probes, each subset of labeled probes comprises multiple labeled probes, each labeled probe in the subset of labeled probes comprises a labeling and binding site for one of the amplifiers in the subset of labeled probes, the labeling in each second subset of labeled probes is distinguishable between the second subsets of labeled probes, the melting temperature between the labeled probes and the amplifiers is lower than the melting temperature between the target probe, pre-pre-amplifier, pre-amplifier and amplifier, and the second set of labeled probes can specifically label a second subset of target nucleic acids different from the first subset of target nucleic acids; and (F) an acid reagent, the acid reagent causing disruption of hybridization between the target probes and their respective target nucleic acids (see, for example, Figures 2B and 6B).

[0176] In one embodiment, the kit further comprises a third set of labeled probes, the third set of labeled probes comprising a plurality of third subsets of labeled probes, each subset of labeled probes being specific to one of the subsets of amplifiers, the third subset of labeled probes being specific to an amplifier of a subset of amplifiers different from the first and second subsets of labeled probes, each subset of labeled probes comprising a plurality of labeled probes, each labeled probe of the subset of labeled probes comprising a labeling and binding site for one of the subsets of amplifiers, the labeling in each third subset of labeled probes being distinguishable among the third subsets of labeled probes, the melting temperature between the labeled probe and the amplifier being lower than the melting temperature between the target probe, the pre-pre-amplifier, the pre-amplifier, and the amplifier, and the third set of labeled probes being able to specifically label a third subset of the target nucleic acid different from the first and second subsets of the target nucleic acid.

[0177] In some embodiments of the kit of the present invention, which includes a set of target probes, the kit includes one or more sets of target probes, each set of target probes includes a pair of target probes that specifically hybridize to a target nucleic acid. In one embodiment, each set of target probes includes two or more pairs of target probes that can specifically hybridize to the same target nucleic acid.

[0178] In some embodiments of the kit of the present invention, the kit includes at least one reagent for fixing and / or permeabilizing cells.

[0179] In some embodiments of the kit of the present invention, the acid reagent comprises 5-40% or 20-30% of an acid, or other concentrations disclosed herein. In one embodiment, the acid is selected from the group consisting of acetic acid, formic acid, propionic acid, butyric acid, valeric acid, oxalic acid, malonic acid, succinic acid, malic acid, tartaric acid, and citric acid.

[0180] In some embodiments of the kit of the present invention, the acid reagent comprises a salt. In one embodiment, the acid reagent comprises SSC. In one embodiment, the acid reagent comprises 1 to 13 times SSC or 3.2 to 12.8 times SSC.

[0181] The present invention also provides a sample comprising cells or a plurality of cells, wherein an acid reagent is applied to the cells and present on the cells. The cells may optionally be fixed. The cells may optionally be permeabilized. Cell fixation and / or permeabilization are particularly applicable to in situ hybridization assays. Optionally, the cells may contain one or more target nucleic acids bound to any of the probe configurations disclosed herein.

[0182] The present invention further provides a slide containing cells or a plurality of cells, wherein an acid reagent is applied to the cells on the slide and present on the cells on the slide. Optionally, the cells are fixed to the slide. Optionally, the cells are permeabilized. In certain embodiments, the cells on the slide are fixed and / or permeabilized for an in situ assay. Optionally, the cells on the slide may contain one or more target nucleic acids bound to any of the probe configurations disclosed herein.

[0183] It should be understood that modifications within the definition of the present invention as shown herein may be made that do not substantially affect the activity of the various embodiments of the invention. Therefore, the following examples are illustrative and are not intended to limit the invention.

[0184] Example I Acid treatment effectively removes probes hybridized to target nucleic acids, minimizing impact on cellular RNA and tissue morphology. This example describes the effective removal of probes hybridized to target nucleic acids within cells, while preserving cellular RNA and tissue morphology.

