A method of analyzing an RNA-binding protein substrate

Abstract

The application belongs to the technical field of biology, and particularly relates to a method for analyzing RNA binding protein substrates, a method for capturing RNA interacting with a target RNA binding protein (RBP), and a method and library for analyzing RNA or RBP. The application no longer uses traditional ultraviolet light crosslinking, but only uses a CLICK reaction for chemical labeling and crosslinking, effectively eliminates structural bias, reduces background binding, and has higher specificity.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a method for capturing RNA that interacts with a target RNA-binding protein (RBP), and a method for analyzing RNA or RBP. Background Technology

[0002] RNA-binding proteins (RBPs) play a vital role in almost every aspect of cell biology. The binding of RBPs to specific targets affects the expression of functionally coordinated mRNAs and involves their combination and dynamic interactions with other RBPs.

[0003] RBPs coordinate the most fundamental cellular processes. Some RBP-RNA interactions form stable ribonucleoprotein (RNP) particles with specific functions, while others (and RBPs and RNPs) interact transiently to process, regulate, and control the fate of almost all RNAs in the cell. Importantly, RBPs play a crucial role in the posttranscriptional regulation of mRNA and non-coding RNA, including RNA splicing, transport, modification, stability, and translation. RBPs interact directly with RNA through sequence-specific, structure-specific, and non-specific binding mechanisms. RBPs extensively utilize these precise mechanisms to spatiotemporally and specifically regulate mRNA expression during development and homeostasis. Regulation of RBP activity, often through increased post-translational modifications, can influence gene expression by altering mRNA translation and stability, thereby fine-tuning mRNA targets to respond appropriately to external or internal stimuli. The importance of RBP gene regulation in homeostasis and disease is increasingly recognized.

[0004] In recent years, significant progress has been made in understanding how RBPs exert regulatory effects within the transcriptome. Researchers have developed various techniques for high-throughput sequencing of RBP-bound RNA sequences to obtain important information about RBP function and regulatory mechanisms, achieving numerous important breakthroughs. The traditional RIP-seq (RNA Immunoprecipitation Sequencing) technique, which uses RBP-specific antibodies to enrich RBP-bound RNA fragments, is one of the basic methods for studying RBP regulation. However, this method has inherent drawbacks: (1) the RNA enrichment efficiency is low and affected by antibody specificity; (2) it can only target a single RBP protein and cannot screen and analyze the entire protein-protein interaction genome; (3) the captured RNA fragments are long, making it difficult to accurately determine the conserved RBP binding motifs.

[0005] Building upon RIP-seq, a series of novel sequencing methods utilizing ultraviolet light irradiation to crosslink RBPs and binding RNA, thereby enhancing enrichment efficiency, known as CLIP-seq (Cross-Linking and Immuno-Precipitation High Throughput Sequencing), have also developed rapidly, with numerous improvements emerging. These techniques all utilize short-wavelength ultraviolet light to catalyze covalent crosslinking between pyrimidine bases in RNA and adjacent amino acid residues in RBPs, significantly stabilizing the structure of the RBP-RNA complex and enabling more efficient enrichment and more accurate motif inference. However, these methods still rely on specific antibodies and bear the systematic errors introduced by differences in antibody efficacy, and cannot perform comprehensive interactome analysis of all interactions within the entire cell. Moreover, ultraviolet crosslinking has its own unique drawbacks; nucleotides and amino acids each have crosslinking preferences based on their molecular structures, and ultraviolet irradiation has low crosslinking efficiency for double-stranded RNA. These limitations restrict the further application of CLIP-series technologies.

[0006] Therefore, in order to solve the above problems, a new RBP analysis method needs to be developed. Summary of the Invention

[0007] To address the aforementioned issues, the inventors of this application, through extensive experimentation and repeated exploration, have developed a method that does not rely on traditional ultraviolet crosslinking or antibody enrichment.

[0008] Therefore, in a first aspect, this application provides a method for capturing RNA interacting with a target RBP, the method comprising:

[0009] Step (I): Provide a cell sample and transfer a plasmid containing the target RBP into the cells;

[0010] Step (II): Provide a first reagent having an alkyne group to the cell so that the first reagent having an alkyne group forms a first complex with the RNA in the cell;

[0011] Step (III): Provide a second reagent having an azide (-N3, azido) group and contact it with the cells so that the second reagent having the azide group forms a second complex with the target RBP in the cells;

[0012] In step (IV), the first and second complexes within the cell are contacted under conditions that allow for copper-catalyzed cycloaddition of alkynes and azide (CUAAC) to form a third complex.

