Method for rapidly detecting RNA (Ribonucleic Acid) and RNA interaction in cells

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for rapidly detecting RNA (Ribonucleic Acid) and RNA interaction in cells. The invention provides a reagent combination for RNA (Ribonucleic Acid) near-end connection. According to the reagent combination, the 5'terminal of an oligonucleotide linker can be connected with the 3 'terminal of RNA at one end of an RNA-RNA complex through a first ligase, and the 3' terminal of the oligonucleotide linker can be connected with the 5 'terminal of RNA at the other end of the RNA-RNA complex through a second ligase, so that two RNAs of the RNA-RNA complex are quickly and efficiently connected through the oligonucleotide linker to obtain chimeric RNA; compared with an existing RNA near-end connection method, the method has the advantages that the experimental operation process is reduced, RRIs information loss caused by tedious operation steps is reduced, the obtained chimeric RNA data is high in proportion, and a global RRIs map in cells can be better revealed.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for rapidly detecting RNA-RNA interactions in cells. Background Art

[0002] RNA plays a vital role in various biological processes. RNA usually binds to chromatin or proteins to play a regulatory role. Highly structured RNA binds to various RNA-binding proteins and then interacts with other RNAs through base complementary pairing or proximal contacts to regulate key biological functions, including transcription, RNA processing, localization and translation. In order to identify the higher-level structure and interaction of RNA, various detection methods have been rapidly developed in the past few decades, which can be mainly divided into three categories: low-throughput methods based on the physicochemical properties of RNA molecules, high-throughput sequencing technologies based on target detection of RNA-RNA interactions (RRIs), and high-throughput sequencing technologies for detecting global RRIs in cells.

[0003] However, due to the limitations of existing technologies and the complexity of RRIs, RNA structures and interactions in cells remain largely unknown. For example, traditional RRIs detection technology is a low-throughput method based on the physicochemical properties of RNA molecules, including gel shift assay (EMSA), surface plasmon resonance (SRP), single-molecule fluorescence resonance energy transfer, sucrose gradient co-precipitation and yeast RNA hybridization system. Traditional RRIs detection technology is mainly used to detect whether two known RNAs interact with each other, with high accuracy: for example: EMSA experiment: Since the fragments with RNA interaction have a large molecular weight, their migration speed in gel electrophoresis is slower than that of the fragments without RNA interaction. Therefore, the migration speed can be used to determine whether two RNA molecules interact with each other; SPR experiment: RNA fragments are fixed on the sensor chip by coupling streptavidin and biotin, and whether RNA fragments interact with potential target fragments can be monitored in real time; single-molecule fluorescence resonance energy transfer experiment: RNA molecules are fixed on the quartz surface or coated in lipid vesicles, and the labeling of two fluorescent dyes can be used to monitor in real time whether two RNAs interact with each other; sucrose gradient co-precipitation experiment: RNA extracts are subjected to sucrose / glycerol gradient centrifugation, and different gradient layers are detected by Northern blot. RNA in the same gradient layer is considered to interact; yeast RNA hybridization system: two RNAs are respectively coupled with MS2 RNA and m26 constitute a fusion gene. If the two RNAs interact, the reporter gene will be activated and expressed. These methods have high detection accuracy, but can only detect whether the two RNA molecules interact with each other, and cannot detect the interaction site. In addition, the single-molecule fluorescence resonance energy transfer experiment has high requirements for sample purification and labeling, and is difficult to operate. In the yeast RNA hybridization system, RNA with weak interactions is easy to separate, resulting in false negative results.

[0004] Advances in sequencing technology have led to major breakthroughs in the study of RNA-RNA interactions (RRIs). Currently, a series of technical methods have been developed to detect RRIs. These technologies can be used to discover thousands of RRIs in a variety of species, which potentially regulate various aspects of gene expression. With the development of high-throughput sequencing technology, technologies for target-based detection of the entire RRIs network have also emerged, including CLASH, RIL-seq, HiCLIP, RIA-seq, and RAP. These technologies are the earliest high-throughput technologies used to detect RRIs, and can detect a specific RNA or protein-interacting RNA group, as well as specific interaction sites: Among them, CLASH, RIL-seq, and HiCLIP technologies are similar in principle, and the experimental steps include cross-linking cells, fixing intracellular protein-mediated RRIs, and then enriching specific proteins through antibodies, proximally connecting RNA-RNA complexes, eluting RNA-RNA complexes, and high-throughput library construction and sequencing. Data analysis can obtain specific protein-mediated RRIs; HiCLIP is used in the connection When connecting RNA-RNA, the addition of a linker can improve the specificity of the data and also discover the secondary structure of RNA; RIA-seq and RAP technologies are similar in principle, and the experimental steps include designing probes, cross-linking cells, lysing cells, hybridization and elution, high-throughput library construction and sequencing, and data analysis. The probe is a biotin-labeled antisense DNA probe designed for a specific RNA. After the probe is incubated with the target RNA, it will also bring down the RNA that interacts with the target RNA. The eluted RNA is sequenced for library construction, and the RNA group that interacts with the target RNA can be obtained by analyzing the data. The difference is that the probe length of RIA-seq is 50-120bp, and the probe length of RAP is 120bp. Therefore, RAP can greatly reduce nonspecific interactions. However, among the high-throughput sequencing technologies based on target detection of RRIs, CLASH, RIL-seq, and HiCLIP can only detect RNA groups that interact with specific proteins. CLASH is the first high-throughput technology used to detect RRIs, and the connection efficiency and sensitivity are limited. RIA-seq and RAP can only detect RNA groups that interact with a specific RNA.

[0005] In the past few years, some technologies for detecting global RRIs in cells have also been developed, including PARIS, SPLSH, LIGR-seq, MARIO and RIC-seq (the schematic diagram of the library construction process is shown in Figure 1As shown in the figure, these technologies can capture global RRIs in cells. The experimental steps mainly include cross-linking, proximal connection of RNA-RNA complexes to form chimeric RNAs, enrichment of chimeric RNAs, high-throughput library construction and sequencing, and data analysis. The main differences are: the first three technologies use AMT and its derivatives to cross-link cells, and the last two technologies use formaldehyde to cross-link cells; PARIS and LIGR-seq use two-dimensional gel electrophoresis and RNase R to enrich chimeric RNAs, respectively, and SPLSH, MARIO, and RIC-seq all capture chimeric RNAs through magnetic beads; RIC-seq connects RNA-RNA complexes in situ, and the remaining technologies connect RNA-RNA complexes in dilute solutions. These technologies have established RRIs maps in different species and multiple cell lines, enabling people to have a deeper understanding of the role of RNA structure and interactions in various biological processes. However, these technologies still have some limitations. For example, the data coverage obtained by PARIS, SPLSH, LIGR-seq, and MARIO are all low, and the proximal ligation is performed in a diluted solution, which will cause false positives. The latest RIC-seq technology uses formaldehyde to cross-link cells and uses biotin-labeled pCp to connect the two ends of adjacent RNA in situ, which increases the proportion of chimeric RNA sequences. However, the experimental cycle is long and takes 6 days to complete. The more centrifugation and washing steps will cause the loss of some RRIs information. Therefore, simplifying the experimental process, improving the efficiency of RNA proximal ligation, and capturing more comprehensive RRIs information in cells are the core points of RRIs detection technology. Summary of the invention

[0006] The purpose of the first aspect of the present invention is to provide a reagent combination for RNA proximal end ligation.

[0007] The second aspect of the present invention aims to provide a kit.

[0008] The third aspect of the present invention aims to provide a method for RNA proximal end ligation.

[0009] The fourth aspect of the present invention aims to provide a method for enriching chimeric RNA.

[0010] The fifth aspect of the present invention aims to provide a method for constructing a library.

[0011] The sixth aspect of the present invention aims to provide a library.

[0012] The seventh aspect of the present invention aims to provide a method for identifying RNA-RNA interactions or capturing RNA higher-order structures.

[0013] The purpose of the eighth aspect of the present invention is to provide an application of the reagent combination of the first aspect of the present invention and / or the kit of the second aspect.

