CN117343993A

CN117343993A - Method for in situ detection of target nucleic acid sequences in a sample

Info

Publication number: CN117343993A
Application number: CN202311632892.2A
Authority: CN
Inventors: 段力辉; 李芽; 黄元
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2023-12-01
Filing date: 2023-12-01
Publication date: 2024-01-05

Abstract

The present disclosure provides a method for in situ detection of a target nucleic acid sequence in a sample. The method comprises the following steps: providing at least one pair of split probe pairs and at least one padlock probe; contacting the sample with the pair of cleavage probes to hybridize the pair of cleavage probes to the target nucleic acid; adding the padlock probe, and enabling the padlock probe to be hybridized with the split probe pair part to form a nucleic acid loop with a notch; using a ligase to form a closed nucleic acid loop on the nicked nucleic acid loop; rolling circle amplification is carried out by taking the closed nucleic acid loop as a template in the presence of polymerase, and rolling circle amplification products are obtained. The method disclosed by the invention has the advantages of high resolution, high signal-to-noise ratio and high sensitivity.

Description

Method for in situ detection of target nucleic acid sequences in a sample

Technical Field

The present disclosure is in the field of biological detection, and in particular relates to a method for in situ detection of a target nucleic acid sequence in a sample.

Background

In recent years, single cell transcriptome sequencing (scRNA-seq) technology has evolved rapidly, providing a powerful tool for obtaining transcriptome information in single cells. Although single cell transcriptome sequencing techniques can obtain single cell gene expression information with high throughput, spatial dimension information of gene expression cannot be obtained. Positional information of expressed genes is critical for studying scientific problems such as tissue and organ development, intercellular communication and heterogeneity.

The existing fluorescence in situ hybridization technologies, such as smFISH, RNAscope, MERFISH, osmFISH, HCR and the like, have obvious defects. For example, smFISH has limitations in terms of low signal-to-noise ratio, low sensitivity to short transcripts, false positive signals caused by non-specific binding of labeled probes, and the lack of ability to detect multiple RNA molecules simultaneously. The principles of seqFISH and MERFISH are similar to smFISH in that they require a large number of gene-specific probes, confocal or super-resolution microscopes to acquire signals, and complex algorithms for probe design and analysis. However, low signal-to-noise ratio remains a major technical challenge for these methods, especially for tissue sections with autofluorescence intensity. Furthermore, because seqFISH and MERFISH rely on high resolution imaging, the imaging time is long, thus limiting their ability to process large area samples. Commercial methods such as RNAscope have limitations such as high cost of use, low throughput, etc., which also make it difficult to detect 10 or more genes simultaneously. The HCR technology has high signal amplification efficiency, but the number of genes which can be detected simultaneously is limited, and high-throughput detection of gene expression cannot be realized. In addition to the above methods, there are in situ hybridization techniques based on padlock probes and rolling circle amplification, such as HybISS. This method first reverses the RNA to cDNA in situ, then targets the cDNA using padlock probes, then adds the primers required for rolling circle amplification, and performs in situ rolling circle amplification to amplify the signal. Such as SCRINSHOT, which uses padlock probes to directly target RNA, and then uses the enzyme Sprint R to ligate the padlock probes into a circle and perform an in situ rolling circle amplification reaction.

In situ reverse transcription in HybISS technology is low in efficiency and high in cost, and the process has high requirements on RNA quality and is easy to cause RNA information loss. Sprint R enzyme used in SCRINSHOT technology can catalyze the ligation reaction between two adjacent DNA single strands and complementary RNA single strands, and the ligation activity and specificity of the enzyme are influenced by the type of base of the ligation site, the number of complementary bases, the experimental temperature and the ion concentration in the experimental system.

The above two techniques require the synthesis of large numbers of phosphorylated padlock probes, and these probes are only directed to one target sequence and cannot be applied to other target sequences, which greatly increases the cost.

Disclosure of Invention

In view of the problems in the prior art, it is an object of the present disclosure to provide a novel, high resolution, high signal to noise ratio, high sensitivity, reusable (low cost) method for in situ detection of a target nucleic acid sequence in a sample.

An aspect of the present disclosure provides a method of detecting a target nucleic acid sequence in a sample, the method comprising the steps of:

(a) Providing at least one pair of split probes and at least one padlock probe,

(b) Contacting the sample with the pair of cleavage probes, hybridizing the pair of cleavage probes to the target nucleic acid,

(c) Adding the padlock probe, hybridizing the padlock probe with the split probe pair part to form a nucleic acid loop with a notch,

(d) Forming a closed nucleic acid loop from the nicked nucleic acid loop using a ligase,

(e) Rolling circle amplification is carried out by taking the closed nucleic acid loop as a template in the presence of polymerase, and rolling circle amplification products are obtained.

In some embodiments, the split probe pair comprises a first split probe and a second split probe.

In some embodiments, the first split probe and/or the second split probe comprises a 5 'single-stranded region, a stem-strand region, and a 3' single-stranded region.

In some embodiments, the 5 'or 3' single stranded region of the first split probe hybridizes to a first portion of the target nucleic acid to form a first complementary binding region and the 5 'or 3' single stranded region of the second split probe hybridizes to a second portion of the target nucleic acid to form a second complementary binding region.

In some embodiments, the 5 'or 3' single-stranded region of the first split probe hybridizes to the 5 'and 3' single-stranded regions of the padlock probe to form a third complementary binding region, and the 5 'or 3' single-stranded region of the second split probe hybridizes to the padlock probe portion to form a fourth complementary binding region.

In some embodiments, the 5 'or 3' single-stranded region of the second split probe hybridizes to the 5 'and 3' single-stranded regions of the padlock probe to form a third complementary binding region, and the 5 'or 3' single-stranded region of the first split probe hybridizes to the padlock probe portion to form a fourth complementary binding.

In some embodiments, in step (e), the rolling circle amplification uses one of the split probes as a primer, or an additional added primer probe as a primer, wherein the primer probe specifically binds to a portion of the padlock probe. Preferably, the primer probe also binds specifically to the second split probe moiety. More preferably, the primer probe further specifically binds to a target nucleic acid.

In some embodiments, the notch is located in the third complementary binding region.

In some embodiments, the first split probe and/or the second split probe is at least 20 nucleotides, preferably 20-80 nucleotides in length, e.g., can be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 nucleotides in length.

In some embodiments, the 5' single-stranded region is 8-40 nucleotides in length, e.g., can be 10, 15, 20, 25, 30, 35, or 40 nucleotides in length.

In some embodiments, the length of the stem-chain region is 2-6 nucleotides, e.g., can be 2, 3, 4, 5, or 6 nucleotides.

In some embodiments, the 3' single-stranded region is 8-40 nucleotides in length, e.g., can be 8, 10, 15, 20, 25, 30, 40 nucleotides in length.

In some embodiments, the first complementary binding region and/or the second complementary binding region is at least 10 nucleotides, preferably 10-40 nucleotides in length, e.g., can be 10, 14, 15, 20, 25, 30, 35, or 40 nucleotides in length.

In some embodiments, the second split probe is not modified or modified between the last 3-5 bases of the 3' end to block 3' to 5' exonuclease activity of the polymerase. In some embodiments, the second split probe is thio-modified between the last 3-5 bases of the 3' end.

In some embodiments, the 3' terminal base of the second split probe is not modified, or is modified with an inverted dT/dG and/or other modification that blocks polymerase extension reactions.

In some embodiments, the 3' end of a portion of the second split probes in the at least one pair of split probes is modified.

In some embodiments, the second split probe is thio-modified between the last 3-5 bases of the 3' end. Such thio modification generally refers to the introduction of phosphorothioate linkages between bases (or nucleotides) such that the internucleotide linkages are resistant to the 3 'to 5' exonuclease activity of the nucleic acid polymerase, i.e., avoid the exonuclease activity of the Phi29 polymerase. Can be selected according to the actual situation.