[0013] Step (V) achieves the capture of RNA interacting with the target RBP by capturing the third complex.

[0014] In some embodiments, in step (II), the first reagent having an alkyne group is incorporated into the intracellular RNA transcript by an RNA polymerase in the cell to form a first complex.

[0015] In some embodiments, in step (III), the second reagent having an azide group binds to the target RBP to form a second complex.

[0016] In some embodiments, total protein in live cells is labeled using the strong binding of the succinimide ester group of NHS-azide (azido-succinimide ester) to the amino group on the side chain of RBP amino acid residues. In some embodiments, after the RBP is labeled with the succinimide ester group of NHS-azide, the RBP is contaminated with an azide group (see reaction labeling route). Figure 2 ).

[0017] In some implementations, methionine analogs are incorporated into newly synthesized peptide chains during protein translation to label newly synthesized proteins in living cells (metabolic labeling routes are described in [link]). Figure 3 In some embodiments, the methionine analogue is L-azidohomoalanine (L-AHA).

[0018] In some embodiments, in step (III), N-ethylmaleimide (NEM, C6H7NO2) is used to bind free thiol groups in living cells to improve the efficiency of the CuAAC reaction in living cells.

[0019] In some embodiments, in step (IV), the RBP with an azide group and the RNA with an alkyne group undergo a copper-catalyzed cycloaddition reaction (CUAAC) under copper ion catalysis, generating a third complex.

[0020] In some implementations, the RNA-binding protein is chemically cross-linked with its recognized RNA motif by utilizing the CuAAC reaction between the azide group and the alkynyl group (under the catalysis of copper ions, the azide group and the alkynyl group undergo an addition reaction to generate a stable 1,2,3-triazole five-membered ring, thereby completing the cross-linking of the biomolecules to which they are attached).

[0021] It is understood that, in this document, the first reagent having an alkyne group is not limited to 5-alkynyluridine specifically used in the examples. As long as the first reagent is a ribonucleotide analog with a terminal alkyne group substituted and a relative molecular mass between 200 and 400 Da (e.g., 250 Da to 300 Da, 200 Da to 250 Da), such that the first reagent can be absorbed by cells and utilized by RNA polymerase II, it can be incorporated into the RNA in the cell to label the RNA.

[0022] Similarly, in this article, the second reagent with an azide group is not limited to NHS-azide or L-AHA specifically used in the examples. As long as the second reagent has an azide group and a relative molecular mass of less than 300 Da (e.g., 250 Da to 300 Da, 200 Da to 250 Da, 100 Da to 200 Da), the second reagent can be rapidly penetrated into the cell by active transport, free diffusion, or through small defects in the cell membrane surface. It can also be used to efficiently covalently bind to specific groups on the protein surface through groups such as NHS (reaction labeling route) or integrate into the nascent peptide chain through translation (metabolic labeling route), thus giving RBP an azide group.

[0023] Furthermore, under the catalysis of copper ions, the alkynyl group and the azide group undergo the CuAAC reaction to generate a stable 1,2,3-triazole five-membered ring, thereby completing the cross-linking of the RNA and RBP they are connected to, and thus generating a third complex, namely the RNA-protein complex.

[0024] In some embodiments, the plasmid also contains a first ligand (e.g., biotin).

[0025] In some embodiments, in step (V), a second ligand (e.g., streptavidin) is provided to capture the third complex through the binding of the first ligand to the second ligand, thereby achieving the capture of RNA interacting with the target RBP.

[0026] In some embodiments, in step (V), the third complex (RNA-protein complex) is enriched using streptavidin-magnetic beads.

[0027] In some implementations, the method includes:

[0028] Step (i): Capture the third complex by means of the method described above;

[0029] Step (ii): Sequencing the RNA in the third complex to obtain RNA information, thereby obtaining information about the target RBP;

[0030] Optionally, prior to step (ii), the RNA in the third complex is processed (e.g., an RNA library is constructed) to sequence the RNA in step (ii).

