Composition, detection method and kit for SpaceAS variable splicing space in-situ detection of nucleic acid

Abstract

The invention discloses a composition, a detection method and a kit for SpaceAS variable splicing space in-situ detection of nucleic acid. The composition comprises a pi-type probe, a U-type probe, an O-type probe, an S-type probe and a signal probe, and the O-type probe adopts an in-vitro cyclization strategy, so that the problem of low hybridization efficiency caused by dependence on enzymatic cyclization reaction in a traditional hybridization method is effectively solved; meanwhile, an efficient probe hybridization system, a high-sensitivity signal amplification mechanism and a multi-round iterative imaging process are combined, so that the detection sensitivity and specificity are remarkably improved. The detection method has the advantages of high efficiency, high sensitivity, high specificity, good repeatability, strong expandability and the like through the synergistic effect of five-step single-stranded DNA probe hybridization reaction and one-step DNA amplification reaction, and can be widely applied to space in-situ detection and analysis of variable splicing events.

Description

Technical Field

[0001] This application belongs to the field of biotechnology for spatial in situ detection of variable splicing, specifically referring to a composition, detection method, and kit containing the composition for spatial in situ detection of nucleic acids using SpaceAS variable splicing. Background Technology

[0002] Alternative splicing (AS) is a core mechanism of post-transcriptional regulation. Through different splicing patterns of pre-mRNA, a single gene can encode multiple proteins, thus greatly enriching the diversity of protein types and functions. In different species, alternative splicing is one of the core molecular bases for the formation of biological phenotypic diversity and functional complexity. Its variants exhibit high spatiotemporal specificity in different tissue types, developmental stages, and specific physiological or pathological environments. Its abnormal regulation is widely considered to be closely related to the occurrence and progression of genetic diseases, neurological disorders, and malignant tumors. Currently, the biological significance of AS is widely recognized and highly valued.

[0003] However, research on in-situ detection methods targeting the spatial dimension of AS remains relatively scarce. Current technological limitations significantly restrict in-depth analysis and systematic research on the functional characteristics and regulatory networks of AS events in the tissue microenvironment. Therefore, there is an urgent need to develop an in-situ spatial detection technology for AS to achieve efficient and specific detection of AS events in organisms. Summary of the Invention

[0004] The purpose of this application is to provide a composition, a detection method, and a kit containing the composition for in-situ spatial detection of nucleic acids using SpaceAS (Spatial Alternative Splicing). This composition, detection method, and kit enable efficient and specific high-precision detection of in-situ spatial alternative splicing.

[0005] In a first aspect, embodiments of this application provide a composition for in-situ spatial detection of nucleic acids via variable splicing, comprising: A π-type probe, used in the first hybridization step, consists of two single-stranded DNA molecules, left and right, forming a π-type structure. The π-type probe includes a π-foot region, a π-top region, and a partially complementary π-middle region. The π-foot region is used to specifically bind to the target nucleic acid sequence. A U-shaped probe, used for the second hybridization step, is a single-stranded DNA molecule; the U-shaped probe includes an intermediate region complementary to the π-top region, and two end regions I and II located on both sides of the intermediate region; The O-type probe, used in the third hybridization and fifth amplification reactions, is an in vitro circularized single-stranded circular DNA molecule; the O-type probe includes a signal recognition region and an O-type probe bottom region that is complementary to the two end regions I; The S-type probe, used in the fourth hybridization step and the fifth amplification step, is a single-stranded DNA molecule; the S-type probe includes a bottom region of the S-type probe that is complementary to the two end regions II, and a top region of the S-type probe that is complementary to the signal recognition region. The signal probe, used in the sixth hybridization step, is a single-stranded DNA molecule; the signal probe is used to complementarily pair with the amplified signal recognition region.

[0006] Secondly, embodiments of this application provide a kit for in-situ detection of nucleic acids in a variable splice space, comprising the composition provided in the above embodiments.

[0007] Thirdly, embodiments of this application provide a method for detecting nucleic acids in situ using the compositions or kits provided in the above embodiments, comprising: S100) provides pre-treated tissue samples; S200) First step hybridization: Hybridize the π-type probe with the target nucleic acid sequence of the tissue sample; S300) Second step hybridization: Hybridize the U-shaped probe with the π-shaped probe; S400) Third step of hybridization: Hybridize the O-type probe with the U-type probe; S500) Step 4 Hybridization: Hybridize the S-type probe with the O-type probe and the U-type probe; The fifth step of the S600 amplification reaction: Under the action of DNA polymerase, using the O-type probe as a template and the S-type probe as a primer, a rolling circle amplification reaction is performed. S700) Step 6 Hybridization: Hybridize the signal probe with the nucleic acid sequence obtained from the amplification reaction in Step 5.

[0008] The beneficial effects of this application are as follows: (1) The detection efficiency of the signal in this application is high. The in vitro cyclization O-type probe hybridization strategy adopted effectively avoids the dependence on inefficient enzymatic cyclization reaction in the traditional hybridization process, and can improve the efficiency of traditional inefficient enzymatic cyclization hybridization to the level of single-molecule hybridization efficiency. It has the advantages of high detection sensitivity, good repeatability and strong scalability.

[0009] (2) The signal amplification capability of this application is strong. Since the differential sequences of different splice isoforms of the same gene are usually short, the space available for designing specific probes is limited. However, the DNA amplification of this application can achieve exponential signal amplification, and in situ detection of variable splice signals can be achieved under hybridization of a single π-type probe. (3) The signal detection of this application has high resolution. By using the hybridization method of stepwise composition, high-precision in-situ spatial detection of variable splicing at the single cell and single molecule level can be achieved. (4) The signal detection of this application is highly specific. On the one hand, the probe that binds to the target gene is a π-type probe, which is composed of two single-stranded DNA probes on the left and right. Only when the two single-stranded DNA probes on the left and right bind to the target sequence at the same time can a positive signal be specifically formed. On the other hand, in the hybridization process of the stepwise combination, only U-type, O-type and S-type probes can specifically bind to form a positive signal. (5) This application has high detection throughput. This application can not only achieve simultaneous imaging through multiple distinguishable multicolor fluorescence, but also combine multiple rounds of color development, elution and USER enzyme treatment in a multi-round iterative detection process, which can realize high-throughput in-situ detection and analysis of multiple target alternative splicing events in the same sample.

[0010] (6) The composition used in this application is simple to synthesize, all of which are DNA probes, are not easily degraded, and are easy to store. Attached Figure Description

[0011] Figure 1 A schematic diagram illustrating the usage principle of the composition designed for SpaceAS variable splicing in-situ detection of nucleic acids in this application; Figure 2 This application demonstrates the in-situ detection of a single SpaceAS probe array in HeLa cells using Example 1 of this application. ACTB Results of gene mRNA; Figure 3 The SpaceAS monochromatic signal probe in Example 2 of this application was used to detect the presence of [unclear - possibly a specific substance or signal] in mouse brain tissue. Gad1 Results of gene mRNA; Figure 4 In Example 3 of this application, the SpaceAS dual-color signal probe was used to locate mouse brain tissue. Sst Results of gene mRNA; Figure 5 In Example 4 of this application, two rounds of hybridization were used to detect the presence of [certain substances] in mouse brain tissue. VIP and Gad1 The results of multiple rounds of SpaceAS detection are presented to demonstrate the gene mRNA. Figure 6 For example, in Example 5 of this application, SpaceAS was used to detect [something] in mouse brain tissue. Nrxn1-SS4 and Nrxn2-SS4 The result of a variable splice body. Detailed Implementation

[0012] The embodiments of this application are described in detail below, with examples of the embodiments shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0013] This application provides a composition, detection method, and kit containing the composition for in-situ spatial detection of nucleic acids using SpaceAS (Space-based Alternative Splicing Assay). Based on the need for specific in-situ spatial detection of alternative splicing events, this application constructs a SpaceAS system centered on multi-round nucleic acid hybridization and amplification. This system, through a five-step probe hybridization and one-step polymerase amplification reaction design, can efficiently and specifically achieve high-resolution in-situ spatial detection of different splice isoforms. The technical principles of this application are: 1) Specific recognition: π-type probes are used to accurately identify the specific sequences of target gene alternative splice variants, ensuring that the probe binds only to the target isoform; 2) Hierarchical hybridization of the composition: Through sequential hybridization of U-type, O-type, and S-type probes, a hierarchical nucleic acid complex structure is formed, providing a stable template for subsequent amplification. The in vitro circularization O-type probe hybridization strategy employed can improve the efficiency of traditional inefficient enzymatic circularization hybridization to the level of single-molecule hybridization efficiency; 3) Circular amplification mechanism: Under the action of DNA polymerase, the S-type probe will use the O-type probe as a template to extend and amplify a large number of nucleic acids, thereby achieving exponential signal amplification; 4) Multi-round iterative imaging: Finally, the fluorescently labeled signal probe is hybridized with the amplification product, and through multiple rounds of color development and elution, spatial resolution visualization detection of different alternative spliceosomes is achieved. Through the above principles, this application effectively avoids dependence on inefficient enzymatic circularization reactions during hybridization, and significantly improves the detection signal intensity and detection sensitivity through an efficient probe hybridization system, thereby achieving precise revelation of the spatial distribution and molecular characteristics of alternative splicing events in the tissue microenvironment at the single-cell and single-molecule level.

