Method for detecting chemical modification sites and interaction sites of nucleic acid based on nucleic acid grafting and nucleic acid reverse transcription template conversion technology

Abstract

The invention provides a method for detecting a chemical modification site and an interaction site of nucleic acid based on nucleic acid grafting and a nucleic acid reverse transcription template conversion technology, which comprises the following steps of: obtaining an RNA (Ribonucleic Acid) branch chain before detecting the chemical modification site and the interaction site of the nucleic acid; then performing reverse transcription from the RNA branched chain by using a reverse transcription primer complementarily paired with the RNA branched chain, performing grafting template conversion when the reverse transcription is performed to the connection position of the RNA branched chain and the modification or interaction connection position, and continuing the reverse transcription by taking the main chain connected with the RNA branched chain as a template to obtain cDNA (complementary deoxyribonucleic acid) containing modification site or interaction site information; and finally, constructing a high-throughput library by using the cDNA containing the modification site or interaction site information, and performing high-throughput sequencing to determine the modification site or interaction site. Or detecting the cDNA containing the modification site or interaction site information by adopting PCR (Polymerase Chain Reaction) and TA-cloring technologies, so as to realize the recognition of the modification site or interaction site. The method can be applied to multiple gene sequencing methods.

Description

Technical Field

[0001] The invention belongs to the field of nucleic acid chemistry, and in particular to a method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template switching technology. Background Art

[0002] In the body, different RNA molecules have different functions, which ensure the normal expression of genetic information in an orderly manner: messenger RNA (mRNA) transmits genetic information, transfer RNA (tRNA) transfers translation materials, ribosomal RNA (rRNA) forms ribosomes for translation, and small nuclear RNA (snRNA) involved in RNA splicing, small nucleolar RNA (snoRNA) involved in RNA modification, and small RNA (miRNA) involved in post-transcriptional expression regulation also regulate the expression of genetic information at different stages. The realization of these functions is inseparable from the recognition and interaction between RNA and other biological molecules.

[0003] RNA can interact directly with RNA through base pairing to achieve sequence-specific biological functions, and play an important role in many important biochemical processes such as miRNA-guided gene regulation, tRNA-involved translation, and crRNA-guided viral RNA degradation. In addition to direct interactions, RNA can also act indirectly through RNA-binding proteins as a medium, which often implies the functional correlation between RNAs. At present, there are many methods to study RNA-RNA interactions, including low-throughput methods based on physicochemical properties and high-throughput methods based on proximity ligation. They have good detection capabilities for stable strong interactions, giving us a preliminary understanding of the RNA interactome. With the deepening of research, the dynamics and cell specificity of RNA interactions have attracted the attention of researchers. We urgently need a new tool to explore the response of the RNA interactome to different physiological and environmental conditions.

[0004] RNA and proteins have extensive interactions and extremely important biological functions. It can almost be said that the realization of any RNA function requires the participation of proteins. In the past, a variety of methods represented by cross-linking immunoprecipitation sequencing (CLIP-seq) have been developed to characterize RNA-protein interactions. With the help of these methods, many RNA-binding proteins that directly interact with RNA via RNA binding domains have been identified, greatly deepening our understanding of RNA functions. Recently, the discovery of non-classical interactions represented by the liquid-liquid phase separation phenomenon has shown that RNA and proteins can have a more indirect and dynamic mode of action. If this phenomenon can be explored in depth, it will greatly promote our understanding of life systems. However, current methods make it difficult to characterize these non-classical interactions, which hinders the progress of research.

[0005] The interaction between RNA and other RNA and proteins can be changed by dynamic RNA modification, thereby achieving the regulation of gene expression, which has been a research hotspot in the past decade. RNA modification can participate in physiological processes such as biological clock regulation, immune regulation, cancer disease formation, embryonic and neural development regulation, stem cell differentiation, RNA virus replication, and sex determination by regulating RNA transcription, splicing, transport, translation, and degradation. These achievements are inseparable from the development of high-throughput sequencing methods. With the deepening and refinement of research, researchers are no longer satisfied with the modification level averaged by a large number of cells, and have turned their attention to the study of single cells. However, current sequencing methods make it difficult to characterize the modification group of single cells, which has become an obstacle to research work. Summary of the invention

[0006] The object of the present invention is to provide a method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template switching technology.

[0007] The present invention adopts the following technical solutions:

[0008] A method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template switching technology, comprising the following steps:

[0009] (1) Before detecting nucleic acid chemical modification sites and interaction sites, it is necessary to first obtain an RNA branch chain. Specifically, chemical and / or biological methods can be used to graft an RNA branch chain with a known sequence at the RNA modification site or RNA-protein interaction site, or two RNA chains can be cross-linked at the RNA-RNA interaction site and one of the chains can be used as the RNA branch chain;

[0010] (2) using a reverse transcription primer complementary to the RNA branch to start reverse transcription from the RNA branch, and when reverse transcription reaches the RNA branch and the modified junction or the interactive junction, performing grafting template switching, and continuing reverse transcription with the main chain (RNA or DNA main chain) connected to the RNA branch as a template to obtain a cDNA containing the modification site or the interactive site information;

[0011] (3) constructing a high-throughput library using the cDNA containing the modification site or interaction site information, and performing high-throughput sequencing to determine the modification site or interaction site; or using PCR and TA-cloning technology to detect the cDNA containing the modification site or interaction site information, thereby realizing the identification of the modification site or interaction site.

[0012] In the above technical solution, further, the chemical modification is N 6 -Methyladenosine m 6A.N 6 -methyldeoxyadenosine 6mA, pseudouridine Ψ, N 6 -Propargyl adenosine p 6 A or N 1 -Methyladenosine m 1 Any one of A, wherein the interaction is any one of RNA-RNA intramolecular interaction, RNA-RNA direct interaction, RNA-RNA indirect interaction, protein-RNA classical interaction, protein-RNA liquid-liquid phase separation interaction and RNA polymerase-nascent RNA interaction.

[0013] Furthermore, detection of N 6 -methyladenosine (m 6 A) and / or N 6 -methyldeoxyadenosine (6mA) modification method, comprising the following steps:

[0014] (a) The fat mass and obesity-associated protein (FTO) demethylase was used to treat cells containing m 6 For RNA samples modified with mA or DNA samples containing 6mA, dithiothreitol (DTT) was added after 5-30 minutes to make the mA 6 A / or 6mA with thiol modification;

[0015] (b) adding dibenzylcyclooctyl maleimide (DBCO-Maleimide) to react with the sulfhydryl group on the RNA or DNA to obtain RNA or DNA labeled with a carbon-carbon triple bond;

[0016] (c) adding an RNA branch chain modified with an azide group at 5' to react with the RNA or DNA labeled with a carbon-carbon triple bond to obtain an RNA or DNA grafted with an RNA branch chain of known sequence;

[0017] (d3) adding a reverse transcription primer complementary to the RNA branch and a template switching probe of a known sequence, starting reverse transcription from the RNA branch, performing grafting template switching when reverse transcription reaches the modified junction, and finally obtaining a cDNA containing the modification site information by means of traditional template switching;

[0018] (e3) constructing a high-throughput library using the cDNA and performing high-throughput sequencing to determine the connection site, thereby obtaining m 6 A modification site or a 6mA modification site in the DNA.

[0019] Further, the method for detecting pseudouridine (Ψ) modification comprises the following steps:

[0020] (a) RNA samples containing Ψ modification were reacted with 1-cyclohexyl-2-(morpholinoethyl)carbodiimide propargyl p-toluenesulfonate (proCMC) at 37°C for 20 min;

[0021] (b) placing the RNA obtained in (1) under alkaline conditions (pH 10-11) at 37° C. for more than 6 hours to obtain RNA specifically labeled with propargyl Ψ;

[0022] (c) adding an RNA branch chain modified with an azide group at 5' to react with the propargyl-labeled RNA to obtain an RNA grafted with a branch chain;

[0023] (d) adding a reverse transcription primer complementary to the RNA branch and a template switching probe of a known sequence, starting reverse transcription from the RNA branch, performing grafting template switching when reverse transcription reaches the modified junction, and finally obtaining a cDNA containing the modification site information by means of traditional template switching;

[0024] (e) constructing a high-throughput library using the cDNA and combining it with high-throughput sequencing technology to analyze the connection sites, thereby obtaining the Ψ modification sites in the RNA.

[0025] Furthermore, detection of N 6 -Propargyl adenosine (p 6 A) A modification method comprising the following steps:

[0026] (a) In the presence of p 6 A modified RNA sample, adding a 5' RNA branch with an azide group modification, and 6 The carbon-carbon triple bond of A reacts to obtain RNA grafted with an RNA branch of known sequence;

[0027] (b) adding a reverse transcription primer complementary to the RNA branch and a template switching probe of a known sequence, starting reverse transcription from the RNA branch, and when reverse transcription reaches the modified junction, performing grafting template switching, and finally obtaining a cDNA containing the modification site information by means of traditional template switching;

[0028] (c) constructing a high-throughput library using the cDNA, and performing high-throughput sequencing to determine the connection site, that is, obtaining the p 6 A modification site.