[0014] In order to achieve the above object, the technical solution adopted by the present invention is:

[0015] The first aspect of the present invention provides a reagent combination for RNA proximal ligation, comprising:

[0016] a first ligase that can ligate the 3' phosphate terminus of the oligonucleotide to the 5' hydroxyl terminus of the oligonucleotide (3'P to 5'OH); and

[0017] a second ligase that can ligate the 5' phosphate terminus of the oligonucleotide to the 3' hydroxyl terminus of the oligonucleotide (5'P to 3'OH); and

[0018] An oligonucleotide linker comprising a 5' hydroxyl terminus and a 3' hydroxyl terminus.

[0019] Preferably, the first ligase comprises at least one of: RtcB ligase, an active fragment of RtcB ligase, a derivative of RtcB ligase, and an analog of RtcB ligase.

[0020] Preferably, the second ligase comprises at least one of: T4 RNA ligase 1, TS2126 RNA ligase, single-stranded DNA / RNA circular ligase (ssDNA / RNA CircLigase), and T4 RNA ligase 2.

[0021] Preferably, one or more nucleotides in the oligonucleotide linker are modified with a first affinity tag.

[0022] Preferably, the length of the oligonucleotide linker is 10 to 25 nt; further 18 to 22 nt.

[0023] Preferably, the reagent combination further comprises: a first cofactor, wherein the first cofactor comprises: triphosphate and manganese ions.

[0024] Preferably, the reagent combination further comprises: a second cofactor, wherein the second cofactor comprises: at least one of ATP and NAD and metal ions.

[0025] Preferably, the metal ions include at least one of magnesium ions and manganese ions.

[0026] Preferably, the reagent combination further comprises: a ligation reaction mixture.

[0027] Preferably, the ligation reaction mixture comprises at least one of a ligation reaction buffer, a coagulant, and an RNase inhibitor.

[0028] Preferably, the reagent combination is used to connect two RNAs of the RNA-RNA complex in the same system to form a chimeric RNA; wherein the same system is specifically carried out in the same reaction container; further specifically, it is carried out in the same reaction container and at the same time.

[0029] Preferably, the reagent combination further comprises instructions, which describe the method for using the reagent combination.

[0030] Preferably, the method for using the reagent combination is: mixing the RNA-RNA complex with the oligonucleotide linker, the first ligase, the second ligase, the first auxiliary factor, and the second auxiliary factor, and performing a ligation reaction on the obtained ligation reaction system.

[0031] Preferably, the final concentration of the triphosphate in the first auxiliary factor in the ligation reaction system is 0.1-0.8 mM; further 0.1-0.5 mM; further 0.1-0.3 mM; further 0.2 mM.

[0032] Preferably, the final concentration of the manganese ions in the first auxiliary factor in the ligation reaction system is 0.5-3.5 mM; further 0.5-3 mM; further 0.5-1.5 mM; further 1 mM.

[0033] Preferably, the final concentration of ATP and / or NAD in the second auxiliary factor in the ligation reaction system is 0.5-3.5 mM; further 0.5-3 mM; further 0.5-1.5 mM; further 1 mM.

[0034] Preferably, the final concentration of the metal ion in the second auxiliary factor in the ligation reaction system is 3.5-22.5 mM; further 3.5-31 mM; further 3.5-10.5 mM; further 7 mM.

[0035] Preferably, the reagent combination further comprises: a polynucleotide kinase that loses 3' terminal phosphatase activity.

[0036] The second aspect of the present invention provides a kit, comprising: the reagent combination of the first aspect of the present invention.

[0037] Preferably, the kit further comprises at least one of a free RNA (i.e., RNA not protected by proteins) interruptor, a cell membrane perforator (e.g., permeabilization solution), and a cell cross-linking agent (i.e., a substance that fixes protein-mediated RNA-RNA interactions).

[0038] Preferably, the free RNA break agent comprises: micrococcal nuclease.

[0039] Preferably, the cell membrane perforating agent comprises at least one of NP-40, Triton X-100 and Tween 20.

[0040] Preferably, the cell cross-linking agent comprises: formaldehyde.

[0041] Preferably, the kit is used for any one of a1) to a5):

[0042] a1) RNA proximal end ligation (preferably RNA in situ proximal end ligation);

[0043] a2) enrichment of chimeric RNA;

[0044] a3) Library construction;

[0045] a4) identifying RNA-RNA interactions (preferably identifying RNA-RNA interactions in situ);

[0046] a5) Capturing RNA higher-order structures (preferably capturing RNA higher-order structures in situ).

[0047] Preferably, when the kit is used for any one of a2) to a5), the kit further comprises: at least one of a cell lysis reagent, a nucleic acid (e.g., RNA) extraction reagent, a nucleic acid (e.g., RNA) purification reagent, an endonuclease (e.g., DNase I), a substance for fragmenting nucleic acids, and a solid phase carrier with a second affinity tag (preferably a streptavidin tag) that can affinity bind to a first affinity tag (preferably a biotin tag).

[0048] Preferably, when the kit is used for any one of a3) to a5), the kit further comprises: RNA library preparation reagents.

[0049] Preferably, when the kit is used for any one of a4) to a5), the kit further comprises: at least one of a cyclization reagent, a nucleic acid nanosphere preparation reagent, and a sequencing reagent.

[0050] The third aspect of the present invention provides an RNA proximal end ligation method, comprising: using the reagent combination of the first aspect of the present invention and / or the kit of the second aspect of the present invention.

[0051] Preferably, the method comprises the following steps: mixing the RNA-RNA complex with the first ligase, the second ligase, and the oligonucleotide linker in the reagent combination of the first aspect of the present invention, and performing a ligation reaction on the obtained ligation reaction system;

[0052] The RNA in the RNA-RNA complex comprises a 5' phosphate terminus and a 3' phosphate terminus.

[0053] Preferably, the ligation reaction ligates two RNAs of the RNA-RNA complex in the same system to form a chimeric RNA; wherein the same system is specifically carried out in the same reaction vessel; further specifically, it is carried out in the same reaction vessel and at the same time.

[0054] Preferably, the ligation reaction system further comprises: the first auxiliary factor and the second auxiliary factor in the reagent combination of the first aspect of the present invention.

[0055] Preferably, the final concentration of the triphosphate in the first auxiliary factor in the ligation reaction system is 0.1-0.8 mM; further 0.1-0.5 mM; further 0.1-0.3 mM; further 0.2 mM.

[0056] Preferably, the final concentration of the manganese ions in the first auxiliary factor in the ligation reaction system is 0.5-3.5 mM; further 0.5-3 mM; further 0.5-1.5 mM; further 1 mM.

[0057] Preferably, the final concentration of ATP and / or NAD in the second auxiliary factor in the ligation reaction system is 0.5-3.5 mM; further 0.5-3 mM; further 0.5-1.5 mM; further 1 mM.

[0058] Preferably, the final concentration of the metal ion in the second auxiliary factor in the ligation reaction system is 3.5-22.5 mM; further 3.5-31 mM; further 3.5-10.5 mM; further 7 mM.

[0059] Preferably, the method for preparing the RNA-RNA complex comprises the following steps:

[0060] 1) Treating cells or tissue samples to fix protein-mediated RNA-RNA interactions;

[0061] 2) performing membrane perforation;

[0062] 3) Randomly interrupt free RNA in cells to obtain protein-protected 3'-terminal phosphorylated RNA;

[0063] 4) Phosphorylating the 5' end of the RNA protected by the protein and protecting the 3' phosphate end of the RNA protected by the protein to obtain an RNA-RNA complex.

[0064] Preferably, in step 1), the processing of the cell or tissue sample comprises the following steps: performing formaldehyde cross-linking on the cell or tissue sample.

[0065] The fourth aspect of the present invention provides a method for enriching chimeric RNA, comprising the steps of the RNA proximal end ligation method of the third aspect of the present invention.

[0066] Preferably, the method for enriching chimeric RNA further comprises the following steps: 1) lysing cells; 2) extracting total RNA and performing fragmentation treatment; 3) enriching chimeric RNA.

[0067] The fifth aspect of the present invention provides a library construction method, comprising the steps of the method for enriching chimeric RNA according to the fourth aspect of the present invention.

[0068] Preferably, the method further comprises the step of constructing a library of the enriched chimeric RNA.

[0069] The sixth aspect of the present invention provides a library obtained by the method of the fifth aspect of the present invention.