[0014] This detection method is accomplished through a five-step hybridization reaction of single-stranded DNA composition and a one-step DNA amplification reaction. The multi-step hybridization reaction involves compositions composed of various specific single-stranded DNA probes; the DNA amplification reaction is achieved through a DNA polymerase-mediated circular amplification reaction. Specifically, the detection process of this invention includes the following steps in sequence: First, hybridization with π-type probes; second, hybridization with U-type probes; third, hybridization with O-type probes; fourth, hybridization with S-type probes; fifth, amplification of the S-type probe using the O-type probe as a template under the action of DNA polymerase to amplify the signal; sixth, hybridization and detection of the signal probe. The probe hybridization and signal amplification are performed sequentially, thereby enabling efficient and specific in-situ spatial detection of the alternative spliceosome.

[0015] This detection method effectively overcomes the key technical bottlenecks of existing methods in detecting in-situ spatial alternative splicing, such as low resolution, low detection efficiency, and insufficient signal amplification, thereby achieving high-precision detection of in-situ spatial alternative splicing with high efficiency and specificity. This application employs an in vitro circular single-stranded DNA (cssDNA) probe hybridization strategy, effectively avoiding dependence on inefficient enzymatic circularization reactions during hybridization. By combining an efficient probe hybridization system, a highly sensitive signal amplification mechanism, and a multi-round iterative imaging process, efficient and specific detection of in-situ spatial alternative spliceosomes can be achieved.

[0016] This application provides a composition for in-situ spatial detection of nucleic acids using SpaceAS (Spatial Alternative Splicing) alternative splicing, such as... Figure 1 As shown, the composition comprises: The π-type probe, used in the first hybridization step, consists of two single-stranded DNA molecules, left and right, forming a π-type structure. The π-type probe includes a π-foot region, a π-top region, and a partially complementary π-middle region. The π-foot region is used to specifically bind to the target nucleic acid sequence. The U-shaped probe, used for the second hybridization step, is a single-stranded DNA molecule. The U-shaped probe includes an intermediate region that is complementary to the π-top region, and two end regions I and II located on both sides of the intermediate region. O-type probes, used in the third hybridization and fifth amplification reactions, are in vitro circularized single-stranded circular DNA molecules; O-type probes include a signal recognition region and a bottom region of the O-type probe that is complementary to the two end regions I; S-type probes, used in the fourth hybridization and fifth amplification reactions, are single-stranded DNA molecules. The S-type probe includes a bottom region that is complementary to the two end regions II, and a top region that is complementary to the signal recognition region. The signal probe, used in the sixth step of hybridization, is a single-stranded DNA molecule; the signal probe is used to pair complementaryly with the amplified signal recognition region.

[0017] In this embodiment, partial base pairing in the π region can be used to maintain structural stability and improve probe hybridization efficiency. The signal probe is used for visualization of the hybridization signal.

[0018] The compositions of this application embodiment can perform probe hybridization and signal amplification in sequence, thereby enabling efficient and specific in-situ spatial detection of the variable spliceosome.

[0019] In some embodiments, the composition further includes: DNA polymerase; the DNA polymerase is used in the fifth amplification reaction; using nucleotide raw materials, the DNA polymerase is used to catalyze a rolling circle amplification reaction using an O-type probe as a template and an S-type probe as a primer. Signal amplification is achieved through the rolling circle amplification reaction.

[0020] In some embodiments, the composition further includes: nucleotide raw materials; the nucleotide raw materials comprise a first nucleotide raw material and / or a second nucleotide raw material; the first nucleotide raw material includes deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate (dTTP); the second nucleotide raw material includes deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxyuridine triphosphate (dUTP). The first nucleotide raw material can undergo an amplification reaction under the action of DNA polymerase, thereby achieving single-round signal detection. The second nucleotide raw material can form an amplification product containing dUTP bases through an amplification reaction, and the dUTP bases in the amplification product are specifically cleaved under the action of USER enzyme. After washing, multiple rounds of probe hybridization can be performed again, thereby achieving multi-round cyclic signal detection.

[0021] In some embodiments, the length of the π-foot region is 16-25 bases; the length of the π-top region is 12-16 bases; the length of the π-middle region is 6-8 bases, and there are 2-5 base complementarities between the two single-stranded DNA sequences.

[0022] In some embodiments, the total length of the U-shaped probe is 109 to 171 bases; the length of the middle region is 27 to 29 bases; the length of the two end regions I is 16 to 22 bases; and the length of the two end regions II is 16 to 22 bases.

[0023] In some embodiments, the total length of the O-type probe is 40 to 70 bases; wherein the length of the bottom region of the O-type probe is 16 to 22 bases; and the length of the signal probe recognition region is 16 to 22 bases.

[0024] In some embodiments, the total length of the S-type probe is 24 to 38 bases; wherein the length of the bottom region of the S-type probe is 16 to 22 bases; and the length of the top region of the S-type probe is 8 to 16 bases.

[0025] In some embodiments, the U-shaped probe has at least one end region I and one end region II on each side of the middle region. In other embodiments, the U-shaped probe has at least a plurality of end regions I and a plurality of end regions II on each side of the middle region, and the end regions I and end regions II are arranged alternately.

[0026] In this embodiment, the U-shaped probe has only one central region. On each side of the central region (i.e., the two end regions), there may be only one end region I and one end region II, or multiple end regions I and multiple end regions II. In some embodiments, the U-shaped probe has only one central region, and on each side of the central region (i.e., the two end regions), there is one end region I and one end region II. For example, the arrangement from the 5' end to the 3' end is: end region II, end region I, central region, end region I, end region II. In other embodiments, the U-shaped probe has only one central region, and each side of the central region has at least multiple end regions I and multiple end regions II. End regions I and end regions II are arranged alternately, but the side immediately adjacent to the central region is end region I, and the side furthest from the central region is end region II. For example, the arrangement from the 5' end to the 3' end is: ...end region II, end region I, end region II, end region I, central region, end region I, end region II, end region I, end region II, end region I, end region II... By using multiple two-end regions I and multiple two-end regions II, the signal can be cascaded amplified and stably anchored by increasing the binding sites, thereby improving detection sensitivity.

[0027] In some embodiments, the O-type probe includes multiple signal recognition regions. This amplifies the signal and improves the flexibility of detection.

[0028] In some embodiments, the O-type probe includes one, two, or more signal recognition regions, which are used to combine with monochromatic fluorescent signal probes, dual-color fluorescent signal probes, or multi-color fluorescent signal probes, respectively. This enables multi-channel signal recognition and detection.

[0029] In some embodiments, both the 5' and 3' ends of the signal probe are labeled with fluorescent groups; the fluorescent groups are selected from one or more of Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 700, Alexa Fluor 750, and Alexa Fluor 790.

[0030] In some embodiments, different fluorescent groups carried by the signal probe can be imaged simultaneously during the same detection process, thereby increasing the signal throughput of the detection of alternative splicing variants.

[0031] In some embodiments, the composition can detect alternative splice variants formed from long and short nucleic acid sequences, as well as miRNA, LncRNA, mRNA, ssDNA, dsDNA, and DNA sequences. A single π-type probe can achieve in-situ detection and amplification of the signal.

[0032] This application provides an application of a composition for SpaceAS variable splicing in-situ detection of nucleic acids.

[0033] This application provides a kit for in-situ detection of nucleic acids in a variable splice space, comprising the composition provided in any of the above embodiments.