[0031] In some embodiments, the first reagent comprises 5-ethynyluridine.

[0032] In some embodiments, the second reagent comprises NHS-azide and / or L-AHA. Optionally, the second reagent further comprises N-ethylmaleimide and / or maleimide polyethylene glycol (Maleimide-PEG).

[0033] According to Siheng Li et al., Chem. Sci., 2017, the main inhibitory factor for the CuAAC reaction in living cells is the metal ion-affinity protein in the cytoplasm, primarily reduced glutathione (GSH). GSH binds to Cu(I) in the CuAAC reaction catalyst with its free thiol groups, thus competing with the THPTA ligand and severely reducing catalytic activity. To block the free thiol groups in the cytoplasm, cells are pretreated with a reagent containing a maleimide group (e.g., N-ethylmaleimide) before the cross-linking reaction. The maleimide group can react rapidly and specifically with the thiol groups, rendering them unable to bind Cu(I). This step can greatly improve the efficiency of the in vivo CuAAC reaction, increasing the yield of the cross-linking product by an average of 8-20 times. This reagent can also be replaced with other reagents containing N-substituted maleimide groups and with chemically inert molecular weights of the substituents (allowing them to permeate through the cell membrane via free diffusion), such as maleimide-PEG.

[0034] In some embodiments, in step (IV), a reagent containing copper ions is provided; optionally, in step (IV), a reagent to accelerate the reaction, a reducing agent, or any combination thereof is also provided.

[0035] In some embodiments, the cells are treated with Trypsin-EDTA prior to step (III).

[0036] In some embodiments, the second reagent comprises NHS-azide and N-ethylmaleimide.

[0037] In some embodiments, the second reagent comprises L-AHA and N-ethylmaleimide.

[0038] In some embodiments, in step (IV), a reagent containing copper ions, a reaction-accelerating reagent, and a reducing agent are provided.

[0039] In some embodiments, a reagent containing copper ions and a reagent that accelerates the reaction are first brought into contact with the cells, followed by a reducing agent being brought into contact with the cells.

[0040] In some embodiments, the accelerating reaction reagent is selected from BTTAA (2-(4-((bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)acetic acid), MBHTM ((1-(4-methoxybenzyl)-1-H-1,2,3-triazol-4-yl)methanol), THPTA (Tris(3-hydroxypropyltriazolylmethyl)amine), aminoguanidine, or any combination thereof.

[0041] In some embodiments, the reagents for accelerating the reaction are THPTA and aminoguanidine.

[0042] In some embodiments, the copper-containing reagent is selected from copper sulfate, copper tetra(acetonitrile)hexafluorophosphonate, copper acetate, and copper bromide.

[0043] In some embodiments, the reagent containing copper ions is copper sulfate.

[0044] In some embodiments, the reducing agent is selected from sodium ascorbate, TCEP (Tris(2-carboxyethyl)phosphine), DTT (dithiothreitol), β-mercaptoethanol, or any combination thereof.

[0045] In some embodiments, the reducing agent is sodium ascorbate.

[0046] In some embodiments, the method includes step (a): providing a cell sample and transferring a plasmid containing the target RBP into the cells, the plasmid also containing a tag capable of binding to streptavidin.

[0047] In some embodiments, the method includes step (b): providing 5-alkynyluridine and contacting cells with 50 μM-1000 μM (e.g., 50 μM-200 μM, 200 μM-500 μM, 500 μM-800 μM, 800 μM-1000 μM) of 5-alkynyluridine for 5 minutes to 16 hours (e.g., 5 minutes to 1 hour, 1 to 4 hours, 4 to 8 hours, 8 to 12 hours, 12 to 16 hours) such that the 5-alkynyluridine forms a first complex with RNA in the cells.

[0048] In some embodiments, in step (b), 200 μM of 5-alkynyluridine is contacted with the cells for 12 hours.

[0049] In some embodiments, the method includes step (c): treating the cells obtained in step (a) with Trypsin-EDTA.

[0050] In some embodiments, the method includes step (d): providing 100 μM-2000 μM (e.g., 100 μM-500 μM, 500 μM-1000 μM, 1000 μM-1500 μM, 1500 μM-2000 μM) NHS-azide and N-ethylmaleimide to the cells, such that the NHS-azide forms a second complex with proteins within the cells.