In some embodiments, the 3' end of the second split probe is subjected to an inverted dT/dG modification or other modification that blocks polymerase extension reactions. When the content of the detected target nucleic acid is high, inverted dT/dG is added to the 3 '-end of the second split probe to cause 3' -3 'ligation, thereby inhibiting degradation of 3' exonuclease and extension of DNA polymerase.

In some embodiments, the padlock probe is at least 40 nucleotides, preferably 40-160 nucleotides in length, e.g., 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 nucleotides in length.

In some embodiments, the third and/or fourth complementary binding regions are 8-40 nucleotides in length, e.g., can be 8, 10, 15, 20, 25, 30, 35, 40 nucleotides in length.

In some embodiments, the padlock probe includes at least one readout sequence and at least one bridging sequence that spaces the at least one readout sequence apart. In some embodiments, the padlock probe comprises multiple read sequences for a single gene.

In some embodiments, the read sequence comprises at least 15 nucleotides, preferably 15-30 nucleotides, e.g., 15, 20, 25, 30 nucleotides.

In some embodiments, the interval between read sequences is 2-6 nucleotides, e.g., can be 2, 3, 4, 5, or 6 nucleotides.

In some embodiments, the padlock probe further comprises a bridging sequence.

In some embodiments, the 5' end of a portion of the at least one padlock probe is modified.

In some embodiments, the 5' end of the padlock probe is or is not phosphorylated.

In some embodiments, a portion of the padlock probe is modified at its 5' end by phosphorylation.

In some embodiments, the 5' ends of all padlock probes are phosphorylated.

When the target nucleic acid content is high, in order to ensure the detection efficiency, the 5' end of the padlock probe aiming at the same gene can be partially phosphorylated, namely, the phosphorylated (which can be connected into a ring) padlock probe and the non-phosphorylated (which cannot be connected into a ring) padlock probe are mixed according to a certain proportion for use, so that a certain proportion of target nucleic acid is blocked, and the target nucleic acid cannot be detected. Therefore, the microscope can collect signal points with clear boundaries, and the quantification is more facilitated.

In some embodiments, the method further comprises: and (f) contacting the rolling circle amplification product with a reading probe and/or an amplifying probe to obtain the spatial position and/or sequence information of the target nucleic acid. In some embodiments, the readout probe hybridizes to the readout sequence.

In some embodiments, the magnifying probe has a binding region that hybridizes to the padlock probe and a non-binding region at one or both ends of the binding region that does not hybridize to the readout sequence.

In some embodiments, the magnifying probe has a binding region that hybridizes to the readout sequence and/or the bridging sequence.

In some embodiments, the readout probe and/or amplification probe comprises at least one of A, T, C or G bases, preferably comprises only at least two of A, T, C bases, more preferably comprises only A, T and C bases.

In some embodiments, the readout probe and/or amplification probe is provided with at least one fluorescent labeling group, magnetic particle, or radiolabel.

In some embodiments, the readout probe has a disulfide bond or other chemical linkage with a fluorescent labeling group, magnetic particle, or radiolabel. In some embodiments, the other chemical attachment means may be selected from amine reactive linkers, thiol reactive linkers, or click chemical linkers (e.g., NHS-azide linkers, NHS-DBCO linkers, maleimide-azide linkers, and maleimide-DBCO linkers) having a carbon chain (e.g., an issp 6 (C6 carbon chain)) containing a succinimidyl ester group, and the like.

In some embodiments, treatment with tris- (2-carboxyethyl) phosphine hydrochloride (TCEP) can break disulfide bonds in the readout probe, i.e., the fluorophore is detached from the readout probe, facilitating rapid multiple rounds of imaging.

In some embodiments, the readout probe carries a variety of fluorescent labeling groups, including, but not limited to: the groups of Alexa Fluor488, alexa Fluor546, alexa Fluor594, alexa Fluor647, alexa Fluor750, IRDye800cw, FITC, cy, cy5, cy7, etc. can be arranged and combined to represent different genes by different combinations, so that detection of a plurality of genes can be performed simultaneously in one experiment. After the image is acquired, decoding can be carried out according to the fluorescent signal types of the signal points, and gene expression information can be obtained. In the present disclosure, the detected signals may be represented by four, three, two or one fluorescent signals, i.e., four, three, two or one color codes. In the methods of the present disclosure, an amplification probe may further be used to amplify the fluorescent signal.

In some embodiments, the method further comprises, prior to step (f), subjecting the tissue sample containing the rolling circle amplification product to a tissue transparentization treatment. In the present disclosure, by combining tissue transparentization techniques, the background signal caused by autofluorescence and non-specific binding in tissue is further reduced.

In some embodiments, the tissue transparentization process includes: firstly, fixing a rolling circle amplification product and tissues around the rolling circle amplification product, then, reacting the fixed tissue sample with melphalan X, acryl-X and SE, and then, contacting the reacted product with gel to further fix the rolling circle amplification product on the gel; and, subjecting the gel immobilized with the rolling circle amplification product to enzymolysis treatment.

In some embodiments, the gel is a polyacrylamide gel.

In some embodiments, the tissue transparentization process includes: after rolling circle amplification, the rolling circle amplification product is immobilized onto the surrounding protein using an immobilization agent comprising 4% pfa and/or BS (PEG) ₉ . The rolling circle amplification product is then immobilized using melphalan X, one end of which may bind to the rolling circle amplification product and the other end of which may bind to the gel such that the rolling circle amplification product is immobilized on the gel. Then, the tissue is treated with protease K, SDS, triton X-100, etc., and the protein lipid and other components in the tissue are removed, followed by hybridization with a readout probe, and finally image acquisition. The background can be reduced by combining the tissue transparentization technology, the nonspecific signals are removed, and the signal to noise ratio is improved.

In some embodiments, the ligase is a T4 DNA ligase.

In some embodiments, the polymerase is Phi29 DNA polymerase.

In some embodiments, the target nucleic acid is RNA or DNA, preferably RNA, complementary DNA (cDNA), or genomic DNA.

In some embodiments, the nicked nucleic acid loop is a single-stranded DNA structure.

In some embodiments, the sample is from a biological sample, preferably from a biological tissue section or cell. In some embodiments, the sample is from an immobilized permeabilized biological tissue section or cell.

Another aspect of the present disclosure provides a probe set for in situ detection of a target nucleic acid sequence in a sample, comprising at least one pair of split probe pairs and at least one padlock probe.

In some embodiments, the 5 'single-stranded region or the 3' single-stranded region of the first split probe is capable of hybridizing to a first portion of the target nucleic acid to form a first complementary binding region, and the 5 'single-stranded region or the 3' single-stranded region of the second split probe is capable of hybridizing to a second portion of the target nucleic acid to form a second complementary binding region.

In some embodiments, the 5 'or 3' single-stranded region of the first split probe is capable of hybridizing to the 5 'and 3' single-stranded regions of the padlock probe to form a third complementary binding region, and the 5 'or 3' single-stranded region of the second split probe is capable of hybridizing to the padlock probe portion to form a fourth complementary binding region.

In some embodiments, the 5' single-stranded region or the 3' single-stranded region of the second split probe is capable of hybridizing to the 5' single-stranded region and the 3' single-stranded region of the padlock probe to form a third complementary binding region, and the 5' single-stranded region of the first split probe is capable of hybridizing to the padlock probe portion to form a fourth complementary binding region.

In some embodiments, the 5' single-stranded region is 8-40 nucleotides in length, e.g., can be 8, 10, 15, 20, 25, 30, 35, or 40 nucleotides in length.

In some embodiments, the phosphate bond between the last 3-5 bases of the 3' end of the second split probe is not modified or modified, preferably thio modified, to block 3' to 5' exonuclease activity of the polymerase.