[0034] In some embodiments, the kit further includes: a variety of prehybridization solutions and a variety of elution buffers; the prehybridization solutions contain dextran sulfate, SSC buffer, deionized formamide, lithium dodecyl sulfate (LDS), Denhardt's solution, and vanadate ribonucleoside complex (RVC); the elution buffers contain SSC buffer, deionized formamide, and lithium dodecyl sulfate.

[0035] In some embodiments, the multiple prehybridization solutions include prehybridization solution A, prehybridization solution B, prehybridization solution C, and prehybridization solution D; and the multiple elution buffers include elution buffer A, elution buffer B, and elution buffer C.

[0036] Prehybridization solutions A through D all used 20 × SSC solution as the base buffer system. Dextran sulfate was added to increase the viscosity of the hybridization solution, prevent liquid evaporation, and increase hybridization efficiency. An appropriate amount of deionized formamide was added to adjust the suitable Tm value for nucleic acid hybridization. 3% lithium dodecyl sulfate (LDS) was added to enhance membrane permeability. Denhardt's solution was added to reduce probe nonspecific binding. Furthermore, vanadate ribonucleoside complex (RVC) was added to inhibit RNase and prevent RNA degradation.

[0037] In some embodiments, the prehybridization solution A is prepared as follows: 0.01–0.02 g dextran sulfate, 20–30 μL 20 × SSC buffer, 25–30 μL deionized formamide, 6–8 μL 3% LDS, 5–10 μL Denhardt's solution, 1–2 μL RVC, and RNase-free water to a final volume of 100 μL.

[0038] In some embodiments, the prehybridization solution B is prepared as follows: 0.01–0.02 g dextran sulfate, 20–25 μL 20 × SSC buffer, 15–20 μL deionized formamide, 8–10 μL 3% LDS, 8–10 μL Denhardt's solution, 1–2 μL RVC, and RNase-free water to a final volume of 100 μL.

[0039] In some embodiments, the prehybridization solution C is prepared as follows: containing 0.01–0.02 g dextran sulfate, 20–25 μL 20 × SSC buffer, 10–25 μL deionized formamide, 8–10 μL 3% LDS, 8–10 μL Denhardt's solution, 1–2 μL RVC, and RNase-free water to a final volume of 100 μL.

[0040] In some embodiments, the prehybridization solution D is prepared as follows: containing 0.01–0.02 g dextran sulfate, 20–25 μL 20 × SSC buffer, 5–10 μL deionized formamide, 8–10 μL 3% LDS, 8–10 μL Denhardt's solution, 1–2 μL RVC, and RNase-free water to a final volume of 100 μL.

[0041] Elution buffers A, B, and C are used for tiered elution of the hybridization signal. Elution buffer A uses a high ionic strength to remove non-specific hybridization; elution buffer B reduces the concentration of SSC buffer and formamide to achieve gentle elution; elution buffer C is the final wash system used to retain the target signal.

[0042] In some embodiments, the elution buffer A is prepared as follows: 10 μL of 20 × SSC buffer, 20–25 μL of deionized formamide, 1–2 μL of 3% LDS, and RNase-free water to a final volume of 100 μL.

[0043] In some embodiments, the elution buffer B is prepared as follows: 5 μL 20 × SSC buffer, 10–12 μL deionized formamide, 1–2 μL 3% LDS, and RNase-free water to a final volume of 100 μL.

[0044] In some embodiments, the elution buffer C is prepared as follows: 1 μL 20 × SSC buffer, 1–2 μL 3% LDS, and RNase-free water to a final volume of 100 μL.

[0045] This application provides a method for detecting nucleic acids in situ using the composition or kit provided in any of the above embodiments, the detection method comprising: S100) provides pre-treated tissue samples; S200) First step hybridization: Hybridize the π-type probe with the target nucleic acid sequence of the tissue sample; S300) Second step hybridization: Hybridize the U-shaped probe with the π-shaped probe; S400) Third step of hybridization: Hybridize the O-type probe with the U-type probe; S500) Step 4 Hybridization: Hybridize the S-type probe with the O-type probe and the U-type probe; The fifth step of the S600 amplification reaction: Under the action of DNA polymerase, using the O-type probe as a template and the S-type probe as a primer, a rolling circle amplification reaction is performed. S700) Step 6 Hybridization: Hybridize the signal probe with the nucleic acid sequence obtained from the amplification reaction in Step 5.

[0046] In an optional embodiment, step S100) involves preprocessing the tissue sample, including: S110) Tissue fixation: Fix the tissue sample with 4% paraformaldehyde (PFA) for 5-10 min; S120) Tissue permeation: Place tissue sample sections in 0.2 M hydrochloric acid solution for permeation for 5–10 min; S130) Tissue digestion: Digest the tissue with 5-10 μg / mL proteinase K at room temperature for 2-5 min to remove proteins; S140) Tissue washing: Wash tissue sections with 1 × PBS solution 1 to 3 times, 5 min each time.

[0047] In some embodiments, the first step of hybridization (200) includes: adding the π-type probe to the prehybridization solution A, mixing thoroughly, and then adding it to the tissue sample, and incubating at 40 °C for 3 to 16 hours; subsequently, adding elution buffer A, elution buffer B, and elution buffer C to the tissue sample in sequence, and washing the tissue sample in a shaker at 40 °C and a speed of 60 to 70 r / min for 5 minutes each time.

[0048] In some embodiments, the second hybridization step (300) includes: adding the U-shaped probe to the prehybridization solution B, mixing thoroughly, and then adding it to the tissue sample that has undergone the first hybridization treatment, and incubating at 40 °C for 1 to 4 hours; adding elution buffer A, elution buffer B, and elution buffer C to the tissue sample in sequence, and washing the tissue sample in a shaker at 40 °C and a speed of 60 to 70 r / min for 3 to 5 minutes each time.

[0049] In some embodiments, the third hybridization step (400) includes: adding the O-type probe to the prehybridization solution C, mixing thoroughly, and then adding it to the tissue sample that has undergone the second hybridization treatment, and incubating at 40 °C for 1 to 4 hours; adding elution buffer A, elution buffer B, and elution buffer C to the tissue sample in sequence, and washing the tissue sample in a shaker at 40 °C and a speed of 60 to 70 r / min for 3 to 5 minutes each time.

[0050] In some embodiments, the fourth hybridization step (500) includes: adding the S-type probe to the prehybridization solution D, mixing thoroughly, and then adding it to the tissue sample that has undergone the third hybridization treatment, and incubating at 40 °C for 1 to 4 hours; adding elution buffer A, elution buffer B, and elution buffer C to the tissue sample in sequence, and washing the tissue sample in a shaker at 40 °C and a speed of 60 to 70 r / min for 3 to 5 minutes each time.

[0051] In some embodiments, the fifth step of the amplification reaction (600) includes: adding DNA polymerase to the tissue sample after the fourth step of hybridization treatment, incubating at 30 °C for 1 to 4 hours, so that the S-type probe uses the O-type probe as a template and performs rolling circle amplification under the action of DNA polymerase, thereby achieving effective signal amplification; then washing three times with 1 × DEPC-PBS, each time for 3 to 5 min.

[0052] In some embodiments, the sixth step of hybridization (700) includes: adding the signal probe to the prehybridization solution D, mixing thoroughly, and then adding it to the tissue sample that has undergone the fifth step amplification reaction, and incubating at 40 °C for 1 to 4 hours; adding elution buffer A, elution buffer B, and elution buffer C to the tissue sample in sequence, and washing the tissue sample in a shaker at 40 °C and a speed of 60 to 70 r / min for 3 to 5 minutes each time.

[0053] In some embodiments, the fifth step of the amplification reaction (S600) includes: S610): DNA polymerase and nucleotide raw materials are added to the tissue sample system after the fourth step of hybridization. Under the action of DNA polymerase, a single round of amplification reaction is carried out using an O-type probe as a template and an S-type probe as a primer. The nucleotide raw materials include either the first nucleotide raw material or the second nucleotide raw material. The first nucleotide raw material includes deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate (dTTP). The second nucleotide raw material includes deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxyuridine triphosphate (dUTP).