[0029] Furthermore, the preparation method of proCMC is:

[0030] Step S1, N-(2-aminoethyl)morpholine (6.48 g, 49.6 mmol) was added to dichloromethane, and then cyclohexyl isocyanate (6.23 g, 49.6 mmol) was added dropwise in an amount equimolar to N-(2-aminoethyl)morpholine, and the mixture was reacted at room temperature for 3 to 5 hours. The dichloromethane was removed by rotary evaporator to obtain a crude product, and the crude product was slurried with petroleum ether to obtain an intermediate 1. The chemical structure of the intermediate 1 is:

[0031]

[0032] Step S2, triphenylphosphine (15.10g, 57.8mmol) is fully dissolved in 100mL dichloromethane under ice-water bath conditions, and then liquid bromine (9.25g, 57.8mmol) and three times the molar amount of liquid bromine are added to the reaction bottle by syringe, and then the intermediate 1 (12.30g, 48mmol) is dissolved in 20mL dichloromethane, and the reaction bottle is slowly added with a syringe, and the reaction is carried out at 10-15°C for 3 hours. After the reaction is completed, the reaction system is washed three times with 500mL of water, and the lower organic phase liquid is retained, and then dried and filtered with anhydrous sodium sulfate to obtain a crude product. The crude product is concentrated by rotary evaporator and distilled under reduced pressure to obtain intermediate 2, and the chemical structure of intermediate 2 is:

[0033]

[0034] Step S3, using acetonitrile as solvent, adding intermediate 2 (8.67 g, 36.6 mmol) and an equimolar amount of propargyl p-toluenesulfonate (7.70 g, 36.6 mmol) dropwise therein, heating to 60-70°C for reaction for 3 hours. After the reaction, the reaction solution was concentrated by rotary evaporator to obtain a crude product, which was then separated by high performance liquid chromatography (HPLC), with acetonitrile and water as the mobile phase, to obtain the 1-cyclohexyl-2-(morpholineethyl)carbodiimide propargyl p-toluenesulfonate (proCMC), whose chemical structure is:

[0035]

[0036] Furthermore, the method for grafting an RNA branch of known sequence is a click chemistry reaction, a biotin-streptavidin bioorthogonal reaction, UV crosslinking, chemical crosslinking agent crosslinking or antibody crosslinking method.

[0037] Furthermore, the RNA is RNA synthesized by in vitro transcription or total RNA, mRNA, rRNA or lncRNA extracted from common mammalian cells, mammalian cancer cells, mammalian stem cells, bacteria, virus host cells and various types of tissues and organs; the DNA is synthetic DNA or DNA extracted from common mammalian cells, mammalian cancer cells, mammalian stem cells, bacteria, virus host cells and various types of tissues and organs.

[0038] Furthermore, the reverse transcriptase is HIV reverse transcriptase and its various mutants, M-MLV reverse transcriptase and its various mutants, AMV reverse transcriptase and its various mutants, preferably Maxima H Minus reverse transcriptase and RevertAid reverse transcriptase; the reverse transcription temperature is 37-65°C, preferably 50°C.

[0039] Furthermore, the RNA purification method in each step is TRIzol TM Reagent, chloroform-phenol extraction, Proteinase K digestion, silica gel membrane centrifugal column method, magnetic bead method, ethanol, isopropanol precipitation and other techniques or a combination thereof, preferably magnetic bead method and silica gel membrane centrifugal column method.

[0040] Furthermore, the method for constructing a high-throughput library is specifically as follows: i) using traditional reverse transcription template conversion or using Tn5 transposase to attach a known sequence to the 3' end of the reverse transcription product cDNA to obtain a cDNA product with known sequences at both ends; ii) designing and synthesizing PCR primers that are complementary to the RNA branch and the template switching probe TSO with a known sequence, respectively, and amplifying the cDNA product obtained in step i) by PCR to obtain double-stranded DNA with known sequences at both ends; iii) designing and synthesizing PCR primers containing sequence information required for second-generation sequencing, and amplifying the double-stranded DNA obtained in step ii) by PCR to construct a high-throughput library.

[0041] Furthermore, the library construction and sequencing step can also use PCR and TA-cloning technology to identify and verify the connection sites on the specific RNA, and can also use a single-cell sequencing method.

[0042] Furthermore, the method for detecting RNA-RNA intramolecular interaction sites comprises the following steps:

[0043] (a) Fixing two RNA chains interacting in cells by UV crosslinking, chemical crosslinking, etc.

[0044] (b) extracting and purifying RNA from cells, and using one of the RNA chains as an RNA branch;

[0045] (c) adding a reverse transcription primer complementary to the RNA branch and a template switching probe of a known sequence, and performing reverse transcription from the RNA branch. If there is an intramolecular interaction between RNA molecules, the reverse transcription will skip the interaction region, perform grafting template switching, and continue reverse transcription with another RNA chain as a template, and finally obtain a cDNA containing RNA-RNA intramolecular interaction information;

[0046] (d) constructing a high-throughput library using the cDNA, and performing high-throughput sequencing to determine the RNA-RNA intramolecular interaction sites.

[0047] Furthermore, the method for detecting direct interaction sites between RNA-RNA molecules comprises the following steps:

[0048] (a) Fixing the interacting RNA in cells by UV crosslinking, chemical crosslinking, etc.

[0049] (b) extracting and purifying RNA from cells, and using one of the RNA chains as an RNA branch;

[0050] (c) adding a reverse transcription primer complementary to the RNA branch and a template switching probe of a known sequence, and performing reverse transcription from the RNA branch. If there is a direct interaction between RNA-RNA molecules, the reverse transcription will skip the interaction region, perform grafting template switching, and continue reverse transcription with another RNA chain as a template, and finally obtain cDNA containing information about direct interaction between RNA-RNA molecules;

[0051] (d) constructing a high-throughput library using the cDNA, and performing high-throughput sequencing to determine the interaction regions between RNA molecules, thereby obtaining an RNA-RNA direct interaction map.

[0052] Further, the method for detecting the indirect interaction site between RNA-RNA molecules comprises the following steps:

[0053] (a) Fixing the interacting RNA in cells by UV crosslinking, chemical crosslinking, etc.

[0054] (b) extracting and purifying RNA from cells, and using one of the RNA chains as an RNA branch;

[0055] (c) adding a reverse transcription primer complementary to the RNA branch and a template switching probe of a known sequence, and performing reverse transcription from the RNA branch. If there is an indirect interaction between RNA-RNA molecules, the reverse transcription will skip the interaction region, perform grafting template switching, and continue reverse transcription with another RNA chain as a template, and finally obtain a cDNA containing information on indirect interactions between RNA-RNA molecules;

[0056] (d) constructing a high-throughput library using the cDNA, and performing high-throughput sequencing to determine the RNA-RNA molecular interaction region, and then obtaining the RNA-RNA indirect interaction site.

[0057] Further, the method for detecting protein-RNA classical interaction sites comprises the following steps:

[0058] (a) Fixing RNA-protein interactions in cells by UV crosslinking, chemical crosslinking, etc.

[0059] (b) using an antibody against the target protein to bind to the target protein;

[0060] (c) using protein A connected to an RNA branch of known sequence to combine with an antibody against the target protein to achieve grafting of the RNA branch;

[0061] (d) adding a reverse transcription primer complementary to the RNA branch and a template switching probe of a known sequence, and starting reverse transcription from the RNA branch. When reverse transcription reaches the interaction site, the reverse transcription reaction will cross the connection between the branch and the main chain, perform grafting template switching, and continue reverse transcription with the target RNA chain as a template to obtain a cDNA containing RNA-protein interaction site information;

[0062] (e) constructing a high-throughput library using the cDNA, and performing high-throughput sequencing to determine the connection sites, thereby obtaining RNA-protein interaction sites.

[0063] Furthermore, the method for detecting protein-RNA liquid-liquid phase separation interaction sites comprises the following steps:

[0064] (a) Fixing RNA-protein interactions in cells by methods such as formaldehyde fixation;

[0065] (b) using an antibody against the target protein to bind to the target protein;

[0066] (c) using protein A connected with an RNA branch of known sequence to combine with an antibody against the target protein to achieve grafting of the RNA branch;

[0067] (d) adding a reverse transcription primer complementary to the RNA branch and a template switching probe of a known sequence, and starting reverse transcription from the RNA branch. When reverse transcription reaches the interaction site, the reverse transcription reaction will cross the connection between the branch and the main chain, perform grafting template switching, and continue reverse transcription with the target RNA chain as a template to obtain a cDNA containing RNA-protein interaction site information;

[0068] (e) constructing a high-throughput library using the cDNA, and performing high-throughput sequencing to determine the connection sites, thereby obtaining RNA-protein interaction sites.

[0069] Further, the method for detecting RNA polymerase-nascent RNA interaction sites comprises the following steps:

[0070] (a) Through the method of intracellular transcription restart, p 6 A is incorporated into the RNA polymerase-nascent RNA interaction site;

[0071] (b) extracting and purifying RNA, adding a 5' RNA branch with a known sequence modified with an azide group to the p 6 The carbon-carbon triple bond of A reacts to obtain RNA grafted with an RNA branch of known sequence;

[0072] (c) adding a reverse transcription primer complementary to the RNA branch and a template switching probe of a known sequence, and starting reverse transcription from the RNA branch. When reverse transcription reaches the modified junction, the reverse transcription reaction will skip the junction between the branch and the main chain, perform grafting template switching, and continue reverse transcription with the target RNA chain as a template to obtain a cDNA containing the modification site information;

[0073] (d) constructing a high-throughput library using the cDNA and performing high-throughput sequencing to determine the junction site, i.e., the p 6 A site, the RNA polymerase-nascent RNA interaction site can be obtained.

[0074] In the above technical solution, further, the cells are common mammalian cells, mammalian cancer cells, mammalian stem cells, bacteria, virus host cells or various types of tissues and organs.

[0075] Furthermore, the reverse transcriptase is HIV reverse transcriptase and its various mutants, M-MLV reverse transcriptase and its various mutants, AMV reverse transcriptase and its various mutants, preferably Maxima H Minus reverse transcriptase and RevertAid reverse transcriptase; the reverse transcription temperature is 37-65°C, preferably 50°C.

[0076] Furthermore, for RNA-RNA intramolecular interactions, direct interactions between RNA-RNA molecules, and indirect interactions between RNA-RNA molecules, the reverse transcription primers used in the detection process are random hexamers with linkers or endogenous RNA complementary primers.

[0077] The beneficial effects of the present invention are:

[0078] The present invention discovered for the first time that RNA that has been chemically cross-linked can be read through by reverse transcriptase, namely the phenomenon of grafting template switching, which provides the possibility of detecting modification sites and interaction sites by means of nucleic acid grafting and nucleic acid reverse transcription template switching.