[0070] The seventh aspect of the present invention provides a method for identifying RNA-RNA interactions or capturing RNA higher-order structures, comprising the following steps: sequencing the library of the sixth aspect of the present invention.

[0071] Preferably, a circularization reaction may also be included before sequencing.

[0072] Preferably, the cyclization reaction further includes a step of preparing nucleic acid nanospheres.

[0073] The eighth aspect of the present invention provides the use of the reagent combination of the first aspect of the present invention and / or the kit of the second aspect in any one of a1) to a5);

[0074] a1) RNA proximal end ligation (preferably RNA in situ proximal end ligation);

[0075] a2) enrichment of chimeric RNA;

[0076] a3) Library construction;

[0077] a4) identifying RNA-RNA interactions (preferably identifying RNA-RNA interactions in situ);

[0078] a5) Capturing RNA higher-order structures (preferably capturing RNA higher-order structures in situ).

[0079] The beneficial effects of the present invention are:

[0080] The present invention provides a reagent combination for RNA proximal end ligation, wherein the reagent combination can connect the 5' end of an oligonucleotide linker to the 3' end of RNA at one end of an RNA-RNA complex (preferably, the RNA in the RNA-RNA complex comprises a 5' phosphate end and a 3' phosphate end) through a first ligase, and connect the 3' end of the oligonucleotide linker to the 5' end of RNA at the other end of the RNA-RNA complex through a second ligase, so that the two RNAs of the RNA-RNA complex are quickly and efficiently connected through the oligonucleotide linker (i.e., the RNA-RNA complex is connected by simultaneously performing a ligation reaction through the first ligase, the second ligase, and the oligonucleotide linker), to obtain a chimeric RNA; compared with the existing RNA proximal end ligation method (such as the proximal end ligation method in RIC-seq), the experimental operation process is reduced (correspondingly, the time and reagent costs are reduced), the loss of RRIs information caused by cumbersome operation steps is reduced, the obtained chimeric RNA data accounts for a high proportion, which is convenient for better revealing the global RRIs map in the cell, and the entire experimental cycle takes 5 days; at the same time, the added oligonucleotide linker can improve the specificity of the data and the accuracy of bioinformatics analysis, and realize the precise positioning of the RNA-RNA interaction position. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 This is the RIC-seq library construction flow chart.

[0082] Figure 2 It is a flow chart of the library construction method of the present invention.

[0083] Figure 3 This is a diagram showing the optimization results of the RtcB ligase reaction system in Example 1.

[0084] Figure 4 This is a graph showing the results of the RtcB ligase and T4 RNA ligase 1 reaction system in Example 2.

[0085] Figure 5 This is the Agilent 2100 Bioanalyzer test result of RNA-RNA interaction library. DETAILED DESCRIPTION

[0086] In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0087] The first aspect of the present invention provides a reagent combination for RNA proximal ligation, comprising:

[0088] A first ligase, which can connect the 3' phosphate terminus (i.e., 3' phosphorylation-modified oligonucleotide) of an oligonucleotide (e.g., RNA) to the 5' hydroxyl terminus (3'P to 5'OH) of an oligonucleotide (e.g., RNA), i.e., has 3'P to 5'OH ligase activity; and

[0089] A second ligase, which can connect the 5' phosphate terminus (i.e., 5' phosphorylation-modified oligonucleotide) of an oligonucleotide (e.g., RNA) to the 3' hydroxyl terminus (5'P to 3'OH) of an oligonucleotide (e.g., RNA), i.e., has 5'P to 3'OH ligase activity; and

[0090] An oligonucleotide linker comprising a 5' hydroxyl terminus and a 3' hydroxyl terminus.

[0091] The above-mentioned reagent combination for RNA proximal end ligation can connect the 5' end of the oligonucleotide adapter to the 3' end of one end RNA of the RNA-RNA complex (preferably the RNA in the RNA-RNA complex comprises a 5' phosphate end and a 3' phosphate end) by a first ligase, and connect the 3' end of the oligonucleotide adapter to the 5' end of the other end RNA of the RNA-RNA complex by a second ligase, so that the two RNAs of the RNA-RNA complex are quickly and efficiently connected through the oligonucleotide adapter (that is, the first ligase, the second ligase, and the oligonucleotide adapter are connected simultaneously, and the RNA-RNA complex is connected). RNA complex), and obtain chimeric RNA; compared with the existing RNA proximal ligation method (such as the proximal ligation method in RIC-seq), the experimental operation process is reduced (correspondingly, the time and reagent costs are reduced), and the loss of RRIs information caused by cumbersome operation steps is reduced. The obtained chimeric RNA data accounts for a high proportion, which is convenient for better revealing the global RRIs map in the cell. The entire experimental cycle takes 5 days (while the entire experimental cycle of RIC-seq takes 6 days); at the same time, the added oligonucleotide linker can improve the specificity of the data and the accuracy of bioinformatics analysis, and realize the precise positioning of the RNA-RNA interaction position.

[0092] Preferably, one or more nucleotides in the oligonucleotide linker are modified with a first affinity tag (preferably a biotin tag), so that the RNA-RNA complex (i.e., chimeric RNA) connected to the oligonucleotide linker can be captured by a solid phase carrier, wherein the solid phase carrier carries a second affinity tag (preferably a streptavidin tag) that can affinity bind to the first affinity tag; further preferably, one nucleotide in the oligonucleotide linker is modified with a first affinity tag.

[0093] Preferably, the nucleotide modified with the first affinity tag is not located at the 5' end or the 3' end of the oligonucleotide adaptor.

[0094] Preferably, the length of the oligonucleotide linker is 10 to 25 nt; further 18 to 22 nt.

[0095] Preferably, the oligonucleotide linker is RNA.

[0096] Preferably, the sequence of the oligonucleotide linker is as shown in SEQ ID NO.5.

[0097] Preferably, the 12th ribonucleotide of the oligonucleotide linker having a sequence as shown in SEQ ID NO.5 is modified with a biotin label.

[0098] Preferably, the RNA proximal ligation is RNA in situ proximal ligation.

[0099] Preferably, the proximal end is within 25 angstroms.

[0100] Preferably, the first ligase is a type of enzyme that can connect the 3' phosphate end of an oligonucleotide (e.g., RNA) to the 5' hydroxyl end of an oligonucleotide (e.g., RNA), and can be selected from at least one of: RtcB ligase, an active fragment of RtcB ligase, a derivative of RtcB ligase, and an analog of RtcB ligase; further comprising: RtcB ligase: wherein the RtcB ligase can be an RtcB ligase from any species; "active fragment of RtcB ligase, derivative of RtcB ligase, analog of RtcB ligase" refers to a polypeptide that substantially maintains the same biological function or activity as the RtcB ligase of the present invention. The derivatives or analogs of the present invention may be (i) polypeptides in which one or more (e.g., 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 1-3, 1-2) conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more (e.g., 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 1-3, 1-2) amino acid residues, or (iii) polypeptides formed by the fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein). According to the definition herein, these active fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0101] In the present invention, the active fragment of RtcB ligase means that as a polypeptide, it can still maintain all or part of the functions of the full-length RtcB ligase. Generally, the active fragment maintains at least 50% of the activity of the full-length RtcB ligase. Under more preferred conditions, the active fragment can maintain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length RtcB ligase.

[0102] In the present invention, RtcB ligase also includes variants having the same function as RtcB ligase. These variants include (but are not limited to): deletion, insertion and / or substitution of several (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, and even more preferably 1 to 8, 1 to 5) amino acids, and addition or deletion of one or several (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, and even more preferably 1 to 8, 1 to 5) amino acids at the C-terminus and / or N-terminus (especially the N-terminus). For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus (especially the N-terminus) does not usually change the function of the protein.

[0103] Any protein with high homology to the RtcB ligase (e.g., 60% or higher, 70% or higher, 80% or higher homology to the sequence of the RtcB ligase; preferably, 85% or higher homology; more preferably, 90% or higher homology, such as 95%, 98% or 99% homology) and having the same function as the RtcB ligase is also included in the present invention. "Homology" refers to the level of similarity (i.e., sequence similarity or identity) between two or more nucleic acids or polypeptides according to the percentage of identical positions. In this article, variants of the gene can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutagenesis, etc.

