Probe sets, kits, and methods for high-throughput in situ detection of nucleic acid molecules in biological samples

Abstract

The application discloses a high-throughput in-situ detection probe set, kit and method for nucleic acid molecules in a biological sample, and belongs to the technical field of molecular biological imaging. The biological sample to be detected is contacted with a first split probe pair, a second dual-arm probe and a hairpin probe with different detectable markers. The first split probe pair anchors target nucleic acid molecules, the second dual-arm probe is connected to the split probe, and the priming sequence in the second dual-arm probe initiates a hybridization chain reaction with the hairpin probe pair to amplify polymerization. The analysis of the target nucleic acid molecules is realized by detecting the detectable markers. Since two different priming sequences are simultaneously mounted on the second dual-arm probe, the "logical superposition" of the physical channel is realized, the high-throughput target detection in the limited fluorescence channel is realized, and the specificity and signal-to-noise ratio are enhanced.

Description

Technical Field

[0001] This application belongs to the field of molecular biological imaging technology, specifically relating to probe sets, kits, and methods for high-throughput in-situ detection of nucleic acid molecules in biological samples. Background Technology

[0002] In situ detection of nucleic acid molecules (such as mRNA) is an important technique for studying the spatiotemporal distribution of gene expression, cellular heterogeneity, and tissue function. Currently, commonly used in situ detection methods for nucleic acid molecules in biological samples mainly include in situ sequencing (ISS), in situ hybridization (ISH), and various signal amplification techniques derived therefrom.

[0003] Taking mRNA as an example, to improve detection sensitivity, hybridization chain reaction (HCR), as a nucleic acid signal amplification method that does not require enzyme participation, is widely used in the field of in-situ detection of nucleic acid molecules due to its advantages such as mild reaction conditions and low background signal. Third-generation HCR utilizes a "split initiator" design, requiring paired probes (usually two from a set of probes) to bind together to adjacent mRNA regions to form a complete initiation sequence; this design achieves automatic background suppression. Even if the probes bind non-specifically, amplification will not be triggered as long as they do not bind in pairs, thus significantly reducing background noise and significantly improving the signal-to-noise ratio. However, each target molecule typically corresponds to one physical fluorescence channel. To achieve multiplex detection in a single round of imaging, n orthogonal initiation sequences and n different colored fluorophores are needed; to increase throughput, extremely complex "cyclic HCR" or "sequential imaging" is required, making it difficult to achieve high-throughput detection targets within a limited number of fluorescence channels.

[0004] In summary, how to achieve in-situ detection methods for nucleic acid molecules with high signal-to-noise ratio, high throughput, and high coding freedom while ensuring in-situ detection resolution remains a pressing technical problem to be solved in this field. Summary of the Invention

[0005] In view of this, the primary objective of this application is to provide a probe set for high-throughput in-situ detection of nucleic acid molecules in biological samples. By optimizing the probe system and combining primary splitting probe pairs and secondary double-arm probes, two different initiation sequences are simultaneously mounted on the secondary double-arm probes, achieving "logical superposition" of physical channels. This not only enables high-throughput target detection within a limited fluorescence channel but also enhances specificity and signal-to-noise ratio.

[0006] To achieve the above objectives, this application adopts the following technical solution:

[0007] One aspect of this application discloses a probe set for high-throughput in-situ detection of nucleic acid molecules in biological samples, including:

[0008] A first-level splitting probe pair includes a first recognition probe and a second recognition probe, wherein the first recognition probe has a first target sequence, a first interval sequence and a first random sequence in sequence, and the second recognition probe has a second random sequence, a second interval sequence and a second target sequence in sequence.

[0009] A two-armed probe, which sequentially includes a first initiation sequence, a binding sequence, and a second initiation sequence;

[0010] And a first hairpin probe pair modified with a first detectable marker and a second hairpin probe pair modified with a second detectable marker, wherein the first detectable marker and the second detectable marker are different;

[0011] Wherein, the first target sequence and the second target sequence are specifically complementary to two sequences on the target nucleic acid molecule, the first random sequence and the second random sequence are specifically complementary to two sequences on the binding sequence, the first initiation sequence can initiate the amplification and polymerization of the first hairpin probe pair, and the second initiation sequence can initiate the amplification and polymerization of the second hairpin probe pair.