[0185] Figures 3A and 3B show acid treatment for sequential rounds of target nucleic acid detection. Figure 3A shows that acid treatment effectively removed the target probes and amplification complexes in fresh frozen mouse brain. Detection of four highly expressed positive control genes—glyceraldehyde-3-phosphate dehydrogenase (Gapdh), phosphoglycerate kinase 1 (Pgk1), basic helix-loop helix family member E22 (Bhlhe22), and complexin 2 (Cplx2)—is shown in mouse brain prepared as fresh frozen sections. Target probes (ZZ probes) for the four genes were hybridized together, and the signals were amplified together using the RNAscope® HiPlex amplification system. The four genes were detected in the first round of repeated detection using fluorescently labeled probes corresponding to the signal amplification systems assigned to these four target probes. Alexa 488, ATTO 550, ATTO 647N, and Alexa 750 fluorophores were used to detect Gapdh, Pgk1, Bhlhe22, and Cplx2, respectively. Nuclei were stained blue with DAPI (4′,6-diamidino-2-phenylindoline) (upper panel). After signal detection, tissue sections were treated with an acidic solution (20% acetic acid, 6.4x SSC) for 5 minutes at room temperature (RT), and the acid treatment was repeated two more times. The sections were then used for a second round of hybridization and amplification without the addition of target probes. In the second round after acid treatment, little to no signal was detected (lower panel), thus indicating complete removal of the previously hybridized target probes and signal amplification components.

[0186] Figure 3B shows that acid treatment had minimal effect on cellular RNA and tissue morphology in fresh frozen mouse brains. As shown in Figure 3A (top panel), four positive control genes (Gapdh, Pgk1, Bhlhe22, and Cplx2) were detected in mouse brains prepared as fresh frozen sections. After signal detection, sections were treated with acid solution as shown in Figure 3A, except that the acid treatment was repeated four times instead of twice. The treated sections were then used for a second round of hybridization and amplification to detect the same four genes. Comparing the signals detected in the second round of hybridization (bottom panel) with those detected in the first round of hybridization (top panel), the second round of hybridization and signal amplification of the target probe yielded similar expression patterns, indicating minimal RNA loss from acid treatment.

[0187] These results indicate that treatment of cells with acid reagents is effective in removing probes hybridized to target nucleic acids within cells while preserving cellular RNA and tissue morphology.

[0188] Example II Multiple rounds of acid treatment and sequential rounds of probe hybridization preserve cell morphology and detectable nucleic acids. This example demonstrates that multiple rounds of acid treatment can be applied to cells to remove probes bound to the target nucleic acid, and that the target nucleic acid can be detected after acid treatment.

[0189] The experiment was carried out essentially as shown in Figure 2B. As shown in Figure 2B, two "rounds" are shown, "K" and "L," where K refers to a sequential round of N-target probe hybridization, and L refers to an iterative round of labeled probe hybridization within each K round. In this experiment, three sequential rounds of target probe hybridization were performed, and within each sequential round, three iterative rounds of labeled probe hybridization and imaging were performed. For demonstration purposes in this experiment, 12 target probes were used in each "K" round: for round K=1, RNA polymerase II subunit A (Polr2A), peptidyl prolyl isomerase B (Ppib), ubiquitin C (Ubc), hypoxanthine phosphoribosyltransferase 1 (Hprt1), actin beta (ActB), tubulin beta 3 class III (Tubb3), crosslinking integrator 1 (Bin1), lactate dehydrogenase A (Ldha), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), phosphoglycerate kinase 1 (Pgk1), and basic helix loop helix The following are members of the sulcus family: E22 (Bhlhe22), and complexin 2 (Cplx2); round K=3, 5-hydroxytryptamine receptor 7 (Htr7), protocadherin 8 (Pcdh8), solute carrier family 32 member 1 (Slc32a1), tyrosine hydroxylase (Th), synaptoporin (Synpr), crystallin mu (Crym), wolframin ER transmembrane glycoprotein (Wfs1), calbindin 1 (Calb1), (Drd1a), dopamine receptor D1 (Drd2), cannabinoid receptor 1 (Cnr1), and forkhead box P1 (Foxp1).

[0190] Each of the 12 target probe groups was hybridized in sequential rounds 1 and 3, respectively, and 12 "dummy" target probes or blank probe buffers were used in the second sequential round. An acid treatment step was performed between the sequential rounds (K rounds) of target probe hybridization. In Figure 4, the upper panel of the image corresponds to the third iterative detection round (L=3) within the first sequential round (K=1). In Figure 4, the image in the lower panel was from the iterative detection (L=1) of the first round within the third sequential round (K=3) of target probe hybridization.