[0104] The specific principle of RtcB ligase (first ligase) ligation is as follows: (1) In the presence of Mn ions, RtcB ligase (first ligase) reacts with triphosphate (such as GTP) to generate a covalent RtcB-histidine-GMP intermediate and releases pyrophosphate (Ppi); (2) GMP is then transferred to the 3' phosphate group at the end of the oligonucleotide to obtain an activated 3'-P oligonucleotide; (3) Then the oligonucleotide with 5'-OH attacks the activated 3'-P oligonucleotide to form a 3', 5'-phosphodiester bond and release GMP; therefore, any enzyme that can complete oligonucleotide ligation according to the above process belongs to the protection scope of the first ligase described in the present invention.

[0105] Preferably, the first ligase comprises: thermophilic RNA ligase RtcB.

[0106] Preferably, the reagent combination further comprises: a first auxiliary factor, wherein the first auxiliary factor comprises: triphosphate and manganese ion, which is used to assist the first ligase in connecting the 3' phosphate end of the oligonucleotide to the 5' hydroxyl end of the oligonucleotide.

[0107] The first auxiliary factor is used by the first ligase (e.g., RtcB ligase) to catalyze the ligation reaction between the 3' phosphate end of the oligonucleotide (3'P oligonucleotide) and the 5' hydroxyl end of the oligonucleotide (5'OH oligonucleotide) through a three-step nucleic acid transfer process: (1) RtcB reacts with triphosphate (e.g., GTP) to form a covalent RtcB-histidine-GMP intermediate and release PPi; (2) GMP is transferred to the 3' phosphate group of the oligonucleotide to obtain an activated 3'-P oligonucleotide; (3) 5'OH oligonucleotide attacks the activated 3'P oligonucleotide to form a 3', 5'-phosphodiester bond and releases GMP.

[0108] Preferably, the triphosphate comprises at least one of NTP and dNTP; further comprises at least one of ATP, CTP, GTP, TTP, UTP, dATP, dCTP, dGTP, dTTP, and dUTP; and further comprises GTP.

[0109] Preferably, the manganese ion is a divalent manganese ion.

[0110] Preferably, the second ligase comprises: at least one of T4 RNA ligase 1, TS2126 RNA ligase, single-stranded DNA / RNA circular ligase (ssDNA / RNA CircLigase), and T4 RNA ligase 2; and further comprises: T4 RNA ligase 1.

[0111] Preferably, the reagent combination further comprises: a second auxiliary factor, wherein the second auxiliary factor comprises: at least one of ATP and NAD and metal ions, for assisting the second ligase in connecting the 5' phosphate end of the oligonucleotide to the 3' hydroxyl end of the oligonucleotide.

[0112] Preferably, the metal ions include at least one of magnesium ions and manganese ions.

[0113] Preferably, the manganese ion is a divalent manganese ion.

[0114] Preferably, the reagent combination further comprises: a ligation reaction mixture; which is used for the ligation reaction catalyzed by the first ligase and / or the second ligase.

[0115] Preferably, the ligation reaction mixture comprises: at least one of a ligation reaction buffer, a coagulant, and an RNase inhibitor; and further comprises: a ligation reaction buffer, a coagulant, and an RNase inhibitor.

[0116] Preferably, the coagulant comprises PEG; and further comprises PEG8000.

[0117] Preferably, the reagent combination is used for RNA proximal ligation of an RNA-RNA complex (the RNA in the RNA-RNA complex preferably comprises a 5' phosphate terminus and a 3' phosphate terminus).

[0118] Preferably, the reagent combination is used to connect two RNAs of the RNA-RNA complex in the same system to form a chimeric RNA; wherein the same system is specifically carried out in the same reaction container; further specifically, it is carried out in the same reaction container and at the same time.

[0119] Preferably, the reagent combination further comprises instructions, which describe the method for using the reagent combination.

[0120] Preferably, the method for using the reagent combination is: mixing the RNA-RNA complex with the oligonucleotide linker, the first ligase, the second ligase, the first auxiliary factor, and the second auxiliary factor, and performing a ligation reaction on the obtained ligation reaction system.

[0121] Preferably, the ligation reaction system further comprises: the ligation reaction mixture mentioned above.

[0122] Preferably, the final concentration of the triphosphate in the first auxiliary factor in the ligation reaction system is 0.1-0.8 mM; further 0.1-0.5 mM; further 0.1-0.3 mM; further 0.2 mM.

[0123] Preferably, the final concentration of the manganese ions in the first auxiliary factor in the ligation reaction system is 0.5-3.5 mM; further 0.5-3 mM; further 0.5-1.5 mM; further 1 mM.

[0124] Preferably, the final concentration of ATP and / or NAD in the second auxiliary factor in the ligation reaction system is 0.5-3.5 mM; further 0.5-3 mM; further 0.5-1.5 mM; further 1 mM.

[0125] Preferably, the final concentration of the metal ion in the second auxiliary factor in the ligation reaction system is 3.5-22.5 mM; further 3.5-31 mM; further 3.5-10.5 mM; further 7 mM.

[0126] Preferably, the reagent combination further comprises: a polynucleotide kinase that loses 3' terminal phosphatase activity (preferably a T4 polynucleotide kinase that loses 3' terminal phosphatase activity), which is used to phosphorylate the 5' end of the RNA in the RNA-RNA complex, while protecting the 3' phosphate end of the RNA to avoid dephosphorylation of the 3' end of the RNA, so that the RNA in the RNA-RNA complex contains both a 5' phosphate end and a 3' phosphate end.

[0127] Preferably, the various components in the reagent combination exist independently or in mixture.

[0128] The second aspect of the present invention provides a kit, comprising: the reagent combination of the first aspect of the present invention.

[0129] Preferably, the kit further comprises: at least one of a free RNA (i.e., RNA not protected by proteins) interrupting agent, a cell membrane perforating agent (e.g., a permeabilizing solution), and a cell cross-linking agent (i.e., a substance that fixes protein-mediated RNA-RNA interactions); further comprises: a free RNA (i.e., RNA not protected by proteins) interrupting agent, a cell membrane perforating agent (e.g., a permeabilizing solution), and a cell cross-linking agent (i.e., a substance that fixes protein-mediated RNA-RNA interactions), which are used to treat cell or tissue samples to obtain protein-protected RNA (i.e., RNA-RNA complex), as follows:

[0130] Treat cells or tissue samples with cell crosslinkers to fix protein-mediated RNA-RNA interactions;

[0131] Treating cross-linked cells or tissue samples with cell membrane perforators to perform membrane perforation (perforating the cytoplasm and nucleus) while keeping the cells intact, so that subsequent reagents can enter the cells for reaction;

[0132] The membrane-perforated cells or tissue samples are treated with a free RNA-breaking agent to break free RNA not protected by proteins, thereby obtaining RNA protected by proteins (ie, RNA-RNA complex).

[0133] Preferably, the free RNA break agent comprises: micrococcal nuclease.

[0134] Preferably, the cell membrane perforating agent comprises: at least one of NP-40, Triton X-100, and Tween 20; and further comprises: NP-40, Triton X-100, and Tween 20.

[0135] Preferably, the cell membrane perforator further comprises: at least one of a protease inhibitor, an RNase inhibitor (such as SUPERase), and a buffer (such as Tris-HCl and NaCl); and further comprises: a protease inhibitor, an RNase inhibitor (such as SUPERase), and a buffer (such as Tris-HCl and NaCl).

[0136] Preferably, the cell cross-linking agent comprises: formaldehyde; and further comprises: 0.5-1.5 v / v% formaldehyde solution (the solvent is PBS).

[0137] Preferably, the kit further comprises: a substance for terminating the cross-linking reaction of the cell or tissue sample.

[0138] Preferably, the substance used to terminate the cross-linking reaction of cells or tissue samples comprises: glycine.

[0139] Preferably, the kit is used for any one of a1) to a5):

[0140] a1) RNA proximal end ligation (preferably RNA in situ proximal end ligation);

[0141] a2) enrichment of chimeric RNA;

[0142] a3) Library construction;

[0143] a4) identifying RNA-RNA interactions (preferably identifying RNA-RNA interactions in situ);

[0144] a5) Capturing RNA higher-order structures (preferably capturing RNA higher-order structures in situ).