[0054] In some embodiments, the fifth step of the amplification reaction (S600) includes: S620): DNA polymerase and second nucleotide raw materials are added to the tissue sample system after the fourth step of hybridization. Under the action of DNA polymerase, using an O-type probe as a template and an S-type probe as a primer, an amplification product containing dUTP bases is formed. The second nucleotide raw materials include deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxyuridine triphosphate (dUTP). The detection methods also include: S800): USER enzyme is added to the tissue sample system containing dUTP bases for amplification. Under the action of USER enzyme, the dUTP bases in the amplification product are specifically cleaved and washed to facilitate the next round of hybridization and amplification reaction.

[0055] In this embodiment, steps S100), S200), S300), S400), S500), S620), and S700) are performed sequentially to complete the first round of amplification. Then, after step S800), steps S100), S200), S300), S400), S500), S620), and S700) are performed sequentially to complete the second round of amplification. This process is repeated to achieve multiple rounds of amplification. In step S800), the dU bond-containing junctions of the previous round hybridization probe are cleaved using the USER enzyme. After washing, the DNA polymer and signal probe are effectively removed. Subsequently, the next round of hybridization and amplification can be performed, thus enabling second, third, and subsequent rounds of amplification and cyclic hybridization detection of the probe. Through this multi-round iterative detection process, high-throughput in-situ detection and imaging of multiple target alternative splicing events can be achieved in the same sample.

[0056] In an optional embodiment, the detection method further includes: 900) Signal detection: Tissue samples were stained with DAPI to stain cell nuclei, and then the tissue samples with signals were photographed to observe the results.

[0057] After each round of hybridization and amplification, the results are photographed and observed. If the detection method involves only a single round of hybridization and amplification, single-round signal detection can be performed. If the detection method involves multiple rounds of hybridization and amplification, multiple rounds of signal detection can be performed. During multiple rounds of signal detection, after each round, USER enzyme is added to the tissue sample and incubated at 37 °C for 15–20 min. This cleaves the dU bond-containing junctions of the previous round's hybridization probe, effectively removing the signal probe and facilitating the next round of hybridization and amplification.

[0058] The following specific embodiments provide a more detailed description of this application, but should not be construed as limiting the application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application are within the scope of this application.

[0059] Example 1 Example 1: SpaceAS in situ detection of HeLa cells ACTB The method for detecting gene mRNA includes the following steps: 1.1 Preprocessing of whole brain tissue samples ① Place the cell slides in a 48-well culture plate and sterilize with 70% alcohol for 1 min; let them air dry, then wash with 1 × DEPC-PBS for 1 min; ② Next, the HeLa cells were seeded on a cell slide and cultured for 24 hours; ③ Next, wash the cells on the cell slide three times with 1 × DEPC-PBS, 5 min each time; ④ Next, fix with 4% paraformaldehyde / 1 × DEPC-PBS at room temperature for 5 min; ⑤ Next, wash the cells on the cell slide three times with 1 × DEPC-PBS, 5 min each time; ⑦ Next, permeabilize the cells with 0.2 M HCl at room temperature for 5 min; ⑧ Next, wash the cells with 1 × DEPC-PBS for 5 min; ⑨ Next, after treatment with 5 μg / mL proteinase K / 1 × DEPC-PBS for 2 min, fixation was performed again with 4% paraformaldehyde / 1 × DEPC-PBS at room temperature for 5 min. ⑩ Next, wash the cells on the cell slide three times with 1 × DEPC-PBS for 5 min each time. Finally, perform SpaceAS hybridization on the treated samples.

[0060] 1.2 Hybridization procedure for cell samples: ① First step: Hybridization and elution of the π-type probe: Add the π-type probe with a final concentration of 10 nM to 500 μL of prehybridization solution A, mix thoroughly, and preheat in a 40 ℃ metal bath for 10 min; then, add the preheated mixture to the HeLa cell sample to be tested and incubate at 40 ℃ for 8–16 h to allow the π-type probe to fully hybridize with the target nucleic acid sequence; after hybridization, discard hybridization solution A, add 500 μL of elution buffer A preheated at 40 ℃, and elute evenly in a shaker at 40 ℃ and a speed of 60–70 r / min for 5 min, then discard elution buffer A; next, add 500 μL of preheated elution buffer B and elute for 5 min under the same conditions. Finally, add 500 μL of elution buffer C and elute evenly for 5 min to remove non-specifically bound probe residues, obtaining the hybridized cell sample; the π-shaped probe consists of two single-stranded DNA probes, and the sequence information of the π feet, π tops, and π segments on both sides is shown below: Sequence of the left π-leg region of the π-type probe: 5'-GCGACGTAGCACAGCTTCTCCTTAA-3' (SEQ ID No. 1); Sequence of the left π-top region of the π-type probe: 5'-CCATAACCATTCGC-3' (SEQ ID No. 2); Sequence of the left π region of the π-type probe: 5'-TGGACTTA-3'; Sequence of the right π-leg region of the π-type probe: 5'-TGGCCATCTCTTGCTCGAAGTCCAG-3' (SEQ ID No. 3); Sequence of the right π-top region of the π-type probe: 5'-CGCTTACCAATACC-3' (SEQ ID No. 4); Sequence of the right π region of the π-type probe: 5'-ATTCACCT-3'.

[0061] ② Second step: Hybridization and elution of the U-shaped probe: Add 500 μL of prehybridization buffer B to a final concentration of 50 nM, mix thoroughly, and preheat on a 40 ℃ metal bath for 10 min. Then, add the preheated mixture to the HeLa cell sample to be tested and incubate at 40 ℃ for 2–3 h to allow the U-shaped probe and π-shaped probe to form a specific hybridization complex. After hybridization, discard hybridization buffer B, add 500 μL of elution buffer A preheated at 40 ℃, and elute evenly for 5 min on a shaker at 40 ℃ and a speed of 60–70 r / min. Then discard elution buffer A. Next, under the same conditions, add 500 μL of preheated elution buffer B and elution buffer C sequentially, and elute for 5 min each. The sequence information of the U-shaped probe is shown below: U-shaped probe middle region sequence: 5'-GGTATTGGTAAGCGTGCGAATGGTTATGG-3' (SEQ ID No. 5); The sequence of region I at both ends of the U-shaped probe is: 5'-GCGTGCGAAGGAGTTTAGCG-3' (SEQ ID No. 6); The sequence of region II at both ends of the U-shaped probe is: 5'-TGCTACGATAGGATTCGA-3' (SEQ ID No. 7).

[0062] ③ Step 3: Hybridization and elution of the O-type probe: Add 50 nM of the O-type probe to 500 μL of prehybridization solution C, mix thoroughly, and preheat on a 40 ℃ metal bath for 10 min. Then, add the preheated mixture to the HeLa cell sample to be tested and incubate at 40 ℃ for 2–3 h to achieve specific hybridization between the O-type and U-type probes. After hybridization, discard hybridization solution C, add 500 μL of elution buffer A preheated at 40 ℃, and elute evenly for 5 min on a shaker at 40 ℃ and 60–70 r / min. Then discard elution buffer A. Next, under the same conditions, add 500 μL of preheated elution buffer B and elution buffer C sequentially, and elute for 5 min each. The sequence information of the O-type probe is shown below: O-type probe bottom region sequence: 5'-CGCTAAACTCCTTCGCACGC-3' (SEQ ID No. 8); O-type probe lateral region sequence: 5'-CATTGAGAC-3'; O-type probe signal recognition region sequence: 5'-AGTACGGTCCATTGAGAC-3' (SEQ ID No. 9).

[0063] ④ Fourth step: Hybridization and elution of the S-type probe: Add the S-type probe to 500 μL of prehybridization solution D to a final concentration of 50 nM, mix thoroughly, and preheat on a 40 ℃ metal bath for 10 min; then, add the preheated mixture to the HeLa cell sample to be tested and incubate at 40 ℃ for 2–3 h to allow the S-type probe to form a stable hybridization structure with the O-type and U-type probes; after hybridization, discard the hybridization solution D, add 500 μL of elution buffer A preheated at 40 ℃, and elute evenly for 5 min on a shaker at 40 ℃ and a speed of 60–70 r / min, then discard the elution buffer A; next, under the same conditions, add 500 μL of preheated elution buffer B and elution buffer C sequentially, and elute for 5 min each; the sequence information of the S-type probe is shown below: The sequence of the bottom region of the S-type probe is: 5'-TCGAATCCTATCGTAGCA-3' (SEQ ID No. 10); S-type probe tip region sequence: 5'-GTCTCAATG-3'.