[0012] Another aspect of this application discloses a kit containing the probe set described in this application.

[0013] Another aspect of this application discloses a method for high-throughput in-situ detection of nucleic acid molecules in biological samples, comprising the following steps:

[0014] Provide the probe set described in this application;

[0015] The biological sample to be tested is brought into contact with the first-level splitting probe pair and the second-level double-arm probe in sequence, and finally a first hairpin probe pair modified with a first detectable marker and a second hairpin probe pair modified with a second detectable marker are added.

[0016] By detecting the first and second detectable markers, imaging analysis of the target nucleic acid molecules is performed, enabling in-situ detection of nucleic acid molecules within biological samples.

[0017] The beneficial effects of this application are:

[0018] This application introduces a secondary two-armed probe as the core component for relay recognition and combinatorial encoding in the probe set. The resulting probe set can achieve high-throughput in-situ detection of nucleic acid molecules in biological samples, and it has the following significant advantages:

[0019] (1) Extremely high coding freedom and detection throughput: A two-stage dual-arm probe design is adopted. By attaching hairpin-induced sequences with different detectable biomarkers to the left and right arms of a single two-stage probe, logical superposition of signals is achieved, thereby breaking through the limitation of the number of targets in optical detection channels. Specifically, theoretically, with n fluorescence channels, only a single round of hybridization imaging is needed to distinguish n + n(n-1) / 2 types of targets, thus significantly improving the single-round detection capability and detection throughput.

[0020] (2) Significantly improved signal-to-noise ratio and detection sensitivity: A dual-gating system based on "first-level splitting recognition - second-level double-arm relay" was constructed. The initiation sequence can only be exposed when the first-level splitting probe pair accurately binds to the target nucleic acid molecule and the second-level double-arm probe is stably bound to the docking site. This design greatly suppresses the spontaneous uncollapse of hairpin probes in complex biological tissues and the non-specific signal generated by the non-specific binding of a single probe, thereby significantly enhancing specificity and signal-to-noise ratio. Attached Figure Description

[0021] Figure 1 This is an experimental flowchart of the dual-arm-based HCR coding strategy in this application.

[0022] Figure 2 The images show fluorescence imaging results of different channels of the SST gene in a 10-micrometer frozen section of the coronal plane of a mouse brain, as described in the example. The scale bar in the small image is 5 μm, and the scale bar in the large image is 50 μm. Detailed Implementation

[0023] The embodiments of this application will be clearly and completely described below. The technical solutions in the embodiments described below are exemplary and only possible technical implementations of this application, not all possible implementations. Those skilled in the art can combine the embodiments of this application to obtain other embodiments without creative effort, and these embodiments are also within the protection scope of this application.

[0024] The first aspect of this application discloses a probe set for high-throughput in-situ detection of nucleic acid molecules in biological samples, aiming to provide a multi-level probe system that achieves high specificity, high sensitivity and high throughput in-situ detection of single or multiple target nucleic acid molecules through multi-level probe arithmetic and special design of each level of probe, combined with cascaded signal amplification.

[0025] In this application, the biological sample can be any sample suitable for imaging in the art, such as paraffin-embedded or frozen biological tissue sections, or cell samples such as cell smears and adherent cells. These biological samples undergo appropriate processing before formal imaging, such as fixation and clearing, to maintain cell morphology while allowing probe entry and facilitating subsequent imaging analysis. These processing methods are conventional techniques in the art and are therefore not particularly limited. In some specific examples, the biological sample is a biological tissue section.

[0026] There is no particular limitation on the thickness of biological samples; the thickness is the conventional thickness used in this field. For example, it is 8-20 μm, and can be any thickness or a range between any two of the following: 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, and 20 μm.