[0145] Preferably, the library is a chain-specific library.

[0146] Preferably, when the kit is used for enriching chimeric RNA, constructing a library, identifying RNA-RNA interactions, or capturing RNA higher-order structures, the kit also comprises: at least one of a cell lysis reagent, a nucleic acid (e.g., RNA) extraction reagent, a nucleic acid (e.g., RNA) purification reagent, a nuclease (e.g., DNase I), a substance for breaking nucleic acids, and a solid phase carrier with a second affinity tag (preferably a streptavidin tag) that can affinity bind to a first affinity tag (preferably a biotin tag); further comprises: a cell lysis reagent, a nucleic acid (e.g., RNA) extraction reagent, a nucleic acid (e.g., RNA) purification reagent, a nuclease (e.g., DNase I), a substance for breaking nucleic acids, and a solid phase carrier with a second affinity tag that can affinity bind to a first affinity tag; used for lysing cells, extracting and purifying nucleic acids (e.g., RNA), digesting DNA, and fragmenting RNA to enrich chimeric RNA.

[0147] Preferably, the cell lysis reagent comprises: proteinase K; more preferably, the cell lysis reagent further comprises: proteinase K buffer; which is used to lyse (digest) cells.

[0148] Preferably, the nucleic acid extraction reagent comprises at least one of Trizol and chloroform.

[0149] Preferably, the nucleic acid purification reagent comprises: at least one of isopropanol and ethanol; more preferably, the nucleic acid purification reagent further comprises: GlycoBlue.

[0150] Preferably, the substance used to shear nucleic acid is a substance selected from one or more of the following shearing methods: chemical shearing method, physical shearing method.

[0151] Preferably, the chemical disruption method comprises alkali treatment, metal ions (such as Mg 2+ and / or Zn 2+ ) at least one of solution treatment and enzyme treatment.

[0152] Preferably, the physical breaking method comprises at least one of a high temperature (90-100° C.) breaking method, an ultrasonic breaking method and a mechanical breaking method; further preferably, the physical breaking method comprises a high temperature (90-100° C.) breaking method.

[0153] Preferably, the system of the high temperature interruption method comprises an alkaline solution of metal ions, First-strand Buffer.

[0154] Preferably, the solid phase carrier comprises beads; further preferably, the solid phase carrier comprises magnetic beads.

[0155] Preferably, when the kit is used for library construction, identification of RNA-RNA interactions, or capture of RNA higher-order structures, the kit also comprises: RNA library preparation reagents; further comprises: RNA directional library preparation reagents; which are used for the preparation of RNA (preferably chain-specific RNA) libraries.

[0156] Preferably, when the kit is used to identify RNA-RNA interactions or capture RNA higher-order structures, the kit also comprises: at least one of a cyclization reagent, a nucleic acid nanoball preparation reagent, and a sequencing reagent; further comprises: a cyclization reagent, a nucleic acid nanoball preparation reagent, and a sequencing reagent; which is used for single-strand cyclization, preparation of nucleic acid nanoballs (e.g., DNBs), and sequencing.

[0157] The third aspect of the present invention provides an RNA proximal end ligation method, comprising: using the reagent combination of the first aspect of the present invention and / or the kit of the second aspect of the present invention.

[0158] Preferably, the method comprises the following steps: mixing the RNA-RNA complex with the first ligase, the second ligase and the oligonucleotide linker in the reagent combination of the first aspect of the present invention, and subjecting the obtained ligation reaction system to a ligation reaction; the RNA in the RNA-RNA complex comprises a 5' phosphate end and a 3' phosphate end.

[0159] The RNA proximal ligation method connects the 5' end of the oligonucleotide linker to the 3' end of the RNA at one end of the RNA-RNA complex through a first ligase, and connects the 3' end of the oligonucleotide linker to the 5' end of the RNA at the other end of the RNA-RNA complex through a second ligase, so that the two RNAs of the RNA-RNA complex are quickly and efficiently connected through the oligonucleotide linker (that is, the RNA-RNA complex is connected by the first ligase, the second ligase, and the oligonucleotide linker at the same time) to obtain a chimeric RNA; compared with the existing RNA proximal ligation method (such as the proximal ligation method in RIC-seq), the experimental operation process is reduced (correspondingly, the time and reagent costs are reduced), the loss of RRIs information caused by cumbersome operation steps is reduced, and the proportion of chimeric RNA data obtained is high, which is convenient for better revealing the global RRIs map in the cell. The entire experimental cycle takes 5 days (while the entire experimental cycle of RIC-seq takes 6 days); at the same time, the added oligonucleotide linker can improve the specificity of the data and the accuracy of bioinformatics analysis, and realize the precise positioning of the RNA-RNA interaction position.

[0160] Preferably, the ligation reaction ligates two RNAs of the RNA-RNA complex in the same system to form a chimeric RNA; wherein the same system is specifically carried out in the same reaction vessel; further specifically, it is carried out in the same reaction vessel and at the same time.

[0161] Preferably, the ligation reaction system further comprises: the first auxiliary factor and the second auxiliary factor in the reagent combination of the first aspect of the present invention.

[0162] Preferably, the final concentration of the triphosphate in the first auxiliary factor in the ligation reaction system is 0.1-0.8 mM; further 0.1-0.5 mM; further 0.1-0.3 mM; further 0.2 mM.

[0163] Preferably, the final concentration of the manganese ions in the first auxiliary factor in the ligation reaction system is 0.5-3.5 mM; further 0.5-3 mM; further 0.5-1.5 mM; further 1 mM.

[0164] Preferably, the final concentration of ATP and / or NAD in the second auxiliary factor in the ligation reaction system is 0.5-3.5 mM; further 0.5-3 mM; further 0.5-1.5 mM; further 1 mM.

[0165] Preferably, the final concentration of the metal ion in the second auxiliary factor in the ligation reaction system is 3.5-22.5 mM; further 3.5-31 mM; further 3.5-10.5 mM; further 7 mM.

[0166] Preferably, the ligation reaction system further comprises: a ligation reaction mixture (preferably the ligation reaction mixture in the reagent combination of the first aspect of the present invention).

[0167] Preferably, the temperature of the ligation reaction is 33-40°C, further 37°C.

[0168] Preferably, the conditions of the ligation reaction are: 800-1200 rpm, 10-20 s / 2-4 min overnight (10-20 s shaking every 2-4 min).

[0169] Preferably, the RNA proximal ligation is RNA in situ proximal ligation.

[0170] Preferably, the proximal end is within 25 angstroms.

[0171] Preferably, the method for preparing the RNA-RNA complex comprises the following steps:

[0172] 1) Treating cells or tissue samples to fix protein-mediated RNA-RNA interactions;

[0173] 2) performing membrane perforation (preferably performing membrane perforation (perforating the cytoplasm and nucleus) while keeping the cells intact) so that subsequent reagents can enter the cells for reaction;

[0174] 3) Randomly interrupt free RNA in cells (i.e., RNA not protected by proteins) to obtain RNA with 3' end phosphorylated and protected by proteins;

[0175] 4) Phosphorylating the 5' end of the RNA protected by the protein and protecting the 3' phosphate end of the RNA protected by the protein to obtain an RNA-RNA complex.

[0176] Preferably, in step 1), the processing of the cell or tissue sample comprises the following steps: performing formaldehyde cross-linking on the cell or tissue sample; further comprising: performing processing using the cell cross-linking agent in the kit of the second aspect of the present invention; further comprising: mixing the cell or tissue sample with the cell cross-linking agent in the kit of the second aspect of the present invention, and reacting at room temperature for 8 to 12 minutes.

[0177] Preferably, in step 1), the processing of the cell or tissue sample further comprises the following step: terminating the cross-linking reaction of the cell or tissue sample, specifically: mixing the cell or tissue sample with the substance for terminating the cross-linking reaction of the cell or tissue sample in the kit of the second aspect of the present invention, and reacting at room temperature for 8 to 12 minutes.

[0178] Preferably, in step 2), the method for membrane perforation comprises the following steps: mixing the cell or tissue sample treated in 1) with a cell membrane perforator (preferably the cell membrane perforator in the kit of the second aspect of the present invention) for reaction.