In some embodiments, the 3' end of a portion of the second split probes of the at least one pair of split probes is modified to block the exonuclease activity of the polymerase. In some embodiments, the second split probe is thio-modified, preferably phosphorothioate-modified, between the last 3-5 bases of the 3' end.

In some embodiments, the 3' terminal base of the second split probe is subjected to an inverted dT/dG modification or other modification that blocks the polymerase extension reaction.

In some embodiments, the padlock probe is at least 40 nucleotides, preferably 40-160 nucleotides in length, e.g., 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 nucleotides in length.

In some embodiments, the padlock probe includes at least one readout sequence and at least one bridging sequence that spaces the at least one readout sequence apart.

In some embodiments, the readout sequence comprises at least 15 nucleotides, preferably 15-30 nucleotides, e.g., 15, 20, 25, 30 nucleotides.

In some embodiments, the padlock probe further comprises a bridging sequence.

In some embodiments, the 5' ends of all padlock probes are phosphorylated.

In some embodiments, the probe set further comprises a readout probe and/or an amplification probe.

In some embodiments, the readout probe hybridizes to the readout sequence.

In some embodiments, the amplification probe has a binding region that hybridizes to the padlock probe and a non-binding region that does not hybridize to the readout sequence at one or both ends of the binding region, preferably the amplification probe has a binding region that hybridizes to the readout sequence and/or the bridging sequence.

In some embodiments, the readout probe hybridizes to the readout sequence.

In some embodiments, the readout probe comprises at least one of A, T, C or G bases, preferably comprises only at least two of A, T, C bases, more preferably comprises only A, T and C bases.

In some embodiments, the readout probe carries at least one fluorescent labeling group, magnetic particle, or radiolabel.

In some embodiments, the readout probe carries a variety of fluorescent labeling groups, including, but not limited to: alexa Fluor488, alexa Fluor546, alexa Fluor594, alexa Fluor647, alexa Fluor750, IRDye800cw, FITC, cy3, cy5, cy7, and the like.

In some embodiments, the ligase is a T4 DNA ligase.

In some embodiments, the polymerase is Phi29 DNA polymerase.

Yet another aspect of the present disclosure provides a kit comprising the aforementioned probe set and a ligase and/or polymerase.

In some embodiments, the ligase is a T4 DNA ligase.

In some embodiments, the polymerase is Phi29 DNA polymerase.

Yet another aspect of the present disclosure provides a system for in situ detection of a target nucleic acid sequence in a sample, the system comprising:

a split probe design module for designing a split probe, wherein the split probe pair hybridizes to the target nucleic acid;

a padlock probe design module for designing padlock probes, wherein the padlock probes partially hybridize with the split probe pair to form a nucleic acid loop with a nick;

A hybridization module for contacting the sample with the split probe pair, hybridizing the split probe pair with the target nucleic acid, then adding the padlock probe, hybridizing the padlock probe with the split probe pair portion to form a nucleic acid loop with a nick,

a ligation module for forming a closed nucleic acid loop on the nicked nucleic acid loop by using a ligase;

and the amplification module is used for rolling circle amplification by taking the closed nucleic acid loop as a template in the presence of polymerase to obtain a rolling circle amplification product.

The split probe and padlock probe are as defined hereinbefore.

In some embodiments, the system further comprises: a read probe design module for designing read probes and/or amplification probes; and a signal generating module for contacting the rolling circle amplification product with a readout probe and/or an amplification probe to generate a signal of the spatial position and/or sequence of the target nucleic acid.

In some embodiments, the system further comprises a detection module for detecting the generated signal. In some embodiments, the detection module may be an imaging device and/or a sequencing device.

Yet another aspect of the present disclosure provides the use of the method according to the foregoing, the probe set of the foregoing, the kit of the foregoing, or the system of the foregoing in situ detection and flow cytometry.

In some embodiments, the in situ detection is tissue or cell in situ detection.

In some embodiments, for obtaining spatial position and/or sequence information of a target nucleic acid in a sample, e.g., for detection of a spatial transcriptome.

The present disclosure uses at least one pair of a split probe and padlock probe to target a target nucleic acid, preferably avoiding the use of a controversial Sprint R ligase while using a highly specific T4 DNA ligase, and without the need for in situ reverse transcription. This also reduces the likelihood of loss of RNA information and reduces costs. At the same time, the use of split probes ensures the specificity of the detection signal. Only when the first split probe and the second split probe are present at the same time will the target signal be amplified and subjected to rolling circle amplification to produce a rolling circle amplification product which is then recognized by the read probe. Without the first split probe, padlock probes cannot be ligated into a circle and subsequent rolling circle amplification cannot be performed. In the absence of the second split probe, rolling circle amplification does not initiate the primers required for amplification nor does rolling circle amplification products result. The presence of split probes also eliminates the need for additional primers necessary for rolling circle amplification.

Padlock probes consist of a bridging sequence and at least one or more identical or different read-out sequences. Therefore, the rolling circle amplification product can be identified by at least one or more same or different readout probes with fluorescent groups, so that the background noise can be effectively removed, and the signal-to-noise ratio can be improved. Further, padlock probes with different read-out sequences and bridging sequences and split probes with their corresponding bridging sequences can be used simultaneously in one experiment to detect multiple genes, achieving high throughput hybridization.

The design of the reading probe effectively avoids the generation of a secondary structure and improves the signal detection efficiency. The padlock probe and the reading probe in the method are designed in advance, can be repeatedly used through programs and manual screening, and when a new gene is detected, only a split probe of the target nucleic acid is generated and then synthesized through a split probe generation program, so that the cost is greatly reduced and the time is saved.

The method also has the characteristics of high sensitivity and high resolution, in-situ rolling circle amplification can generate rolling circle amplification products with the size of 10 Kb in a short time, the signals detected by the split probes are greatly amplified, and the rolling circle amplification products display signal points with clear boundaries under a 20X lens. The method not only can realize qualitative detection of gene expression in subcellular structure, but also can quantitatively detect gene expression. The method has simple and rapid experimental flow, can simultaneously detect the expression of a plurality of genes in the tissue slice within 24 hours, and has higher specificity and signal amplification efficiency. The method can be used simultaneously with immunofluorescent staining.

Drawings

Fig. 1 shows a schematic flow chart of example 1.

FIG. 2 shows the different binding patterns (A to H) of the split probe, padlock probe and target nucleic acid.

FIG. 3 shows a schematic representation of hybridization with rolling circle amplification products using different fluorescently labeled read probes.

FIG. 4 shows a schematic representation of the detection of multiple genes in one experiment using either a single or dual color coding scheme.

FIG. 5 shows a schematic representation of hybridization with rolling circle amplification products using both read probes and amplification probes.

FIG. 6 shows the examination of adult mouse brain tissue sections in example 1SstThe result of the signal, where A is the combined image of AF488, AF546 acquired and DAPI stained, B is the DAPI stained image, C is the image of AF488 acquired, and D is the combined image of AF546 acquired.

FIG. 7 shows a schematic of the addition of an inverted dT modification (A) to the 3 'end of a portion of the second split probe and a partial phosphorylation modification (B) to the 5' end of the padlock probe.

FIG. 8 shows the detection of adult mouse brain tissue sections in example 2SstThe result of the signal, wherein A (blocker+) is detected in a one-to-one mixture using split probe P2 with and without inverted dT added at the 3' endSstAs a result of the signal, B (Blocker-) is a split probe P2 detection using dT without addition of inversionSstAs a result of the signal, C is detected in the two ways SstStatistical analysis of the signal results.

Fig. 9 shows a schematic flow chart of example 3.

Fig. 10 shows the results of the tissue transparency process performed in example 3, where a is a combined image of AF488, AF546, and DAPI staining, B is an image of AF488, and C is a combined image of AF 546.

Fig. 11 shows the results of example 3 without tissue transparency treatment, where a is the combined image of AF488, AF546 and DAPI staining, B is the image of AF488, and C is the combined image of AF 546.