[0064] ⑤ Fifth step DNA amplification: After completing the fourth step of hybridization, add 1:10 diluted PhI29 DNA polymerase, 1 × PhI29 DNA polymerase reaction buffer, 0.4 mg / mL recombinant albumin, 1 mM dTTP, 1 mM dGTP, 1 mM dATP, and 1 mM dCTP to the tissue sample; incubate at 30 ℃ for 1–4 hours, so that the S-type probe uses the O-type probe as a template and undergoes an amplification reaction under the action of DNA polymerase, thereby achieving effective signal amplification.

[0065] ⑥ Step 6: Hybridization and Elution of Signal Probes: Add the signal probe with a final concentration of 100 nM to 500 μL of prehybridization solution D, mix thoroughly, and preheat on a 40 ℃ metal bath for 10 min. Then, add the preheated mixture to the HeLa cell sample to be tested and incubate at 40 ℃ for 2–3 h to hybridize the signal probe with the O-type probe nucleic acid sequence amplified in Step 5. After hybridization, discard hybridization solution D, add 500 μL of elution buffer A preheated at 40 ℃, and elute evenly for 5 min on a shaker at 40 ℃ and a speed of 60–70 r / min. Then discard elution buffer A. Next, under the same conditions, add 500 μL of preheated elution buffer B and elution buffer C sequentially, and elute for 5 min each. Both ends of the signal probe are modified with Alexa Fluor 488 fluorescence, and their sequence information is shown below: 5'-AGTACGGTCCATTGAGAC-3 (SEQ ID No. 11)'.

[0066] Meanwhile, the aforementioned π-type probe, U-type probe, O-type probe, S-type probe and signal probe can all be synthesized using conventional methods. Among them, the π-type probe, U-type probe, S-type probe and signal probe are all single-stranded linearized DNA probes, while the O-type probe is a single-stranded circular DNA probe.

[0067] ⑦ Stain the cell nuclei with DAPI for 2 min, then mount the slide for microscopic photography. Results are as follows: Figure 2 As shown in the in-situ detection results of this application, it can be seen that HeLa cells can be effectively detected using only one SpaceAS probe set. ACTB The expression of gene mRNA showed that the SpaceAS method has a good ability to detect short alternative splicing sequences.

[0068] In Example 1, the prehybridization solution A was prepared as follows: 0.01–0.02 g dextran sulfate, 20–30 μL 20× SSC buffer, 25–30 μL deionized formamide, 6–8 μL 3% LDS, 5–10 μL Denhardt's solution, 1–2 μL RVC, and RNase-free water to a final volume of 100 μL. The prehybridization solution B was prepared as follows: containing 0.01–0.02 g dextran sulfate, 20–25 μL 20 × SSC buffer, 15–20 μL deionized formamide, 8–10 μL 3% LDS, 8–10 μL Denhardt's solution, 1–2 μL LVC, and RNase-free water to a final volume of 100 μL. The prehybridization solution C was prepared as follows: containing 0.01–0.02 g dextran sulfate, 20–25 μL 20 × SSC buffer, 10–25 μL deionized formamide, 8–10 μL 3% LDS, 8–10 μL Denhardt's solution, 1–2 μL LVC, and RNase-free water to a final volume of 100 μL. The prehybridization solution D was prepared as follows: containing 0.01–0.02 g dextran sulfate, 20–25 μL 20 × SSC buffer, 5–10 μL deionized formamide, 8–10 μL 3% LDS, 8–10 μL Denhardt's solution, 1–2 μL LVC, and RNase-free water to a final volume of 100 μL. The preparation system of elution buffer A is as follows: containing 10 μL 20 × SSC buffer, 20-25 μL deionized formamide, 1-2 μL 3% LDS, and RNase-free water to a final volume of 100 μL; In some embodiments, the preparation system of elution buffer B is: 5 μL 20 × SSC buffer, 10-12 μL deionized formamide, 1-2 μL 3% LDS, and RNase-free water adjusted to 100 μL. In some embodiments, the elution buffer C is prepared as follows: 1 μL 20 × SSC buffer, 1–2 μL 3% LDS, and RNase-free water to a final volume of 100 μL.

[0069] Example 2 Example 2: SpaceAS single-round detection of mouse brain tissue Gad1 The method for detecting gene mRNA includes the following steps: 2.1 Pretreatment of tissue samples ① Take the brains of 6-8 week old mice, embed them with OCT, and freeze them at -80 ℃ for later use.

[0070] ② Next, the mouse brain that has been embedded in OCT was cut into tissue sections of 10-12 μm using a cryostat and attached to a glass slide.

[0071] ③ Next, place the slide containing the tissue into the tissue sectioning box and wash the tissue three times with 1 × DEPC-PBS, 5 min each time; ④ Next, fix the tissue with 4% paraformaldehyde / 1 × DEPC-PBS at room temperature for 10 min; ⑤ Next, wash the tissue three times with 1 × DEPC-PBS, 5 min each time; ⑥ Next, permeate the tissue with 0.2 M HCl at room temperature for 5 min.

[0072] ⑦ Next, wash the tissue with 1 × DEPC-PBS for 5 min.

[0073] ⑧ Next, treat the tissue with 10 μg / mL proteinase K preheated at 40 ℃ for 2 min; ⑨ Next, fix the solution again for 5 min at room temperature with 4% paraformaldehyde / 1 × DEPC-PBS.

[0074] ⑩ Next, the tissue was washed three times with 1 × DEPC-PBS for 5 min each time. Then, the mouse brain tissue slices prepared above were subjected to SpaceAS hybridization.

[0075] 2.2 Hybridization steps for tissue samples ① First step: Hybridization and elution of the π-type probe: Wipe the prepared mouse brain tissue sections dry with lint-free paper, draw hybridization cells with a hydrophobic pen, and then add the π-type probe with a final concentration of 10 nM to 500 μL of prehybridization solution A. Mix thoroughly and preheat in a 40 ℃ metal bath for 10 min. Subsequently, add the preheated mixture to the mouse brain tissue sections to be tested and incubate at 40 ℃ for 8–16 h for complete hybridization. After hybridization, discard hybridization solution A, and add elution buffer A, elution buffer B, and elution buffer C (preheated to 40 ℃) sequentially to the sample. Wash three times at 40 ℃ and a shaking speed of 60–70 r / min for 5 min each time. The sample is then tested for π-type probes. Gad1 The sequence information of the π-legs, π-apex, and π-sections on both sides of the π-shaped probe of the gene is shown below: Sequence of the left π-leg region of the π-type probe: 5'-ACAGGTTGGAGAAGTCGGTCTCTGT-3' (SEQ ID No. 12); Sequence of the left π-top region of the π-type probe: 5'-TTGCACGTAGATCG-3' (SEQ ID No. 13); Sequence of the left π region of the π-type probe: 5'-TGGACTTA-3'; Sequence of the right π-leg region of the π-type probe: 5'-CTTAGCTGGAAGCAGATCTTGAGCA-3' (SEQ ID No. 14); Sequence of the right π-top region of the π-type probe: 5'-GCTAGATGCACGTT-3' (SEQ ID No. 15); Sequence of the right π region of the π-type probe: 5'-ATTCACCT-3'.

[0076] ② Second step: Hybridization and elution of the U-shaped probe: Add the U-shaped probe to a final concentration of 50 nM into 500 μL of prehybridization solution B, mix thoroughly, and preheat in a 40 ℃ metal bath for 10 min; then, add the preheated mixture to the mouse brain tissue slices to be tested, and incubate at 40 ℃ for 3–4 h to allow the U-shaped probe and π-shaped probe to form a specific hybridization complex; after hybridization, discard hybridization solution B, and add elution buffer A, elution buffer B, and elution buffer C (preheated to 40 ℃) sequentially to the sample, and wash the tissue sample in a shaker at 40 ℃ and a speed of 60–70 r / min for 5 min each time; among which, the mouse brain was tested... Gad1 The sequence information of the gene-compatible U-shaped probe is shown below: U-shaped probe middle region sequence: 5'-AACGTGCATCTAGCACGATCTACGTGCAA-3' (SEQ ID No. 16); The sequence of region I at both ends of the U-shaped probe is: 5'-AACTAGTCGACCGAACGCCA-3' (SEQ ID No. 17); The sequence of region II at both ends of the U-shaped probe is: 5'-ATCGATGCTCTAACGGTA-3' (SEQ ID No. 18).