[0027] In this application, the nucleic acid molecule to be tested is a target nucleic acid molecule present or suspected to be present in a biological sample, specifically whether it is RNA or DNA. Subsequent probes are designed based on these target nucleic acid molecules. It should be understood that there can be one or more target nucleic acid molecules, which can be determined according to the experimental purpose and research needs, and therefore there is no particular limitation.

[0028] In this application, the probe set includes:

[0029] A primary splitting probe pair, comprising a first recognition probe and a second recognition probe;

[0030] Secondary dual-arm probe;

[0031] And a first hairpin probe pair modified with a first detectable marker and a second hairpin probe pair modified with a second detectable marker.

[0032] It should be understood that there is no particular limit to the number of probe sets; there can be one or multiple sets, depending on the experimental purpose and research needs, such as the number of genes being analyzed.

[0033] As described in this application, a "first-order split probe pair" refers to a probe combination that needs to bind to the same target nucleic acid molecule to function, including a first recognition probe and a second recognition probe. This "split" design (i.e., the two probes target two adjacent or neighboring sequences on the nucleic acid molecule respectively) significantly improves specificity and reduces non-specific binding to non-target sequences compared to a single long probe, making it particularly suitable for distinguishing highly homologous nucleic acid sequences. Only when both probes bind to the correct target site simultaneously can the correct template be provided for subsequent signal amplification steps.

[0034] In this application, the "identification probe" (including a first identification probe and a second identification probe) is the basic unit constituting a first-order split probe pair. Each identification probe contains three functional regions: a target sequence, a spacer sequence, and a random sequence. However, the functional regions of the first identification probe and the second identification probe are in different order. From the 5' end to the 3' end, the first identification probe has a first target sequence, a first spacer sequence, and a first random sequence in sequence, while the second identification probe has a second random sequence, a second spacer sequence, and a second target sequence in sequence.

[0035] As described in this application, the "target sequence" is located at the end of the recognition probe and is used to specifically recognize and bind to a predetermined region on the target nucleic acid molecule. The target sequence of the first recognition probe (first target sequence) and the target sequence of the second recognition probe (second target sequence) are specifically complementary to two selected sequences (usually adjacent or spaced-apart sequences) on the target nucleic acid molecule. Only when the target sequences of the first and second recognition probes bind complementary to the target nucleic acid molecule simultaneously can a linear docking platform that can be recognized by the secondary two-arm probes be provided. This split design effectively suppresses background noise caused by non-specific binding of a single probe. It is understood that the length and specific sequence of the target sequence can be designed and optimized according to the sequence of the target nucleic acid molecule. For example, in a preferred example, the length of the target sequence can be independently set to about 25 nucleotides (nt) to balance binding specificity and affinity.

[0036] As described in this application, the "spacer sequence" is located between the target sequence and the random sequence, and its base composition is not complementary to that of the target nucleic acid molecule. Its main functions include: (1) providing steric hindrance or flexible connection, which is beneficial for the random sequence at the end of the two recognition probes to be in a conformation more favorable to binding with the secondary double-arm probe after binding to the nucleic acid molecule; (2) adjusting the Tm value or spatial effect of the entire probe. The length of the spacer sequence can be very short, for example, in one embodiment, its length is independently about 2 nt. In this application, the spacer sequence is preferably AA or AT. Specifically, (1) only two hydrogen bonds are formed between A and T (compared to three hydrogen bonds of GC), and its binding energy is lower. Using AA or AT sequences can minimize non-specific hybridization caused by the spacer sequence inside or between probes. If a spacer sequence with high GC content is used, the probe may fold incorrectly or form stable byproducts with other probes, resulting in false positive signals. (2) In order for the two split initiator fragments to come together smoothly and trigger the opening of the downstream fluorescent hairpin, the probe needs a certain degree of spatial freedom. Regions with high concentrations of adenine (A) and thymine (T) are generally more flexible in the single-stranded state than GC-rich regions. (3) In in situ hybridization, experiments are usually conducted at specific temperatures and formamide concentrations. The melting temperatures (Tm) of AA or AT are extremely low, ensuring that the spacer sequence remains in an open single-stranded state at the hybridization temperature and does not obscure the initiation sequence due to the formation of local double strands.