FIG. 12 shows the use of the present disclosure to detect SST proteins while using SST antibodies in example 4SstResults of mRNA signal, wherein A is DAPI stained image, B is SST protein signal image, C isSstImages of mRNA signal, D is DAPI staining, SST protein signal andSstcombined image of mRNA signals.

Detailed Description

In order to make the objects, technical solutions and advantages of the present disclosure more apparent, the present disclosure will be further described in detail with reference to the following examples. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the disclosure in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.

In this method, referring to fig. 1, a pair of split probes are first used to target RNA or DNA, and then padlock probes are combined with the split probes to form a stable nicked DNA double-stranded structure. The 5' phosphorylated padlock probes are then ligated into a circle using T4 DNA ligase, providing a template for rolling circle amplification. Finally, the loop rolling amplification is started under the action of Phi29 DNA polymerase, namely high-fidelity polymerase with chain replacement, so that the detected signal is amplified with higher efficiency. The padlock probe comprises at least one readout sequence and at least one bridging sequence separating the at least one readout sequence. The present method uses padlock probes with different read-out sequences and bridging sequences in combination with corresponding split probes to identify different genes. The fluorescent-bearing read probe binds to the rolling circle amplification product, allowing the amplified signal to be detected by a microscope. The genes represented by each signal point are decoded according to the read fluorescent probe combination. The padlock probe and the reading probe are designed in advance and can be reused.

Fig. 2A illustrates an embodiment according to the present disclosure. In this embodiment, the 3 'single-stranded region of the first split probe hybridizes to a first portion of the target nucleic acid to form a first complementary binding region, the 5' single-stranded region of the second split probe hybridizes to a second portion of the target nucleic acid to form a second complementary binding region, the 5 'single-stranded region of the first split probe hybridizes to the 5' single-stranded region and the 3 'single-stranded region of the padlock probe to form a third complementary binding region, and the 3' single-stranded region of the second split probe hybridizes to the padlock probe portion to form a fourth complementary binding region.

Fig. 2B illustrates an embodiment according to the present disclosure. In this embodiment, the 3 'single-stranded region of the first split probe hybridizes to a first portion of the target nucleic acid to form a first complementary binding region, the 5' single-stranded region of the second split probe hybridizes to a second portion of the target nucleic acid to form a second complementary binding region, the 3 'single-stranded region of the second split probe hybridizes to the 5' single-stranded region and the 3 'single-stranded region of the padlock probe to form a third complementary binding region, and the 5' single-stranded region of the first split probe hybridizes to the padlock probe portion to form a fourth complementary binding region.

Fig. 2C illustrates an embodiment according to the present disclosure. In this embodiment, the 5 'single-stranded region of the first split probe hybridizes to a first portion of the target nucleic acid to form a first complementary binding region, the 5' single-stranded region of the second split probe hybridizes to a second portion of the target nucleic acid to form a second complementary binding region, the 3 'single-stranded region of the first split probe hybridizes to the 5' single-stranded region and the 3 'single-stranded region of the padlock probe to form a third complementary binding region, and the 3' single-stranded region of the second split probe hybridizes to the padlock probe portion to form a fourth complementary binding region.

Fig. 2D illustrates an embodiment according to the present disclosure. In this embodiment, the 5 'single-stranded region of the first split probe hybridizes to a first portion of the target nucleic acid to form a first complementary binding region, the 5' single-stranded region of the second split probe hybridizes to a second portion of the target nucleic acid to form a second complementary binding region, the 3 'single-stranded region of the second split probe hybridizes to the 5' single-stranded region and the 3 'single-stranded region of the padlock probe to form a third complementary binding region, and the 3' single-stranded region of the first split probe hybridizes to the padlock probe portion to form a fourth complementary binding region.

Fig. 2E illustrates an embodiment according to the present disclosure. In this embodiment, the 3 'single-stranded region of the first split probe hybridizes to a first portion of the target nucleic acid to form a first complementary binding region, the 3' single-stranded region of the second split probe hybridizes to a second portion of the target nucleic acid to form a second complementary binding region, the 5 'single-stranded region of the first split probe hybridizes to the 5' single-stranded region and the 3 'single-stranded region of the padlock probe to form a third complementary binding region, and the 5' single-stranded region of the second split probe hybridizes to the padlock probe portion to form a fourth complementary binding region.

Fig. 2F illustrates an embodiment according to the present disclosure. In this embodiment, the 3 'single-stranded region of the first split probe hybridizes to a first portion of the target nucleic acid to form a first complementary binding region, the 3' single-stranded region of the second split probe hybridizes to a second portion of the target nucleic acid to form a second complementary binding region, the 5 'single-stranded region of the second split probe hybridizes to the 5' single-stranded region and the 3 'single-stranded region of the padlock probe to form a third complementary binding region, and the 5' single-stranded region of the first split probe hybridizes to the padlock probe portion to form a fourth complementary binding region.

Fig. 2G illustrates an embodiment according to the present disclosure. In this embodiment, the 5 'single-stranded region of the first split probe hybridizes to a first portion of the target nucleic acid to form a first complementary binding region, the 3' single-stranded region of the second split probe hybridizes to a second portion of the target nucleic acid to form a second complementary binding region, the 3 'single-stranded region of the first split probe hybridizes to the 5' single-stranded region and the 3 'single-stranded region of the padlock probe to form a third complementary binding region, and the 5' single-stranded region of the second split probe hybridizes to the padlock probe portion to form a fourth complementary binding region.

Fig. 2H illustrates an embodiment according to the present disclosure. In this embodiment, the 5 'single-stranded region of the first split probe hybridizes to a first portion of the target nucleic acid to form a first complementary binding region, the 3' single-stranded region of the second split probe hybridizes to a second portion of the target nucleic acid to form a second complementary binding region, the 5 'single-stranded region of the second split probe hybridizes to the 5' single-stranded region and the 3 'single-stranded region of the padlock probe to form a third complementary binding region, and the 3' single-stranded region of the first split probe hybridizes to the padlock probe portion to form a fourth complementary binding region.

Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the art to which this disclosure belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.

The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.

The term "about" as used herein means a range of + -20% of the numerical values thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value following that. In some embodiments, the term "about" means a range of ±5% of the numerical value following that.

The term "target nucleic acid" as used herein refers to any polynucleotide nucleic acid molecule (e.g., DNA molecule, RNA molecule, modified nucleic acid, etc.) present in a single cell. In some embodiments, the target nucleic acid is a coding RNA (e.g., mRNA). In some embodiments, the target nucleic acid is a non-coding RNA (e.g., tRNA, rRNA, microrna (miRNA), mature miRNA, immature miRNA, etc.). In some embodiments, the target nucleic acid is a splice variant of an RNA molecule (e.g., mRNA precursor, etc.) in a cellular environment. Thus, a suitable target nucleic acid can be an unspliced RNA (e.g., a pre-mRNA, an mRNA), a partially spliced RNA, or a fully spliced RNA, etc. The target nucleic acids of interest may be expressed to varying degrees, i.e., with varying abundance, in a population of cells, wherein the methods of the present disclosure allow for analysis and comparison of the expression levels of nucleic acids (including but not limited to RNA transcripts) in individual cells. The target nucleic acid may also be a DNA molecule, e.g., denatured genomic DNA, viral DNA, plasmid DNA, and the like. In some embodiments, the target nucleic acid is RNA. In these embodiments, the target nucleic acid may be mRNA. In other embodiments, the target nucleic acid is DNA, such as complementary DNA (cDNA) or genomic DNA.