[0077] ③ Step 3: Hybridization and elution of the O-type probe: Add the O-type probe to 500 μL of prehybridization solution C to a final concentration of 50 nM, mix thoroughly, and preheat on a 40 ℃ metal bath for 10 min; then, add the preheated mixture to the mouse brain tissue sections to be tested and incubate at 40 ℃ for 2–4 h; after hybridization, discard the hybridization solution C, and add elution buffer A, elution buffer B, and elution buffer C (preheated to 40 ℃) sequentially to the sample, and wash the tissue sample in a shaker at 40 ℃ and a speed of 60–70 r / min for 5 min each time; among which, the mouse brain was tested... Gad1 The sequence information of the gene-matching type O probe is shown below: O-type probe bottom region sequence: 5'-TGGCGTTCGGTCGACTAG-3' (SEQ ID No. 19); O-type probe lateral region sequence: 5'-TCCGATAT-3'; O-type probe signal identification region sequence: 5'-CCAATGATCGTCCGATAT-3' (SEQ ID No. 20).

[0078] ④ Fourth step: Hybridization and elution: Add the S-type probe to 50 nM concentration in 500 μL of prehybridization solution D, mix thoroughly, and preheat in a 40 ℃ metal bath for 10 min; then, add the preheated mixture to the mouse brain tissue slices to be tested, and incubate at 40 ℃ for 2–4 h to allow the S-type probe to form a stable hybridization structure with the O-type and U-type probes; after hybridization, discard hybridization solution D, and add elution buffer A, elution buffer B, and elution buffer C (preheated to 40 ℃) sequentially to the sample, and wash the tissue sample in a shaker at 40 ℃ and a speed of 60–70 r / min for 5 min each time; among which, the mouse brain was tested... Gad1 The sequence information of the gene-matched S-type probe is shown below: The sequence of the S-shaped probe's basal region is: 5'-TACCGTTAGAGCATCGAT-3' (SEQ ID No. 21). The sequence of the tip region of the S-type probe is: 5'-ATATCGGA-3'.

[0079] ⑤ Fifth step DNA amplification: After completing the fourth step of hybridization, add the DNA polymerase amplification system to the tissue sample, including 1:10 diluted Phi29 DNA polymerase, 1 × Phi29 DNA polymerase reaction buffer, 0.4 mg / mL recombinant albumin, 1 mM dTTP, 1 mM dGTP, 1 mM dATP and 1 mM dCTP. Incubate at 30 ℃ for 1 to 4 hours, so that the S-type probe uses the O-type probe as a template and undergoes an amplification reaction under the action of DNA polymerase, thereby achieving effective signal amplification.

[0080] ⑥ Step 6: Hybridization and Elution of Signal Probe: Add the signal probe with a final concentration of 100 nM to 500 μL of prehybridization solution D, mix thoroughly, and preheat in a 40 ℃ metal bath for 10 min. Then, add the preheated mixture to the mouse brain tissue slices to be tested and incubate at 40 ℃ for 2–3 h to hybridize the signal probe with the O-type probe nucleic acid sequence obtained in Step 5. After hybridization, discard hybridization solution D, and add elution buffer A, elution buffer B, and elution buffer C (preheated to 40 ℃) sequentially to the sample. Wash the tissue sample in a shaker at 40 ℃ and a speed of 60–70 r / min for 5 min each time. Both ends of the signal probe are modified with Alexa Fluor 594 fluorescence, and its sequence information is shown below: 5'-CCAATGATCGTCCGATAT-3' (SEQ ID No. 22).

[0081] ⑦ Stain cell nuclei in the tissue with DAPI for 2 min, then dehydrate with a gradient of 70%, 85%, and 100% alcohol, and finally mount the slide for microscopic photography; the results are as follows. Figure 3 As shown in the in-situ detection results of this application, it can be seen that mouse brain tissue sections can be effectively detected using only one SpaceAS probe set. Gad1 The expression of gene mRNA was analyzed, and the results showed that the SpaceAS strategy has a good ability to detect alternative splicing short sequences in tissues.

[0082] In Example 2, the prehybridization solution and elution buffer are the same as in Example 1.

[0083] Example 3 Example 3: SpaceAS dual-color signal probe for locating mouse brain tissue Sst The method for detecting gene mRNA includes the following steps: 3.1 Pretreatment of tissue samples Dual-color signal probes locate mouse brain tissue Sst The sample preprocessing for the gene method is the same as the steps in step 2.1 of Example 2.

[0084] 3.2 Hybridization steps for tissue samples Dual-color signal probes locate mouse brain tissue Sst The hybridization steps for tissue samples using the gene method are consistent with step 2.2 in Example 2. There are two differences: first, in the third hybridization step, the O-type probe has recognition regions for both signal probes; second, in the sixth hybridization step, two fluorescently modified signal probes, signal probe A and signal probe B, need to be added simultaneously to increase the throughput of the detection signal. SpaceAS dual-color signal needle localization in mouse brain tissue... Sst The sequence information of the π-type probe, U-type probe, O-type probe, S-type probe, and signal probe in the gene composition is as follows: Sequence information of the π-type probe: Sequence of the left π-leg region of the π-type probe: 5'-AGTACTTGGCCAGTTCCTGTTTCCC-3' (SEQ ID No. 23); Sequence of the left π-top region of the π-type probe: 5'-CAAGATCACGATGC-3' (SEQ ID No. 24); Sequence of the left π region of the π-type probe: 5'-TGGACTTA-3'; Sequence of the right π-leg region of the π-type probe: 5'-GCTCGGACAGCAGCTCTGCCAAG-3' (SEQ ID No. 25); Sequence of the right π-top region of the π-type probe: 5'-CGTAGCACTAGAAC-3' (SEQ ID No. 26); Sequence of the right π region of the π-type probe: 5'-ATTCACCT-3'.

[0085] Sequence information of the U-shaped probe: U-shaped probe middle region sequence: 5'-GTTCTAGTGCTACGAGCATCGTGATCTTG-3' (SEQ ID No. 27); The sequence of region I at both ends of the U-shaped probe is: 5'-CTAACTATCGATCGTGCATC-3' (SEQ ID No. 28); The sequence of region II at both ends of the U-shaped probe is: 5'-AACTCGTTCTAGCCTAGA-3' (SEQ ID No. 29).

[0086] Sequence information of type O probes: O-type probe bottom region sequence: 5'-GATGCACGATCGATAGTTAG-3' (SEQ ID No. 30); O-type probe lateral region sequence: 5'-TTGAGAC-3'; O-type probe signal identification region sequence: 5'-AGTACGGTCCATTGAGAC-3' (SEQ ID No. 31); 5'-CCAATGATCGTCCGATAT-3' (SEQ ID No. 32).

[0087] Sequence information of the S-type probe: The sequence of the bottom region of the S-type probe is: 5'-TCTAGGCTAGAACGAGTT-3' (SEQ ID No. 33). S-type probe tip region sequence: 5'-GTCTCAA-3'.

[0088] Sequence information of the signal probe: Both ends of signal probe A are modified with Alexa Fluor 488 fluorescence. Its sequence information is as follows: 5'-AGTACGGTCCATTGAGAC-3' (SEQ ID No. 34); Both ends of signal probe B are modified with Alexa Fluor 594 fluorescence, and its sequence information is as follows: 5'-CCAATGATCGTCCGATAT-3' (SEQ ID No. 35).

[0089] The results are as follows Figure 4 As shown in the in situ detection results of this application, the overlap rate of signals of single genes located by the SpaceAS probe group in two colors is very high, indicating that SpaceAS can effectively utilize the design of single and dual channels to realize multi-channel signal recognition and detection, thereby improving the detection throughput.

[0090] Example 4 Example 4: SpaceAS two-round hybridization detection of mouse brain tissue VIP and Gad1 Gene mRNA was used to demonstrate a multi-round detection method, which includes the following steps: 4.1 Pretreatment of tissue samples Multiple rounds of testing of mouse brain tissue VIP and Gad1 The sample pretreatment for the gene mRNA method is the same as the steps in step 2.1 of Example 2.

[0091] 3.2 Multi-round hybridization steps for tissue samples: ① First round of hybridization process: First, in mouse brain tissue... VIPThe mRNA of the gene is hybridized. The hybridization steps are the same as those in step 2.2 of Example 2, except that in the fifth step of DNA amplification, dTTP in the nucleotide raw materials is replaced with dUTP, that is, dATP, dUTP, GTP and CTP are used as nucleotide raw materials for amplification, so that the USER enzyme in the second round of hybridization can specifically cleave the dUTP bases in the amplification product. After washing, the next round of hybridization can be carried out, thereby realizing multi-round cyclic signal detection.