[0037] As described in this application, the "random sequence" is located at the other end of the recognition probe. Its sequence is independent of the target nucleic acid molecule and is used to couple the first and second recognition probes to the secondary two-arm probes, respectively. Specifically, the random sequence of the first recognition probe (first random sequence) and the random sequence of the second recognition probe (second random sequence) are designed to be specifically complementary to two predetermined regions within the "binding sequence" on the secondary two-arm probe. This ensures that only when both recognition probes correctly bind to the target nucleic acid molecule can they juxtapose their respective random sequences, thereby hybridizing together accurately with the binding sequence of a secondary two-arm probe. In one embodiment, the length of the random sequence can be independently approximately 10 nt.

[0038] As described in this application, the "secondary two-arm probe" is a key intermediate for signal transduction and amplification. It has three main regions in sequence (from the 5' end to the 3' end): a first initiation sequence, a binding sequence, and a second initiation sequence.

[0039] The "first initiation sequence" and "second initiation sequence" serve as the two arms of the secondary double-arm probe, located at opposite ends of the binding sequence. Their function is to act as initiators, triggering subsequent hybridization chain reactions (HCR) of different hairpin probe pairs. Once the secondary double-arm probe is correctly "anchored" to the primary splitting probe pair that has bound the target nucleic acid molecule through its binding sequence, its free first and second initiation sequences can then freely initiate signal amplification.

[0040] The "binding sequence," located between the first and second initiating sequences, comprises two specific regions for specific hybridization with the first and second random sequences, respectively. This design ensures that a secondary two-armed probe must simultaneously and correctly hybridize with the recognition probes of a pair of bound nucleic acid molecules. Only when the random sequences of the first and second recognition probes simultaneously bind to the binding sequence of the two-armed probe can a platform for initiating hairpin amplification be provided, automatically suppressing nonspecific background and further increasing the system's specificity. The length of the binding sequence can be designed as needed, for example, approximately 20 nt in one embodiment. Correspondingly, the length of the "initiating sequence" must also be sufficient to initiate HCR, for example, approximately 36 nt in one embodiment.

[0041] As described in this application, the "hairpin probe pair" is the core of the signal amplification module, typically comprising two or more specially designed hairpin-shaped DNA probes. These hairpin probes are initially in a "metastable state," meaning they form a stable stem-loop structure within their own sequences, are in a kinetically repressed state, and do not undergo significant non-specific hybridization reactions with each other or with the triggering sequence at room temperature. The "metastable state" refers to the hairpin probes maintaining their closed hairpin structure for an extended period without a specific triggering sequence; only when they encounter a perfectly matching triggering sequence will a strand displacement reaction occur, opening the hairpin structure. In this application, the hairpin probe pair includes a first hairpin probe pair modified with a first detectable biomarker and a second hairpin probe pair modified with a second detectable biomarker, wherein the first and second detectable biomarkers are different. The first and second hairpin probe pairs undergo hybridization chain reactions initiated by the first and second triggering sequences, respectively. In some typical examples, the hairpin probe pair comprises two hairpin probes, namely hairpin probe H1 and hairpin probe H2. Their sequence design satisfies the following: the initiating sequence can completely hybridize with the "foot" region of H1, thus opening H1; the opened H1 exposes a new sequence region that can completely hybridize with the "foot" region of H2, thus opening H2; the opened H2 then exposes a sequence identical to (or functionally equivalent to) the "foot" region of H1, thus opening another H1 molecule; this cycle repeats, initiating alternating hybridization and strand substitution between hairpin probes H1 and H2, ultimately self-assembling into a long double-stranded DNA polymer nanowire. This process is called the "hybridization chain reaction (HCR)".