[0079] The method of the present invention is based on grafting template conversion starting from RNA branches specifically modified at nucleic acid modification sites. Compared with existing gene sequencing technologies used for modification detection, it improves the accuracy of currently commonly used antibody immunoprecipitation and large-scale parallel sequencing methods for detecting modification sites, reduces false positive signals, and is a direct high-throughput, low-false-positive single-base identification method.

[0080] The method of the present invention grafts an RNA branch of known sequence at the interaction site, so that the modification site or interaction site can be enriched by grafting template conversion initiated by the RNA branch, thereby bypassing the traditional antibody enrichment model and eliminating the need for repeated purification. It can be used for single-cell sequencing to characterize the modification sites or interaction sites of cell differences.

[0081] The present invention uses Maxima H Minus reverse transcriptase to perform reverse transcription on the grafted RNA. The inventors selected a number of commercial reverse transcriptases, including HIV reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, RevertAid reverse transcriptase, SuperScriptⅡ reverse transcriptase, SuperScriptⅢ reverse transcriptase, TGIRT III reverse transcriptase, etc., and finally found that Maxima H Minus reverse transcriptase has strong grafting template conversion and read-through capabilities in the grafting template conversion process of grafted RNA.

[0082] Based on nucleic acid grafting and nucleic acid reverse transcription template conversion, the present invention can be applied to various gene sequencing methods, such as the detection of various modification sites on various types of nucleic acids, and the study of RNA-RNA interaction and RNA-protein interaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 It is a schematic diagram of nucleic acid grafting and nucleic acid reverse transcription template conversion;

[0084] Figure 2 It is a schematic diagram of a method for detecting nucleic acid modification groups based on nucleic acid grafting and nucleic acid reverse transcription template switching technology;

[0085] Figure 3 Is Dot-blot verification m 6 A grafting of branches onto modified RNA;

[0086] Figure 4 is m 6A. Result diagram of grafting template switching start site obtained by high-throughput sequencing of modified RNA probe;

[0087] Figure 5 is m 6 A graph of the number of randomly incorporated bases during the conversion of the grafted template obtained by high-throughput sequencing of the modified RNA probe;

[0088] Figure 6 28S rRNA 4220m 6 A. Result diagram of the grafting template switching start site obtained by high-throughput sequencing at the modified site;

[0089] Figure 7 It is the transcriptome obtained by 293T mRNA high-throughput sequencing. 6 A site result map;

[0090] Figure 8 It is the transcriptome obtained by 293T mRNA high-throughput sequencing. 6 The conserved sequence of the A site;

[0091] Fig. 9 This is the result diagram of the grafting template switching start site obtained by low-throughput sequencing of 6mA modified DNA probe;

[0092] Fig.10 This is a graph showing the result of grafting template switching start site obtained by high-throughput sequencing of pseudouridine-modified RNA probes;

[0093] Fig.11 Yes 6 A. Result diagram of grafting template switching start site obtained by high-throughput sequencing of modified RNA probe;

[0094] Fig.12 It is 1-cyclohexyl-2-(morpholinoethyl)carbodiimide propargyl p-toluenesulfonate (proCMC) 1 H NMR spectroscopy;

[0095] Fig.13 It is a high-resolution mass spectrum of 1-cyclohexyl-2-(morpholinoethyl)carbodiimide propargyl p-toluenesulfonate (proCMC);

[0096] Fig.14 This is a schematic diagram of the detection of RNA-RNA interactions based on nucleic acid grafting and nucleic acid reverse transcription template switching technology;

[0097] Fig.15 Schematic diagram of the detection of RNA-protein interactions based on nucleic acid grafting and nucleic acid reverse transcription template switching technology. DETAILED DESCRIPTION

[0098] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0099] This application proposes a new approach to solve the above problems, which is that the current characterization tools for RNA-RNA interaction, RNA-protein interaction and RNA and / or DNA modification are difficult to meet the current research needs. The present invention finds that RNA can be read through by reverse transcriptase after chemical cross-linking, and this phenomenon is called graft template switching phenomenon ( Figure 1 ). Based on this phenomenon, the present invention grafts RNA of known sequence at the interaction or modification site, then obtains complementary DNA (cDNA) through grafting template conversion, adds 3' adapter through traditional template switching (Template-switch) or transposase fragmentation, and then can be amplified by PCR and then high-throughput sequencing can be performed to obtain RNA interaction or modification sites. This method can bypass the purification step in the library construction process and can be used for single-cell library construction; its specific grafting and library construction method can enrich the target sequence from the total RNA, reduce the interference of background signals, and reduce sequencing costs, which will provide an important tool for biological function research.

[0100] The present invention hopes to utilize nucleic acid grafting and nucleic acid reverse transcription template switching technology to achieve RNA and / or DNA nucleic acid modification sequencing by performing grafting template switching at modification or interaction sites, and study the interaction between non-classical RNA and other macromolecules.

[0101] like Figure 1 The figure is a schematic diagram of nucleic acid grafting and nucleic acid reverse transcription template switching in the present invention. Reverse transcription starts from the grafted RNA branch. When reverse transcription reaches the connection between the RNA branch and the target RNA main chain, due to the steric hindrance of the chemical group, the reverse transcription reaction will skip the connection and perform template switching, and continue reverse transcription with the target RNA main chain as a template.

[0102] like Figure 4-11 The figure is a result diagram of the grafting template switching start site obtained by high-throughput sequencing of RNA and / or DNA with various nucleic acid modifications, wherein the abscissa is the distance from the grafting template switching start site to the modification site.

[0103] like Figure 12-13 The figures are the relevant characterization data of 1-cyclohexyl-2-(morpholineethyl)carbodiimide propargyl p-toluenesulfonate (proCMC) prepared by the method of the present invention. Fig.12 1-cyclohexyl-2-(morpholinoethyl)carbodiimide propargyl p-toluenesulfonate (proCMC) 1 H NMR spectroscopy: 1H NMR (400MHz, CDCl3): δ7.75(d,J=8.2Hz,2H),7.18(d,J=8.1Hz,2H),4.76(s,2H),4.00(s,2H),3.91(d,J=13.6Hz,4H),3.73(s,6H),3.47(t,J=10.5Hz ,1H),2.85(s,1H),2.35(s,3H),1.81(d,J=9.5Hz,2H),1.65(d,J=13.3Hz,2 H), 1.54 (d, J = 12.8Hz, 1H), 1.27 (d, J = 17.2Hz, 2H), 1.10 (q, J = 12.2Hz, 3H). Fig.13 High resolution mass spectrum of 1-cyclohexyl-2-(morpholinoethyl)carbodiimide propargyl p-toluenesulfonate (proCMC): HRMS (ESI), m / z = 276.2064 ([M+H] + ,calcd 276.2070).

[0104] The present invention will be further described below in conjunction with specific embodiments.

[0105] Example 1 Extracellular RNA m 6 Detection of A modification sites (such as Figure 2 )

[0106] 1. RNA m 6 A site labels RNA branches and verifies branch grafting

[0107] (1) Using HiScribe TM T7 High Yield RNA Synthesis Kit (NEB) was used to prepare RNA with m 6 A modified RNA probe with a length of 460 nt, the sequence of which is shown in Table 9;

[0108] (2) Take the number with m 6 A modified RNA probe, FTO demethylation reaction system was prepared according to Table 1, and the reaction was carried out at 37°C for 10 minutes. 6 A is oxidized to hm 6 A(N 6 -hydroxymethyladenosine);

[0109] Table 1 FTO demethylation reaction system

[0110]

[0111] (3) Prepare the dithiothreitol (DTT) labeling reaction system according to Table 2 and react at 37°C for 3 hours to allow hm 6A reacts with DTT to generate adenosine with thiol modification;

[0112] Table 2 DTT labeling reaction system

[0113]

[0114] (4) Add 250 μL TRIzol to the above 50 μL solution. TM Reagent, shake the centrifuge tube vigorously for 15 seconds, let it stand at room temperature for 5 minutes, add 50 μL of chloroform to the above centrifuge tube, shake the centrifuge tube vigorously for 15 seconds, extract at room temperature for 5 minutes, centrifuge at 4°C and 12000 rpm for 15 minutes, separate the solution, transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of isopropanol, 1 / 10 volume of 3M sodium acetate solution and 1 μL of glycogen, mix and blow to mix, precipitate at -20°C overnight, centrifuge at 15000 rpm at 4°C for 1 hour, wash the precipitate with an equal volume of 80% ethanol, centrifuge at 15000 rpm at 4°C for 15 minutes, remove the supernatant again, air-dry for 5 minutes, and dissolve RNA with RNase-free water;

[0115] (5) The above-mentioned RNA modified with thiol was added to the modification system shown in Table 3, and reacted at 37°C for 2 hours. 6 A site modifies the carbon-carbon triple bond;

[0116] Table 3 Carbon-carbon triple bond labeling reaction system

[0117]

[0118] (6) Add 90 μL RNAClean XP beads (Beckman Coulter) to the reaction system, pipette and mix 6-10 times, incubate at room temperature for 15 min, separate using a magnetic rack and discard the supernatant, wash the beads twice with 80% ethanol for 30 seconds each time, let stand for 5-10 minutes to evaporate the ethanol, elute RNA with 42 μL resuspension buffer, incubate at room temperature for 2 minutes, and separate using a magnetic rack;

[0119] (7) The above-mentioned RNA modified with DBCO was added to the modification system shown in Table 4 and reacted at 37°C for 2 hours. 6 An RNA branch is grafted into the A site;

[0120] Table 4 Branched chain labeling reaction system

[0121]

[0122] (8) Add 90 μL RNAClean XP beads to the reaction system, pipet and mix 6 to 10 times, incubate at room temperature for 15 minutes, separate with a magnetic rack and discard the supernatant, wash the beads twice with 80% ethanol for 30 seconds each time, let stand for 5 to 10 minutes to evaporate the ethanol, elute the RNA with 50 μL resuspension buffer, incubate at room temperature for 2 minutes, separate with a magnetic rack, repeat the purification steps, and finally elute with 10 μL resuspension buffer to obtain the branched chain labeled product;

[0123] (9) Take the same amount with m 6 A modified RNA probe, repeat the above steps, wherein step (2) does not add FTO, as a control group;

[0124] (10) Take 1 μg of branched-chain labeled product and control product, denature at 95°C for 2 min, quickly quench on ice, spot on nylon membrane, dry completely, and crosslink in UV crosslinker, select UV lamp with wavelength of 254 nm and energy of 150-400 mJ / cm 2 , cross-linked twice, blocked with 5% skim milk (dissolved in PBST) for 1 hour, washed three times with PBST for 5 minutes each time, applied with 10 ml of antibody solution (anti-biotin-HRP, diluted 1:10000 in PBST), incubated for 1 hour, washed three times with PBST for 5 minutes each time, developed, and finally stained with methylene blue;

[0125] (11) Dot-blot results are as follows Figure 3 Compared with the control group, m 6 The branch-chain labeled product of RNA probe A showed a spot, indicating that it had been labeled with a biotin-modified branch.