[0179] Preferably, the reaction conditions are 0-4°C for 12-18 min.

[0180] Preferably, in step 3), the method for randomly disrupting free RNA in cells comprises the following steps: mixing the cells or tissue samples treated in step 2) with a free RNA disrupting agent (preferably the free RNA disrupting agent in the kit of the second aspect of the present invention) for reaction.

[0181] Preferably, the reaction conditions are incubation at 32-42° C. for 8-12 min.

[0182] Preferably, the method for phosphorylating the 5' end of the protein-protected RNA and protecting the 3' phosphate end of the protein-protected RNA in step 4) is: mixing the cell or tissue sample treated in step 3) with a polynucleotide kinase that loses 3' terminal phosphatase activity (preferably the polynucleotide kinase that loses 3' terminal phosphatase activity in the kit of the second aspect of the present invention) and reacting.

[0183] Preferably, the reaction conditions are 32-42° C. for 35-55 min.

[0184] Preferably, the cell is derived from an animal, a plant or a cellular microorganism.

[0185] Preferably, the tissue is derived from an animal or a plant.

[0186] The fourth aspect of the present invention provides a method for enriching chimeric RNA, comprising the steps of the RNA proximal end ligation method of the third aspect of the present invention.

[0187] Preferably, the method for enriching chimeric RNA further comprises the following steps: 1) lysing cells; 2) extracting total RNA and performing fragmentation treatment; 3) enriching chimeric RNA.

[0188] Preferably, the method for lysing cells in step 1) is: using a cell lysis reagent (preferably the cell lysis reagent in the kit of the second aspect of the present invention) to lyse the cells treated with RNA proximal end ligation.

[0189] Preferably, the method for extracting total RNA in step 2) is: extracting using a nucleic acid extraction reagent (preferably the nucleic acid extraction reagent in the kit of the second aspect of the present invention).

[0190] Preferably, the extraction of total RNA in step 2) further comprises a purification step.

[0191] Preferably, the purification method is purification using a nucleic acid purification reagent (preferably the nucleic acid purification reagent in the kit of the second aspect of the present invention).

[0192] Preferably, the purification further comprises a step of digesting the DNA.

[0193] Preferably, the method for digesting DNA is: treating with an endonuclease (preferably the endonuclease in the kit of the second aspect of the present invention).

[0194] Preferably, the fragmentation treatment method in step 2) comprises at least one of a chemical fragmentation method and a physical fragmentation method.

[0195] Preferably, the chemical disruption method comprises alkali treatment, metal ions (such as Mg2+ 、Zn 2+ ) at least one of solution treatment and enzyme treatment.

[0196] Preferably, the physical breaking method comprises at least one of a high temperature (90-100°C) breaking method, an ultrasonic breaking method, and a mechanical breaking method; further preferably, the physical breaking method comprises a high temperature (90-100°C) breaking method.

[0197] Preferably, the system of the high temperature interruption method comprises an alkaline solution of metal ions, First-strand Buffer.

[0198] Preferably, the length of the fragmented RNA is about 200 nt (eg, 180 to 220 nt).

[0199] Preferably, the method for enriching chimeric RNA in step 3) is: enrichment using a solid phase carrier with a second affinity tag that can affinity bind to the first affinity tag (preferably a solid phase carrier with a second affinity tag that can affinity bind to the first affinity tag in the kit of the second aspect of the present invention).

[0200] The fifth aspect of the present invention provides a library construction method, comprising the steps of the method for enriching chimeric RNA according to the fourth aspect of the present invention.

[0201] Preferably, the method further comprises the step of constructing a library of the enriched chimeric RNA.

[0202] Preferably, the library construction of the enriched chimeric RNA comprises the following steps: synthesizing cDNA, performing end repair on the cDNA, linker ligation, and PCR amplification to obtain a library.

[0203] Preferably, the library is a chain-specific library.

[0204] Preferably, dUTP is replaced with dTTP when synthesizing the second strand of cDNA.

[0205] Preferably, the method further comprises library quality control.

[0206] The sixth aspect of the present invention provides a library obtained by the method of the fifth aspect of the present invention.

[0207] The seventh aspect of the present invention provides a method for identifying RNA-RNA interactions or capturing RNA higher-order structures, comprising the following steps: sequencing the library of the sixth aspect of the present invention.

[0208] Preferably, the sequencing may further include: a cyclization reaction and a step of preparing nucleic acid nanoballs (DNBs).

[0209] The eighth aspect of the present invention provides the use of the reagent combination of the first aspect of the present invention and / or the kit of the second aspect in any one of a1) to a5);

[0210] a1) RNA proximal end ligation (preferably RNA in situ proximal end ligation);

[0211] a2) enrichment of chimeric RNA;

[0212] a3) Library construction;

[0213] a4) identifying RNA-RNA interactions (preferably identifying RNA-RNA interactions in situ);

[0214] a5) Capturing RNA higher-order structures (preferably capturing RNA higher-order structures in situ).

[0215] The present invention is further described in detail below through specific examples.

[0216] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0217] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0218] Example 1 Optimization of RtcB ligase reaction system

[0219] 1) Mn 2+ Concentration Optimization

[0220] 120 bp RNA1 (5'-rArArArArArArArArArArACATGGTCAGCCTGGAGGTGAGGCCAGGAAGACTGGGGCTAGAGGGTTTAGCGGGGGAGGGTAAGGGAAATAATTCATTCCTGTAAGCAAGAGTGAGCrArArArArArArArArArArA-3', SEQ ID NO.1, r represents the modification of ribonucleotide) was mixed with 25 bp 5Ada (5'NH 2C6-GAACGACATGGCTACGATCCGACTrU (SEQ ID NO. 2)-P3', 5' end with a blocking group modification, 3' end with a phosphate group modification, r represents a modified ribonucleotide) was connected in the following reaction system: RtcB ligase (NEB, M0458) 2μL, 10X RtcB Reaction Buffer 2μL, 30mM MnCl 2 0.67μL, 1.3μL, 2μL (i.e. Mn 2+ The final concentrations were approximately 1, 2, and 3 mM, respectively), 1 mM GTP 2 μL (i.e., the final concentration of GTP was 0.1 mM), 3 μM RNA1 1 μL, 100 μM 5Ada 1 μL, RNase free H 2 O To 20μL; reaction procedure: 37℃ reaction for 30min; ligation product run 12% denaturing PAGE gel, the result is as follows Figure 3 Shown: Mn 2+ The ligation efficiency is highest when the final concentration is 1 mM.

[0221] 2) Optimization of GTP concentration

[0222] The 120 bp RNA1 was connected with the 25 bp 5Ada in the following reaction system: RtcB ligase (NEB, M0458) 2 μL, 10X RtcB Reaction Buffer 2 μL, 10 mM MnCl 2 2μL (i.e. Mn 2+ The final concentration is 1mM), 5mM GTP 0.8μL, 2μL (i.e. the final concentration of GTP is 0.2 and 0.5mM respectively), 3μM RNA1 1μL, 100μM 5Ada 1μL, RNasefree H 2 O To 20μL; reaction procedure is the same as 1); the ligation product is run on 12% denaturing PAGE gel, the result is as follows Figure 3 Shown: Mn 2+ When the final concentration was 1 mM and the final concentration of GTP was 0.2 mM, the ligation effect was the highest, about 50%.

[0223] Example 2 Confirmation of the RtcB ligase and T4 RNA ligase 1 reaction system

[0224] The 36 bp RNA2 (5'-rArArArArArAAAAAACATGGTCAGCCTGGAGGTGrArArArAr ArA-3', r represents the modification of ribonucleotide, SEQ ID NO.3) was mixed with 25 bp 5Ada and 18 bp 3Ada (5'-rAAGTCGGAGGCCAAGCGG (SEQ ID NO.4)-NH 2 C6, 5-terminal with phosphate group modification, 3-terminal with blocking group modification, r represents ribonucleotide modification) was connected under the following connection system: 10X RtcB Reaction Buffer 20μL, 20mM GTP 2μL, 70mM MgCl 2 20 μL, 100 mM MnCl 2 2μL, 100mM ATP 2μL, 50% PEG8000 50μL, RtcB ligase (NEB, M0458) 10μL, T4 RNA ligase 1 (NEB, M0204) 10μL, RNase inhibitor 10μL, 10μM RNA2 1μL, 100μM 5Ada 1μL, 100μM 3Ada 1μL, RNase free H 2 OTo 200μL; reaction procedure: 37℃ reaction for 30min; ligation product run 12% denaturing PAGE gel, the result is as follows Figure 4 As shown: The connection effect is as expected, and the efficiency of simultaneous connection at both ends is about 40%.