[0042] Specifically, in this application, since the left and right arms of the secondary dual-arm probe carry the initiation sequences of two hairpin probes (i.e., the first initiation sequence and the second initiation sequence), the colors are encoded by pairwise combinations based on the different fluorescence carried by the different hairpin probes. Therefore, if n fluorescence channels are used, and through the monochromatic and bichromatic combination design of the secondary dual-arm probe, the theoretically distinguishable number m of target genes in a single-round imaging is:

[0043] ;

[0044] This enables high-throughput target detection within a limited number of fluorescence channels.

[0045] More importantly, each pair of secondary double-arm probes can generate a linear polymer carrying a large number of fluorescent groups in situ, thereby converting the molecular recognition event of a single nucleic acid molecule into a high-contrast optical signal.

[0046] As described in this application, a "detectable marker" refers to a molecule or group attached to a hairpin probe for ultimately generating a detection signal. It is understood that the detectable marker can be directly modified during the synthesis of the hairpin probe, or it can be coupled later through a chemical reaction. The detectable marker includes, but is not limited to:

[0047] (1) Fluorescent groups (such as FAM, Cy3, Cy5, Alexa Fluor series, ATTO series, etc.) are the most commonly used labeling methods in in situ imaging, which can achieve high-resolution optical detection. The long-chain polymer formed by the hairpin probe polymerization can carry a large number of fluorescent molecules. The different fluorescence carried by different hairpin probes can be combined and encoded with colors, which can not only achieve effective signal amplification, but also achieve high-throughput detection in limited channels.

[0048] (2) Other markers, such as biotin (which can be detected by streptavidin-enzyme or fluorescent substances), chemiluminescent groups, radioactive isotopes, etc., can also be selected according to different detection platforms (such as colorimetric, chemiluminescent, and autoradiography).

[0049] The second aspect of this application discloses a kit containing the probe set described in this application. As described in this application, a "kit" refers to a product formed by combining and packaging the components required to achieve the method of this application. It should be understood that the kit may also include: buffer solutions (such as fixatives, permeabilizing solutions, hybridization solutions, washing solutions) for sample processing, blocking agents for background reduction, substrates for signal detection (if using an enzyme-linked immunosorbent assay system), positive and negative control samples, and detailed instructions for use, but is not limited thereto. The probe set or other auxiliary reagents of the kit may be provided in liquid or dry powder (such as lyophilized) form for easy storage, transportation, and use.

[0050] The third aspect of this application discloses a method for high-throughput in-situ detection of nucleic acid molecules in biological samples, the specific process of which is as follows: Figure 1 As shown, it includes the following steps:

[0051] Provide the probe set described in this application;

[0052] The biological sample to be tested is brought into contact with the first-level splitting probe pair and the second-level double-arm probe in sequence, and finally a first hairpin probe pair modified with a first detectable marker and a second hairpin probe pair modified with a second detectable marker are added.

[0053] By detecting the first and second detectable markers, imaging analysis of the target nucleic acid molecules is performed, enabling in-situ detection of nucleic acid molecules within biological samples.

[0054] The specific steps can be detailed as follows:

[0055] (1) Sample preparation: Provide the biological sample to be tested and fix the sample (e.g., paraformaldehyde), clear it, etc., to maintain the cell morphology while allowing the probe to enter. The specific method can be referred to in the field according to the sample condition and experimental purpose and needs.

[0056] (2) Hybridization and Assembly: The treated sample is contacted with the "primary splitting probe pair" under suitable hybridization conditions (e.g., incubation at 37-55°C for several hours in a hybridization buffer containing formamide). The target sequences of the first and second recognition probes hybridize to specific sites on the target nucleic acid molecule. After washing to remove unbound primary splitting probe pairs, secondary double-arm probes are added. Under hybridization conditions, the binding sequence of the secondary double-arm probes hybridizes with the random sequence of the primary splitting probe pair that has been correctly anchored to the nucleic acid molecule, thereby immobilizing the secondary double-arm probes at the nucleic acid molecule site. Washing is performed again to remove unbound secondary double-arm probes.