[0126] 2. Grafting template switching and traditional template switching of branched modified RNA

[0127] Take the above-mentioned branch-labeled products and control group products to PCR tubes respectively, mix with 1 μL 10 μM reverse transcription primer (RT-primer) and 5 μL RNase-free water, mix well by pipetting, heat denaturation at 65°C for 5 minutes, cool on ice, configure the grafting template switching-traditional template switching reaction system according to the reaction system in Table 5, react at 50°C for 1.5 hours, and then heat at 85°C for 5 minutes to inactivate the reverse transcriptase.

[0128] Table 5 Grafting template switching-traditional template switching reaction system

[0129]

[0130] 3. PCR amplification to construct high-throughput library

[0131] (1) Take 5 μL of the above reaction solution and prepare the PCR amplification system according to the reaction system in Table 6. The PCR primers at both ends have adapter sequences, the sequences are shown in Table 9. After pipetting and mixing, run the program in the PCR instrument as shown in Table 7;

[0132] Table 6 PCR amplification reaction system

[0133]

[0134] Table 7 PCR amplification operation program

[0135]

[0136] (2) Purification of the double-stranded DNA fragments obtained above: add 40 μL of AMPureXP beads (Beckman Coulter) that have been restored to room temperature in advance to the reaction system, pipette and mix 6 to 10 times, incubate at room temperature for 15 minutes, separate using a magnetic rack and discard the supernatant, wash the beads twice with 80% ethanol for 30 seconds each time, let stand for 5 to 10 minutes to evaporate the ethanol, elute the double-stranded DNA fragments with 50 μL resuspension buffer, incubate at room temperature for 2 minutes, separate using a magnetic rack, repeat the purification steps, and finally elute with 22 μL resuspension buffer;

[0137] (3) The double-stranded DNA obtained above is configured into a PCR amplification system according to the reaction system in Table 8, and the PCR primers at both ends carry high-throughput sequencing primer sequences, wherein PCR2-R can be selected from any one of PCR2-R-1, PCR2-R-2, PCR2-R-3 and PCR2-R-4, and the sequences are shown in Table 9. After pipetting and mixing, the program running in the PCR instrument is shown in Table 7;

[0138] Table 8 PCR amplification reaction system

[0139]

[0140]

[0141] Table 9 RNA template, RNA branch, reverse transcription primer, TSO and PCR primer sequences

[0142]

[0143] (5) Purification of the double-stranded DNA fragments obtained above: add 40 μL of AMPureXP beads (Beckman Coulter) that have been restored to room temperature in advance to the reaction system, pipette and mix 6 to 10 times, and incubate at room temperature for 15 minutes. Separate with a magnetic rack and discard the supernatant. Wash the beads twice with 80% ethanol for 30 seconds each time, let stand for 5 to 10 minutes to evaporate the ethanol, elute the double-stranded DNA fragments with 50 μL resuspension buffer, incubate at room temperature for 2 minutes, separate with a magnetic rack, repeat the purification steps, and finally elute with 20 μL resuspension buffer to obtain a high-throughput library.

[0144] 4. High-throughput sequencing analysis to verify m 6 A modification site

[0145] The obtained high-throughput library was sequenced with double-end 150 using the Illumina NovaS4 platform. The obtained data was filtered for low quality and adapters and then compared with the original RNA sequence to obtain the connection site, i.e., m 6 A modification site. High-throughput sequencing results are shown in Figure 4 , 5 As shown, the connection sites are concentrated in m 6 A modification site is located 1 to 2 bases from the 5' end, and a base may be randomly incorporated during the conversion of the grafting template, indicating that this method can detect m on RNA 6 A modification site.

[0146] Example 2: mRNA expression in HEK293T cells 6 Detection of A modification sites

[0147] 1. HEK293T cell culture, total RNA extraction and fragmentation

[0148] (1) After HEK293T cells were cultured under normal culture conditions until the confluence reached 80%, the culture medium was aspirated, the residual culture medium was washed with phosphate buffered saline (PBS), and TRIzol was added to the culture dish. TM Reagent to cover the bottom of the culture dish, wash all the attached HEK293T cells into the solution, transfer to a centrifuge tube and lyse at room temperature for 5 minutes;

[0149] (2) Per 1mL TRIzol TMReagent, add 0.2 mL of chloroform to the above centrifuge tube, shake the centrifuge tube vigorously for 15 seconds, extract at room temperature for 5 minutes, centrifuge at 4°C and 12000 rpm for 15 minutes, separate the solution into layers, the upper clear liquid is RNA, the middle white precipitate is DNA, and the lower red liquid is protein. Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of isopropanol, incubate on ice for 10 minutes, centrifuge at 4°C and 12000 rpm for 15 minutes to obtain a total RNA white precipitate;

[0150] (3) Remove the supernatant, leaving the total RNA white precipitate, wash the precipitate with 1 mL 75% ethanol / 1 mL TRIzol, centrifuge at 4°C and 12,000 rpm for 15 minutes, remove the supernatant again, air-dry for 3 minutes, dissolve the total RNA with 250 μL RNase-free water, heat at 70°C for 10 minutes to fully dissolve the total RNA, and measure the RNA concentration with Bio Drop;

[0151] (4) Take 20 μg of total RNA sample, add 2 μL 10× RNA Fragmentation Buffer (NEB), add RNase-free water to a total volume of 20 μL, mix, pipette and mix, heat at 94°C for 2 minutes, and then add 2 μL 10× RNA Fragmentation Stop Solution (NEB) to stop fragmentation;

[0152] (5) Add RNase-free water to the reaction system to a total volume of 50 μL, add 90 μL RNACleanXP beads, pipette and mix 6 to 10 times, incubate on ice for 15 min, separate using a magnetic rack and discard the supernatant, wash the beads twice with 80% ethanol for 30 seconds each, let stand for 5 to 10 minutes to evaporate the ethanol, elute the RNA with 20 μL resuspension buffer, incubate at room temperature for 2 minutes, and separate using a magnetic rack to obtain fragmented RNA with a length of 200-300 nt.

[0153] 2. Same as RNA m in Example 1 6 The RNA branch chain was labeled at site A, wherein the control RNA was not added with FTO, and the reaction system was supplemented with RNase-free water, and the RNA branch chain used was the 5'N3-RNA branch chain.

[0154] 3. Grafting template conversion

[0155] (1) Take the branched-chain labeled product and transfer it to a PCR tube. Mix it with 1 μL of 10 μM reverse transcription primer (RT-primer) and 5 μL of RNase-free water, pipette and mix well, heat and denature at 65°C for 5 minutes, and cool on ice.

[0156] (2) The grafting template conversion reaction system was prepared according to the reaction system in Table 10, and the reaction was carried out at 50°C for 1.5 hours, and then heated at 85°C for 5 minutes to inactivate the reverse transcriptase.

[0157] Table 10 Grafting template switching reaction system

[0158]

[0159] 4. The high-throughput library was constructed by PCR amplification as in Example 1, wherein the first PCR primers were PCR1-28S and PCR1-F.

[0160] 5. High-throughput sequencing analysis to detect 28S rRNA 4220m 6 A modification site

[0161] The obtained high-throughput library was sequenced with double-end 150 using the Illumina NovaS4 platform. The obtained data was filtered for low quality and adapters and then compared with the human rRNA sequence to obtain the junction site, i.e., m 6 A modification site. High-throughput sequencing results are shown in Figure 6 As shown in Figure 2, there is a template switching signal at the 28SrRNA 4220 site that is significantly different from that of the control group, and the ligation sites are concentrated in m 6 A modification site 1 to 2 bases 5', indicating that this method can identify m on real samples 6 A modification site.

[0162] Example 3: mRNA expression in HEK293T cells 6 Detection of A modification sites

[0163] 1. HEK293T cell culture and mRNA extraction

[0164] (1) After HEK293T cells were cultured under normal culture conditions until the confluence reached 80%, the culture medium was aspirated, the residual culture medium was washed with phosphate buffered saline (PBS), and TRIzol was added to the culture dish. TM Reagent to cover the bottom of the culture dish, wash all the attached HEK293T cells into the solution, transfer to a centrifuge tube and lyse at room temperature for 5 minutes;

[0165] (2) Per 1mL TRIzol TMReagent, add 0.2 mL of chloroform to the above centrifuge tube, shake the centrifuge tube vigorously for 15 seconds, extract at room temperature for 5 minutes, centrifuge at 4°C and 12000 rpm for 15 minutes, separate the solution into layers, the upper clear liquid is RNA, the middle white precipitate is DNA, and the lower red liquid is protein. Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of isopropanol, incubate on ice for 10 minutes, centrifuge at 4°C and 12000 rpm for 15 minutes to obtain a total RNA white precipitate;

[0166] (3) Remove the supernatant, leaving the total RNA white precipitate, wash the precipitate with 1 mL 75% ethanol / 1 mL TRIzol, centrifuge at 4°C and 12,000 rpm for 15 minutes, remove the supernatant again, air-dry for 3 minutes, dissolve the total RNA with 250 μL RNase-free water, heat at 70°C for 10 minutes to fully dissolve the total RNA, and measure the RNA concentration with Bio Drop;

[0167] (4) Use GenElute mRNA Miniprep Kit (Sigma) to further extract mRNA. Take 250 μg of total RNA in a 1.5 mL centrifuge tube, add 250 μL of 2x Binding Solution, and gently shake the centrifuge tube to mix the solution;

[0168] (5) Add 20 μL of evenly dispersed oligo(dT) beads and shake to mix the system thoroughly. Heat at 70°C for 3 minutes to denature the RNA, and then incubate at room temperature for 10 minutes to allow oligo(dT) to fully bind to RNA. Centrifuge the mixed system at RCF 15,000g for 2 minutes to obtain oligo(dT) beads bound to mRNA. Carefully remove the supernatant, leaving about 50 μL of solution to prevent beads loss.