[0191] Figure 4 shows good morphology and signal detection after two rounds of acid treatment and sequential hybridization. In Figure 4, the top panel shows the detection of four positive control genes, Polr2A, Ppib, Ubc, and Hprt1, in fresh frozen mouse brain sections after the first round of target probe hybridization (K=1) and the third round of repeated detection (L=3), substantially performed as outlined in the workflow in Figure 2B. Alexa 488, ATTO 550, ATTO 647N, and Alexa 750 fluorophores were used to detect Polr2A, Ppib, Ubc, and Hprt1, respectively, and the nuclei were stained with blue DAPI. In Figure 4, the lower panel shows the detection of four different low-expression targets, Htr7, Pcdh8, Slc32a, and Th, in the striatal region of the mouse brain during the third round of target probe hybridization and amplification (K=3) and the first round of repeated detection (L=1). The acid treatment, target hybridization, and amplification steps were performed after target hybridization in rounds 1 and 2, as shown in Figure 3A.

[0192] These results demonstrate that multiple rounds of acid treatment can be applied to cells to remove probes bound to target nucleic acids. The results also show that the target nucleic acids can be detected after acid treatment.

[0193] Example III Exemplary acid treatment reagents and conditions This embodiment describes experiments to test various acid treatment reagents and conditions.

[0194] In a series of experiments to explore optimized acid treatment conditions, fresh frozen mouse brains or formalin-fixed paraffin-embedded HeLa cells were stained for various positive control genes using the RNAscope® HiPlex protocol (Advanced Cell Diagnostics, Newark CA). After imaging, slides were treated with acetic acid under various conditions to remove bound target probes and signal-generating complexes. Following acid treatment, the RNAscope® HiPlex signal amplification step was performed without adding any target probes to detect any signals from residually bound target probes from previous rounds. Slides were imaged and visually evaluated. The results are shown in Table 1. [Table 1]

[0195] These results demonstrate that various acid reagents can be used to remove probes bound to target nucleic acids within cells.

[0196] Throughout this application, various publications are referenced. The entirety of the disclosures of these publications is incorporated herein by reference for the purpose of further providing a comprehensive description of the current state of the art to which the present invention pertains. While the present invention has been described with reference to the above embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. [Brief explanation of the drawing]

