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

Abstract

The present invention provides a method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template conversion technology. Before detecting the nucleic acid chemical modification sites and interaction sites, an RNA branch chain needs to be obtained; then, using a reverse transcription primer that is complementary to the RNA branch chain, reverse transcription is carried out starting from the RNA branch chain. When the reverse transcription proceeds to the junction of the RNA branch chain and the modification or the interaction junction, grafting template conversion is performed, and the main chain connected to the RNA branch chain is used as a template to continue reverse transcription to obtain cDNA containing modification site or interaction site information; finally, a high-throughput library is constructed using the cDNA containing modification site or interaction site information and high-throughput sequencing is carried out to determine the modification site or interaction site; or the cDNA containing modification site or interaction site information is detected by PCR and TA-cloning technology, so as to realize the identification of the modification site or interaction site. This method can be applied to a variety of gene sequencing methods.

Description

Technical Field

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

[0002] In vivo, different ribonucleic acid macromolecules (RNAs) undertake different functions, ensuring the normal expression of genetic information in an orderly manner: messenger RNA (mRNA) transmits genetic information, transfer RNA (tRNA) transfers translation raw 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, small RNA (miRNA) involved in post-transcriptional expression regulation, etc. also regulate the expression of genetic information at different links. The realization of these functions is inseparable from the recognition and interaction between RNA and other biomolecules.

[0003] RNAs can directly interact with each other through base pairing to achieve sequence-specific biological functions, and play important roles in many important biochemical processes such as miRNA-guided gene regulation, tRNA-involved translation process, and crRNA-guided viral RNA degradation. In addition to direct interactions, RNAs can also act indirectly via RNA-binding proteins as mediators, which often implies the functional correlation between RNAs. Currently, a variety of methods have been developed to study RNA-RNA interactions, including low-throughput methods based on physical and chemical properties and high-throughput methods based on proximity ligation. They have good detection capabilities for stable strong interactions, enabling us to have a preliminary understanding of the RNA interactome. With the in-depth study, 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] There are extensive interactions between RNA and proteins, which have extremely important biological functions. Almost all RNA functions require the participation of proteins. In the past, a variety of methods represented by immunocrosslinking precipitation 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 through RNA-binding domains have been identified, greatly deepening our understanding of RNA functions. Recently, the discovery of non-classical interactions represented by liquid-liquid phase separation phenomena indicates that there can be more indirect and dynamic interaction modes between RNA and proteins. If this phenomenon can be explored in depth, it will greatly promote our understanding of the life system. However, current methods are difficult to characterize these non-classical interactions, hindering the progress of research work.

[0005] The interactions between RNA and other RNAs and proteins can be altered by dynamic RNA modifications, thereby regulating gene expression, which has been a research hotspot in the past decade. RNA modifications can participate in physiological processes such as circadian regulation, immune regulation, cancer disease formation, embryonic and neural development regulation, stem cell differentiation, RNA virus replication, and sex determination through the regulation of RNA transcription, splicing, transportation, translation, degradation, etc. 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 levels averaged by a large number of cells and have turned their attention to single-cell research. However, current sequencing methods are difficult to characterize the single-cell modification group, which has become an obstacle to research work. Summary of the Invention

[0006] The purpose 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 conversion technologies.

[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 conversion technologies, comprising the following steps:

[0009] (1) Before detecting nucleic acid chemical modification sites and interaction sites, an RNA branch chain needs to be obtained first. Specifically, a chemically and / or biologically method can be used to graft an RNA branch chain with a known sequence at the RNA modification site or RNA-protein interaction, or cross-link two RNA chains at the RNA-RNA interaction site and use one of the chains as the RNA branch chain;

[0010] (2) Using a reverse transcription primer complementary to the RNA branch chain, reverse transcription is carried out starting from the RNA branch chain. When the reverse transcription proceeds to the junction of the RNA branch chain and the modification or interaction, grafting template conversion is performed, and the main chain (RNA or DNA main chain) connected to the RNA branch chain is used as a template to continue reverse transcription to obtain cDNA containing the modification site or interaction site information;

[0011] (3) Using the cDNA containing the modification site or interaction site information to construct a high-throughput library and perform high-throughput sequencing to determine the modification site or interaction site; or using PCR and TA-cloning technologies 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 -Methyl deoxyadenosine 6mA, pseudouridine Ψ, N 6 -Propargyl adenosine p 6 A or N 1 -Methyl adenosine m 1 Any one of those in A, and the interaction is any one of RNA-RNA intramolecular interaction, direct RNA-RNA intermolecular interaction, indirect RNA-RNA intermolecular interaction, classical protein-RNA interaction, protein-RNA liquid-liquid phase separation interaction, and RNA polymerase-nascent RNA interaction.

[0013] Furthermore, to detect N 6 -Methyl adenosine (m 6 A) and / or N 6 -Methyl deoxyadenosine (6mA) modification method, comprising the following steps:

[0014] (a) Treat an RNA sample containing m 6 A modification or a DNA sample containing 6mA modification with the demethylase fat mass and obesity-associated protein (FTO), and add dithiothreitol (DTT) after 5 - 30 minutes to make the m 6 A / or 6mA carry a thiol modification;

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

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

[0017] (d3) Add a reverse transcription primer complementary to the RNA branch chain and a template switching probe of a known sequence, start reverse transcription from the RNA branch chain, when reverse transcription proceeds to the modification junction, perform grafting template switching, and finally obtain a cDNA containing modification site information with the aid of traditional template switching;

[0018] (e3) Use the cDNA to construct a high-throughput library and perform high-throughput sequencing to determine the ligation site, and then the m 6 A modification site in the RNA or the 6mA modification site in the DNA can be obtained.

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

[0020] (a) React the RNA sample containing Ψ modification with 1 - cyclohexyl - 2 - (morpholinoethyl)carbodiimide propynyl p - toluenesulfonate (proCMC) at 37 °C for 20 minutes;

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

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

[0023] (d) Add a reverse transcription primer complementary to the RNA branch chain and a template - switching probe with a known sequence, start reverse transcription from the RNA branch chain, when reverse transcription proceeds to the modification junction, perform grafting template switching, and finally obtain cDNA containing modification site information by means of traditional template switching;

[0024] (e) Use the cDNA to construct a high - throughput library and combine with high - throughput sequencing technology to analyze the ligation site, that is, obtain the Ψ modification site in the RNA.

[0025] Furthermore, to detect the method for N 6 - propargyladenosine (p 6 A) modification, includes the following steps:

[0026] (a) In an RNA sample containing p 6 A modification, add an RNA branch chain modified with an azide group at the 5'-end and react with the carbon - carbon triple bond of p 6 A to obtain RNA grafted with an RNA branch chain of a known sequence;

[0027] (b) Add a reverse transcription primer complementary to the RNA branch chain and a template - switching probe with a known sequence, start reverse transcription from the RNA branch chain, when reverse transcription proceeds to the modification junction, perform grafting template switching, and finally obtain cDNA containing modification site information by means of traditional template switching;

[0028] (c) Use the cDNA to construct a high - throughput library and perform high - throughput sequencing to determine the ligation site, that is, obtain the p 6 A modification site in the RNA.