[0169] (6) Add 500 μL of Wash Solution to the centrifuge tube and pipette to fully suspend the oligo(dT), transfer to the GenElute centrifugal filter column / collection tube, centrifuge at RCF 15,000 g for 2 minutes, and discard the liquid in the collection tube;

[0170] (7) Add 500 μL of Wash Solution to the filter column again, centrifuge at 15,000 g for 2 min, and discard the liquid in the collection tube;

[0171] (8) Finally, transfer the filter column to a new collection tube, pipette 50 μL of Elution Solution preheated at 70°C and add it to the center of the centrifugal filter column filter membrane to fully contact the mRNA complex in the column pores, incubate at 70°C for 5 minutes, and centrifuge at RCF15,000g for 1 minute to obtain the mRNA solution in the collection tube. The mRNA concentration is measured using Bio Drop. In addition, another 50 μL of 70°C Elution Solution can be pipetted and repeated to fully dissolve the mRNA in the column pores of the filter column.

[0172] (9) Repeat the above mRNA extraction steps, and finally elute with 15 μL Elution Solution to obtain mRNA;

[0173] (10) Take 500 ng of mRNA sample, add 2 μL 10× RNA Fragmentation Buffer (NEB), add RNase-free water to a total volume of 20 μL, mix, pipette and mix, heat at 94°C for 4 minutes, and then add 2 μL 10× RNA Fragmentation Stop Solution (NEB) to stop fragmentation;

[0174] (11) Add RNase-free water to the reaction system to a total volume of 50 μL, add 90 μL RNACleanXP beads, mix by pipetting 6 to 10 times, incubate on ice for 15 min, separate using a magnetic rack and discard the supernatant, wash the beads twice with 80% ethanol for 30 seconds each, let stand for 5 to 10 minutes to evaporate the ethanol, elute the RNA with 10 μL resuspension buffer, incubate at room temperature for 2 minutes, and separate using a magnetic rack to obtain fragmented mRNA with a length of 200 to 300 nt.

[0175] 2. Same as RNA m in Example 1 6 The RNA branch chain was labeled at site A, wherein the control RNA was not added with FTO, and the reaction system was supplemented with RNase-free water, and the RNA branch chain used was the 5'N3-RNA branch chain.

[0176] 3. The grafting template switching and traditional template switching of the branched modified RNA in Example 1.

[0177] 4. Construct a high-throughput library by PCR amplification as in Example 1.

[0178] 5. High-throughput sequencing analysis to detect mRNA on HEK293T cells 6 A modification site

[0179] The obtained high-throughput library was sequenced with double-end 150 using the Illumina NovaS4 platform. The obtained data was compared with the human transcriptome sequence after low-quality filtering and adapter filtering. The site where the end of the branched sequence is connected is the m 6 A modification site. High-throughput sequencing results are shown in Figure 7 As shown, the method of the present invention identified a total of 50907 mRNAs in HEK293T cells. 6 A modification site. The sequence characteristics before and after the connection site were analyzed to obtain the m 6 A conservative sequence of modification sites (such as Figure 8 ), which is different from the common m 6 The conserved sequences obtained by immunoprecipitation with antibody A and sequencing technology are roughly the same, indicating that the site identified by this method is m 6 A site.

[0180] Example 4 Detection of extracellular DNA 6mA modification sites

[0181] 1. DNA 6mA site labeling RNA branch

[0182] (1) Prepare a DNA probe with 6mA modification, the sequence of which is shown in Table 9;

[0183] (2) Same as RNA m in Example 1 6 The A site labels the RNA branch, and the RNA branch used is the 5'N3-RNA branch.

[0184] 2. The grafting template is converted as in Example 2.

[0185] 3. PCR Amplification

[0186] (1) Take 5 μL of the above reaction solution and prepare the PCR amplification system according to the reaction system in Table 11. The PCR primers at both ends have adapter sequences and sequences corresponding to the 5' end of the DNA probe. The sequences are shown in Table 9.

[0187] Table 11 PCR amplification reaction system

[0188]

[0189] (2) After mixing the above system by pipetting, run the program in the PCR instrument as shown in Table 12;

[0190] Table 12 PCR amplification operation program

[0191]

[0192] (3) Purification of the double-stranded DNA fragments obtained above: add 40 μL of AMPureXP beads (Beckman Coulter) that have been restored to room temperature in advance to the reaction system, pipette and mix 6 to 10 times, and incubate at room temperature for 15 minutes. Separate with a magnetic rack and discard the supernatant. Wash the beads twice with 80% ethanol for 30 seconds each time, let stand for 5 to 10 minutes to evaporate the ethanol, elute the double-stranded DNA fragments with 50 μL of resuspension buffer, incubate at room temperature for 2 minutes, and separate with a magnetic rack to obtain a purified PCR product.

[0193] 4. Low-throughput sequencing to verify 6mA sites on DNA

[0194] (1) 10-40 ng of the above DNA product was ligated to a plasmid vector using the ABclonal Zero TOPO-TA / Blunt Cloning Kit (ABclonal) at 25°C for 5 minutes. The vector ligation system is shown in Table 13;

[0195] Table 13 Vector connection system

[0196]

[0197] (2) Thaw 100 μL of DH5α competent cells on ice for 5 minutes until the cells are evenly suspended, add the above-mentioned ligation solution, gently tap to mix, and place on ice for 25 minutes;

[0198] (3) Heat shock in a 42°C water bath for 45 seconds, place on ice for 2 minutes, add 700 μL LB medium, and culture at 37°C and 220 rpm for 1 hour;

[0199] (4) Centrifuge at 5000 rpm for 1 min, remove 700 μL of supernatant with a pipette tip, and suspend the cells with the remaining culture medium;

[0200] (5) Prepare 50 mL LB plates containing 50 μL 1000× ampicillin, 15 mL per plate, and evenly spread the above bacterial suspension on the LB plates;

[0201] (6) The plate was first incubated at 37°C for 1 hour, then inverted and incubated for 16 hours, and a single white colony was selected to extract the plasmid for Sanger sequencing to obtain low-throughput sequencing results;

[0202] (7) Low-throughput sequencing results such as Fig. 9 As shown, according to the sequencing results, there is an obvious template switching signal at the 6mA site, indicating that the method of the present invention can detect the 6mA modification site on DNA.

[0203] Example 5 Detection of extracellular RNAΨ modification sites

[0204] 1. RNA Ψ site labeling RNA branches

[0205] (1)Use HiScribe TM T7 High Yield RNA Synthesis Kit (NEB) was used to prepare an RNA probe with a length of 83 nt. At the same time, UTP was replaced with ΨTP to prepare an RNA probe with a length of 83 nt containing Ψ modification. The sequence is shown in Table 9.

[0206] (2) Prepare proCMC reaction buffer I (BEU buffer) according to Table 14 and adjust its pH to 8.5. Take 5 μg of RNA probes with and without Ψ modification, respectively, and prepare proCMC reaction system I according to Table 15. Incubate at 37°C for 30 minutes to label the U, G, and Ψ sites on the RNA probe with proCMC.

[0207] Table 14proCMC reaction buffer I

[0208]

[0209] Table 15 proCMC reaction system

[0210]

[0211] (3) Add 10 μL of sodium acetate solution with pH = 5.2 to the reaction system and mix well by pipetting. Then add 110 μL of isopropanol, mix well by inversion, and precipitate at -20°C for more than 3 hours. After precipitation, centrifuge at 4°C and 12000 rpm for 1 hour, discard the supernatant, then add 100 μL of 80% ethanol, centrifuge for 20 minutes under the same conditions, collect the precipitate and dissolve it in RNase-free water;

[0212] (4) Prepare proCMC reaction buffer II (Buffer II) according to Table 16, and adjust its pH to 10.4. Take the RNA probe purified in (3), prepare proCMC reaction system II according to Table 17, incubate at 37°C for 6 hours to remove the proCMC labeled at the U and G sites on the RNA probe, thereby achieving proCMC specific labeling of the RNA probe Ψ site. After the reaction is completed, purify the RNA probe according to the steps described in (3);

[0213] Table 16 proCMC reaction buffer II

[0214]

[0215] Table 17proCMC reaction system

[0216]

[0217] (5) Add 50 ng of RNA probe and 1 μL of 50 μM 5'N3-RNA branch to a PCR tube, denature at 95°C for 5 minutes, and then cool on ice for 1 minute; add 1 μL of 20 mM copper sulfate solution and 1 μL of 100 mM tris(3-hydroxypropyltriazinemethyl)amine (THPTA) solution to another PCR tube, then add 1 μL of 200 mM sodium ascorbate solution, and let stand at room temperature for 8 minutes to obtain click chemistry reaction buffer; mix the solutions in the two PCR tubes thoroughly, and add RNase-free water to the system to 20 μL, react at 37°C for 3 hours, and add 1 μL of 0.5 M EDTA solution to the reaction system to terminate the reaction;

[0218] (6) Expand the reaction system to 50 μL, add 90 μL RNAClean XP beads, mix by pipetting for more than 10 times, incubate at room temperature for 15 min, separate with a magnetic rack and discard the supernatant, wash the beads twice with 80% ethanol for 30 seconds each time, let stand for 5 to 10 minutes to evaporate the ethanol, elute the RNA with 50 μL resuspension buffer, incubate at room temperature for 2 minutes, separate with a magnetic rack, repeat the purification steps, and finally elute with 10 μL resuspension buffer to obtain the branched-chain labeled product.