[0051] In some embodiments, in step (d), 500 μM NHS-azide is provided to contact the cells.

[0052] In some embodiments, the method includes step (e): providing copper sulfate, THTTA, aminoguanidine, and sodium ascorbate to cells for 10 to 30 minutes (e.g., 10 minutes, 20 minutes, 30 minutes) to allow the first and second complexes to contact and form a third complex.

[0053] In some embodiments, in step (e), copper sulfate, THPTA, aminoguanidine, and sodium ascorbate are provided to contact the cells for 20 minutes.

[0054] In some embodiments, the method includes step (f): lysing the cells with a cell lysis buffer.

[0055] In some embodiments, the method includes step (g): capturing RNA in the third complex using streptavidin magnetic beads.

[0056] In some embodiments, the method includes step (h): treating the RNA in the third complex with RNase for 20-60 minutes (e.g., 20-30 minutes, 30-40 minutes, 40-50 minutes, 50-60 minutes) to produce an RNA fragment.

[0057] In some implementations, in step (h), the RNA in the third complex is treated with RNase I for 30 minutes.

[0058] In some embodiments, the method includes step (i): phosphorylating the 5' end of the RNA fragment via the T4 PNK enzyme.

[0059] In some embodiments, the method includes step (j): digesting impurities in the RNA fragment using proteinase K.

[0060] In some implementations, the method includes step (k): constructing an RNA library using the RNA fragment.

[0061] In some implementations, the method includes step (l): sequencing the RNA library to obtain intracellular RNA information, thereby obtaining information about the target RBP.

[0062] In some embodiments, the plasmid contains one or more target RBPs derived from the cell. In some embodiments, the RBP is the PTBP1 protein. In some embodiments, the RBP has a sequence as described in SEQ ID NO:1 or 2.

[0063] In some embodiments, the cell is a single cell or a group of cells. In some embodiments, the cell is a single cell.

[0064] In some embodiments, the cell is a mammalian cell (e.g., human, mouse, rat).

[0065] In some implementations, the cells are early embryonic cells.

[0066] On the other hand, this application provides a library containing the sequence of RNA captured by the methods described above.

[0067] On the other hand, this application provides a kit for capturing or analyzing RNA interacting with a target RBP, the kit comprising:

[0068] (a) A vector containing a nucleic acid molecule encoding a target RBP; in some embodiments, the vector further contains a first ligand (e.g., biotin);

[0069] (b) A first reagent having an alkynyl group;

[0070] (c) a second reagent or a combination of second reagents having an azido (-N3, azido) group; and,

[0071] (d) Reagents containing copper ions.

[0072] In some embodiments, the RBP is the PTBP1 protein. In some embodiments, the RBP has the sequence as described in SEQ ID NO:1 or 2.

[0073] In some embodiments, the kit also includes reagents for constructing RNA libraries.

[0074] In some implementations, the kit also includes reagents for sequencing.

[0075] In some embodiments, the kit also includes reagents for processing cell samples (e.g., Trypsin-EDTA).

[0076] In some embodiments, the kit also contains N-ethylmaleimide.

[0077] In some embodiments, the kit also contains maleimide-PEG.

[0078] In some embodiments, the kit also contains reagents that accelerate the reaction (e.g., BTTAA, MBHTM, Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), aminoguanidine).

[0079] In some embodiments, the kit also contains a reducing agent (e.g., sodium ascorbate, tris(2-carboxyethyl)phosphine, dithiothreitol, β-mercaptoethanol).

[0080] In some embodiments, the kit also includes magnetic beads with a second ligand (e.g., streptavidin).

[0081] In some embodiments, the second reagent is a combination of NHS-azide and N-ethylmaleimide.

[0082] In some embodiments, the combination of the second reagent is L-AHA and N-ethylmaleimide.

[0083] In some embodiments, the first reagent is 5-ethynyluridine.

[0084] In some implementations, the vector is an expression vector.

[0085] In some embodiments, the copper-containing reagent is selected from copper sulfate, copper tetra(acetonitrile)hexafluorophosphonate, copper acetate, and copper bromide.