[0197] [Figure 1]A schematic diagram of the workflow for a multiplex assay (RNAscope® HiPlex assay workflow) is shown. N target sequences are hybridized to target probes (shown as double Z probes), and the signals are simultaneously amplified by an amplification system such as RNAscope®. In the shown embodiment, the first four targets are detected via four non-spectral overlapping fluorescent dye-conjugated oligonucleotides (labeled probes) and imaged using a conventional fluorescence microscope or scanner. The fluorophores are then cleaved from the labeled probes, and the next four targets are labeled and imaged using the same method. After detection of L rounds of each of the four targets, the images are registered using an image registration software algorithm to create a final composite image at single-cell resolution. [Figure 2A]A schematic diagram of sequential hybridization of nucleic acid probes using an acid reagent to remove target probes is shown. A schematic diagram of acid treatment and removal of target(s) bound to target(s) nucleic acids(s) for sequential hybridization is shown. The N target probe is hybridized to the target nucleic acid, for example, in an in situ hybridization assay. The diagram shows arbitrary signal amplification of the target probe hybridized to the target nucleic acid. Cells may be counterstained to facilitate cell visualization; for example, the nucleus may be stained with 4',6-diamidino-2-phenylindole (DAPI). The target probe and counterstained cells are visualized, for example, by imaging, thereby detecting and imaging the target nucleic acid. When using an RNAscope® assay, the RNAscope® double Z probe and signal are simultaneously amplified by the RNAscope® amplification system. An acid treatment step is performed to remove the target(s) bound to each(s) target(s). One or more additional sets of N target nucleic acids can be detected by repeating the entire Nplex workflow for one or more rounds. After all targets have been detected, the images are registered using image registration software algorithms to create a final composite image superimposed at single-cell resolution. The total level of multiplexing available in this method is K rounds of N targets (multiple) × N plexing per round. Generally, N is greater than or equal to 1, and K is greater than or equal to 2 if an acid removal step is included. In the diagram shown in Figure 2A, if K=1, only one round of target probe hybridization and imaging is required, and therefore no acid removal step is necessary. [Figure 2B]A schematic diagram of sequential hybridization of nucleic acid probes using an acid reagent to remove the target probe is shown. A schematic diagram of acid treatment and removal of the probe bound to the target nucleic acid for sequential hybridization is shown. The N target probe is hybridized to the target nucleic acid using an in situ hybridization assay, such as the RNAscope® HiPlex assay. For example, the N target probe is hybridized to the target sequence using the RNAscope® double Z probe, and the signal is simultaneously amplified, for example, by the RNAscope® amplification system. The diagram shows arbitrary signal amplification of the target probe hybridized to the target nucleic acid. Cells may be counterstained to facilitate cell visualization; for example, the nucleus may be stained with 4′,6-diamidino-2-phenylindole (DAPI). The N target nucleic acid is detected by labeling, e.g., fluorescent labeling, imaging, and cleavage of the detectable label, e.g., repeated rounds of cleavage of the fluorescent label. In the diagram, N-target nucleic acids are hybridized with N-target probes and repeatedly detected to label and detect a subset of N-targets (N-target subset 1), cleave the label from the labeled nucleic acid subset (L-round labeled probe hybridization, L=1), then label and detect a second subset of N-targets (N-target subset 2), cleave the label from the labeled nucleic acid subset (L-round labeled probe hybridization, L=2), and so on, until all N-target nucleic acids are detected. After all N-target nucleic acids have been detected for the desired number of labels (L=desired label rounds), an acid treatment step is performed to remove the hybridized N-target probes (e.g., ZZ probe signal-generating complexes). One or more additional sets of N-target nucleic acids (e.g., N'-target nucleic acids, N''-target nucleic acids, etc.) can be detected by repeating the entire Nplex workflow for one or more rounds. After all targets have been detected, the images are registered using image registration software algorithms to create a final composite image superimposed at single-cell resolution.The total number of multiplex levels available in this method is K rounds of N-target nucleic acids × N-plexing per round (where "N-plexing" refers to the flow from "hybridization of the N-target probe" to "acid removal of the probe and amplifier" or, in the final round, to the final "counterstaining and imaging" step). Generally, N=1 or greater, and K=2 or greater if the acid removal step is included. In the diagram shown in Figure 2B, if K=1, only L rounds of labeled probe hybridization, imaging, and fluorophore cleavage are required, and therefore the acid removal step of the probe and amplifier is not necessary. [Figure 3A]This section describes acid treatment for a sequential round of target nucleic acid detection. Acid treatment effectively removed target probes and amplification complexes in fresh, frozen mouse brain. The section also shows the detection of four highly expressed positive control genes—glyceraldehyde-3-phosphate dehydrogenase (Gapdh), phosphoglycerate kinase 1 (Pgk1), basic helix-loop helix family member E22 (Bhlhe22), and complexin 2 (Cplx2)—in mouse brain prepared as fresh, frozen sections. Target probes (ZZ probes) for the four genes were hybridized together, and the signals were amplified together using the RNAscope® HiPlex amplification system. The four genes were detected in the first round of repeated detection using fluorescently labeled probes corresponding to the signal amplification systems assigned to these four target probes. Alexa 488, ATTO 550, ATTO 647N, and Alexa 750 fluorophores were used to detect Gapdh, Pgk1, Bhlhe22, and Cplx2, respectively, and the nuclei were stained blue with DAPI (4′,6-diamidino-2-phenylindoline) (upper panel). After signal detection, tissue sections were treated with an acidic solution (20% acetic acid, 6.4x SSC) for 5 minutes at room temperature (RT), and the acid treatment was