[0219] 2. The grafting template is converted as in Example 2.

[0220] 3. The high-throughput library was constructed by PCR amplification as in Example 1, wherein the first PCR primers were PCR1-83 and PCR1-F.

[0221] 4. High-throughput sequencing analysis to verify the Ψ modification sites on RNA

[0222] The obtained high-throughput library was sequenced with double-end 150 using the Illumina NovaS4 platform. The obtained data was filtered for low quality and adapters and then compared with the original RNA sequence to obtain the connection site, i.e., the Ψ modification site. The high-throughput sequencing results are shown in Fig.10 As shown, the ligation sites are concentrated at the Ψ modification sites, indicating that this method can detect the Ψ modification sites on RNA.

[0223] Example 6 Extracellular RNA p 6 Detection of A modification sites

[0224] 1. RNA p 6 A site labels RNA branch

[0225] (1) Using HiScribe TM T7 High Yield RNA Synthesis Kit (NEB) was used to prepare RNA with p 6 A modified RNA probe with a length of 460 nt, the sequence of which is shown in Table 9;

[0226] (2) Take 1 μg of p 6 A RNA probe and 2 μL of 100 μM 5'N3-RNA branched chain were added to a PCR tube, denatured at 95°C for 5 minutes, and then chilled on ice for 1 minute; 1 μL of 20 mM copper sulfate solution and 1 μL of 100 mM tris(3-hydroxypropyltriazinemethyl)amine (THPTA) solution were added to another PCR tube, followed by 1 μL of 200 mM sodium ascorbate solution. After observing that the reaction system turned from yellow turbidity to red solution and then to light yellow transparent solution, it was allowed to stand at room temperature for 5 minutes to obtain click chemistry reaction buffer; the solutions in the two PCR tubes were fully mixed, reacted at 37°C for 2 hours, and then 2 μL of 0.5 M EDTA solution was added to the reaction system to terminate the reaction;

[0227] (3) Expand the reaction system to 50 μL, add 90 μL RNAClean XP beads, mix by pipetting 6 to 10 times, incubate at room temperature for 15 minutes, separate with a magnetic rack and discard the supernatant, wash the beads twice with 80% ethanol for 30 seconds each time, let stand for 5 to 10 minutes to evaporate the ethanol, elute the RNA with 50 μL resuspension buffer, incubate at room temperature for 2 minutes, separate with a magnetic rack, repeat the purification steps, and finally elute with 10 μL resuspension buffer to obtain the branched-chain labeled product.

[0228] 2. The grafting template switching and traditional template switching of the branched modified RNA in Example 1.

[0229] 3. Construct a high-throughput library by PCR amplification as in Example 1.

[0230] 4. High-throughput sequencing analysis to verify the p 6 A modification site

[0231] The obtained high-throughput library was sequenced with double-end 150 sequencing using the Illumina NovaS4 platform. The obtained data was filtered for low quality and adapters and then compared with the original RNA sequence to obtain the connection site, i.e., p 6 A modification site. High-throughput sequencing results are shown in Fig.11 As shown, the connection sites are concentrated in p 6A modification site is 1 to 2 bases 5', indicating that this method can detect p on RNA 6 A modification site.

[0232] Example 7 Detection of RNA polymerase-nascent RNA interaction sites in HeLa cells

[0233] 1. HeLa cell nucleus extraction

[0234] (1) After HeLa cells were cultured in a 15 cm culture dish under normal culture conditions until the confluence reached 80%, the culture medium was aspirated and the residual culture medium was washed away with phosphate buffered saline (PBS);

[0235] (2) Add 5 mL of 0.25% trypsin solution to each 15 cm culture dish and digest at 37°C for 5 min;

[0236] (3) Add 5 mL of culture medium to terminate digestion, collect the harvested cells in a 50 mL centrifuge tube, and centrifuge at 1000 g for 5 minutes to harvest the cells;

[0237] (4) Wash the cells twice with PBS to remove residual culture medium;

[0238] (5) Oscillate for 5 seconds to loosen the cell clusters;

[0239] (6) Cells were cultured at 5×10 7 The cells were suspended in sucrose solution I precooled on ice and the cell concentration was determined using a phase contrast microscope. The composition of sucrose solution I is shown in Table 18.

[0240] Table 18 Sucrose solution I composition table

[0241]

[0242] (7) After incubation on ice for 5 minutes, the cell suspension was transferred to a pre-cooled Dounce homogenizer on ice to disrupt the cells. Phase contrast microscopy was used to confirm that the cells were completely disrupted.

[0243] (8) Transfer the disrupted cell suspension to a 50 mL centrifuge tube, add an equal volume of ice-cold sucrose solution II, and mix by inversion. The composition of sucrose solution II is shown in Table 19;

[0244] Table 19 Composition of sucrose solution II

[0245]

[0246] (9) Add 4.4 mL of sucrose solution II to the bottom of the ultracentrifuge tube and carefully add the cell lysate to its upper layer. No more than 2 × 10 8 cells;

[0247] (10) Add sucrose solution I to the top of the tube for sealing and centrifuge at 4°C and RCF 30,000 g for 45 min;

[0248] (11) The upper layer of liquid is aspirated, and the cell nuclei form a compact sediment in the lower layer;

[0249] *If cells are not lysed, no precipitate will form; if nuclei are also lysed, a gel-like chromatin precipitate will form.

[0250] (12) Shake for 5 seconds to loosen the cell nuclear pellet;

[0251] (13) 5×10 7 Add ice-cold glycerol stock solution at a concentration of 100 nuclei per ml and mix by pipetting up and down. The composition of the glycerol stock solution is shown in Table 20.

[0252] Table 20 Glycerol storage solution composition

[0253]

[0254] (14) Place 100 μL of cell nucleus suspension into a pre-cooled 1.5 mL centrifuge tube, freeze it with liquid nitrogen, and store it in a -80°C refrigerator until use.

[0255] 2. Chromatin Access and RNA Polymerase Restart

[0256] (1) Add 10 μL of 5 M sodium chloride solution to every 100 μL of cell nucleus suspension and mix well;

[0257] (2) After the solution is clarified, add 110 μL of RNase-free water, mix well, and centrifuge at 4°C and RCF 12,000g for 3 min;

[0258] (3) Transfer the supernatant, which contains the nuclear RNA and can be used for subsequent quality control. The chromatin forms a dense gel-like precipitate at the bottom;

[0259] (4) Wash the pellet twice with 500 μL of 50 mM Tris-HCl solution (pH = 7.5) to remove residual nuclear RNA and sodium chloride;

[0260] (5) Dissolve the chromatin precipitate with 100 μL of glycerol storage solution, then add an equal volume of transcription restart buffer preheated at 37°C. The experimental group used 80% p 6 ATP, the control group used ATP, mixed and reacted at 37°C for 5 minutes. The composition of the transcription restart buffer is shown in Table 4;

[0261] Table 21 Transcription restart buffer composition

[0262]

[0263]

[0264] (6) Add 600 μL LC-Trizol, mix by pipetting, and terminate the reaction;

[0265] (7) Add 0.16 mL of chloroform, shake the centrifuge tube vigorously for 15 seconds, extract at room temperature for 3 minutes, centrifuge at 4°C and RCF 12,000g for 15 minutes, and separate the solutions into layers. The upper supernatant is RNA, the middle white precipitate is mainly protein, and the lower red liquid is mainly DNA;

[0266] (8) Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of phenol-chloroform solution (pH 5.3), shake the centrifuge tube vigorously for 15 seconds, extract at room temperature for 3 minutes, and centrifuge at 4°C and RCF 12,000g for 15 minutes to remove residual DNA and protein impurities. Repeat twice;

[0267] (9) Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of chloroform, shake the centrifuge tube vigorously for 15 seconds, extract at room temperature for 3 minutes, centrifuge at 4°C and RCF 12,000g for 15 minutes to remove residual phenol, and repeat twice;

[0268] (10) Transfer the upper aqueous phase to a new centrifuge tube, add 1 μL of 20 mg / mL glycogen solution, mix well, add an equal volume of isopropanol, incubate at -20°C for 30 min, and centrifuge at 4°C and RCF 15,000g for 45 min to obtain a white precipitate of chromatin RNA;

[0269] (11) Remove the supernatant, leaving the chromatin RNA white precipitate, wash the precipitate with 200 μL 80% ethanol, and centrifuge at 4°C and RCF 15,000g for 15 min. Repeat twice to remove residual isopropanol and salt;

[0270] (12) Remove the supernatant again, air-dry for 5 minutes, dissolve the chromatin RNA with 100 μL of RNase-free water, and heat at 70°C for 10 minutes to fully dissolve the chromatin RNA. A total of about 20 μg of RNA was removed, and part of the RNA was used for quality control;

[0271] 3. rRNA Removal

[0272] (1) Transfer 5 μg of chromatin RNA into a PCR tube, add 5 μL of DNaseI reaction solution, 2U of DNaseI, add RNase-free water to 50 μL, react at 37°C for 10 minutes to remove trace DNA;

[0273] (2) Add 150 μL LC-Trizol, mix by pipetting, terminate the reaction, and transfer to a 1.5 mL centrifuge tube;

[0274] (3) Add 40 μL of chloroform, shake the centrifuge tube vigorously for 15 seconds, extract at room temperature for 3 minutes, centrifuge at 4°C and RCF 12,000g for 15 minutes, separate the solutions, and the supernatant is RNA;

[0275] (4) Transfer the upper aqueous phase to a new centrifuge tube, add 1 μL of 20 mg / mL glycogen solution, mix well, add an equal volume of isopropanol, incubate at -20°C for 30 min, and centrifuge at 4°C and RCF 15,000g for 45 min to obtain a white RNA precipitate;

[0276] (5) Remove the supernatant, leaving the RNA white precipitate, wash the precipitate with 200 μL 80% ethanol, and centrifuge at 4°C and RCF 15,000g for 15 min. Repeat twice to remove residual isopropanol and salt;

[0277] (6) Remove the supernatant again, air-dry for 5 minutes, dissolve the RNA in 50 μL of RNase-free water, heat at 70°C for 10 minutes to fully dissolve the chromatin RNA, measure the RNA concentration, take out part of the RNA and perform qPCR together with the RNA in 2(12) to determine the residual amount of DNA;

[0278] (7) Use NEBNext rRNA Depletion Kit to remove rRNA. Mix 1 μL NEBNext rRNA Depletion Solution and 2 μL Probe Hybridization Buffer with 12 μL chromatin RNA (not more than 1 μg RNA) in a PCR tube. After pipetting and mixing, place the tube in a PCR instrument and run the program as shown in Table 22.