[0057] (3) Signal amplification: Add a first hairpin probe pair modified with a first detectable label (such as a fluorescent group) and a second hairpin probe pair modified with a second detection label (including hairpin probes H1 and H2, respectively). In a suitable buffer, the free first and second initiation sequences of the secondary double-arm probes open the corresponding hairpin probe H1, triggering the HCR cascade reaction, and generating DNA polymers with different detectable labels in situ on the target mRNA.

[0058] (4) Washing and Detection: Thoroughly wash to remove hairpin probes that did not participate in the reaction, and then use appropriate equipment for detection. For example, if the label is a fluorescent group, use a fluorescence microscope, confocal microscope, or tissue section scanner to image the sample. By analyzing the signal points under specific fluorescence channels, the target nucleic acid molecules can be located, counted, and relatively quantified, realizing in-situ detection of nucleic acid molecules in biological samples.

[0059] This application introduces a secondary two-armed probe and simultaneously mounts two different initiation sequences on the probe, achieving a "logical superposition" of physical channels and overcoming the limitations of the number of optical channels. This allows a single gene locus to be presented in a "composite color" form, increasing not only discrimination but also significantly improving detection throughput, achieving high-throughput target detection within a limited number of fluorescence channels. Furthermore, the two-armed probe ensures that the signals of the two colors originate from the same secondary relay molecule, achieving high co-localization at the physical level, reducing false positives caused by random background, and enhancing specificity and signal-to-noise ratio.

[0060] The present application will be further illustrated below with reference to specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present application in any way.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0062] In addition, unless otherwise specified, methods without detailed conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0063] Example 1: High-throughput in situ detection of mRNA in biological samples

[0064] In this embodiment, a 10 μm thick frozen section of the coronal surface of a mouse brain was used as the experimental sample to detect the SST gene (Somatostatin gene) in situ. The process of high-throughput in situ detection of mRNA in this application is described in detail, and the principle of the two-arm-based HCR strategy is verified.

[0065] 1.1 Probe Design

[0066] 1.1.1 Design of First-Order Splitting Probe Pairs

[0067] In this embodiment, the SST gene, a hallmark gene of inhibitory neurons, was selected as the target gene. First, the transcript information of the target mouse gene was retrieved from the ensemble database, its cDNA sequence was downloaded, and exported in FASTA format as a template sequence for subsequent probe design. Then, the Oligominer online sequence design platform was used to perform high-throughput screening of primary probe target sequences for the target cDNA sequence. The following key parameters were adjusted and optimized to ensure probe specificity: sequence length: 48-52 nt; melting temperature (Tm): 45-80℃; GC content: 35%-65%. This screened RNA sequences with high specificity, moderate GC content, and no significant secondary structure as target sequences. In this embodiment, the target sequence used as an example has a length of 50 nt. After the target sequence was reverse-complemented and split into left and right halves, the sequences of the primary split probe pairs were designed. The sequences of the first recognition probe are shown in SEQ ID NO.1-SEQ ID NO.6, and the sequences of the second recognition probe are shown in SEQ ID NO.7-SEQ ID NO.12. The specific sequence structures are shown in Table 1 below.

[0068] Table 1. Sequence display of SST gene primary division probe pairs

[0069]

[0070] 1.1.2 Design of a two-stage double-arm probe

[0071] 20nt random DNA sequences with moderate GC content and no significant secondary structure were screened, and the high specificity of the random sequences was confirmed by NCBI BLAST. The secondary double-arm probe (SEQ ID NO.13) was composed of B5 hairpin initiation sequence (B5I1) and B2 hairpin initiation sequence (B2I2) added to the left and right ends of the random sequence, respectively. The sequence composition is shown in Table 2 below.