The terms "oligonucleotide," "polynucleotide," and "nucleic acid molecule" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, i.e., ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA; genome DNA, cDNA, DNA-RNA hybrid; or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The backbone of the polynucleotide may include sugar and phosphate groups (typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide may comprise a polymer composed of synthetic subunits (e.g., phosphoramidites and/or phosphorothioates) and thus may be an oligodeoxynucleoside phosphoramidate or a mixed phosphoramidate-phosphodiester oligomer. The polynucleotide may comprise one or more L-nucleosides. Polynucleotides may include modified nucleotides, for example, methylated nucleotides and nucleotide analogs, uracil, other sugars, linking groups (e.g., fluororibose and thioester), and nucleotide branching. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be modified to include N3'-P5' (NP) phosphoramidates, morpholino phosphorodiamidates (MF), locked Nucleic Acids (LNA), 2 '-O-Methoxyethyl (MOE), or 2' -fluoroarabinonucleic acids (FANA), which may enhance the resistance of the polynucleotide to nuclease degradation. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. Other types of modifications included in this definition are capping, substitution of one or more naturally occurring nucleotides with an analog, and the introduction of a manner in which a polynucleotide is attached to a protein, metal ion, labeling component, other polynucleotide, or solid support. The immune modulatory nucleic acid molecules can be provided in a variety of formulations, e.g., associated with liposomes, microencapsulated, etc., as described in detail herein. The polynucleotide used in amplification is usually a single-stranded polynucleotide to maximize amplification efficiency, but may be a double-stranded polynucleotide. In the case of double-stranded polynucleotides, the polynucleotides may be treated to separate their double strands prior to use in preparing the extension products. This denaturation step is generally effected by high temperature, but it is also possible to carry out the relevant operation with a base and then to carry out the neutralization.

The term "nick" refers to a nick formed by the fact that the 5 '-phosphate of the 5' -terminal nucleotide of a nucleic acid loop is not covalently linked to the 3'-OH of the 3' -terminal nucleotide or the phosphodiester bond between two adjacent nucleotides is broken.

The term "thio modification" refers to the replacement of a non-bridging oxygen atom in the oligophosphate backbone with a sulfur atom to introduce a phosphorothioate linkage.

The term "total phosphorylation" refers to the total phosphorylation of probes directed to the same gene. In some embodiments, padlock probes directed against the same gene are all phosphorylated, and the phosphorylated padlock probes may be ligated into a loop to form a nucleic acid loop.

The term "inverted dT/dG modification" refers to the addition of an inverted dT/dG at the end of the probe, see FIG. 7A.

The term "partially phosphorylated" refers to the partial phosphorylation of probes directed to the same gene, i.e., the simultaneous presence of phosphorylated (which may be ligated into a loop) probes and non-phosphorylated (which may not be ligated into a loop) probes, see FIG. 7B.

The terms "hybridization" and "hybridization" as used herein refer to the formation of a complex between nucleotide sequences that are appropriately complementary to form a complex via Watson-Crick base pairing. In the case of "hybridization" of a primer to a target (template), such complexes (or hybrids) are sufficiently stable to exert priming effects such as those required to cause DNA polymerase to prime DNA synthesis. It will be appreciated that the hybrid sequences need not have perfect complementarity in providing stable hybrids. In many cases, stable hybrids are formed in which the proportion of mismatched bases is less than about 10% and loops consisting of four or more nucleotides are ignored. Thus, the term "complementary" as used herein refers to an oligonucleotide that forms a stable duplex with its "complement" under assay conditions (typically about 90% or more homology).

The term "melting temperature" or "Tm" as used herein refers to the temperature at which half of the helical structure of the nucleic acid is lost due to heating or otherwise dissociating hydrogen bonds between base pairs (e.g., by acid or base treatment, etc.). The Tm of a nucleic acid molecule depends on its length and its base composition. The Tm of a GC base pair-rich nucleic acid molecule is higher than the melting temperature of a nucleic acid molecule having a large number of AT base pairs. Upon the temperature falling below Tm, the separated complementary strands of the nucleic acid spontaneously reassociate or anneal to form a double-stranded nucleic acid. The highest nucleic acid hybridization rate occurs at about 25℃below the Tm. Tm can be estimated by the following relation: tm=69.3+0.41 (GC)%.

In some embodiments, the one or more first and second cleavage probes bind to different regions or target sites of the target nucleic acid. In one target site pair, each target site is different, and the target sites are adjacent sites on the target nucleic acid (e.g., typically no more than 15 nucleotides apart, e.g., no more than 10, 8, 6, 4, or 2 nucleotides apart from another site), and may be contiguous sites. The target sites are typically present on the same strand of the target nucleic acid in the same orientation. In addition, the target site will also be selected relative to other nucleic acids present in the cell to provide a unique binding site. Each target site is typically about 19 to about 25 nucleotides in length, e.g., about 19 to 23 nucleotides, about 19 to 21 nucleotides, or about 19 to 20 nucleotides. The first and second split probes are selected such that each split probe in the split probe pair has a similar melting temperature for binding to its cognate target site, e.g., the Tm may be no less than about 50 ℃, no less than about 52 ℃, no less than about 55 ℃, no less than about 58 ℃, no less than about 62 ℃, no less than about 65 ℃, no less than about 70 ℃, or no less than about 72 ℃. The GC content of the target site is typically selected to be no more than about 20%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%.

The terms "label" and "detectable label" as used herein refer to molecules capable of detection, including but not limited to radioisotopes, fluorescers, chemiluminescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens), and the like. The term "fluorescent labeling group" refers to a substance or portion thereof capable of exhibiting fluorescence in a detectable range. Specific examples of labels that may be used in conjunction with the present disclosure include, but are not limited to, phycoerythrin, alexa dye, fluorescein, YPet, cytot, cascade blue, allophycocyanin, cy3, cy5, cy7, rhodamine, dansyl, umbelliferone, texas Red, luminol, acridinium ester, biotin, green Fluorescent Protein (GFP), enhanced Green Fluorescent Protein (EGFP), yellow Fluorescent Protein (YFP), enhanced Yellow Fluorescent Protein (EYFP), blue Fluorescent Protein (BFP), red Fluorescent Protein (RFP), firefly luciferase, renilla luciferase, NADPH, β -galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenicol acetyl transferase, and urease. In some embodiments, the fluorescent labeling groups include, but are not limited to: the groups of Alexa Fluor488, alexa Fluor546, alexa Fluor594, alexa Fluor647, alexa Fluor750, IRDye800cw, FITC, cy, cy5, cy7, etc. can be arranged and combined to represent different genes by different combinations, so that detection of a plurality of genes can be performed simultaneously in one experiment. After the image is acquired, decoding can be carried out according to the fluorescent signal types of the signal points, and gene expression information can be obtained. In some embodiments, the detected signal may be represented by four, three, two, or one fluorescent signal, i.e., four, three, two, or single color coding, see fig. 3 and 4. In the methods of the present disclosure, an amplification probe may be further used to amplify the fluorescent signal, see fig. 5.

The term "ligase" as used herein refers to enzymes commonly used to ligate polynucleotides together or to ligate the ends of individual polynucleotides. Ligases include ATP-dependent double-stranded polynucleotide ligases, NAD-i-dependent double-stranded DNA or RNA ligases, and single-stranded polynucleotide ligases, e.g., any of the ligases described in EC 6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2 (NAD+ dependent ligases), EC 6.5.1.3 (RNA ligases). Specific examples of ligases include bacterial ligases, e.g., E.coli DNA ligases and Taq DNA ligases, thermostable DNA ligases and phage ligases, e.g., T3 DNA ligases, T4 DNA ligases and T7 DNA ligases and mutants thereof.

Rolling circle amplification techniques are well known in the art (see, e.g., baner et al, nucleic acids research, 26:5073-5078, 1998; lizardi et al, natl. Acad. Sci., 97:10113-119, 2000; faruqi et al, BMC genomics, 2:4, 2000; nallur et al, nucleic acids research, 29:el18, 2001; dean et al, genome research, 11:1095-1099, 2001; schweitzer et al, natl. Biotech, 20:359-365, 2002; U.S. Pat. Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329 and 6,368,801). In some embodiments, the polymerase is Phi29 DNA polymerase.