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

[0030] Step S1: Add N-(2-aminoethyl)morpholine (6.48 g, 49.6 mmol) into dichloromethane, and then dropwise add cyclohexyl isocyanate (6.23 g, 49.6 mmol) with an equimolar amount to N-(2-aminoethyl)morpholine. React at room temperature for 3 to 5 hours. After removing dichloromethane by a rotary evaporator, a crude product is obtained. The crude product is slurried with petroleum ether to obtain Intermediate 1. The chemical structural formula of Intermediate 1 is:

[0031]

[0032] Step S2: Dissolve triphenylphosphine (15.10 g, 57.8 mmol) in 100 mL of dichloromethane under an ice-water bath condition. Subsequently, dropwise add bromine (9.25 g, 57.8 mmol) with an equimolar amount to triphenylphosphine and triethylamine (17.53 g, 173.4 mmol) with three times the molar amount of bromine into the reaction flask in sequence using a syringe. Then dissolve Intermediate 1 (12.30 g, 48 mmol) in 20 mL of dichloromethane and slowly add it to the reaction flask using a syringe. React at 10 - 15 °C for 3 hours. After the reaction is completed, wash the reaction system three times with 500 mL of water, retain the lower-layer organic phase liquid, and then dry it with anhydrous sodium sulfate and filter to obtain a crude product. The crude product is concentrated by a rotary evaporator and then distilled under reduced pressure to obtain Intermediate 2. The chemical structural formula of Intermediate 2 is:

[0033]

[0034] Step S3: Using acetonitrile as a solvent, dropwise add Intermediate 2 (8.67 g, 36.6 mmol) and propargyl p-toluenesulfonate (7.70 g, 36.6 mmol) with an equimolar amount. Heat up to 60 - 70 °C and react for 3 hours. After the reaction is completed, concentrate the reaction solution by a rotary evaporator to obtain a crude product. Subsequently, separate the crude product using a high-performance liquid chromatograph (HPLC). The mobile phase is acetonitrile and water to obtain the 1-cyclohexyl-2-(morpholinoethyl)carbodiimide propargyl p-toluenesulfonate (proCMC). Its chemical structural formula is:

[0035]

[0036] Furthermore, the method for grafting an RNA branch chain with a known sequence is a click chemical reaction, a biotin-streptavidin bioorthogonal reaction, ultraviolet cross-linking, chemical cross-linking agent cross-linking, or antibody cross-linking method.

[0037] Further, the RNA is in vitro transcribed and synthesized RNA or total RNA, mRNA, rRNA or lncRNA extracted from normal mammalian cells, mammalian cancer cells, mammalian stem cells, bacteria, host cells of viruses, and various types of tissues and organs; the DNA is synthesized DNA or DNA extracted from normal mammalian cells, mammalian cancer cells, mammalian stem cells, bacteria, host cells of viruses, and various types of tissues and organs.

[0038] Further, 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] Further, the purification method of RNA in each step is TRIzol TM Reagent, chloroform-phenol extraction, Proteinase K digestion, silica gel membrane centrifugal column method, magnetic bead method, ethanol and isopropanol precipitation and other techniques, or a combination of one or more of them, preferably the magnetic bead method and the silica gel membrane centrifugal column method.

[0040] Further, the method for constructing a high-throughput library is specifically as follows: i) Use traditional reverse transcription template conversion or use Tn5 transposase to ligate 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) Design and synthesize PCR primers that are complementary to the RNA branch chain and the template switching probe TSO of the known sequence respectively, and amplify the cDNA product obtained in step i) by PCR to obtain double-stranded DNA with known sequences at both ends; iii) Design and synthesize PCR primers containing the sequence information required for next-generation sequencing, and amplify the double-stranded DNA obtained in step ii) by PCR to construct a high-throughput library.

[0041] Further, the library construction and sequencing steps can also use PCR and TA-cloning techniques to identify and verify the ligation sites on specific RNAs, and can also use single-cell sequencing methods.

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

[0043] (a) Fix the two interacting RNA strands in the cell by methods such as ultraviolet cross-linking and chemical cross-linking agent cross-linking;

[0044] (b) Extract and purify the RNA in the cell, and use one of the strands as the RNA branch chain;

[0045] (c) Add a reverse transcription primer complementary to the RNA branch and a template-switching probe with a known sequence, and perform reverse transcription from the RNA branch. If there is an intramolecular RNA interaction, reverse transcription will bypass the interaction region, perform grafting template switching, and continue reverse transcription using the other RNA strand as a template, ultimately obtaining cDNA containing information on RNA-RNA intramolecular interactions;

[0046] (d) Use the cDNA to construct a high-throughput library and perform high-throughput sequencing to determine the RNA-RNA intramolecular interaction sites.

[0047] Furthermore, a method for detecting RNA-RNA intermolecular direct interaction sites includes the following steps:

[0048] (a) Fix the interacting RNAs in cells by methods such as ultraviolet cross-linking and chemical cross-linking agent cross-linking;

[0049] (b) Extract and purify the RNAs in cells and use one of the strands as the RNA branch;

[0050] (c) Add a reverse transcription primer complementary to the RNA branch and a template-switching probe with a known sequence, and perform reverse transcription from the RNA branch. If there is a direct RNA-RNA intermolecular interaction, reverse transcription will bypass the interaction region, perform grafting template switching, and continue reverse transcription using the other RNA strand as a template, ultimately obtaining cDNA containing information on direct RNA-RNA intermolecular interactions;

[0051] (d) Use the cDNA to construct a high-throughput library and perform high-throughput sequencing to determine the RNA-RNA intermolecular interaction region, and further obtain a direct RNA-RNA interaction map.

[0052] Furthermore, a method for detecting RNA-RNA intermolecular indirect interaction sites includes the following steps:

[0053] (a) Fix the interacting RNAs in cells by methods such as ultraviolet cross-linking and chemical cross-linking agent cross-linking;

[0054] (b) Extract and purify the RNAs in cells and use one of the strands as the RNA branch;

[0055] (c) Add a reverse transcription primer complementary to the RNA branch and a template-switching probe with a known sequence, and perform reverse transcription from the RNA branch. If there is an indirect RNA-RNA intermolecular interaction, reverse transcription will bypass the interaction region, perform grafting template switching, and continue reverse transcription using the other RNA strand as a template, ultimately obtaining cDNA containing information on indirect RNA-RNA intermolecular interactions;

[0056] (d) Construct a high-throughput library using the cDNA and perform high-throughput sequencing to determine the RNA-RNA intermolecular interaction region, and then obtain the RNA-RNA indirect interaction sites.

[0057] Furthermore, a method for detecting protein-RNA classical interaction sites includes the following steps:

[0058] (a) Fix the RNA-protein interaction in cells by methods such as ultraviolet cross-linking and cross-linking with chemical cross-linking agents;

[0059] (b) Combine the antibody of the target protein with the target protein;

[0060] (c) Combine protein A linked to an RNA branch with a known sequence with the antibody of the target protein to achieve the grafting of the RNA branch;

[0061] (d) Add a reverse transcription primer complementary to the RNA branch and a template switching probe with a known sequence, and start reverse transcription from the RNA branch. When the reverse transcription proceeds to the interaction site, the reverse transcription reaction will cross the junction of the branch and the main chain, perform grafting template switching, and continue reverse transcription using the target RNA strand as a template to obtain cDNA containing information on the RNA-protein interaction site;

[0062] (e) Construct a high-throughput library using the cDNA and perform high-throughput sequencing to determine the junction site, and then the RNA-protein interaction site can be obtained.