[0225] Example 3: RNA-RNA interaction detection method for Hela cells

[0226] 1. Experimental Materials

[0227] HeLa cells.

[0228] 2. Experimental steps (process as follows Figure 2 (shown)

[0229] 1. Cell pretreatment (cell cross-linking, perforation)

[0230] (1) Prepare about 1x10 7 Hela cells were used for library construction, and the cells were washed three times with pre-cooled PBS. 5 mL of PBS (containing 1 v / v% formaldehyde) was taken to cross-link the cells at room temperature for 10 min. Then 0.25 mL of 2.5 M glycine (final concentration was 0.125 M) was added and mixed, and then gently shaken for 10 min. The cells were washed three times with pre-cooled PBS, centrifuged at 4°C, 2500 rpm for 10 min, and the supernatant was discarded.

[0231] (2) Suspend the cells with 1 mL of permeabilization solution, mix well, place on ice for 15 min, centrifuge at 3500 rpm at 4°C for 5 min, and discard the supernatant. The components and contents of the permeabilization solution are shown in Table 1 (1 mL).

[0232] Table 1 Components and contents of permeabilization solution

[0233] Components volume 1M Tris-HCl (pH 7.5) 10μL 10v / v%NP-40 50μL 5M NaCl 2μL SUPERase(20U / μL)(Thermo Fisher, AM2694) 0.1μL 10v / v%Triton X-100 30μL 10v / v%Tween 20 10μL 1×protease inhibitors(Sigma,P8340) 10μL <![CDATA[RNase free H 2 The]]> to 1mL

[0234] (3) Use 600μL 1×PNK Buffer (50mM Tris-HCl pH7.4, 0.2v / v% NP-40, 10mMgCl 2 ) Wash the precipitate, centrifuge at 3500 rpm at 4°C for 5 min, and discard the supernatant.

[0235] 2. RNA in situ proximal ligation

[0236] (1) Pipette 0.5 μL micrococcal nuclease (Thermo Fisher, catalog number EN0181) and add it to 499.5 μL 1×MN Buffer (50 mM Tris-HCl pH 8.0, 5 mM CaCl 2 ) was used to prepare the diluent, and then 50 μL of the diluent was added to 450 μL of 1× MN Buffer. After mixing, 200 μL of the resuspended precipitate was aspirated and incubated at 37°C for 10 min.

[0237] (2) Wash the precipitate twice with pre-cooled 1× PNK Buffer.

[0238] (3) Prepare 100 μL T4 PNK system (10 μL 10×T4 PNK Reaction Buffer, 15 μL 10 mM ATP, 10 μL T4 PNK Enzyme (NEB, M0236S), 65 μL RNase free H 2 O) Resuspend the precipitate and place it at 37°C for 45 min; then wash the precipitate twice with pre-cooled 1× PNK Buffer (0.05 v / v% NP-40).

[0239] (4) Prepare 200 μL of the ligation system to suspend the precipitate, place in a mixer at 37°C, 1000 rpm, 15 s / 3 min (15 s every 3 min) overnight. The components and contents of the ligation system (200 μL) are shown in Table 2, wherein the RNA linker is: 5'OH-rGrUrUrGrGrArUrUrCrArC / iBiorU / rArGrArCrArCrArG (SEQ ID NO.5)-OH-3', r represents ribonucleotide, and iBiorU represents biotin-labeled rU.

[0240] Table 2 Components and contents of the connection system

[0241]

[0242]

[0243] 3. RNA Extraction and Purification

[0244] (1) Add 200μL Proteinase K Buffer (2μL 1M Tris-HCl pH 7.5, 4μL 0.5MEDTA, 10μL 10% SDS, 184μL RNase free H 2 O) and 50 μL Proteinase K (Takara, 9034) to suspend the precipitate, gently turn it upside down 20 times, react at 37°C for 60 min, react at 56°C for 15 min, and finally cool to room temperature.

[0245] (2) Add 750 μL Trizol LS (Invitrogen, 10296028), vortex for 15 seconds, and leave at room temperature for 5 minutes. Then add 220 μL chloroform, vortex for 15 seconds, and leave at room temperature for 3 minutes. Centrifuge at 4°C, 13,000 rpm for 15 minutes, remove the supernatant and place it in a new centrifuge tube.

[0246] (3) Add 500 μL of isopropanol and 1 μL of GlycoBlue, mix well, and place at -20°C overnight.

[0247] (4) Centrifuge at 4°C, 13,000 rpm for 20 min, and remove the supernatant. Wash the precipitate twice with 600 μL 75% ethanol, and centrifuge at 4°C, 13,000 rpm for 5 min. After air drying at room temperature, dissolve the RNA in 20 μL enzyme-free water. Add 100 μL DNase I system to the RNA solution and place at 37°C for 20 min. The components and contents of the DNase I system are shown in Table 3.

[0248] Table 3 Components and contents of DNase I system

[0249] Components volume 10×RQ1 DNaseI Buffer 10μL RNase Inhibitor (40U / μL) 3μL Total RNA (20 μg) 6μL RQ1 RNase-free DNase I (Thermo Fisher, AM2238) 5μL <![CDATA[RNase free H 2 The]]> 76μL

[0250] (5) Add an equal volume of phenol chloroform, vortex for 15 seconds, and then centrifuge at 13,000 rpm for 15 minutes at 4°C. Remove the supernatant, add 500 μL of anhydrous ethanol and 1 μL of GlycoBlue, mix well, and place at -20°C overnight for precipitation.

[0251] 4. Enrichment of target RNA

[0252] (1) Add 4 μL of 5× First-strand Buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl) to the RNA solution. 2 ), incubate at 94°C for 5 min.

[0253] (2) Pretreatment of streptavidin magnetic beads: Take 20 μL of magnetic beads, wash the beads twice with solution A (0.05 M NaCl, 0.1 M NaOH), and then wash the beads once with solution B (0.1 M NaCl).

[0254] (3) Prepare blocking solution (32 μL yeast RNA (~50 μg, Roche, 10109223001), 100 μL 2×TWB Buffer, 68 μL RNase free H 2 O) was added to the magnetic beads, mixed and placed at room temperature for 1 hour. The magnetic beads were washed three times with 1×TWB Buffer. The components and contents of 2×TWB Buffer (5 mL) are shown in Table 4.

[0255] Table 4 Composition and content of 2×TWB Buffer (5 mL)

[0256] Components volume 1M Tris-HCl (pH 7.5) 50μL 0.5M EDTA 10μL 5M NaCl 2.5mL 10v / v%Tween 20 10μL <![CDATA[RNase free H 2 The]]> to 5mL

[0257] (4) Use 30 μL RNase free H 2 O and 50 μL 2×TWB Buffer to resuspend the magnetic beads, then add the RNA solution, incubate at room temperature for 30 min, and wash the magnetic beads four times with 1×TWB Buffer.

[0258] (5) Add 100 μL PK Buffer (10 μL 1 M Tris-HCl (pH 7.0), 2 μL 0.5 M EDTA, 20 μL 5 M NaCl, 50 μL 10% SDS, RNase free H 2 0 to 5mL), incubate at 95℃ for 10min, place on a magnetic rack, and transfer the supernatant to a new centrifuge tube. Repeat this step once, wash the magnetic beads with 100μL PK Buffer, and collect the supernatant.

[0259] (6) Add an equal volume of phenol chloroform to extract RNA, then add 900 μL of anhydrous ethanol and 1 μL of GlycoBlue, mix well, and place at -20°C overnight to precipitate.

[0260] (7) The solution in the above step was centrifuged at 4°C, 13,000 rpm for 20 min, the precipitate was washed twice with 75% ethanol, air-dried at room temperature, and the RNA was dissolved in 10 μL of enzyme-free water.