Tissue samples suitable for use in the methods described herein generally include any type of tissue sample collected from a living or dead subject, such as biopsy samples and autopsy samples, including but not limited to epithelial tissue, muscle tissue, connective tissue, and nerve tissue. Tissue samples may be collected and processed using the methods described herein and subjected to microscopic analysis immediately after processing, or stored and subjected to microscopic analysis at a later date (e.g., after a longer period of storage). In some embodiments, the methods described herein can be used to preserve tissue samples in a stable, accessible, and intact state for later analysis. In some embodiments, the methods described herein can be used to analyze previously preserved or stored tissue samples. In some embodiments, the intact tissue comprises brain tissue, such as a slice of the visual cortex. In some embodiments, the intact tissue is a thin slice having a thickness of 5-20 μm, including, but not limited to, for example, 5-18 μm, 5-15 μm, or 5-10 μm. In other embodiments, the intact tissue is a thick slice having a thickness of 50-200 μm, including, but not limited to, for example, 50-150 μm, 50-100 μm, or 50-80 μm.

Aspects of the present disclosure include securing intact tissue. The term "immobilization process" or "immobilization" as used herein refers to a process of preserving biological material (e.g., tissue, cells, organelles, molecules, etc.) from decay and/or degradation. The fixing may be accomplished according to any suitable scheme. Immobilization may include contacting the sample with an immobilization reagent (i.e., a reagent containing at least one immobilization agent). The sample may be contacted with the fixing reagent for an extended period of time, depending on the temperature, nature of the sample, and fixing agent. For example, the sample may be contacted with the immobilized reagent for 24 hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less. The sample may be contacted with the immobilized reagent at various temperatures, depending on the protocol and the reagents used. For example, in some cases, the sample may be contacted with the immobilized reagent at a temperature in the range of-80 ℃ to 55 ℃, where specific temperature ranges of interest include, but are not limited to, 50 to 54 ℃, 40 to 44 ℃, 35 to 39 ℃, 28 to 32 ℃, 20 to 26 ℃, 0 to 6 ℃, and-18 to-22 ℃. In some cases, the sample may be contacted with the immobilized reagent at a temperature of-20 ℃, 4 ℃, room temperature (22-25 ℃), 30 ℃, 37 ℃, 42 ℃, or 52 ℃.

Any suitable immobilization reagent may be used. Common fixing agents include crosslinking fixing agents, precipitation fixing agents, oxidation fixing agents, mercury agents, and the like. The crosslinking fixative chemically links two or more molecules through covalent bonds, and various crosslinking reagents may be used. Examples of suitable crosslinking fixatives include, but are not limited to, aldehydes (e.g., formaldehyde, also commonly referred to as "paraformaldehyde" and "formalin"; glutaraldehyde, etc.), imidoesters, NHS (N-hydroxysuccinimide) esters, and the like. Examples of suitable precipitation fixatives include, but are not limited to, alcohols (e.g., methanol, ethanol, etc.), acetone, acetic acid, and the like. In some embodiments, the fixative is formaldehyde (i.e., paraformaldehyde or formalin). Suitable final concentrations of formaldehyde in the fixing reagent are 0.1-10%, 1-8%, 1-4%, 1-2%, 3-5% or 3.5-4.5%, including about 1.6%, for 10 minutes. In some embodiments, the sample is fixed in formaldehyde at a final concentration of 4% (diluted with a stock solution at a higher concentration, e.g., 38%, 37%, 36%, 20%, 18%, 16%, 14%, 10%, 8%, 6%, etc.). In some embodiments, the sample is fixed in formaldehyde at a final concentration of 10%. In some embodiments, the sample is fixed in formaldehyde at a final concentration of 1%.

The term "permeabilization" or "permeabilization" as used herein refers to the process of making cells (cell membranes, etc.) in a sample permeable to an experimental reagent (e.g., a nucleic acid probe, an antibody, a chemical substrate, etc.). Any suitable method and/or reagent for permeabilization may be used. Suitable permeabilizing reagents include detergents (e.g., saponins, triton X-100, tween-20, etc.), organic fixatives (e.g., acetone, methanol, ethanol, etc.), pepsin, proteinase K, etc. Various concentrations of detergent may be used. For example, permeabilization can be achieved using 0.001% -1% detergent, 0.05% -0.5% detergent, or 0.1% -0.3% detergent (e.g., 0.1% saponin, 0.2% tween-20, 0.1-0.3% triton X-100, etc.). In some embodiments, permeabilization is performed using methanol (placed on ice) for at least 10 minutes.

The methods herein include a method for spatially localizing or in situ gene sequencing a target nucleic acid in cells within intact tissue. In certain embodiments, the cell is present in a population of cells. In certain other embodiments, the population of cells comprises any cell type. The cells used in the assays of the present disclosure may be organisms, single cell types derived from organisms, or mixtures of cell types. The present disclosure includes naturally occurring cells and cell populations, genetically engineered cell lines, cells derived from transgenic animals, and the like. Almost all cell types and sizes can be encompassed. Suitable cells include bacterial, fungal, plant and animal cells. In an embodiment of the disclosure, the cell is a mammalian cell, e.g., a complex cell population, e.g., naturally occurring tissue, e.g., blood, liver, pancreas, neural tissue, bone marrow, skin, etc. Some tissues may be dissociated to make a monodisperse suspension. Alternatively, the cells may be a population of cultured cells, e.g., a culture derived from a complex population of cells, a culture derived from a single cell type, wherein the cells have differentiated into multiple lineages, or wherein the cells respond differently to stimuli, etc.

Cell types used in the present disclosure include stem/progenitor cells (e.g., embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural crest cells, etc.), endothelial cells, muscle cells, cardiomyocytes, smooth and skeletal muscle cells, mesenchymal cells, epithelial cells; hematopoietic cells, e.g., lymphocytes, including T cells, e.g., th 1T cells, th 2T cells, thO T cells, cytotoxic T cells; b cells, pre-B cells, etc.; monocytes; dendritic cells; neutrophils; macrophages; natural killer cells; mast cells, and the like; adipocytes, cells associated with a particular organ, such as thymus, endocrine glands, pancreas, brain, such as neurons, glial cells, astrocytes, dendritic cells, and the like, as well as genetically modified cells thereof. Hematopoietic cells may be associated with inflammatory processes, autoimmune diseases, etc., endothelial cells, smooth muscle cells, cardiomyocytes, etc. may be associated with cardiovascular diseases; almost all types of cells may be associated with neoplasias, such as sarcomas, carcinomas, and lymphomas; liver diseases associated with liver cells; kidney disease associated with kidney cells; etc. The cells may be obtained from various tissues of the individual (e.g., blood, bone marrow), solid tissues (e.g., solid tumors), ascites by various techniques known in the art using methods such as extraction, lavage, washing, surgical dissection, etc. Cells may be obtained from fixed or unfixed, fresh or frozen, intact or dissociated samples. The tissue may be dissociated mechanically or enzymatically using known techniques.

The methods herein include imaging the target nucleic acid using any of a number of different types of microscopy techniques, such as confocal microscopy, fluorescence microscopy, two-photon microscopy, light sheet microscopy, whole tissue expansion microscopy, and/or CLARITYTM optimized monolayer light microscopy (COLM).

The following examples and figures are provided to aid in the understanding of the present disclosure. It is to be understood that these examples and drawings are for purposes of illustration only and are not to be construed as limiting the present disclosure in any way. The actual scope of the disclosure is set forth in the following claims. It will be understood that any modifications and variations may be made without departing from the spirit of the disclosure.

Example 1

Fresh brain tissue samples from C57BL/6J adult mice were embedded with a frozen section embedding medium and then rapidly frozen, and then stored in a refrigerator at-80℃and the frozen brain tissue samples were sliced in a frozen microtome to a thickness of 10 to 15. Mu.m, and the sliced tissues were stored in a refrigerator at-80℃for use.