[0092] ② Second round hybridization procedure: After the first round of hybridization, the tissue sections were washed three times with 1×DEPC-PBST for 5 min each time. Then, 50 μL of USER enzyme digestion system was added to the tissue and incubated at 37 ℃ for 30 min to specifically cleave dUTP in the amplified products. The digestion system included 1 μL USER enzyme, 5 μL 10× digestion buffer, 1 μL Murine RNase inhibitor, and 43 μL RNase-free water. Next, the tissue sections were washed three times with 1×DEPC-PBST for 5 min each time. Then, the second round of SpaceAS hybridization was performed, following the same steps as in step 2.2 of Example 2. SpaceAS was used to detect dUTP in mouse brain tissue. Gad1 The gene composition was identical to that in Example 2; SpaceAS was used to detect the gene in mouse brain tissue. VIP The gene composition, except for the π-type probe, is identical to the composition in Example 1, and the detection of [genes] in mouse brain tissue [is also performed]. VIP The sequence information of the π-type probe for the gene is as follows: Sequence information of the π-type probe: Sequence of the left π-leg region of the π-type probe: 5'-TTCTGCTAAGGGATTCTGCAAGATG-3' (SEQ ID No. 36); Sequence of the left π-top region of the π-type probe: 5'-CCATAACCATTCGC-3' (SEQ ID No. 37); Sequence of the left π region of the π-type probe: 5'-TGGACTTA-3'; Sequence of the right π-leg region of the π-type probe: 5'-GACACATCATAATAGGGTGTGCCAT-3' (SEQ ID No. 38); Sequence of the right π-top region of the π-type probe: 5'-CGCTTACCAATACC-3' (SEQ ID No. 39); Sequence of the right π region of the π-type probe: 5'-ATTCACCT-3'.

[0093] The results are as follows Figure 5 As shown, microscopic images of the tissue samples were taken after each round of hybridization. The results clearly show that SpaceAS detected the virus clearly after the first round of hybridization. VIP The signal was clearly detected by SpaceAS after the second round of hybridization. Gad1 The signal indicates that SpaceAS can effectively improve the throughput of detection signals through multiple rounds of iterative hybridization.

[0094] In Example 4, the prehybridization solution and elution buffer are the same as in Example 1.

[0095] Example 5 Example 5: SpaceAS detection of mouse brain tissue Nrxn1-SS4 and Nrxn2-SS4 A method for detecting variable spliceosomes, the method comprising the following steps: The same gene can generate multiple alternative splice variants through different splicing methods. However, different splice variants have high sequence similarity, leaving little room for designing specific probes. Therefore, it is difficult to distinguish different splice variants using existing technologies. The SpaceAS composition in Example 5, however, is not limited by nucleic acid sequence length, enabling effective detection of short nucleic acid sequences. It has the advantages of strong signal amplification and high detection efficiency, requiring only a pair of π-type probes to efficiently and specifically detect alternative splice variants in tissues. Detailed explanations will follow with specific application examples.

[0096] 5.1. Pretreatment of tissue samples Detection of mouse brain tissue Nrxn1-SS4 and Nrxn2-SS4 The sample preprocessing for the variable splice body method is the same as the steps in step 2.1 of Example 2.

[0097] 5.2 Hybridization steps for tissue samples Detection of mouse brain tissue Nrxn1-SS4 and Nrxn2-SS4 The hybridization steps for the alternative spliceosome method are consistent with step 2.2 in Example 2. The only difference is that in the first hybridization reaction, the final concentration of the sample needs to be 10 nM. Nrxn1-SS4 and Nrxn1-SS4 The π-type probe was added together with 500 μL of prehybridization solution A for hybridization. SpaceAS was used to detect π-type probes in mouse brain tissue. Nrxn1-SS4 The composition in the variable spliceosome, except for the π-type probe, is the same as the composition in Example 1; SpaceAS detection of mouse brain tissue Nrxn2-SS4 The composition of the variable splice body is the same as that in Example 2, except for the π-type probe.

[0098] Detection of mouse brain tissue Nrxn1-SS4The sequence information of the π-type probe of the variable spliceosome is as follows: Sequence of the left π-leg region of the π-type probe: 5'-ACCAAGTCGATATGGAATTCGCTGT-3' (SEQ ID No. 40); Sequence of the left π-top region of the π-shaped probe: 5'-CCATAACCATTCGC-3' (SEQ ID No. 41); Sequence of the left π region of the π-type probe: 5'-TGGACTTA-3'; Sequence of the right π-leg region of the π-type probe: 5'-TCGAGTAGCCATTCATCAACTACTC-3' (SEQ ID No. 42); Sequence of the right π-top region of the π-type probe: 5'-CGCTTACCAATACC-3' (SEQ ID No. 43); Sequence of the right π region of the π-type probe: 5'-ATTCACCT-3'.

[0099] Detection of mouse brain tissue Nrxn2-SS4 The sequence information of the π-type probe of the variable spliceosome is as follows: Sequence of the left π-leg region of the π-type probe: 5'-ACCAAGCCGGTAGGGGATTCTCTGT-3' (SEQ ID No. 44); Sequence of the left π-top region of the π-type probe: 5'-TTGCACGTAGATCG-3' (SEQ ID No. 45); Sequence of the left π region of the π-type probe: 5'-TGGACTTA-3'; Sequence of the right π-leg region of the π-type probe: 5'-TCGAGCAGCCACTCATCTACTACTC-3' (SEQ ID No. 46); Sequence of the right π-top region of the π-type probe: 5'-GCTAGATGCACGTT-3' (SEQ ID No. 47); Sequence of the right π region of the π-type probe: 5'-ATTCACCT-3'.

[0100] The results are as follows Figure 6 As shown in the results, the rat brain tissue can be clearly seen. Nrxn1-SS4 and Nrxn2-SS4 The expression of the variable spliceosome demonstrates that SpaceAS can effectively achieve in-situ detection of spatial variable spliceosomes in tissues.

[0101] Furthermore, the prehybridization solution and washing buffer solution described in this embodiment are the same as those in Example 1. It should be understood that the embodiments described in this application are only for illustrating specific implementation methods of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to the embodiments, various modifications, equivalent substitutions, or variations made by those skilled in the art to its technical solutions without departing from the core ideas and substance of this application should all fall within the scope of protection of this application.

Claims

1. A composition for in-situ spatial detection of nucleic acids using variable splicing, characterized in that, include: A π-type probe, used in the first hybridization step, consists of two single-stranded DNA molecules, left and right, forming a π-type structure. The π-type probe includes a π-foot region, a π-top region, and a partially complementary π-middle region. The π-foot region is used to specifically bind to the target nucleic acid sequence. A U-shaped probe, used for the second hybridization step, is a single-stranded DNA molecule; the U-shaped probe includes an intermediate region complementary to the π-top region, and two end regions I and II located on both sides of the intermediate region; The O-type probe, used in the third hybridization and fifth amplification reactions, is an in vitro circularized single-stranded circular DNA molecule; the O-type probe includes a signal recognition region and an O-type probe bottom region that is complementary to the two end regions I; The S-type probe, used in the fourth hybridization step and the fifth amplification step, is a single-stranded DNA molecule; the S-type probe includes a bottom region of the S-type probe that is complementary to the two end regions II, and a top region of the S-type probe that is complementary to the signal recognition region. The signal probe, used in the sixth step of hybridization, is a single-stranded DNA molecule. The signal probe is used to complement the amplified signal recognition region.

2. The composition according to claim 1, characterized in that, Also includes: DNA polymerase is used in the fifth step of the amplification reaction; the DNA polymerase is used to catalyze the rolling circle amplification reaction using an O-type probe as a template and an S-type probe as a primer. and / or Nucleotide raw materials, comprising a first nucleotide raw material and / or a second nucleotide raw material; the first nucleotide raw material includes deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate (dTTP); the second nucleotide raw material includes deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxyuridine triphosphate (dUTP).