[0086] Terminology Definition

[0087] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0088] As used herein, the term "first reagent having an alkynyl group" refers to a compound or combination of compounds containing at least one carbon-carbon triple bond (-C≡C-). A compound containing at least one carbon-carbon triple bond (-C≡C-) can be, for example, acetylene (containing at least two carbons), propyne (containing at least three carbons), butyne (containing at least four carbons), pentyne (containing at least five carbons), hexyne (containing at least six carbons), heptyne (containing at least seven carbons), octyne (containing at least eight carbons), nonyne (containing at least nine carbons), or decyne (containing at least ten carbons). In some embodiments, compounds having an alkynyl group are used to label RNA within cells.

[0089] As used herein, the term "acetylene-azide cycloaddition reaction (CUAAC)" refers to a chemical reaction in which a molecule having an azide group combines with a molecule having an acetylene group to form a cyclic adduct, and is a type of "click chemistry (or CLICK reaction)." In this application, the acetylene-azide cycloaddition reaction catalyzes the conjugation of acetylene-labeled RNA and its interacting azide-labeled RBP to form a complex. In this application, the acetylene-azide cycloaddition reaction may be selected from any of the following: copper-catalyzed azide-acetylene cycloaddition (CuAAc), strain-promoted azide-acetylene cycloaddition (SPAAC), ruthenium-catalyzed azide-acetylene cycloaddition (RuAAc), and silver-catalyzed azide-acetylene cycloaddition (AgAAc). As used herein, the term "cyclic adduct" refers to the structure formed by the acetylene-azide cycloaddition. In some embodiments, a cyclic adduct may be formed between a first complex and a second complex.

[0090] In some embodiments, the alkyne-azide cycloaddition reaction is a copper-catalyzed alkyne-azide cycloaddition reaction (CuAAc). In some embodiments, conditions allowing the copper-catalyzed alkyne-azide cycloaddition reaction (CUAAC) include a reagent containing copper ions, a reaction-accelerating reagent, and a reducing agent. In some embodiments, the copper-containing reagent catalyzes the activation of alkyne-labeled RNA (i.e., the first complex) and its binding to an azide-labeled RBP (i.e., the second complex). In some embodiments, the reducing agent catalyzes the reduction of Cu(II) to Cu(I). In some embodiments, the reaction-accelerating reagent increases the reaction rate.

[0091] As used in this article, the term "RNA binding protein (RBP)" refers to an important class of proteins in cells that interact with RNA by recognizing specific RNA-binding domains and are widely involved in multiple post-transcriptional regulatory processes such as RNA splicing, transport, sequence editing, intracellular localization, and translation control.

[0092] Beneficial effects of the invention

[0093] Compared with the prior art, this application provides a method for capturing RBP-RNA complexes and a method for analyzing RBP or RNA. The method provided in this application does not rely on traditional methods such as ultraviolet crosslinking or antibody enrichment, which have drawbacks.

[0094] The method provided in this application has the following beneficial effects:

[0095] (1) Revolutionary breakthrough in RNA-protein crosslinking technology: Instead of using traditional ultraviolet light crosslinking, the CLICK reaction is used for chemical labeling and crosslinking, which effectively eliminates structure bias, reduces background binding, and has higher specificity;

[0096] (2) Reduce false positives: Biotin-streptavidin purification can efficiently enrich the whole interactome with its superior affinity and specificity compared to antibody immunoprecipitation enrichment, while obtaining binding RNA sequence information and RBP identification information.

[0097] (3) High flexibility: It can be used to study the binding sequence of specific target RBPs. By constructing a plasmid expression vector that can express the fusion protein of streptavidin-binding peptide (SBP) and expressing it exogenously in cells, this method can also obtain the binding sequence information of a single RBP protein.

[0098] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0099] Figure 1 A schematic diagram of the pCMV-flag-SBP-PTBP1 plasmid structure is shown.

[0100] Figure 2 The reaction labeling roadmap shows the formation process of the second complex and its combination with the first complex to form the third complex.

[0101] Figure 3 The metabolic marker roadmap shows the formation process of the second complex and its combination with the first complex to form the third complex.