Detection in adult mouse brain tissue sectionsSstSignal [ (x-ray)SstThe DNA sequence is shown as SEQ ID NO. 1), wherein the RNA signal is detected by two fluorescent probes with different fluorophores. The method comprises the following specific steps:

Sections were removed from the-80℃refrigerator and incubated with 4% paraformaldehyde for 20 minutes at room temperature to immobilize RNA. The reaction was then quenched with 1.25M glycine for 5 minutes at room temperature. After incubation with 0.5% Triton X-100 at room temperatureThe tissue is permeabilized for 15 minutes, and after permeabilization, the tissue is incubated with a blocking mixture (1X Ampligase buffer, 0.05M KCl, 20% deionized formamide, 0.1 mu M Oligo-dT,0.2 mu g/mu L BSA,0.2 mu g/mu L tRNA, 0.4U/mu L Murine RNase inhibitor) at room temperature. Tissues were then incubated with a solution (1X amplinase buffer, 0.05M KCl, 20% deionized formamide, 0.1. Mu.m Oligo-dT, 0.2. Mu.g/. Mu.L BSA, 0.2. Mu.g/mu.L tRNA, 0.4U/mu.L Murine RNase inhibitor, 20 nM split probe) containing split probes (probe pair Sst-73-1A, sst-73B; probe pair Sst-228-2A, sst-228-2B; probe pair Sst-330-3A, sst-330-3B; probe pair Sst-394-4A, sst-394-4B; SEQ ID NO: 2-9). After washing the tissue, the tissue was incubated with a solution (1 XAmpligase buffer, 0.05M KCl, 20% deionized formamide, 0.1 [ mu ] M Oligo-dT,0.2 [ mu ] g/[ mu ] L BSA,0.2 [ mu ] g/[ mu ] L tRNA, 0.4U/[ mu ] L Murine RNase inhibitor, 20 nM padlock probe) containing a padlock probe (SEQ ID NO: 11, wherein read sequence 1 is shown in SEQ ID NO: 12, and read sequence 2 is shown in SEQ ID NO: 13) at 37 ℃. After washing the tissue, the tissue was washed with T4 ligase buffer. The tissue was then incubated with T4 ligase at room temperature. After ligation, the tissues were incubated with a solution containing Phi29 DNA polymerase at 30℃for in situ rolling circle amplification. The amplified product is treated with 4% paraformaldehyde or 10 [ mu ] g/[ mu ] L BS (PEG) ₉ (PEGylated bis (sulfosuccinimidyl) succinate)) the immobilized rolling circle amplification product was incubated at room temperature. The tissue was then washed three times with 65% deionized formamide solution, and then incubated with hybridization buffer (2 XSSC, 20% deionized formamide, 0.2. Mu.L BSA, 0.5. Mu.g/mL PAPI, 2 nM fluorescent probe) containing deionized formamide and fluorescent probes (wherein the sequences of readout probes AF488-C6 spacer-RO25 and RO34-SS-AF546 are shown as SEQ ID NO: 14 and SEQ ID NO: 15, respectively) at room temperature, the fluorescent probes bound to the rolling circle amplification product. After washing the patch, the detected RNA signal was visualized by imaging under a fluorescent microscope (Axio imager.Z2) placed in Germany Cai Sizheng.

The results are shown in FIG. 6. The results indicate that both AF488 (FIG. 6C) and AF546 (FIG. 6D) may represent detectedSstmRNA signal, which can be further confirmed by co-localization of both (FIG. 6A)Recognizing the detected signal asSstmRNA signal. With fluorescent signalSstmRNA is gathered around the nucleus, and the mRNA is in a cell-like form and has a clear boundary. Each of the positive cellsSst mRNA all showed punctate signals.

Information on the partial sequences involved in the examples is provided in table 1 below.

TABLE 1

Note that: * Refers to thio modification; p refers to a phosphorylation modification; inverteddT refers to an Inverted dT modification.

Example 2

Rolling circle amplification was performed in the same manner as in example 1 except that Sst-228-2A (SEQ ID NO: 4) and Sst-228-2B (SEQ ID NO: 5) were used as the cleavage probe pairs, respectively, and Sst-228-2A (SEQ ID NO: 4) and Sst-228-2B blocker (SEQ ID NO: 10) were used as the cleavage probe pairs, i.e., sst-228-2B blocker was modified as shown in FIG. 7A.

As shown in FIGS. 8A-8C, it was revealed that the addition of an inverted dT to the 3' -end of the cleavage probe P2 blocked the target nucleic acid in a proportion such that it could not be detected. Thereby enabling the microscope to collect the images with clear boundariesSst mRNA signal point, improves signal to noise ratio, is convenient for quantitative.

Example 3

Tissue transparentization treatment is carried out on the tissue sample after rolling circle amplification, and the experimental flow is shown in fig. 9.

Rolling circle amplification was performed in the same manner as in example 1, using a split probe pair Sst-228-2A (SEQ ID NO: 4) and Sst-228-2B (SEQ ID NO: 5), followed by incubation of the immobilized rolling circle amplification product with 4% paraformaldehyde at room temperature, incubation of glycine (purchased from Aladin, A110749) covered tissue at room temperature for 5min to terminate the reaction, followed by washing with PBST (PBS buffer containing 0.05% Tween 20), incubation of tissue with a solution containing 1mg/mL MelphaX (from Melphalan (purchased from MedChemExpress, HY-17575) and acryl-X, SE (purchased from Invitrogen, A20770) to form Melphalan X) and 0.1mg/mL of acryl-X, SE (Acryloyl-X, SE) covered tissue at 37℃overnight, washing of tissue with PBST, incubation of tissue with nitrogen-treated monomer buffer (2 XSSC buffer, 4% acrylamide, 0.2% methylene bisacrylamide) at room temperature for 15 min, followed by incubation of the polymerization mixture (0.2% Ammonium Persulfate (APS), 0.2% TEMED, 2XSSC buffer, 4% acrylamide, 0.2% methylene bisacrylamide, and 5% glass slides at room temperature (5% glass slides at room temperature, 2X) covered with 2.37% glass wool (5% glass slides washed at room temperature, 2X, 2% glass slides washed with 2X (X, SE) and 2% glass slides at room temperature, the tissue was then incubated with a hybridization buffer containing deionized formamide (2 XSSC, 20% deionized formamide, 0.2. Mu.g/mu.L BSA, 0.5. Mu.g/mL PAPI, 2 nM fluorescent probe) containing fluorescent probes, which bound to the rolling circle amplification product, at room temperature. After washing the patch, the detected RNA signal was visualized by imaging under a microscope. The results are shown in FIGS. 10A-10C.

The results of not subjecting the rolling circle amplified tissue sample to the tissue transparentization treatment are shown in FIGS. 11A to 11C. Comparing the results of fig. 10A-10C and fig. 11A-11C, it can be seen that after tissue transparentization, the tissue background fluorescence intensity is reduced, i.e. the non-signal/non-specific tissue autofluorescence is reduced, and the signal-to-noise ratio of the technology is effectively improved and the signal detection efficiency is improved when the technology is used in combination with the tissue transparentization technology.

Example 4

The same procedure as in example 1 was followedSstmRNA signal detection using split probe pair Sst-73-1A, sst-73-1B; the probe pair Sst-228-2A, sst-228-2B; the probe pair Sst-330-3A, sst-330-3B; the probe pair Sst-394-4A, sst-394-4B (SEQ ID NO: 2-9) was then used to detect the expression of SST protein in tissue sections in situ using polyclonal antibodies to the SST protein using immunofluorescence methods.