[0279] Table 22 PCR instrument operation program

[0280]

[0281] (8) After the program is completed, place on ice, add 1 μL RNase-free water, 2 μL RNase H Reaction Buffer and 2 μL NEBNext RNase H, mix well by pipetting, place in a PCR instrument, and incubate at 37°C for 30 minutes;

[0282] (9) After the program is completed, place on ice, add 22.5 μL RNase-free water, 5 μL DNase I reaction buffer and 2.5 μL DNase I (RNase-free), mix well by pipetting, place in a PCR instrument, and incubate at 37°C for 30 minutes;

[0283] (10) Purify RNA using NEBNext RNASample Purification Beads. After shaking and mixing, take out 110 μL of NEBNext RNASample Purification Beads and add it to the rRNA removal reaction solution. Mix by pipetting and incubate on ice for 15 minutes.

[0284] (11) Place the PCR tube on a magnetic table and let it stand for 5 minutes until all the magnetic beads are attached to the wall, then discard the supernatant;

[0285] (12) Add 200 μL of 80% ethanol, let stand for 30 seconds, discard the supernatant, and repeat twice;

[0286] (13) Air-dry for 5 minutes, dissolve the RNA in 30 μL of RNase-free water, and measure the RNA concentration;

[0287] 4. Same as RNA p in Example 6 6 A site labeling RNA branch

[0288] 5. The grafting template switching and traditional template switching of the branched modified RNA in Example 1.

[0289] 6. Construct a high-throughput library by PCR amplification as in Example 1.

[0290] 7. The obtained library was subjected to double-end 150 sequencing using the Illumina X-10 platform. The obtained data was compared with the transcriptome of HeLa cells after low-quality filtering and adapter filtering. The enriched RNA was the nascent RNA that interacted with RNA polymerase, and the reverse transcription-blocking junction site was the RNA polymerase-nascent RNA interaction site.

[0291] Example 8 Detection of protein-mediated RNA-RNA indirect interactions in HeLa cells (detection principle as shown in Fig.14 )

[0292] 1. Cross-linking of HeLa cells

[0293] (1) After HeLa cells were cultured in a 15 cm culture dish under normal culture conditions until the confluence reached 80%, the culture medium was aspirated and the residual culture medium was washed away with phosphate buffered saline (PBS);

[0294] (2) Add 5 mL of 0.25% trypsin solution to each 15 cm culture dish and digest at 37°C for 5 min;

[0295] (3) Add 5 mL of culture medium to terminate digestion, collect the harvested cells in a 50 mL centrifuge tube, and centrifuge at 1000 g for 5 minutes to harvest the cells;

[0296] (4) Wash the cells twice with PBS to remove residual culture medium;

[0297] (5) Add 5 mL of PBS to suspend HeLa cells;

[0298] (6) Add 500 μL of the cross-linking buffer shown in Table 23 and incubate the HeLa cells at room temperature for 15 minutes;

[0299] Table 23 Cross-linking buffer formula

[0300]

[0301] (7) Add 300 μL of 2.5 M glycine and incubate HeLa cells at room temperature for 5 minutes;

[0302] (8) RCF was centrifuged at 1000 g for 5 minutes to harvest the cells;

[0303] (9) Wash the cells twice with PBS to remove residual buffer.

[0304] 2. Enrichment of Protein-crosslinked RNA

[0305] (1) Add 1 mL of TRIzol to the cells TM Reagent, mix by pipetting, and terminate the reaction;

[0306] (2) Add 0.2 mL of chloroform, shake the centrifuge tube vigorously for 15 seconds, extract at room temperature for 5 minutes, centrifuge at 4°C and 12,000 rpm for 15 minutes, and separate the solutions into layers. The upper clear liquid is RNA, the middle white precipitate is the protein-RNA cross-linked product, and the lower red liquid is mainly DNA and protein.

[0307] (3) Aspirate the upper aqueous phase and the lower organic phase, add 0.5 mL of water and 0.5 mL of an equal volume of pH 5.3 phenol-chloroform solution, shake the centrifuge tube vigorously for 15 seconds, extract at room temperature for 5 minutes, and centrifuge at 4°C and 12,000 rpm for 15 minutes to remove residual uncross-linked RNA and protein impurities. Repeat twice;

[0308] (4) Discard the upper aqueous phase and the lower organic phase, add 0.5 mL of water and 0.5 mL of chloroform, shake the centrifuge tube vigorously for 15 seconds, extract at room temperature for 3 minutes, centrifuge at 4°C and 12,000 rpm for 15 minutes to remove residual phenol, and repeat twice;

[0309] (5) Discard the upper aqueous phase and the lower organic phase, open the lid and place in a 70°C metal bath to heat for 10 minutes to remove residual chloroform.

[0310] 3. HeLa cell grafting template conversion and traditional template conversion

[0311] (1) Prepare reverse transcription denaturation buffer according to Table 24, incubate at 70°C for 10 minutes to dissolve the protein-RNA cross-linked product;

[0312] Table 24 Reverse transcription denaturation buffer

[0313]

[0314] (2) Add Maxima H Minus reverse transcriptase and the corresponding reverse transcription reaction buffer, mix well, and react at 50°C for 1 hour;

[0315] (3) RNase A and RNase H were added to digest the cellular RNA, and then the cells were dissolved with saturated phenol at pH = 8.0, transferred to a centrifuge tube, and vigorously shaken to mix the aqueous phase and the organic phase thoroughly, and then centrifuged at 4°C and RCF 12,000 rpm for 3 minutes;

[0316] (4) Transfer the upper aqueous phase to a new centrifuge tube, add 1 μL of 20 mg / mL glycogen solution, mix well, add an equal volume of isopropanol, incubate at -20°C for 30 min, and centrifuge at 4°C and RCF 15,000g for 45 min to obtain a white precipitate containing cDNA;

[0317] (5) Remove the supernatant, leaving the white precipitate containing cDNA, wash the precipitate with 200 μL 80% ethanol, and centrifuge at 4°C and RCF 15,000g for 15 min. Repeat twice to remove residual isopropanol and salt;

[0318] (6) Remove the supernatant again, air-dry for 5 minutes, and dissolve the white precipitate containing cDNA in 10 μL of RNase-free water.

[0319] Heat at 70°C for 10 minutes to fully dissolve the cDNA;

[0320] (7) Add the front primer and the back primer complementary to the template switching probe and the RNA probe to the DNA solution, respectively, then add NTP, PCR enzyme and the corresponding buffer solution, pipette and mix, put into the PCR instrument, and run the program as shown in Table 25;

[0321] Table 25 PCR amplification operation program

[0322]

[0323] (8) Add 45 μL of AMPure XP beads that have been restored to room temperature to the reaction system, pipette and mix 6-10 times, and incubate at room temperature for 15 minutes. Separate with a magnetic rack and discard the supernatant. Wash the beads twice with 80% ethanol for 30 seconds each time, let stand for 5-10 minutes to evaporate the ethanol, elute the double-stranded DNA fragments with 23 μL of 0.1X TE Buffer, incubate at room temperature for 2 minutes, separate with a magnetic rack, take 20 μL of the supernatant and transfer it to a new PCR tube, of which 1 μL is used for Qubit concentration determination and stored at -20°C.

[0324] 4. High-throughput sequencing analysis

[0325] The obtained library was subjected to double-end 150 sequencing using the Illumina X-10 platform. The obtained data was compared with the transcriptome of HeLa cells after low-quality filtering and adapter filtering. The enriched RNA is the RNA that interacts with the protein, and the reverse transcription-blocking junction site is the site of protein-mediated RNA-RNA indirect interaction.

[0326] Example 9 Detection of RNA-protein interaction in liquid-liquid phase separation in HeLa cells (detection principle as shown in Fig.15 )

[0327] 1.Cultivation, fixation and permeabilization of HeLa cells

[0328] (1) Coat the coverslip with poly-L-lysine and leave at room temperature for 1 hour;

[0329] (2) Rinse the coverslip thoroughly with sterile water three times, each time for 1 hour;

[0330] (3) Dry the coverslip thoroughly and sterilize it under ultraviolet light for at least 4 hours;

[0331] (4) culturing HeLa cells on glass coverslips under normal culture conditions until they reach 80% confluence;

[0332] (5) Aspirate the culture medium and wash away the residual culture medium with phosphate buffered saline (PBS);

[0333] (6) Add 4% paraformaldehyde (dissolved in PBS, pH = 7.4) and incubate HeLa cells for 15 minutes;

[0334] (7) Remove the paraformaldehyde, add 100% methanol (frozen at -20°C), and incubate the HeLa cells for 10 minutes;

[0335] (8) Wash cells three times with ice-cold PBS;

[0336] (9) Add PBS containing 0.2% TritonX-100 and incubate HeLa cells for 10 minutes;

[0337] (10) Wash the cells three times with PBST (PBS + 0.1% Tween 20).

[0338] 2. Preparation of Protein A for RNA Branching

[0339] (1) using non-natural amino acid incorporation technology to add non-natural amino acids containing triple bond groups into protein A;

[0340] (2) using in vitro transcription technology to synthesize RNA branches with azide group caps;

[0341] (3) using click chemistry to cross-link RNA branches with protein A;

[0342] (4) Purify protein A with RNA branches using protein purification technology.