3. The composition according to claim 1, characterized in that, The length of the π-foot region is 16-25 bases; the length of the π-top region is 12-16 bases; the length of the π-middle region is 6-8 bases, and there are 2-5 complementary bases between the two single-stranded DNA sequences; And / or, the total length of the U-shaped probe is 109–171 bases; the length of the middle region is 27–29 bases; the length of the two end regions I is 16–22 bases; the length of the two end regions II is 16–22 bases; And / or, the total length of the O-type probe is 40 to 70 bases; wherein, the length of the bottom region of the O-type probe is 16 to 22 bases; and the length of the signal probe recognition region is 16 to 22 bases. And / or, the total length of the S-type probe is 24 to 38 bases; wherein, the length of the bottom region of the S-type probe is 16 to 22 bases; and the length of the top region of the S-type probe is 8 to 16 bases.

4. The composition according to claim 1, characterized in that, In the U-shaped probe, each side of the middle region has at least one end region I and one end region II.

5. The composition according to claim 1, characterized in that, The O-type probe contains multiple signal recognition areas; And / or, the O-type probe includes one, two, or more signal recognition regions, which are used to combine a monochromatic fluorescent signal probe, a dual-color fluorescent signal probe, or a multi-color fluorescent signal probe, respectively.

6. The composition according to claim 1, characterized in that, The signal probe has fluorescent groups labeled at both its 5' and 3' ends; the fluorescent groups are selected from one or more of Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 700, Alexa Fluor 750, and Alexa Fluor 790.

7. A kit for in-situ spatial detection of nucleic acids using alternative splicing, characterized in that, Includes the composition as described in any one of claims 1 to 6.

8. The reagent kit according to claim 7, characterized in that, Also includes: Multiple prehybridization solutions and multiple elution buffers; The prehybridization solution contains dextran sulfate, SSC buffer, deionized formamide, lithium dodecyl sulfate (LDS), Denhardt's solution, and vanadate ribonucleoside complex (RVC). The elution buffer contains SSC buffer, deionized formamide, and lithium dodecyl sulfate.

9. The reagent kit according to claim 8, characterized in that, The various prehybridization solutions include prehybridization solution A, prehybridization solution B, prehybridization solution C, and prehybridization solution D; the various elution buffers include elution buffer A, elution buffer B, and elution buffer C; The prehybridization solution A was prepared as follows: 0.01–0.02 g dextran sulfate, 20–30 μL 20 × SSC buffer, 25–30 μL deionized formamide, 6–8 μL 3% LDS, 5–10 μL Denhardt's solution, 1–2 μL RVC, and RNase-free water to a final volume of 100 μL. The prehybridization solution B was prepared as follows: containing 0.01–0.02 g dextran sulfate, 20–25 μL 20 × SSC buffer, 15–20 μL deionized formamide, 8–10 μL 3% LDS, 8–10 μL Denhardt's solution, 1–2 μL LVC, and RNase-free water to a final volume of 100 μL. The prehybridization solution C was prepared as follows: containing 0.01–0.02 g dextran sulfate, 20–25 μL 20 × SSC buffer, 10–25 μL deionized formamide, 8–10 μL 3% LDS, 8–10 μL Denhardt's solution, 1–2 μL LVC, and RNase-free water to a final volume of 100 μL. The prehybridization solution D was prepared as follows: containing 0.01–0.02 g dextran sulfate, 20–25 μL 20 × SSC buffer, 5–10 μL deionized formamide, 8–10 μL 3% LDS, 8–10 μL Denhardt's solution, 1–2 μL LVC, and RNase-free water to a final volume of 100 μL. The preparation system of the elution buffer A is as follows: containing 10 μL 20 × SSC buffer, 20-25 μL deionized formamide, 1-2 μL 3% LDS, and RNase-free water to a final volume of 100 μL; The preparation system of the elution buffer B is as follows: 5 μL 20 × SSC buffer, 10-12 μL deionized formamide, 1-2 μL 3% LDS, and RNase-free water to a final volume of 100 μL. The elution buffer C was prepared as follows: 1 μL 20 × SSC buffer, 1–2 μL 3% LDS, and RNase-free water to a final volume of 100 μL.

10. A method for detecting nucleic acids in situ using the composition as described in any one of claims 1-6 or the kit as described in any one of claims 7-9, characterized in that, include: S100) provides pre-treated tissue samples; S200) First step hybridization: Hybridize the π-type probe with the target nucleic acid sequence of the tissue sample; S300) Second step hybridization: Hybridize the U-shaped probe with the π-shaped probe; S400) Third step of hybridization: Hybridize the O-type probe with the U-type probe; S500) Step 4 Hybridization: Hybridize the S-type probe with the O-type probe and the U-type probe; The fifth step of the S600 amplification reaction: Under the action of DNA polymerase, using the O-type probe as a template and the S-type probe as a primer, a rolling circle amplification reaction is performed. S700) Step 6 Hybridization: Hybridize the signal probe with the nucleic acid sequence obtained from the amplification reaction in Step 5.

11. The detection method according to claim 10, characterized in that, The first step of hybridization in S200 includes: adding the π-type probe to prehybridization solution A, mixing thoroughly, and then adding it to the tissue sample, incubating at 40 ℃ for 3 to 16 hours; subsequently, adding elution buffer A, elution buffer B, and elution buffer C to the tissue sample in sequence, and washing the tissue sample in a shaker at 40 ℃ and a speed of 60 to 70 r / min for 5 min each time; The second hybridization step (S300) includes: adding the U-shaped probe to prehybridization solution B, mixing thoroughly, and then adding it to the tissue sample that has undergone the first hybridization treatment, and incubating at 40 ℃ for 1 to 4 hours; adding elution buffer A, elution buffer B, and elution buffer C to the tissue sample in sequence, and washing the tissue sample in a shaker at 40 ℃ and a speed of 60 to 70 r / min for 3 to 5 minutes each time; The third hybridization step of S400 includes: adding the O-type probe to prehybridization solution C, mixing thoroughly, and then adding it to the tissue sample that has undergone the second hybridization treatment, and incubating at 40 ℃ for 1 to 4 hours; adding elution buffer A, elution buffer B, and elution buffer C to the tissue sample in sequence, and washing the tissue sample in a shaker at 40 ℃ and a speed of 60 to 70 r / min for 3 to 5 minutes each time; The fourth step of hybridization in the S500 method includes: adding the S-type probe to the prehybridization solution D, mixing thoroughly, and then adding it to the tissue sample that has undergone the third step of hybridization treatment, and incubating at 40 ℃ for 1 to 4 hours; adding elution buffer A, elution buffer B, and elution buffer C to the tissue sample in sequence, and washing the tissue sample in a shaker at 40 ℃ and a speed of 60 to 70 r / min for 3 to 5 minutes each time; The fifth step of the S600 amplification reaction includes: adding DNA polymerase to the tissue sample after the fourth step of hybridization treatment, incubating at 30 ℃ for 1 to 4 hours, so that the S-type probe uses the O-type probe as a template and performs rolling circle amplification under the action of DNA polymerase, thereby achieving effective signal amplification; then washing three times with 1 × DEPC-PBS, each time for 3 to 5 min; The sixth step of hybridization in S700 includes: adding the signal probe to prehybridization solution D, mixing thoroughly, and then adding it to the tissue sample that has undergone the fifth step amplification reaction, and incubating at 40 ℃ for 1 to 4 hours; adding elution buffer A, elution buffer B, and elution buffer C to the tissue sample in sequence, and washing the tissue sample in a shaker at 40 ℃ and a speed of 60 to 70 r / min for 3 to 5 minutes each time.

12. The detection method according to claim 10, characterized in that, The fifth step of the S600 amplification reaction includes: S610): DNA polymerase and nucleotide raw materials are added to the tissue sample system. Under the action of DNA polymerase, a single-round amplification reaction is performed using an O-type probe as a template and an S-type probe as a primer. The nucleotide raw materials include a first nucleotide raw material or a second nucleotide raw material. The first nucleotide raw material includes deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate (dTTP). The second nucleotide raw material includes deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxyuridine triphosphate (dUTP).

13. The detection method according to claim 10, characterized in that, The fifth step of the S600 amplification reaction includes: S620): DNA polymerase and second nucleotide raw materials are added to the tissue sample system. Under the action of DNA polymerase, using an O-type probe as a template and an S-type probe as a primer, an amplification product containing dUTP bases is formed. The second nucleotide raw materials include deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxyuridine triphosphate (dUTP). The detection method further includes: S800): USER enzyme is added to the tissue sample system containing dUTP bases for amplification. Under the action of USER enzyme, the dUTP bases in the amplification product are specifically cleaved and washed to facilitate the next round of hybridization and amplification reaction.

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