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

[0064] (a) Fix the RNA-protein interaction in cells by methods such as formaldehyde fixation;

[0065] (b) Combine the antibody of the target protein with the target protein;

[0066] (c) Combine protein A linked to an RNA branch with a known sequence with the antibody of the target protein to achieve the grafting of the RNA branch;

[0067] (d) Add a reverse transcription primer complementary to the RNA branch and a template switching probe with a known sequence, and start reverse transcription from the RNA branch. When the reverse transcription proceeds to the interaction site, the reverse transcription reaction will cross the junction of the branch and the main chain, perform grafting template switching, and continue reverse transcription using the target RNA strand as a template to obtain cDNA containing information on the RNA-protein interaction site;

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

[0069] Furthermore, a method for detecting RNA polymerase-nascent RNA interaction sites includes the following steps:

[0070] (a) By the method of intracellular transcription restart, incorporate p 6 A into the RNA polymerase-nascent RNA interaction sites;

[0071] (b) Extract and purify the RNA, add a RNA branch with a known sequence modified with an azide group at the 5'-end to react with the carbon-carbon triple bond of the p 6 A to obtain an RNA grafted with a RNA branch of a known sequence;

[0072] (c) Add a reverse transcription primer complementary to the RNA branch and a template-switching probe of a known sequence, start reverse transcription from the RNA branch. When the reverse transcription proceeds to the modification junction, the reverse transcription reaction will cross the junction between the branch and the main chain and perform grafting template switching, and continue reverse transcription using the target RNA strand as a template to obtain cDNA containing the modification site information;

[0073] (d) Construct a high-throughput library using the cDNA and perform high-throughput sequencing to determine the ligation sites, that is, the p 6 A sites in the RNA, thereby obtaining the RNA polymerase-nascent RNA interaction sites.

[0074] In the above technical solution, further, the cell is a normal mammalian cell, a mammalian cancer cell, a mammalian stem cell, a bacterium, a host cell of a virus, 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, RNA-RNA direct intermolecular interactions, and RNA-RNA indirect intermolecular interactions, during the detection process, the reverse transcription primer used is a random hexamer with a linker or an endogenous RNA complementary primer.

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

[0078] The present invention for the first time discovers that chemically cross-linked RNA can be read through by reverse transcriptase, that is, the grafting template conversion phenomenon, which provides the possibility for detecting modification sites and interaction sites through nucleic acid grafting and nucleic acid reverse transcription template conversion.

[0079] The method of the present invention is based on grafting template conversion starting from a specifically modified RNA branch at the nucleic acid modification site. Compared with the existing gene sequencing technologies applied to modification detection, it improves the accuracy of detecting modification sites by the currently commonly used method based on antibody immunoprecipitation and massively parallel sequencing, 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 with a known sequence at the interaction site, so that the modification site or interaction site can be enriched through the grafting template conversion starting from the RNA branch, thus bypassing the traditional antibody enrichment mode, without repeated purification, and can be used for single-cell sequencing to characterize cell-differentiated modification sites or interaction sites.

[0081] The present invention uses Maxima H Minus reverse transcriptase to perform reverse transcription on the grafted RNA. Because the inventors selected numerous 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., it was finally found that Maxima H Minus reverse transcriptase has strong grafting template conversion and read-through capabilities during the grafting template conversion 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 research on RNA-RNA interactions and RNA-protein interactions, etc. Brief Description of the Drawings

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

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

[0085] Figure 3 is Dot-blot verification of m 6 A modification of the grafting of the branch on RNA;

[0086] Figure 4 is m 6Results graph of grafting template conversion start sites obtained by high-throughput sequencing of A-modified RNA probes;

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

[0088] Figure 6 is 28S rRNA 4220m 6 Results graph of grafting template conversion start sites obtained by high-throughput sequencing at the A-modification site;

[0089] Figure 7 is the m on the transcriptome obtained by high-throughput sequencing of 293T mRNA 6 Results graph of the A site;

[0090] Figure 8 is the m on the transcriptome obtained by high-throughput sequencing of 293T mRNA 6 Conserved sequence of the A site;

[0091] Figure 9 Results graph of grafting template conversion start sites obtained by low-throughput sequencing of 6mA-modified DNA probes;

[0092] Figure 10 Results graph of grafting template conversion start sites obtained by high-throughput sequencing of pseudouridine-modified RNA probes;

[0093] Figure 11 is p 6 Results graph of grafting template conversion start sites obtained by high-throughput sequencing of A-modified RNA probes;

[0094] Figure 12 is of 1-cyclohexyl-2-(morpholinoethyl)carbodiimide propyl p-toluenesulfonate (proCMC) 1 1H nuclear magnetic resonance spectrum;

[0095] Figure 13 is the high-resolution mass spectrum of 1-cyclohexyl-2-(morpholinoethyl)carbodiimide propyl p-toluenesulfonate (proCMC);

[0096] Figure 14 Schematic diagram of detecting RNA-RNA interactions based on nucleic acid grafting and nucleic acid reverse transcription template conversion technology;

[0097] Figure 15 Schematic diagram of detecting RNA-protein interactions based on nucleic acid grafting and nucleic acid reverse transcription template conversion technology. Specific implementation method

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

[0099] In view of the current situation 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 application proposes a new idea to solve the above problems. The present invention discovers that chemically cross-linked RNA can be read through by reverse transcriptase, and this phenomenon is called the grafting template switching phenomenon ( Figure 1 ). Based on this phenomenon, the present invention grafts RNA with known sequences at the interaction or modification sites, and then obtains complementary DNA (cDNA) via grafting template switching. By adding a 3' adapter through traditional template switching (Template-switch) or transposase fragmentation, it can then be amplified by PCR and subjected to high-throughput sequencing to know the RNA interaction or modification sites. This method can bypass the purification step in the library construction process, so it can be used for single-cell library construction; its specific grafting and library construction methods can enrich the target sequence from total RNA, reduce the interference of background signals, reduce the sequencing cost, and 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 technologies to achieve RNA and / or DNA nucleic acid modification sequencing and study the interaction between non-classical RNA and other macromolecules by performing grafting template switching at the modification or interaction sites.

[0101] As Figure 1 is the 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 proceeds to the junction of the RNA branch and the target RNA backbone, due to the steric hindrance of chemical groups, the reverse transcription reaction will bypass the junction and perform template switching, and continue reverse transcription using the target RNA backbone as a template.

[0102] As Figures 4 - 11 is the result graph of the grafting template switching starting site obtained by high-throughput sequencing of RNA and / or DNA with various nucleic acid modifications, where the abscissa is the distance from the grafting template switching starting site to the modification site.

[0103] As Figures 12 - 13 are the relevant characterization data of 1-cyclohexyl-2-(morpholinoethyl)carbodiimide propynyl p-toluenesulfonate (proCMC) prepared by the method of the present invention. Figure 12 For 1-cyclohexyl-2-(morpholinoethyl)carbodiimide propynyl p-toluenesulfonate (proCMC) 1 1H nuclear magnetic resonance spectrum: 11H NMR (400 MHz, CDCl3): δ 7.75 (d, J = 8.2 Hz, 2H), 7.18 (d, J = 8.1 Hz, 2H), 4.76 (s, 2H), 4.00 (s, 2H), 3.91 (d, J = 13.6 Hz, 4H), 3.73 (s, 6H), 3.47 (t, J = 10.5 Hz, 1H), 2.85 (s, 1H), 2.35 (s, 3H), 1.81 (d, J = 9.5 Hz, 2H), 1.65 (d, J = 13.3 Hz, 2H), 1.54 (d, J = 12.8 Hz, 1H), 1.27 (d, J = 17.2 Hz, 2H), 1.10 (q, J = 12.2 Hz, 3H). Figure 13 High-resolution mass spectrum of 1-cyclohexyl-2-(morpholinoethyl)carbodiimide propynyl 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 Detection of extracellular RNA m 6 A modification sites (such as Figure 2 )