repeated two more times. The sections were then used for a second round of hybridization and amplification without the addition of target probes. In the second round after acid treatment, little to no signal was detected (lower panel), thus indicating complete removal of the previously hybridized target probes and signal amplification components. [Figure 3B]This shows acid treatment for sequential rounds of target nucleic acid detection. It demonstrates that acid treatment had minimal impact on cellular RNA and tissue morphology in fresh, frozen mouse brains. Four positive control genes (Gapdh, Pgk1, Bhlhe22, and Cplx2) were detected in mouse brains prepared as fresh, frozen sections, as shown in Figure 3A (top panel). Sections were treated with acid solution as shown in Figure 3A, except that the acid treatment was repeated four times instead of twice after signal detection. The treated sections were then used for a second round of hybridization and amplification to detect the same four genes. Comparing the signals detected in the second round of hybridization (bottom panel) with those detected in the first round of hybridization (top panel), the two rounds of target probe hybridization and signal amplification yielded similar expression patterns, indicating minimal RNA loss from repeated acid treatment. [Figure 4]The images show good morphology and signal detection after two rounds of acid treatment and sequential hybridization. In Figure 4, the top panel shows the detection of four positive control genes, RNA polymerase II subunit A (Polr2A), peptidyl prolyl isomerase B (Ppib), ubiquitin C (Ubc), and hypoxanthine phosphoribosyltransferase 1 (Hprt1), in fresh frozen mouse brain sections after the first round of target probe hybridization (k=1) and the third round of repeated detection (l=3), substantially performed as outlined in the workflow in Figure 2B. Twelve target probes (RNAscope® HiPlex 12 plex mouse positive controls) were simultaneously hybridized and amplified using the RNAscope® Hiplex assay. Four genes were detected in the first round using Alexa 488, ATTO 550, ATTO 647N, and Alexa 750 fluorophores, and the fluorophores were cleaved after imaging. The next four genes were detected in the second round of detection using the same four fluorophores, and the fluorophores were cleaved after imaging. The third round of repeated detection is shown in the upper panel of Figure 4. Alexa 488, ATTO 550, ATTO 647N, and Alexa 750 fluorophores were used to detect Polr2a, Ppib, Ubc, and Hprt1, respectively, and the nuclei were stained with blue DAPI. In Figure 4, the lower panel shows the detection of four distinct low-expression targets—5-hydroxytryptamine receptor 7 (Htr7), protocadherin 8 (Pcdh8), solute carrier family 32 member 1 (Slc32a1), and tyrosine hydroxylase (Th)—in the third round of target probe hybridization and amplification (k=3) and the first round of repeated detection (l=1) in the striatal region of the mouse brain. The acid treatment, target hybridization, and amplification steps were performed after target hybridization in rounds 1 and 2, as shown in Figure 3A. The second round of target probe hybridization and amplification was performed after acid treatment.The target probe was omitted; instead, a probe diluent was used. A third round of target probe hybridization and amplification was performed after the second acid treatment using 12 different target probes. Four of the 12 target probes were initially detected in the first round of detection, as shown in the lower panel. [Figure 5] Figures A-C show schematic diagrams of the aforementioned detection method for nucleic acid targets using signal-generating complexes (SGCs). PPA: pre-pre-amplifier; PA: pre-amplifier; AMP: amplifier; LP: labeled probe. [Figure 6A] A schematic diagram of orthogonal labeling of target nucleic acids is shown. Orthogonal labeling of target nucleic acids based on the RNAscope® assay is shown in Figure 6A. Three exemplary target nucleic acid labeling using each signal-generating complex (SGC) is shown in Figure 6A. The binding of target probe pair 1 (TP1a and TP1b) to target nucleic acid 1 is shown. It is shown that the pre-amplifier (PA1) is bound to the target probe pair (TP1a and TP1b). It is shown that multiple amplifiers (AMP1) are bound to PA1. Multiple labeled probes (LP1) bound to the amplifiers are shown. Similar configurations are shown for targets 2 and 3, each having SGC components (target probe, pre-amplifier, amplifier, labeled probe) specific to each target. [Figure 6B]A schematic diagram of orthogonal labeling of target nucleic acids is shown. A modification of the configuration shown in Figure 6A is shown. Two exemplary target nucleic acid labelings using each signal-generating complex (SGC) are shown in Figure 6B. The binding of target probe pair 1 (TP1a and TP1b) to target nucleic acid 1 is shown. It is shown that the pre-pre-amplifier (PPA1) is bound to the target probe pair (TP1a and TP1b). It is shown that multiple pre-amplifiers (PA1) are bound to PPA1. It is shown that multiple amplifiers (AMP1) are bound to PA1. For simplicity, the amplifier is shown as being bound to one pre-amplifier, but it should be understood that the amplifier can be bound to all amplifiers. Multiple labeled probes (LP1) bound to the amplifier are shown. A similar configuration for target 2, which has SGC components (target probe, pre-pre-amplifier, pre-amplifier, amplifier, labeled probe) specific to each target, is shown. [Figure 6C] A schematic diagram of orthogonal labeling of target nucleic acids is shown. Orthogonal labeling of target nucleic acids based on the Basescope® assay is shown in Figure 6C. Two exemplary target nucleic acid labelings using each signal-generating complex (SGC) are shown in Figure 6C. The binding of target probe pair 1 (TP1a and TP1b) to target nucleic acid 1 is shown. It is shown that a pair of pre-pre-amplifiers (PPA1a and PPA1b) are bound to each target probe pair (TP1a and TP1b). It is shown that a pre-amplifier (PA1) is bound to the pre-amplifier pair (PPA1a and PPA1b). It is shown that multiple amplifiers (AMP1) are bound to PA1. For simplicity, it is shown that the amplifier is bound to one pre-amplifier, but it should be understood that the amplifier can be bound to all amplifiers. Multiple labeled probes (LP1) bound to the amplifiers are shown. A similar configuration for target 2 is shown, having SGC components (target probe, pre-pre-amplifier, pre-amplifier, amplifier, labeled probe) specific to each of the targets.