[0261] 5. Strand-specific RNA library construction

[0262] Prepare MGIEasy RNA Directional Library Preparation Kit (16RXN) (MGI, 1000006385), perform reverse transcription and duplex synthesis on the enriched RNA according to the kit instructions, purify the duplex product with magnetic beads, repair the ends and add A, connect the adapters, and amplify the library by PCR. Prepare DNBSEQ DNB Preparation Kit (20RXN) (MGI, 1000016115), denature PCR products, circularize single strands, linearize and digest, prepare DNA nanoballs, and after the concentration is qualified, use MGISEQ-2000RS High-throughput Sequencing Kit (FCL PE100) (MGI, 1000006385) to perform PE100 sequencing on the MGISEQ-2000 sequencing platform.

[0263] The PCR amplification products were quality checked using Bioanalyzer 2100. Figure 5 As shown: the main peak is around 330bp, which is in line with expectations.

[0264] The obtained data were analyzed, and the results are shown in Table 5: the proportion of chimeric reads was 18%, which is higher than the existing methods (RIC-seq, PARIS, SPLSH, LIGR-seq, MARIO, data from the literature: RIC-seq for global in situprofiling of RNA–RNA spatial interactions, Zhaokui Cai, Nature volume 582, pages 432–437 (2020)), that is, it is more efficient than the existing methods in capturing RNA-RNA interactions, among which it is much higher than PARIS, SPLSH, LIGR-seq, and MARIO. Although the proportion of chimeric reads is not much higher than that of RIC-seq, the experimental operation process is greatly reduced (correspondingly, the time and reagent costs are reduced), which can reduce the loss of RRIs information caused by cumbersome operation steps, and facilitate better revealing of the global RRIs map in the cell. The entire experimental cycle takes 5 days; at the same time, the addition of RNA adapters also facilitates subsequent data analysis, which can improve the specificity of the data and the accuracy of bioinformatics analysis.

[0265] Table 5 Data comparison of different methods

[0266]

[0267] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A reagent combination for RNA proximal ligation, comprising: a first ligase that can ligate the 3' phosphate terminus of the oligonucleotide to the 5' hydroxyl terminus of the oligonucleotide; and a second ligase that can ligate the 5' phosphate terminus of the oligonucleotide to the 3' hydroxyl terminus of the oligonucleotide; and An oligonucleotide linker comprising a 5' hydroxyl terminus and a 3' hydroxyl terminus.

2. The reagent combination according to claim 1, characterized in that: The first ligase comprises: at least one of RtcB ligase, an active fragment of RtcB ligase, a derivative of RtcB ligase, and an analog of RtcB ligase; Preferably, the second ligase comprises at least one of: T4 RNA ligase 1, TS2126 RNA ligase, single-stranded DNA / RNA circular ligase, and T4 RNA ligase 2.

3. The reagent combination according to claim 1 or 2, characterized in that: One or more nucleotides in the oligonucleotide linker are modified with a first affinity tag; Preferably, the length of the oligonucleotide linker is 10 to 25 nt; Preferably, the reagent combination further comprises: a first cofactor, wherein the first cofactor comprises: triphosphate and manganese ions; Preferably, the reagent combination further comprises: a second cofactor, wherein the second cofactor comprises: at least one of ATP and NAD and metal ions.

4. The reagent combination according to claim 3, characterized in that: The reagent combination is used to connect two RNAs of the RNA-RNA complex in the same system to form a chimeric RNA; wherein the same system is specifically carried out in the same reaction container; further specifically, it is carried out in the same reaction container and at the same time; Preferably, the reagent combination further comprises an instruction manual, which records the method for using the reagent combination; Preferably, the method for using the reagent combination is: mixing the RNA-RNA complex with the oligonucleotide linker, the first ligase, the second ligase, the first auxiliary factor, and the second auxiliary factor, and performing a ligation reaction on the obtained ligation reaction system; Preferably, the final concentration of the triphosphate in the first auxiliary factor in the ligation reaction system is 0.1-0.8 mM; Preferably, the final concentration of manganese ions in the first auxiliary factor in the ligation reaction system is 0.5 to 3.5 mM; preferably, the final concentration of ATP and / or NAD in the second auxiliary factor in the ligation reaction system is 0.5 to 3.5 mM; Preferably, the final concentration of the metal ion in the second auxiliary factor in the ligation reaction system is 3.5 to 22.5 mM; Preferably, the reagent combination further comprises: a polynucleotide kinase that loses 3' terminal phosphatase activity.

5. A kit comprising: the reagent combination according to any one of claims 1 to 4; The kit further comprises: at least one of a free RNA disrupting agent, a cell membrane perforating agent, and a cell cross-linking agent; Preferably, the kit is used for any one of a1) to a5): a1) RNA proximal ligation; a2) enrichment of chimeric RNA; a3) Library construction; a4) Identification of RNA-RNA interactions; a5) Capture RNA higher-order structure.

6. The kit according to claim 5, characterized in that: When the kit is used for any one of a2) to a5), the kit further comprises: at least one of a cell lysis reagent, a nucleic acid extraction reagent, a nucleic acid purification reagent, an endonuclease, a substance for breaking nucleic acids, and a solid phase carrier with a second affinity tag that can affinity bind to the first affinity tag; Preferably, when the kit is used for any one of a3) to a5), the kit further comprises: an RNA library preparation reagent; preferably, when the kit is used for any one of a4) to a5), the kit further comprises: at least one of a cyclization reagent, a nucleic acid nanoball preparation reagent, and a sequencing reagent.

7. A method for RNA proximal ligation, comprising: using the reagent combination according to any one of claims 1 to 4 and / or the kit according to any one of claims 5 to 6, Preferably, the method comprises the following steps: mixing the RNA-RNA complex with the first ligase, the second ligase, and the oligonucleotide linker in the reagent combination according to any one of claims 1 to 4, and performing a ligation reaction on the obtained ligation reaction system; The RNA in the RNA-RNA complex comprises a 5' phosphate terminus and a 3' phosphate terminus.

8. The RNA proximal end ligation method according to claim 7, characterized in that: The ligation reaction ligates two RNAs of the RNA-RNA complex in the same system to form a chimeric RNA; wherein the same system is specifically carried out in the same reaction vessel; further specifically, it is carried out in the same reaction vessel and at the same time; Preferably, the ligation reaction system further comprises: the first auxiliary factor and the second auxiliary factor in the reagent combination according to any one of claims 3 to 4; Preferably, the final concentration of the triphosphate in the first auxiliary factor in the ligation reaction system is 0.1-0.8 mM; Preferably, the final concentration of manganese ions in the first auxiliary factor in the ligation reaction system is 0.5 to 3.5 mM; preferably, the final concentration of ATP and / or NAD in the second auxiliary factor in the ligation reaction system is 0.5 to 3.5 mM; Preferably, the final concentration of the metal ion in the second auxiliary factor in the ligation reaction system is 3.5 to 22.5 mM; Preferably, the method for preparing the RNA-RNA complex comprises the following steps: 1) Treating cells or tissue samples to fix protein-mediated RNA-RNA interactions; 2) performing membrane perforation; 3) Randomly interrupt free RNA in cells to obtain protein-protected 3'-terminal phosphorylated RNA; 4) phosphorylating the 5' end of the RNA protected by the protein, and protecting the 3' phosphate end of the RNA protected by the protein, to obtain an RNA-RNA complex; Preferably, in step 1), the processing of the cell or tissue sample comprises the following steps: performing formaldehyde cross-linking on the cell or tissue sample.

9. A method for enriching chimeric RNA, comprising the steps of the RNA proximal end ligation method according to claim 7 or 8; The method further comprises the following steps: 1) lysing cells; 2) extracting total RNA and performing fragmentation treatment; 3) enriching chimeric RNA.

10. A method for constructing a library, comprising the steps of the method for enriching chimeric RNA according to claim 9; The method further comprises the following step: constructing a library for the enriched chimeric RNA.

11. A library obtained by the method according to claim 10.

12. A method for identifying RNA-RNA interactions or capturing RNA higher-order structures, comprising the steps of: sequencing the library of claim 11; Preferably, before sequencing, the method further comprises: Cyclization reaction; Preferably, the cyclization reaction further includes a step of preparing nucleic acid nanospheres.

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