[0106] 1. RNA m 6 A site-labeled RNA branch and verification of the grafting of the branch

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

[0108] (2) Take the RNA probe with m 6 A modification, configure the FTO demethylation reaction system according to Table 1, react at 37 °C for 10 minutes, and oxidize m 6 A to hm 6 A (N 6 -hydroxymethyladenosine);

[0109] Table 1 FTO demethylation reaction system

[0110]

[0111] (3) Configure the dithiothreitol (DTT) labeling reaction system according to Table 2, react at 37 °C for 3 hours, and make hm 6A reacts with DTT to generate adenosine modified with a mercapto group;

[0112] Table 2 DTT labeling reaction system

[0113]

[0114] (4) Add 250 μL of TRIzol TM Reagent to the above 50 μL solution, 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 12,000 rpm for 15 minutes. The solution will be layered. Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of isopropanol, 1 / 10 volume of 3 M sodium acetate solution and 1 μL of glycogen, mix well by pipetting, precipitate overnight at -20 °C, then centrifuge at 15,000 rpm at 4 °C for 1 hour. Wash the precipitate with an equal volume of 80% ethanol, centrifuge at 15,000 rpm at 4 °C for 15 minutes, remove the supernatant again, air dry for 5 minutes, and dissolve the RNA with RNase-free water;

[0115] (5) Add the above-mentioned RNA modified with a mercapto group to the modification system shown in Table 3, react at 37 °C for 2 hours, and modify the carbon-carbon triple bond at the m 6 A site;

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

[0117]

[0118] (6) Add 90 μL of RNAClean XP beads (Beckman Coulter) to the reaction system, pipette and mix 6 - 10 times, then incubate at room temperature for 15 minutes. Separate with a magnetic stand and discard the supernatant. Wash the beads twice with 80% ethanol, 30 seconds each time. Let it stand for 5 - 10 minutes to volatilize the ethanol, and elute the RNA with 42 μL of resuspension buffer. Incubate at room temperature for 2 minutes and then separate with a magnetic stand;

[0119] (7) Add the above-mentioned RNA modified with DBCO to the modification system shown in Table 4, react at 37 °C for 2 hours, and graft an RNA branch chain at the m 6 A site;

[0120] Table 4 Branch chain labeling reaction system

[0121]

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

[0123] (9) Take an equal amount of the RNA probe with m 6 A modification, repeat the above steps, and do not add FTO in step (2) as a control group;

[0124] (10) Take 1 μg of the branched-chain labeled product and the control group product respectively, denature at 95 °C for 2 minutes, quickly quench on ice, dot onto a nylon membrane, completely dry it, and crosslink it in a UV crosslinker. Select a UV lamp with a wavelength of 254 nm and an energy of 150 - 400 mJ / cm 2 , crosslink twice, block the membrane with 5% non-fat milk (dissolved in PBST) for 1 hour, wash the membrane three times with PBST for 5 minutes each time, apply 10 ml of antibody solution (anti-biotin-HRP, diluted 1:10000 in PBST), incubate for 1 hour, wash the membrane three times with PBST for 5 minutes each time, develop, and finally stain with methylene blue;

[0125] (11) The results of Dot-blot are as Figure 3 shown. Compared with the control group, spots appeared in the branched-chain labeled product of the m 6 A RNA probe, indicating that it has been labeled with a branched chain with biotin modification.

[0126] 2. Grafting template conversion and traditional template conversion of branched-chain modified RNA

[0127] Take the above-mentioned branched-chain labeled product and the control group product into PCR tubes respectively, mix well with 1 μL of 10 μM reverse transcription primer (RT-primer) and 5 μL of RNase-free water by pipetting, heat and denature at 65 °C for 5 minutes, cool on ice, configure the grafting template conversion - traditional template conversion 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 conversion - traditional template conversion reaction system

[0129]

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

[0131] (1) Take 5 μL of the above reaction solution, prepare the PCR amplification system according to the reaction system in Table 6. The PCR primers at both ends carry adapter sequences, and the sequences are shown in Table 9. After pipetting and mixing evenly, 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 Running Program

[0135]

[0136] (2) Purify the double-stranded DNA fragment obtained above: Add 40 μL of AMPure XP beads (Beckman Coulter) that have been pre-restored to room temperature to the reaction system. After pipetting and mixing evenly 6 - 10 times, incubate at room temperature for 15 minutes. Separate with a magnetic stand and discard the supernatant. Wash the beads twice with 80% ethanol, 30 seconds each time. Let stand for 5 - 10 minutes to volatilize the ethanol. Elute the double-stranded DNA fragment with 50 μL of resuspension buffer. After incubating at room temperature for 2 minutes, separate with a magnetic stand. Repeat the purification step, and finally elute with 22 μL of resuspension buffer;

[0137] (3) Prepare the PCR amplification system according to the reaction system in Table 8 with the double-stranded DNA obtained above. The PCR primers at both ends carry high-throughput sequencing primer sequences. Among them, PCR2-R can be 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 evenly, run the program in the PCR instrument as 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) Purify the obtained double-stranded DNA fragment: Add 40 μL of AMPure XP beads (Beckman Coulter) that have been pre-restored to room temperature to the reaction system, pipette and mix well 6 - 10 times, then incubate at room temperature for 15 minutes. Separate and discard the supernatant using a magnetic stand, wash the beads twice with 80% ethanol, 30 seconds each time, let stand for 5 - 10 minutes to volatilize the ethanol, elute the double-stranded DNA fragment with 50 μL of resuspension buffer, incubate at room temperature for 2 minutes, then separate using a magnetic stand. Repeat the purification step, and finally elute with 20 μL of resuspension buffer to obtain a high-throughput library.

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

[0145] Perform paired-end 150 sequencing on the obtained high-throughput library using the illumina NovaS4 platform. The obtained data is filtered for low quality and adapter sequences, and then aligned with the original RNA sequence to obtain the ligation sites, which are the m 6 A modification sites. The high-throughput sequencing results are as Figure 4 、 5 shown. The ligation sites are concentrated at 1 - 2 bases at the 5' end of the m 6 A modification sites, and one base may be randomly incorporated during the grafting template conversion process, indicating that this method can detect the m 6 A modification sites on RNA.

[0146] Example 2 Detection of m 6 A modification sites on transcriptome rRNA in HEK293T cells

[0147] 1. Culture of HEK293T cells, extraction and fragmentation of total RNA

[0148] (1) After culturing HEK293T cells to 80% confluence under normal culture conditions, aspirate the culture medium, wash away the residual culture medium with phosphate-buffered saline (PBS), then add TRIzol TM Reagent to cover the bottom of the culture dish, elute all the adherent HEK293T cells into the solution, and transfer to a centrifuge tube for lysis at room temperature for 5 minutes;

[0149] (2) For every 1 mL of 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 12,000 rpm for 15 minutes. The solution will be layered. 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, and centrifuge at 4 °C and 12,000 rpm for 15 minutes to obtain a white precipitate of total RNA;

[0150] (3) Remove the supernatant, leaving the white precipitate of total RNA. Wash the precipitate with 1 mL of 75% ethanol / 1 mL of 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 of 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 the total RNA sample, add 2 μL of 10×RNA Fragmentation Buffer (NEB), add RNase-free water to a total volume of 20 μL, mix well by pipetting, heat at 94 °C for 2 minutes, and then add 2 μL of 10×RNA Fragmentation Stop Solution (NEB) to terminate the fragmentation;

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

[0153] 2. The same as the RNA m in Example 1 6 Label the RNA branch at the A site. In the control group, no FTO is added to the RNA, and the reaction system is made up with RNase-free water. The RNA branch used is the 5’N3-RNA branch.