Claims

1. A method for disrupting the binding of a probe bound to a nucleic acid within a cell, comprising contacting the cell with an acid reagent, wherein the cell contains one or more first probes hybridized to one or more first target nucleic acids within the cell, and the acid reagent disrupts the hybridization between the one or more first probes and the one or more first target nucleic acids. The method wherein the acid reagent comprises 20 to 30 volume / vol% acetic acid and 3.2 to 12.8 times the amount of physiological saline sodium citrate (SSC).

2. The method according to claim 1, wherein contact of the cells with the acid reagent is repeated one or more times.

3. The method according to claim 1 or 2, further comprising removing the one or more first probes from the cells.

4. The method according to claim 1 or 2, wherein the cells include two or more first probes hybridized to two or more first target nucleic acids.

5. The method according to claim 4, wherein each of the first target nucleic acids is labeled by hybridization to the first probe, and the label on each first target nucleic acid is distinguishable from the label on other first target nucleic acids hybridized to the first probe.

6. The method according to any one of claims 1 to 5, further comprising the step of contacting the cells with one or more second probes, wherein the one or more second probes hybridize to one or more second target nucleic acids in the cells, and the one or more second target nucleic acids are identical to or different from the one or more first target nucleic acids.

7. The method according to claim 6, wherein the cells include two or more second probes hybridized to two or more second target nucleic acids.

8. The method according to claim 7, wherein each of the second target nucleic acids is labeled by hybridization to the second probe, and the label on each second target nucleic acid is distinguishable from the label on other second target nucleic acids hybridized to the second probe.

9. The method according to any one of claims 6 to 8, further comprising the step of contacting the cells with the acid reagent, wherein the acid reagent disrupts hybridization between the second probe and the one or more second target nucleic acids.

10. The method according to claim 9, wherein contact between the cells and the acid reagent is repeated one or more times.

11. The method according to claim 9 or 10, further comprising the step of removing the second probe from the cells.

12. The method according to any one of claims 1 to 11, wherein the target nucleic acid is independently DNA or RNA.

13. The method according to claim 12, wherein the target nucleic acid, which is RNA, is independently selected from the group consisting of messenger RNA (mRNA), microRNA (miRNA), ribosomal RNA (rRNA), mitochondrial RNA, and non-coding RNA.

14. The sample is (a) a tissue specimen or derived from a tissue specimen; (b) a blood sample or derived from a blood sample; or (c) The method according to any one of claims 12 to 13, wherein the material is a cytological specimen or is derived from a cytological specimen.

15. Contains acid reagents, It further comprises one or more probes specific to one or more nucleic acid targets, The acid reagent comprises 20 to 30 volume / vol% acetic acid and 3.2 to 12.8 times the amount of physiological saline sodium citrate (SSC). A kit for use in a method for disrupting the binding of probes bound to nucleic acids within cells.

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