[0102] Figure 4 The results of the sequencing data analysis are shown, in which, Figure 4 A represents the distribution of RNA species corresponding to the target sequences captured by the reporter RNA binding protein PTBP1 using the RCLICK crosslinking method. Figure 4 B and Figure 4 C shows the location distribution characteristics of the PTBP1 target sequence in the mRNA. Figure 4 D shows the conserved motif for PTBP1 binding obtained by RCLICK-Seq detection. Figure 4 E represents the result of the PTBP1 binding peak obtained from RCLICK-Seq data displayed in IGV.

[0103] Figure 5 The results of the comparison of crosslinking efficiency and specificity are shown, among which, Figure 5 A represents the result of the RCLICK crosslinking reaction. Figure 5 B represents the result of ultraviolet crosslinking.

[0104] Figure 6 The results of the implementation of crosslinking reagent concentration and crosslinking time are shown, in which, Figure 6 A represents the optimization of the cross-linking reaction time. Figure 6 B represents the optimization of the crosslinking reagent labeling time. Figure 6 C represents the optimization of the cross-linking reagent concentration.

[0105] Sequence information

[0106] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0107] Table 1: Sequence Description

[0108] Detailed Implementation

[0109] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0110] Unless otherwise specified, the experiments and methods described in the examples are performed in accordance with conventional methods well known in the art and described in various references. For example, conventional techniques such as biochemistry, chemistry, molecular biology, genomics and recombinant DNA used in this invention can be found in Sambrook, Fritsch and Maniatis, *Molecular Cloning: A Laboursory Manual*, 2nd edition (1989); *Current Protocols in Molecular Biology* (edited by F.M. Ausubel et al., (1987)).

[0111] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0112] Example 1. Preparation of key reagents

[0113] 1. Prepare 250mM NHS-Azide (1000×)

[0114] Weigh 0.0099g of NHS-Azide (Thermo, 88902) powder and dissolve it in 200μL of DMSO. Store in the dark at -20℃.

[0115] 2. Prepare 500mM CuSO4

[0116] Weigh 1.2484 g of CuSO4·5H2O (Aladdin, C112409) crystals and dissolve them in 10 mL of Nuclease-free ddH2O. Store at 4 °C.

[0117] 3. Prepare 500mM Tris(3-hydroxypropyltriazolylmethyl)amine(THPTA)

[0118] Weigh 2.1725g of THPTA (Lumiprobe, H4050) powder and dissolve it in 10mL of Nuclease-free ddH2O. Aliquot the solution and store at -20℃.

[0119] 4. Preparation of 1M N-ethylmaleimide

[0120] Weigh 1.2512g of N-ethylmaleimide (Thermo, 23030) powder and dissolve it in 10mL of DMSO. Aliquot the solution and store at -20℃.

[0121] 5. Prepare 500mM Sodium L-ascorbate

[0122] Weigh 0.9906g of Sodium L-ascorbate (Sigma, 11140) crystals and dissolve them in 10mL of Nuclease-free ddH2O. Store at 4℃.

[0123] 6. Prepare 200mM 5-ethynyl uridine (5-EU)

[0124] Weigh 0.0107 g of 5-ethynyl uridine (Abcam, ab146642) powder and dissolve it in 200 μL of DMSO. Store in the dark at -20°C.

[0125] Example 2. Analysis of RNA-binding protein substrates

[0126] This embodiment employs a reactive labeling route, which involves labeling RBP with the succinimide ester group of NHS-azide, thereby giving RBP an azide group (see Appendix for the detailed flow of the reactive labeling route). Figure 2 ).

[0127] 1. Prepare cells and label RNA with 5-ethynyl uridine.

[0128] Approximately 2×10 6 One suspension of HEK293T cells was added to a 10cm disposable culture dish, and 6mL of DMEM (GIBCO, 11995500) medium containing 10% fetal bovine serum (FBS, ExCell, FSS500) was added. The cells were incubated at 37°C for approximately 24 hours in a 5% CO2 incubator. The cells were then transfected with a reporter gene expression plasmid vector carrying the SBP tag, which expresses PTBP1 protein and streptavidin-binding peptide (SBP). The vector map is shown below. Figure 1As shown. After the cells have firmly adhered to the culture medium and grown stably, add 6 μL of 200 mM 5-ethynyluridine (5-ethynyluridine, 5-EU, C11H12N2O6) to each tray according to the experimental requirements for labeling time. The working concentration is 0.2 mM (reaction labeling route). After gently shaking to mix, continue to incubate at 37°C in a 5% CO2 incubator for the specified time. The specific labeling time depends on the experimental requirements and the type of RNA molecule to be labeled. At this point, the first complex is formed.