[0154] 3. Grafting template conversion

[0155] (1) Take the above branched-chain labeled product into 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 then cool on ice;

[0156] (2) Configure the grafting template conversion reaction system according to the reaction system in Table 10, react at 50 °C for 1.5 hours, and then heat at 85 °C for 5 minutes to inactivate the reverse transcriptase.

[0157] Table 10 Grafting template conversion reaction system

[0158]

[0159] 4. Perform PCR amplification to construct a high-throughput library as in Example 1, where the first PCR primers are PCR1-28S and PCR1-F.

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

[0161] Perform paired-end 150 sequencing on the obtained high-throughput library using the illumina NovaS4 platform. The obtained data is filtered for low quality and adapter filtering and then aligned with the human rRNA sequence to obtain the ligation site, that is, m 6 A modification site. The high-throughput sequencing results are as Figure 6 shown. There is a template conversion signal at the 28S rRNA 4220 site that is significantly different from the control group, and the ligation sites are concentrated at 1-2 bases at the 5' end of the m 6 A modification site, indicating that this method can identify the m 6 A modification site on real samples.

[0162] Example 3 Detection of m 6 A modification sites on transcriptome mRNA in HEK293T cells

[0163] 1. Culture of HEK293T cells and extraction of mRNA

[0164] (1) After culturing HEK293T cells to 80% confluence under normal culture conditions, aspirate the culture medium, wash away the residual culture medium with phosphate-buffered saline (PBS), and then add TRIzol TM Reagent to cover the bottom of the culture dish, elute all the adherent HEK293T cells into the solution, and transfer to a centrifuge tube for lysis at room temperature for 5 minutes;

[0165] (2) For every 1 mL of 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 12,000 rpm for 15 minutes. The solution will separate 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, and centrifuge at 4 °C and 12,000 rpm for 15 minutes to obtain a white precipitate of total RNA;

[0166] (3) Remove the supernatant, leaving the white precipitate of total RNA. Wash the precipitate with 1 mL of 75% ethanol / 1 mL of 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 of RNase-free water, and heat at 70 °C for 10 minutes to fully dissolve the total RNA. Measure the RNA concentration with a Bio Drop;

[0167] (4) Further extract mRNA using the GenElute mRNA MiniprepKit (Sigma). 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 fully mix the system. Heat at 70 °C for 3 minutes to denature the RNA, then incubate at room temperature for 10 minutes to allow oligo(dT) to fully bind to the RNA. Centrifuge the mixed system at RCF 15,000 g for 2 minutes to obtain oligo(dT) beads bound to mRNA. Carefully remove the supernatant, leaving about 50 μL of solution to prevent loss of beads;

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

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

[0171] (8)Finally, transfer the filtration column to a new collection tube, pipette 50 μL of the Elution Solution preheated to 70 °C and add it to the exact center of the filter membrane of the centrifugal filtration column to fully contact the mRNA complex in the column pores. Incubate at 70 °C for 5 minutes, centrifuge at RCF 15,000 g for 1 minute, and obtain the mRNA solution in the collection tube. Measure the mRNA concentration using Bio Drop. In addition, 50 μL of the 70 °C Elution Solution can be pipetted again to repeat the above elution process to fully dissolve the mRNA in the column pores of the filtration column;

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

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

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

[0175] 2. The same as RNA m in Example 1 6 Label the RNA branch at the mA site. In the control group RNA, no FTO is added, and the reaction system is supplemented with RNase-free water. The RNA branch used is the 5’N3-RNA branch.

[0176] 3. The same as the grafting template conversion and traditional template conversion of the branched-chain modified RNA in Example 1.

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

[0178] 5. High-throughput sequencing analysis to detect the mA modification sites on the mRNA of HEK293T cells 6 mA modification sites

[0179] The obtained high-throughput library was subjected to paired-end 150 sequencing using the Illumina NovaS4 platform. The data obtained was filtered for low quality and adapter sequences, and then aligned with the human transcriptome sequence. The site where the branched sequence was ligated, i.e., the ligation site, was m 6 A modification site. The high-throughput sequencing results are as Figure 7 shown. A total of 50,907 m 6 A modification sites were identified on the mRNA of HEK293T cells by the method of the present invention. By analyzing the sequence characteristics before and after the ligation site, the conserved sequence of m 6 A modification sites on the HEK293T cell transcriptome was obtained (as Figure 8 ), which was roughly the same as the conserved sequence obtained by the common m 6 A antibody immunoprecipitation and sequencing technology, indicating that the sites identified by this method are m 6 A sites.

[0180] Example 4 Detection of 6mA Modification Sites in Extracellular DNA

[0181] 1. Labeling of RNA Branches at DNA 6mA Sites

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

[0183] (2) Similar to the labeling of RNA branches at RNA m 6 A sites in Example 1, the RNA branch used is the 5' N3-RNA branch.

[0184] 2. The same grafting template conversion as in Example 2.

[0185] 3. PCR Amplification

[0186] (1) Take 5 μL of the above reaction solution and configure the PCR amplification system according to the reaction system in Table 11. The PCR primers at both ends carry adapter sequences and the 5' end sequence of the DNA probe, and the sequence is shown in Table 9;

[0187] Table 11 PCR Amplification Reaction System

[0188]

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

[0190] Table 12 PCR Amplification Running Program

[0191]

[0192] (3) Purify the double-stranded DNA fragment obtained above: Add 40 μL of AMPure XP beads (Beckman Coulter) that have been restored to room temperature in advance to the reaction system, pipette and mix well 6 - 10 times, then incubate at room temperature for 15 minutes. Separate with a magnetic stand and discard the supernatant. Wash the beads twice with 80% ethanol, 30 seconds each time. Let it stand for 5 - 10 minutes to volatilize the ethanol. Elute the double-stranded DNA fragment with 50 μL of resuspension buffer, incubate at room temperature for 2 minutes, and then separate with a magnetic stand to obtain the purified PCR product.

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

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

[0195] Table 13 Vector ligation system

[0196]

[0197] (2) Place 100 μL of DH5α competent cells on ice and thaw for 5 minutes until the cells are evenly suspended. Add the above ligation solution, gently tap and mix well, and place on ice for 25 minutes;

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

[0199] (4) Centrifuge at 5000 rpm for 1 minute, use a pipette tip to aspirate and discard 700 μL of the supernatant, and resuspend the cells with the remaining medium;

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

[0201] (6) First, incubate the plates at 37 °C for 1 hour, then incubate them upside down for 16 hours. Select white single colonies to extract plasmids for Sanger sequencing to obtain the low-throughput sequencing results;

[0202] (7) The low-throughput sequencing results are as Figure 9 shown. According to the sequencing results, there are obvious template conversion signals at the 6mA sites, indicating that the method of the present invention can detect the 6mA modification sites on DNA.