Incubation of tissues with PBS solution containing 5% BSA in wet cartridges for 1 hour at room temperature blocked by non-specific binding of antibodies with SST proteinsPolyclonal antibody 1 of (2): 1000 in PBS containing 5% BSA and 0.5% Triton X-100, incubating the tissue with the solution in a wet box at 4℃for 2 hours, discarding the primary antibody solution, washing the tissue with PBS 3 times for 5 minutes each. At room temperature, the corresponding secondary antibody 1:1000 in PBS containing 5% BSA, the tissue was incubated with this solution in a wet box protected from light for 1 hour. The secondary antibody solution was discarded, and the tissue was washed 3 times with PBS in the absence of light for 5 minutes each, and visualized by imaging under a Germany Cai Sizheng fluorescence microscope (Axio image. Z2) after sealing SstmRNA and SST protein signals.

The results are shown in FIGS. 12A-12D. The results show that the expression of SST proteins detected using SST antibodies and the detection using the present disclosureSstmRNA signals exhibit good co-localization, i.e.SST proteins and SST proteins are detected simultaneously in one cellSstExpression of mRNA, indicative of what is detected by the present disclosureSstThe mRNA signal is correct and the detection method of the present disclosure has high confidence.

The technical solution of the present disclosure is not limited to the above specific embodiments, and all technical modifications made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. A method of detecting a target nucleic acid sequence in a sample in situ, the method comprising the steps of:

(a) Providing at least one pair of split probes and at least one padlock probe,

2. The method of claim 1, wherein the split probe pair comprises a first split probe and a second split probe, and/or

The padlock probe includes at least one readout sequence and at least one bridging sequence that spaces the at least one readout sequence apart.

3. The method of claim 2, wherein the first and/or second split probes comprise a 5 'single-stranded region, a stem-stranded region, and a 3' single-stranded region,

wherein the 5 'single-stranded region or the 3' single-stranded region of the first split probe hybridizes to a first portion of the target nucleic acid to form a first complementary binding region, the 5 'single-stranded region or the 3' single-stranded region of the second split probe hybridizes to a second portion of the target nucleic acid to form a second complementary binding region, and/or

The 5 'end single-chain region or the 3' end single-chain region of the first split probe hybridizes with the 5 'end single-chain region and the 3' end single-chain region of the padlock probe to form a third complementary binding region, and the 5 'end single-chain region or the 3' end single-chain region of the second split probe hybridizes with the padlock probe to form a fourth complementary binding region; or,

the 5' single-stranded region or the 3' single-stranded region of the second split probe hybridizes to the 5' single-stranded region and the 3' single-stranded region of the padlock probe to form a third complementary binding region, the 5' single-stranded region of the first split probe hybridizes to the padlock probe to form a fourth complementary binding region, and/or

The notch is located in the third complementary binding region.

4. A method according to claim 3, wherein the first and/or second split probes are at least 20 nucleotides in length, and/or

The 5' -terminal single-stranded region is 8-40 nucleotides in length, and/or

The length of the stem-chain region is 2-6 nucleotides, and/or

The 3' -terminal single-stranded region is 8-40 nucleotides in length, and/or

The first complementary binding region and/or the second complementary binding region is at least 10 nucleotides in length, and/or

The third complementary binding region and/or the fourth complementary binding region is 8-40 nucleotides in length.

5. The method of claim 2, wherein the padlock probe is at least 40 nucleotides in length, and/or

The read-out sequence comprises at least 15 nucleotides, and/or

The interval between the individual read-out sequences is 2-6 nucleotides, and/or

The 5' -end of the padlock probe is or is not subjected to phosphorylation modification, and/or

The phosphate bond between the last 3-5 bases of the 3' end of the second split probe is not modified or modified to block the 3' to 5' exonuclease activity of the polymerase, and/or

The 3' terminal base of the second split probe is not modified, or is modified with an inverted dT/dG and/or other modification that blocks polymerase extension reactions.

6. The method according to claim 1, wherein the method further comprises: and (f) contacting the rolling circle amplification product with a reading probe and/or an amplifying probe to obtain the spatial position and/or sequence information of the target nucleic acid.

7. The method of claim 6, wherein the read probe hybridizes to the read sequence, and/or

The amplification probe has a binding region hybridized to the padlock probe and a non-binding region at one or both ends of the binding region which does not hybridize to the readout sequence, and/or

The readout probe and/or amplification probe comprises at least one of A, T, C or G bases, and/or

The readout probes and/or amplification probes carry at least one fluorescent labeling group, magnetic particle or radiolabel.

8. The method of claim 6, further comprising, prior to step (f), subjecting the tissue sample comprising the rolling circle amplification product to tissue transparentization.

9. The method of claim 8, wherein the tissue transparentizing treatment comprises: firstly, fixing a rolling circle amplification product and tissues around the rolling circle amplification product, then, reacting the fixed tissue sample with melphalan X, acryl-X and SE, and then, contacting the reacted product with gel to further fix the rolling circle amplification product on the gel; and performing enzymolysis treatment on the gel fixed with the rolling circle amplification product.

10. The method of claim 1, wherein the target nucleic acid is RNA or DNA,

and/or the sample is from a biological sample.

11. A probe set for in situ detection of a target nucleic acid sequence in a sample comprising at least one pair of split probes and at least one padlock probe.

12. The probe set of claim 11, wherein the split probe pair comprises a first split probe and a second split probe, and/or

13. The probe set of claim 12, wherein the first split probe and/or the second split probe comprises a 5 'single-stranded region, a stem-stranded region, and a 3' single-stranded region, wherein the 5 'single-stranded region or the 3' single-stranded region of the first split probe is capable of hybridizing to a first portion of the target nucleic acid to form a first complementary binding region, the 5 'single-stranded region or the 3' single-stranded region of the second split probe is capable of hybridizing to a second portion of the target nucleic acid to form a second complementary binding region, and/or

The 5 'end single-stranded region or the 3' end single-stranded region of the first split probe can be hybridized with the 5 'end single-stranded region and the 3' end single-stranded region of the padlock probe to form a third complementary binding region, and the 5 'end single-stranded region or the 3' end single-stranded region of the second split probe can be hybridized with the padlock probe to form a fourth complementary binding region; or,

The 5' single-stranded region or the 3' single-stranded region of the second split probe is capable of hybridizing to the 5' single-stranded region and the 3' single-stranded region of the padlock probe to form a third complementary binding region, the 5' single-stranded region of the first split probe is capable of hybridizing to the padlock probe portion to form a fourth complementary binding region, and/or

The cut is located in the third complementary binding region, and/or

The first split probe and/or the second split probe is at least 20 nucleotides in length, and/or

The 5' -terminal single-stranded region is 8-40 nucleotides in length, and/or

The length of the stem-chain region is 2-6 nucleotides, and/or

The 3' -terminal single-stranded region is 8-40 nucleotides in length, and/or

The padlock probe is at least 40 nucleotides in length, and/or

The read-out sequence comprises at least 15 nucleotides, and/or

The 3' -end of the second split probe is not modified or modified to block 3' -end to 5' -end exonuclease activity of polymerase, and/or

14. The probe set of claim 11, further comprising a readout probe and/or an amplification probe.

15. The probe set of claim 14, wherein the readout probe hybridizes to the readout sequence, and/or

The amplification probe has a binding region hybridized to the padlock probe and a non-binding region positioned at one or both ends of the binding region that does not hybridize to the readout sequence; and/or

The readout probe comprises only at least one of A, T, C or G bases, and/or

The readout probe carries at least one fluorescent labeling group, magnetic particle or radiolabel.

16. A kit comprising the probe set of any one of claims 11-15 and a ligase and/or polymerase.

17. Use of the method according to any one of claims 1-10, the probe set according to any one of claims 11-15, the kit according to claim 16 in situ detection and flow cytometry.

18. A system for in situ detection of a target nucleic acid sequence in a sample, the system comprising:

a ligation module for forming a closed nucleic acid loop on the nicked nucleic acid loop by using a ligase; and

19. The system of claim 18, wherein the split probe and padlock probe are as defined in the probe set of any one of claims 11 to 15.