[0072] Table 2. Structure of the secondary two-arm probe sequence

[0073]

[0074] 1.1.3 Design of the hairpin probe

[0075] The hairpin probe pair used in this application is the B2 and B5 hairpin probe pair. The fluorescent molecule carried by the B2 hairpin is cy3, which is detected in channel 561, and the fluorescent molecule carried by the B5 hairpin is cy5, which is detected in channel 647. The specific sequences of the hairpin probes are shown in Table 3:

[0076] Table 3. Hairpin probe sequence display

[0077]

[0078] 1.2 Pretreatment of frozen sections

[0079] Section fixation: Treat sections with 4% paraformaldehyde (PFA) for 15 min to fix the sections, then wash the sections 3 times with 2 mM VRC (1×PBSV) supplemented with 1×PBS to thoroughly remove residual PFA.

[0080] Section clearing: 2 mM VRC was added with 1×PBS, sections were treated with 0.1% Triton (1×PBSTV) for 5 min, and sections were washed with 1×PBSV for 10 min.

[0081] 1.3 RNA Anchoring

[0082] Frozen sections were pre-incubated in 20 mM MOPS buffer for 30 min, then incubated overnight at 37°C with 1 mg / ml MelphaX in 20 mM MOPS buffer. Afterwards, sections were washed twice with 2×SSC followed by 2 mM VRC (2×SSCV), 5 min each time.

[0083] 1.4 Gelation and Digestion

[0084] Gelation: The sections were treated with a gelling solution (2×SSC, 4% acrylamide, 0.2% N,N'-methylenebisacrylamide, 0.1% VA-044, 1% RNase inhibitor) at 4°C for 30 min. The previous gelling solution was aspirated, and fresh gelling solution containing 0.1% APS and 0.1% TEMED was added to the brain slice sample. A 12 mm coverslip pretreated with gel slick was then placed on top to form a closed gel solution environment. The sample was incubated at 37°C for 2 hours to form a gel.

[0085] SDS treatment: Gel-coated sections were incubated overnight at 37°C in 8% SDS (supplemented with 1% RNase inhibitor). The sections were washed four times at 37°C for 30 min each time with 2×SSCV.

[0086] Proteinase K digestion: The sections were digested in proteinase K digestion solution at 37°C for 30 min (2×SSC, 1% proteinase K, 1% RNase inhibitor), and then washed twice with 2×SSCV for 15 min each time.

[0087] 1.5 Hybridization primary division probe

[0088] Pre-incubation of sections: The sections were pre-incubated at 37°C for 30 min in 30% probe hybridization buffer (30% formamide, 5×SSC, 0.1% Tween 20, 10% dextran sulfate, 2mM VRC).

[0089] Hybridization of primary division probe: Add 40 nM of primary division probe to 30% probe hybridization buffer and incubate at 37°C for 36 h. Then wash 4 times at 37°C for 15 min each time with 30% probe washing buffer (30% formamide, 5×SSC, 0.1% Tween 20, 2 mM VRC).

[0090] 1.6 Hybridization Secondary Two-Arm Probe

[0091] Pre-incubation of sections: Incubate sections in 10% probe hybridization buffer (10% formamide, 2×SSC, 0.1% Tween 20, 10% dextran sulfate, 2mM VRC) for 30 min.

[0092] Hybridization of secondary two-armed probes: Add 10 nM of secondary two-armed probes to 10% probe hybridization buffer and incubate at room temperature for 6 h. Then wash twice with 10% probe washing buffer (10% formamide, 2×SSC, 0.1% Tween 20, 2 mM VRC), 15 min each time.

[0093] 1.7 Hairpin Amplification

[0094] Hairpin probe pretreatment: B5H1, B5H2, B2H2 and B2H2 hairpin probes were heat-shocked at 95℃ for 90 seconds and then cooled in the dark for 30 minutes.

[0095] Pre-incubation in amplification buffer: The slides were pre-incubated in amplification buffer 5×SSCT (5×SSC, 0.1% Tween 20, 2mM VRC) for 30 min.

[0096] Hairpin probe incubation: Add hairpin probe to the amplification buffer to bring the final concentration to 90 nM, and amplify in the dark for 6 h.