[0203] Example 5 Detection of Ψ modification sites of extracellular RNA

[0204] 1. RNA Ψ-site labeling of RNA branches

[0205] (1) Use HiScribe TM T7 High Yield RNA Synthesis Kit (NEB) kit to prepare an 83-nt RNA probe, and at the same time replace UTP with ΨTP to prepare an 83-nt RNA probe 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 = 8.5. Take 5 μg of RNA probes with and without Ψ modification respectively, configure proCMC reaction system I according to Table 15, and incubate at 37 °C for 30 minutes to label the U, G, and Ψ sites on the RNA probe with proCMC;

[0207] Table 14 proCMC 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, pipette and mix well. Then add 110 μL of isopropanol, invert and mix well, precipitate at -20 °C for more than 3 hours. After precipitation, centrifuge at 4 °C and 12,000 rpm for 1 hour, discard the supernatant, then add 100 μL of 80% ethanol, centrifuge under the same conditions for 20 minutes, 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 = 10.4. Take the RNA probe purified in (3), configure proCMC reaction system II according to Table 17, incubate at 37 °C for 6 hours, remove the proCMC labeled on the U and G sites of the RNA probe, and then achieve the specific labeling of the Ψ site of the RNA probe with proCMC. After the reaction, purify the RNA probe according to the steps described in (3);

[0213] Table 16 proCMC reaction buffer II

[0214]

[0215] Table 17 proCMC reaction system

[0216]

[0217] (5) Take 50 ng of RNA probe and 1 μL of 50 μM 5’N3-RNA branch chain into a PCR tube, denature at 95 °C for 5 minutes and then quickly cool on ice for 1 minute; take 1 μL of 20 mM copper sulfate solution and 1 μL of 100 mM tris(3-hydroxypropyltriazylmethyl)amine (THPTA) solution and add them to another PCR tube, then add 1 μL of 200 mM sodium ascorbate solution, and let it stand at room temperature for 8 minutes to obtain the click chemical reaction buffer; mix the solutions in the two PCR tubes well, and supplement the system to 20 μL with RNase-free water, react at 37 °C for 3 hours, and after the reaction, 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 of RNAClean XP beads, pipette and mix more than 10 times, incubate at room temperature for 15 minutes, separate with a magnetic stand and discard the supernatant, wash the beads twice with 80% ethanol, 30 seconds each time, let it stand for 5 - 10 minutes to volatilize ethanol, elute the RNA with 50 μL of resuspension buffer, incubate at room temperature for 2 minutes and then separate with a magnetic stand, repeat the purification step, and finally elute with 10 μL of resuspension buffer to obtain the branched-chain labeled product.

[0219] 2. The same grafting template conversion as in Example 2.

[0220] 3. The same PCR amplification to construct a high-throughput library as in Example 1, where the first PCR primers are 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 is subjected to paired-end 150 sequencing using the illumina NovaS4 platform, and the obtained data is compared with the original RNA sequence after low-quality filtering and adapter filtering to obtain the ligation sites, that is, the Ψ modification sites. The high-throughput sequencing results are as Figure 10 shown, and 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 Detection of extracellular RNA p 6 A modification sites

[0224] 1. RNA p 6 A site-labeled RNA branch chain

[0225] (1) Use HiScribe TM T7 High Yield RNA Synthesis Kit (NEB) to prepare an RNA probe with p 6 A modification and a length of 460 nt. The sequence 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 branch chain into a PCR tube. Denature at 95 °C for 5 minutes and then quickly cool on ice for 1 minute; Take 1 μL of 20 mM copper sulfate solution and 1 μL of 100 mM tris(3-hydroxypropyltriazylmethyl)amine (THPTA) solution and add them to another PCR tube. Subsequently, add 1 μL of 200 mM sodium ascorbate solution. After observing that the reaction system changes from a brief yellow turbidity to a red solution and then to a pale yellow transparent solution, let it stand at room temperature for 5 minutes to obtain the click chemistry reaction buffer; Mix the solutions in the two PCR tubes thoroughly and react at 37 °C for 2 hours. Then add 2 μL of 0.5 M EDTA solution to the reaction system to terminate the reaction;

[0227] (3) Expand the reaction system to 50 μL, add 90 μL of RNAClean XP beads, pipette and mix 6 - 10 times, then incubate at room temperature for 15 minutes. Separate with a magnetic stand and discard the supernatant. Wash the beads twice with 80% ethanol, 30 seconds each time. Let it stand for 5 - 10 minutes to volatilize the ethanol. Elute the RNA with 50 μL of resuspension buffer, incubate at room temperature for 2 minutes, then separate with a magnetic stand. Repeat the purification step. Finally, elute with 10 μL of resuspension buffer to obtain the branched chain labeled product.

[0228] 2. The grafting template conversion and traditional template conversion of the branched chain modified RNA are the same as in Example 1.

[0229] 3. PCR amplification to construct a high-throughput library is the same as in Example 1.

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

[0231] Use the illumina NovaS4 platform to perform paired-end 150 sequencing on the obtained high-throughput library. The data obtained is filtered for low quality and adapter filtering, and then compared with the original RNA sequence to obtain the ligation sites, that is, the p 6 A modification sites. The high-throughput sequencing results are as Figure 11 shown. The ligation sites are concentrated at p 6At the 1-2 bases at the 5' end of the modification site A, indicating that this method can detect p on RNA 6 Modification site A.

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

[0233] 1. Extraction of HeLa cell nuclei

[0234] (1) After HeLa cells are cultured to 80% confluence in a 15-cm culture dish under normal culture conditions, aspirate the culture medium and wash away the residual culture medium 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 minutes;

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

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

[0238] (5) Shake for 5 seconds to loosen the cell clumps;

[0239] (6) Suspend the cells in ice-cold sucrose solution I at a concentration of 5×10 7 cells per milliliter, and determine the cell concentration using a phase-contrast microscope. The composition of sucrose solution I is shown in Table 18;

[0240] Table 18 Composition of sucrose solution I

[0241]

[0242] (7) After incubating on ice for 5 minutes, transfer the cell suspension to an ice-cold Dounce homogenizer to break the cells, and use a phase-contrast microscope to confirm that the cells are completely broken;

[0243] (8) Transfer the broken cell suspension to a 50-mL centrifuge tube, add an equal volume of ice-cold sucrose solution II, invert and mix well. 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 an ultracentrifuge tube, carefully add the cell lysate to its upper layer, and each centrifuge tube should not exceed 2×10 8 cells;

[0247] (10) Seal with sucrose solution I at the top and centrifuge at 4 °C and RCF 30,000 g for 45 minutes;

[0248] (11) Aspirate the upper liquid, and a tight precipitate of cell nuclei forms in the lower layer;

[0249] * If the cells are not lysed, no precipitate will form; if the cell nuclei are also lysed, a gelatinous chromatin precipitate will form.

[0250] (12) Shake for 5 seconds to loosen the precipitate of cell nuclei;

[0251] (13) Add ice-cold glycerol storage solution pre-cooled on ice at a concentration of 5×10 7 nuclei per milliliter, pipette up and down to mix evenly. The composition of the glycerol storage solution is shown in Table 20;

[0252] Table 20 Composition of glycerol storage solution

[0253]

[0254] (14) Put every 100 μL of the cell nucleus suspension into a pre-cooled 1.5 mL centrifuge tube, quickly cool it with liquid nitrogen, and store it in a -80 °C refrigerator for later use.