[0343] 3. Blocking of HeLa cells, antibody incubation and protein A binding

[0344] (1) Add PBST containing 6% BSA to the permeabilized HeLa cells and incubate for 30 minutes;

[0345] (2) Wash cells three times with PBST;

[0346] (3) Anti-RBM15 antibody was diluted 1:10 in PBST containing 6% BSA, added to the cells, and incubated for 30 min;

[0347] (4) Wash cells three times with PBST;

[0348] (5) Protein A for linking RNA branches was diluted to a concentration of 10 μg / mL in PBST containing 6% BSA, added to the cells, and incubated for 30 minutes;

[0349] (6) Wash the cells three times with PBST.

[0350] 4. Library Construction

[0351] (1) Mix the reverse transcription primer, template switching probe, Maxima H Minus reverse transcriptase and the corresponding reverse transcription buffer, add them to the cells, and react at 50°C for 1 hour;

[0352] (2) RNase A and RNase H were added to digest the cellular RNA, and then the cells were dissolved with saturated phenol at pH 8.0, transferred to a centrifuge tube, and vigorously shaken to mix the aqueous phase and the organic phase thoroughly, and then centrifuged at 4°C and RCF 12,000g for 3 min;

[0353] (3) Transfer the upper aqueous phase to a new centrifuge tube, add 1 μL of 20 mg / mL glycogen solution, mix well, add an equal volume of isopropanol, incubate at -20°C for 30 min, and centrifuge at 4°C and RCF 15,000g for 45 min to obtain a white precipitate containing cDNA;

[0354] (4) Remove the supernatant, leaving the white precipitate containing cDNA, wash the precipitate with 200 μL 80% ethanol, and centrifuge at 4°C and RCF 15,000g for 15 min. Repeat twice to remove residual isopropanol and salt;

[0355] (5) Remove the supernatant again, air-dry for 5 minutes, dissolve the white precipitate containing cDNA with 10 μL of DEPC-treated water, and heat at 70°C for 10 minutes to fully dissolve the cDNA;

[0356] (6) Add the front primer and the back primer complementary to the template switching probe and the RNA probe to the DNA solution, respectively, then add NTP, PCR enzyme and the corresponding buffer solution, pipette and mix, put into the PCR instrument, and run the program as shown in Table 25:

[0357] (7) Add 45 μL of AMPure XP beads that have been restored to room temperature in advance to the reaction system, pipette and mix 6-10 times, and incubate at room temperature for 15 minutes. Separate with a magnetic rack and discard the supernatant. Wash the beads twice with 80% ethanol for 30 seconds each time, let stand for 5-10 minutes to evaporate the ethanol, elute the double-stranded DNA fragments with 23 μL of 0.1X TE Buffer, incubate at room temperature for 2 minutes, separate with a magnetic rack, take 20 μL of the supernatant and transfer it to a new PCR tube, of which 1 μL is used for Qubit concentration determination and stored at -20°C;

[0358] 5. High-throughput sequencing analysis

[0359] The obtained library was subjected to double-end 150 sequencing using the Illumina X-10 platform. The obtained data was compared with the transcriptome of HeLa cells after low-quality filtering and adapter filtering. The enriched RNA was the RNA that interacted with the protein, and the reverse transcription-blocking junction site was the site of RNA-protein interaction.

Claims

1. A method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template switching technology, characterized in that: The method The following steps are involved: First, an RNA branch chain needs to be obtained by grafting an RNA branch chain with a known sequence at the nucleic acid modification site or RNA-protein interaction site, or by cross-linking two RNA chains at the RNA-RNA interaction site and using one of the chains as the RNA branch chain; Then, using a reverse transcription primer complementary to the RNA branch, reverse transcription is started from the RNA branch. When reverse transcription reaches the RNA branch and the modified connection or the interactive connection, the grafting template is switched, and reverse transcription is continued using the main chain connected to the RNA branch as a template to obtain a cDNA containing the modification site or the interactive site information; Finally, a high-throughput library is constructed using the cDNA containing the modification site or interaction site information, and high-throughput sequencing is performed to determine the modification site or interaction site; or PCR and TA-cloning technology are used to detect the cDNA containing the modification site or interaction site information, thereby realizing the identification of the modification site or interaction site.

2. A method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template switching technology according to claim 1, characterized in that: The chemical modification is N 6 -Methyladenosine m 6 A.N 6 -methyldeoxyadenosine 6mA, pseudouridine Ψ, N 6 -Propargyl adenosine p 6 A or N 1 -Methyladenosine m 1 Any one of A, wherein the interaction is any one of RNA-RNA intramolecular interaction, RNA-RNA direct interaction, RNA-RNA indirect interaction, protein-RNA classical interaction, protein-RNA liquid-liquid phase separation interaction and RNA polymerase-nascent RNA interaction.

3. The method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template switching technology according to claim 1, characterized in that: The method for grafting an RNA branch with a known sequence is a click chemistry reaction, a biotin-streptavidin bioorthogonal reaction, an ultraviolet crosslinking, a chemical crosslinking agent crosslinking or an antibody crosslinking method.

4. A method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template switching technology according to claim 1, characterized in that: The reverse transcriptase used in the reverse transcription process is HIV reverse transcriptase and its various mutants, M-MLV reverse transcriptase and its various mutants, AMV reverse transcriptase and its various mutants.

5. The method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template switching technology according to claim 1, characterized in that: The method for constructing a high-throughput library is specifically as follows: i) using traditional reverse transcription template conversion or using Tn5 transposase to connect a known sequence to the 3' end of the reverse transcription product cDNA to obtain a cDNA product with known sequences at both ends; ii) designing and synthesizing PCR primers that are complementary to the RNA branch and the template conversion probe TSO with a known sequence, respectively, and amplifying the cDNA product obtained in step i) by PCR to obtain double-stranded DNA with known sequences at both ends; iii) designing and synthesizing PCR primers containing sequence information required for second-generation sequencing, and amplifying the double-stranded DNA obtained in step ii) by PCR to construct a high-throughput library.

6. A method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template switching technology according to claim 2, characterized in that: For m 6 A or 6mA modification, grafting an RNA branch of known sequence at the modification site, specifically including the following steps: (1) Treatment of fat mass and obesity-related protein FTO with m 6 For RNA samples modified with mA or DNA samples containing 6mA, dithiothreitol was added after 5-30 minutes to make mA 6 A or 6mA is modified with thiol groups; (2) adding dibenzylcyclooctyl-maleimide to react with the sulfhydryl group on the RNA or DNA to obtain RNA or DNA labeled with a carbon-carbon triple bond; (3) adding an RNA branch chain modified with an azide group at 5' to react with the RNA or DNA labeled with a carbon-carbon triple bond to obtain an RNA or DNA grafted with an RNA branch chain of known sequence.

7. A method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template switching technology according to claim 2, characterized in that: For p 6 A modification, grafting an RNA branch of known sequence at the modification site, the specific method is as follows: In the presence of p 6 A modified RNA sample is added with a 5' RNA branch modified with an azide group, and the p 6 The carbon-carbon triple bond of A is reacted to obtain RNA grafted with an RNA branch of known sequence.

8. A method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template switching technology according to claim 2, characterized in that: For pseudouridine Ψ modification, a RNA branch with a known sequence is grafted at the modification site, specifically including the following steps: (1) Mix the RNA sample containing Ψ modification and 1-cyclohexyl-2-(morpholinoethyl) carbodiimide propargyl p-toluenesulfonate evenly and react at 37°C for 20 minutes; (2) adding the RNA obtained in (1) to a sodium carbonate-sodium bicarbonate buffer solution with a pH between 10 and 11, reacting at 37° C. for more than 6 hours to obtain a propargyl-specifically labeled Ψ-modified RNA; (3) adding an RNA branch chain modified with an azide group at 5' to react with the propargyl-labeled RNA to obtain an RNA grafted with a branch chain.

9. A method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template switching technology according to claim 8, characterized in that: In step (1), the preparation method of 1-cyclohexyl-2-(morpholineethyl)carbodiimide propargyl p-toluenesulfonate is: Step S1, adding N-(2-aminoethyl)morpholine to dichloromethane, then dropping cyclohexyl isocyanate in an amount equimolar to N-(2-aminoethyl)morpholine, reacting at room temperature for 3 to 5 hours, removing the solvent by rotary evaporator and beating with petroleum ether to obtain intermediate 1, the chemical structure of intermediate 1 is: Step S2, triphenylphosphine is fully dissolved in dichloromethane under ice-water bath conditions, and then liquid bromine, triethylamine and intermediate 1 are added dropwise in sequence, and reacted at 10-15° C. for 3-5 hours, wherein the molar ratio of liquid bromine, triethylamine, intermediate 1 and triphenylphosphine is 1:1:3:0.83; after the reaction is completed, the reaction system is washed with water, dried, and distilled under reduced pressure to obtain intermediate 2, and the chemical structure of intermediate 2 is: Step S3, using acetonitrile as a solvent, adding equimolar amounts of intermediate 2 and propargyl p-toluenesulfonate thereto, reacting at 60-70° C. for 2-5 hours, and after the reaction is completed, the reaction solution is concentrated by a rotary evaporator, and the product is separated by a high performance liquid chromatography to obtain the 1-cyclohexyl-2-(morpholineethyl)carbodiimide propargyl p-toluenesulfonate, whose chemical structure is:

10. A method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template switching technology according to claim 2, characterized in that: For the classical protein-RNA interaction and the protein-RNA liquid-liquid phase separation interaction in RNA-protein interaction, the method for obtaining an RNA branch is: using the antibody cross-linking method to add an RNA branch of known sequence, and the specific method is to use a protein connected with an RNA branch of known sequence to combine with the antibody of the protein.

11. A method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template switching technology according to claim 2, characterized in that: For the RNA polymerase-nascent RNA interaction in RNA-RNA interaction, the method to obtain an RNA branch is to use click chemistry to add an RNA branch of known sequence, specifically: through the intracellular transcription restart method, p 6 A is incorporated into the RNA polymerase-nascent RNA interaction site and a 5' RNA branch with a known sequence modified with an azide group is used to bind to the p 6 The carbon-carbon triple bond of A undergoes click chemistry reaction.

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