[0097] 1.8 Spatial location of in situ nucleic acid hybridization detection

[0098] The sample was washed five times with 5×SSCT, each time for 5 min. It was then stained with 0.1 μg / ml DAPI in 1×PBSV for 10 min, followed by imaging using a rotary confocal microscope. A Z-stack step of 1 μm was set for multi-slice imaging, and in-situ signal readout was performed.

[0099] 1.9 Results Analysis

[0100] The results are as follows Figure 2 As shown in the diagram, in this embodiment, the dual-arm HCR successfully detected the SST gene signal in channels 561 and 647, and the signals from the two channels perfectly overlapped. Specifically, the SST gene mRNA signal was successfully detected in both red and green physical channels, and the signals from the two channels perfectly overlapped (visually appearing as yellow). This result strongly demonstrates the feasibility of the dual-arm probe encoding strategy. Through this strategy, in addition to distinguishing pure red and pure green targets within only two physical channels, a third independent detection dimension (red + green combination color) was successfully opened up, theoretically improving the single-round detection capability by 50% compared to traditional methods.

[0101] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A probe set for high-throughput in situ detection of nucleic acid molecules in a biological sample, characterized in that, include: A first-level splitting probe pair includes a first recognition probe and a second recognition probe, wherein the first recognition probe has a first target sequence, a first interval sequence and a first random sequence in sequence, and the second recognition probe has a second random sequence, a second interval sequence and a second target sequence in sequence. A two-armed probe, which sequentially includes a first initiation sequence, a binding sequence, and a second initiation sequence; And a first hairpin probe pair modified with a first detectable marker and a second hairpin probe pair modified with a second detectable marker, wherein the first detectable marker and the second detectable marker are different; Wherein, the first target sequence and the second target sequence are specifically complementary to two sequences on the target nucleic acid molecule, respectively; the first spacer sequence and the second spacer sequence are not complementary to the target nucleic acid molecule; the first random sequence and the second random sequence are specifically complementary to two sequences on the binding sequence, respectively; the first initiation sequence can initiate the amplification and polymerization of the first hairpin probe pair, and the second initiation sequence can initiate the amplification and polymerization of the second hairpin probe pair.

2. The probe set of claim 1, wherein The first interval sequence and the second interval sequence are independently AA or AT.

3. The probe set of claim 1, wherein The first and second targeting sequences have independent base lengths of 25 nt; The first spacer sequence and the second spacer sequence are each 2 nt in length independently; The base lengths of the first and second random sequences are independently 10 nt.

4. The probe set of claim 1, wherein The first and second initiating sequences are each 36 nt in length, and the binding sequence is each 20 nt in length.

5. The probe set of claim 1, wherein The first hairpin probe pair and the second hairpin probe pair each independently include two metastable hairpin probes H1 and H2; The first initiation sequence and the second initiation sequence can respectively open the corresponding hairpin probe H1, thereby initiating a cascade reaction. The hairpin probe H1 and the corresponding hairpin probe H2 alternately hybridize to form a long-chain polymer.

6. The probe set of claim 1, wherein The first detectable marker and the second detectable marker are different fluorescent groups.

7. A kit, characterized in that, It contains the probe set according to any one of claims 1-6.

8. A method for high-throughput in situ detection of nucleic acid molecules in a biological sample, which is not aimed at the diagnosis of a disease, characterized in that, The method includes the following steps: Provide a probe set as described in any one of claims 1-6; The biological sample to be tested is brought into contact with the first-level splitting probe pair and the second-level double-arm probe in sequence, and finally a first hairpin probe pair modified with a first detectable marker and a second hairpin probe pair modified with a second detectable marker are added. By detecting the first and second detectable markers, imaging analysis of the target nucleic acid molecules is performed, enabling in-situ detection of nucleic acid molecules within biological samples.

9. The method of claim 8, wherein, The biological sample to be tested is a biological tissue section.

Images