[0255] 2. Chromatin acquisition and RNA polymerase re-initiation

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

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

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

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

[0260] (5) Dissolve the chromatin precipitate with 100 μL of glycerol storage solution, and then add an equal volume of transcription restart buffer pre-warmed to 37 °C. Among them, the experimental group uses 80% p 6 ATP, and the control group uses ATP. Mix well and react with shaking at 37 °C for 5 minutes. The composition of the transcription restart buffer is shown in Table 4;

[0261] Table 21 Composition of transcription restart buffer

[0262]

[0263]

[0264] (6) Add 600 μL of LC-Trizol, pipette and mix well to 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 12,000 g for 15 minutes. The solution will separate into layers. The upper clear liquid 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 with pH = 5.3, shake the centrifuge tube vigorously for 15 seconds, extract at room temperature for 3 minutes, centrifuge at 4 °C and RCF 12,000 g for 15 minutes to remove residual DNA and protein impurities, and 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 12,000 g 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 and then add an equal volume of isopropanol, incubate at -20 °C for 30 minutes, centrifuge at 4 °C and RCF 15,000 g for 45 minutes to obtain a white precipitate of chromatin RNA;

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

[0270] (12) Remove the supernatant again, air-dry for 5 minutes, dissolve the chromatin RNA with 100 μL of RNase-free water, heat at 70 °C for 10 minutes to fully dissolve the chromatin RNA, about 20 μg of RNA in total. Take out a part of the RNA 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, 2 U 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 of LC-Trizol, pipette and mix well to terminate the reaction, and transfer it 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 12,000 g for 15 minutes, and the solution will separate into layers. The upper clear liquid 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, then add an equal volume of isopropanol, incubate at -20 °C for 30 minutes, and centrifuge at 4 °C and RCF 15,000 g for 45 minutes to obtain a white RNA precipitate.

[0276] (5) Remove the supernatant, leaving the white RNA precipitate. Wash the precipitate with 200 μL of 80% ethanol, centrifuge at 4 °C and RCF 15,000 g for 15 minutes, and repeat twice to remove residual isopropanol and salts.

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

[0278] (7) Use the NEBNext rRNA Depletion Kit to remove rRNA. Mix 1 μL of NEBNext rRNA Depletion Solution and 2 μL of Probe Hybridization Buffer with 12 μL of chromatin RNA in a PCR tube (not exceeding 1 μg of RNA), pipette and mix well, then place it in a PCR instrument and run the program as shown in Table 22.

[0279] Table 22 PCR instrument running program

[0280]

[0281] (8) After the program ends, place it on ice, add 1 μL of RNase-free water, 2 μL of RNase H Reaction Buffer and 2 μL of NEBNext RNase H, pipette and mix well, then place it in a PCR instrument and incubate at 37 °C for 30 minutes.

[0282] After the procedure is completed, place it on ice, add 22.5 μL of RNase-free water, 5 μL of DNase I Reaction Buffer, and 2.5 μL of DNase I (RNase-free), pipette and mix well, then place it in a PCR instrument and incubate at 37 °C for 30 minutes;

[0283] (10) Purify the RNA using NEBNext RNA Sample Purification Beads. After vortexing and mixing the NEBNext RNA Sample Purification Beads, take out 110 μL and add it to the reaction solution after rRNA removal, pipette and mix well, and incubate on ice for 15 minutes;

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

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

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

[0287] 4. The same as the RNA p in Example 6 6 RNA branch labeled at the A site

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

[0289] 6. The same as PCR amplification to construct a high-throughput library in Example 1.

[0290] 7. Use the illumina X-10 platform to perform paired-end 150 sequencing on the obtained library. The data obtained is subjected to low-quality filtering and adapter filtering and then compared with the transcriptome of HeLa cells. The enriched RNA is the nascent RNA that interacts with RNA polymerase, and the reverse transcription blocking ligation site is the RNA polymerase-nascent RNA interaction site.

[0291] Example 8 Detection of protein-mediated RNA-RNA indirect interaction in HeLa cells (the detection principle is as Figure 14 )

[0292] 1. Crosslinking of HeLa cells

[0293] (1) After culturing HeLa cells in a 15 cm culture dish under normal culture conditions until they reach 80% confluence, aspirate the culture medium and wash away the residual culture medium 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 minutes;

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

[0296] (4) Wash the cells twice with PBS to remove the residual 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 HeLa cells at room temperature for 15 minutes;

[0299] Table 23 Formulation of Cross-linking Buffer

[0300]

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

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

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

[0304] 2. Enrichment of Protein-crosslinked RNA

[0305] (1) Add 1 mL of TRIzol TM Reagent to the cells, pipette to mix well, 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 12000 rpm at 4 °C for 15 minutes, the solution will separate 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 and discard the upper aqueous phase and the lower organic phase. Add 0.5 mL of water and 0.5 mL of an equal volume of phenol-chloroform solution with pH 5.3, shake the centrifuge tube vigorously for 15 seconds, extract at room temperature for 5 minutes, centrifuge at 12000 rpm at 4 °C for 15 minutes to remove the residual uncross-linked RNA and protein impurities, and repeat twice;

[0308] (4) Aspirate and 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) Aspirate and discard the upper aqueous phase and the lower organic phase, open the lid and place it in a 70°C metal bath for 10 minutes to remove residual chloroform.

[0310] 3. Grafting template conversion and traditional template conversion of HeLa cells

[0311] (1) Prepare the 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) Add RNase A and RNase H to digest cellular RNA, then dissolve the cells with saturated phenol at pH = 8.0, transfer to a centrifuge tube, shake vigorously to fully mix the aqueous phase and the organic phase, and then centrifuge 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 and then add an equal volume of isopropanol, incubate at -20°C for 30 minutes, centrifuge at 4°C and RCF 15,000 g for 45 minutes to obtain a white precipitate containing cDNA;

[0317] (5) Remove the supernatant, leave the white precipitate containing cDNA, wash the precipitate with 200 μL of 80% ethanol, centrifuge at 4°C and RCF 15,000 g for 15 minutes, and repeat twice to remove residual isopropanol and salts;

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

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

[0320] (7) Add the forward primer and reverse primer complementary to the template conversion probe and RNA probe to the DNA solution respectively, then add NTP, PCR enzyme and the corresponding buffer solution, pipette and mix well, place it in a PCR instrument, and run the program as shown in Table 25;

[0321] Table 25 PCR amplification running program

[0322]

[0323] (8) Add 45 μL of AMPure XP beads that have been pre-restored to room temperature to the reaction system, pipette and mix well 6 - 10 times, then incubate at room temperature for 15 minutes. Separate with a magnetic stand and discard the supernatant. Wash the beads twice with 80% ethanol, 30 seconds each time. Let it stand for 5 - 10 minutes to volatilize the ethanol. Elute the double-stranded DNA fragment with 23 μL of 0.1X TE Buffer, incubate at room temperature for 2 minutes, then separate with a magnetic stand. Transfer 20 μL of the supernatant to a new PCR tube, and use 1 μL for Qubit concentration measurement, and store at -20 °C.

[0324] 4. High-throughput sequencing analysis

[0325] Use the obtained library for paired-end 150 sequencing on the illumina X-10 platform. The obtained data is filtered for low quality and adapter filtering, and then compared with the transcriptome of HeLa cells. The enriched RNA is the RNA that interacts with the protein, and the reverse transcription blocking ligation site is the site of protein-mediated indirect RNA-RNA interaction.

[0326] Example 9 Detection of liquid-liquid phase separation RNA-protein interaction in HeLa cells (the detection principle is as Figure 15 )

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

[0328] (1) Coat the cover glass with polylysine and place it at room temperature for 1 hour;

[0329] (2) Rinse the cover glass thoroughly with sterile water 3 times, 1 hour each time;

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

[0331] (4) Seed HeLa cells on the glass cover slip and culture them to 80% confluence under normal culture conditions;

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

[0335] (8) Wash the cells 3 times with ice-cold PBS;

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

[0337] (10) Wash the cells 3 times with PBST (PBS + 0.1% Tween 20).

[0338] 2. Preparation of Protein A Linked with RNA Branches

[0339] (1) Incorporate unnatural amino acids containing triple bond groups into Protein A using unnatural amino acid incorporation technology;

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

[0341] (3) Crosslink the RNA branches with Protein A using click chemistry method;

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

[0343] 3. Blocking, Antibody Incubation and Protein A Binding of HeLa Cells

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

[0345] (2) Wash the cells 3 times with PBST;

[0346] (3) Dilute the anti-RBM15 antibody at 1:10 in PBST containing 6% BSA, add it to the cells, and incubate for 30 minutes;

[0347] (4) Wash the cells 3 times with PBST;

[0348] (5) Dilute the Protein A linked with RNA branches at a concentration of 10 μg / mL in PBST containing 6% BSA, add it to the cells, and incubate for 30 minutes;

[0349] (6) Wash the cells 3 times with PBST.

[0350] 4. Library Construction

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

[0352] (2) Add RNase A and RNase H to digest cellular RNA. Then dissolve the cells with saturated phenol at pH = 8.0, transfer them to a centrifuge tube, shake vigorously to fully mix the aqueous and organic phases, and then centrifuge at 4 °C and RCF 12,000 g for 3 minutes;

[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 minutes, and centrifuge at 4 °C and RCF 15,000 g for 45 minutes to obtain a white precipitate containing cDNA;

[0354] (4) Remove the supernatant, leave the white precipitate containing cDNA, wash the precipitate with 200 μL of 80% ethanol, centrifuge at 4 °C and RCF 15,000 g for 15 minutes, repeat twice to remove residual isopropanol and salts;

[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 forward primer and reverse primer complementary to the template switching probe and RNA probe respectively to the DNA solution, then add NTP, PCR enzyme and the corresponding buffer solution, pipette and mix well, put it into a PCR instrument, and run the program as shown in Table 25:

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

[0358] 5. High-throughput sequencing analysis

[0359] The obtained library was subjected to paired-end 150 sequencing using the Illumina X-10 platform. The data obtained were filtered for low quality and adapter sequences, and then aligned with the transcriptome of HeLa cells. The enriched RNA is the RNA that interacts with the protein, and the reverse transcription blocking ligation site is the site where the RNA interacts with the protein.

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 conversion technology, characterized in that, The method is for non-diagnostic purposes, including the following steps: First, an RNA branch chain needs to be obtained by grafting an RNA branch chain with a known sequence at a nucleic acid modification site or an RNA-protein interaction site, or by cross-linking two RNA strands at an RNA-RNA interaction site and using one of the strands as the RNA branch chain; Then, using a reverse transcription primer that is complementary to the RNA branch chain, reverse transcription is carried out starting from the RNA branch chain. When the reverse transcription proceeds to the modification junction or interaction junction of the RNA branch chain, a grafting template conversion is carried out, and reverse transcription continues using the main chain linked to the RNA branch chain as the template to obtain cDNA containing information on the modification site or interaction site; Finally, a high-throughput library is constructed using the cDNA containing information on the modification site or interaction site and high-throughput sequencing is carried out to determine the modification site or interaction site; or the cDNA containing information on the modification site or interaction site is detected using PCR and TA-cloning techniques, thereby realizing the identification of the modification site or interaction site.

2. The method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template conversion technology according to claim 1, wherein, The chemical modification is N 6 -methyladenosine m 6 A, N 6 -methyldeoxyadenosine 6mA, pseudouridine Ψ, N 6 -propargyladenosine p 6 A or N 1 -methyladenosine m 1 Any one of A, and the interaction is any one of RNA-RNA intramolecular interaction, RNA-RNA intermolecular direct interaction, RNA-RNA intermolecular 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 conversion technology according to claim 1, characterized in that, The method for grafting an RNA branch chain with a known sequence is a click chemical reaction, a biotin-streptavidin bioorthogonal reaction, ultraviolet cross-linking, chemical cross-linking agent cross-linking, or antibody cross-linking method.

4. The method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template conversion technology according to claim 1, wherein, The reverse transcriptase used in the reverse transcription process is Maxima HMinus reverse transcriptase.

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

6. The method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template conversion technology according to claim 2, wherein, For m 6 Modified by A or 6 mA, graft an RNA branch chain with a known sequence at the modification site, specifically including the following steps: (1) Treat the RNA sample containing m 6 A modification or the DNA sample containing 6mA modification with the demethylase fat mass and obesity-associated protein FTO, and add dithiothreitol after 5 - 30 minutes to modify m 6 A or 6mA with a thiol modification; (2) Add dibenzocyclooctyl-maleimide and react with the thiol group on the RNA or DNA to obtain RNA or DNA labeled with a carbon-carbon triple bond; (3) Add an RNA branch chain modified with an azide group at the 5' end and react with the RNA or DNA labeled with a carbon-carbon triple bond to obtain RNA or DNA grafted with an RNA branch chain with a known sequence.

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

8. The method for detecting nucleic acid chemical modification sites and interaction sites based on nucleic acid grafting and nucleic acid reverse transcription template conversion technology according to claim 2, characterized in that, For pseudouridine Ψ modification, grafting an RNA branch chain with a known sequence at the modification site specifically includes the following steps: (1) Mix the RNA sample containing Ψ modification evenly with 1-cyclohexyl-2-(morpholinoethyl)carbodiimide propyl p-toluenesulfonate and react at 37 °C for 20 minutes; (2) Add the RNA obtained in (1) to a sodium carbonate-sodium bicarbonate buffer solution with a pH between 10 and 11 and react at 37 °C for more than 6 hours to obtain RNA specifically labeled with Ψ modification by propargyl group; (3) Add an RNA branch chain modified with an azide group at the 5' end and react with the RNA labeled with a propargyl group to obtain 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 conversion technology according to claim 8, characterized in that, In step (1), the preparation method of 1-cyclohexyl-2-(morpholinoethyl)carbodiimide propargyl p-toluenesulfonate is as follows: Step S1: Add N-(2-aminoethyl)morpholine to dichloromethane, and then dropwise add cyclohexyl isocyanate in an equimolar amount to N-(2-aminoethyl)morpholine. React at room temperature for 3 to 5 hours. Remove the solvent by a rotary evaporator and slurry with petroleum ether to obtain intermediate 1. The chemical structural formula of intermediate 1 is: Step S2: Dissolve triphenylphosphine in dichloromethane under an ice-water bath condition. Then, dropwise add bromine, triethylamine, and intermediate 1 in sequence. React at 10 - 15 °C for 3 - 5 hours. The molar ratio of bromine, triethylamine, intermediate 1 to triphenylphosphine is 1:1:3:0.

83. After the reaction, wash the reaction system with water, dry it, and perform vacuum distillation to obtain intermediate 2. The chemical structural formula of intermediate 2 is: Step S3: Using acetonitrile as a solvent, dropwise add intermediate 2 and propargyl p-toluenesulfonate in an equimolar amount to it. React at 60 - 70 °C for 2 - 5 hours. After the reaction, concentrate the reaction solution by a rotary evaporator, and separate the product using a high-performance liquid chromatograph to obtain the 1-cyclohexyl-2-(morpholinoethyl)carbodiimide propargyl p-toluenesulfonate. Its chemical structural formula 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 conversion 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 as follows: Use the method of antibody cross-linking to add an RNA branch with a known sequence. Specifically, combine a protein linked with an RNA branch with a known sequence with the antibody of the protein.

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

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