[0129] 2 Collect cells and label RBP

[0130] Discard the culture medium, wash adherent cells twice with 3 mL of 1×PBS, add 1 mL of 0.25% Trypsin-EDTA (GIBCO, 25200072), digest at 37°C for 3 minutes, add 4 mL of DMEM medium containing 10% FBS to stop digestion, gently resuspend the cells by pipetting, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 200×g at 37°C for 3 minutes, discard the supernatant, wash the cell pellet once with 2 mL of 1×PBS, resuspend the cells in 400 μL of 1×PBS, add 50 μL of 1M N-ethylmaleimide (NEM, C6H7NO2) and 50 μL of 250 mM NHS-azide (C6H6N4O4), gently mix, incubate at 37°C for 15 minutes, gently invert the centrifuge tube several times every 3-5 minutes to keep the cells suspended, then centrifuge at 200×g. Centrifuge at 37°C for 3 minutes, discard the supernatant, wash the cell pellet twice with 2 mL of 1×PBS, gently pipetting to resuspend each time, centrifuge at 37°C for 3 minutes at 200×g, discard the supernatant, and the second complex is formed.

[0131] 3. CLICK crosslinking reaction (acetylene and azide cycloaddition reaction CUAAC)

[0132] Prepare CLICK solution: Take 0.5 mL of 1×PBS, add 1 μL of 500 mM CuSO4 and 2 μL of 500 mM MHPTA in sequence, gently shake to mix after each addition, and finally add 20 μL of 500 mM Sodium L-ascorbate and mix well.

[0133] After cell washing, resuspend the cells in CLICK solution and incubate at room temperature for 20 minutes, gently inverting the centrifuge tube every 3-5 minutes to keep the cells suspended. This forms the third complex. For details on the formation of the first to third complexes in steps 1-3, please refer to [link to documentation]. Figure 2 .

[0134] 4 Lyse the cells and centrifuge to obtain the supernatant.

[0135] Aspirate the cell suspension into a 1.5 mL Eppendorf tube, centrifuge at 200×g for 3 minutes at room temperature, discard the supernatant, wash the cell pellet twice with 1 mL 1×PBS + 5 mM EDTA to terminate the CuAAC reaction, gently resuspending the cells by pipetting after each wash, centrifuge at 200×g at 37°C for 3 minutes, and discard the supernatant. Prepare lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.5% Triton X-100, 200 U / ml RNase inhibitor, 25 U / ml DNase). Add 500 μL of pre-chilled lysis buffer to the cell pellet, mix by pipetting, and incubate on ice for 10 minutes. Use an ultrasonic disruptor at 15 W power, 10 / 15 sec working / interval time, to disrupt the cell pellet for 3 minutes to obtain the cell lysate. Subsequently, the lysis buffer was centrifuged at 13,000 rpm at 4°C for 15 minutes, and the supernatant was transferred to a new 1.5 mL Eppendorf tube as the cell lysis supernatant. 20 μL of this supernatant was used as the protein sample input, and 40 μL as the RNA sample input.

[0136] 5. preparation of streptavidin magnetic beads

[0137] Take 2 μL of Dynabead MyOne magnetic beads (Invitrogen: 65002) into a 1.5 mL low-adsorption centrifuge tube. Wash the magnetic beads with 50 μL of BW buffer (5 mM Tris-HCl (pH 7.5), 500 μM EDTA, 1 M NaCl, 0.05% Triton X-100), gently tap the centrifuge tube to mix, let stand for 2 minutes, place the centrifuge tube on a magnetic rack, and let stand for 5 minutes until the solution becomes clear. Aspirate the supernatant to complete one wash. Repeat the wash 3 times.

[0138] The magnetic beads were cleaned once with 50 μL of Bead-solution buffer A (50 mM NaCl, 100 mM NaOH, 0.1% Triton X-100), and then cleaned once with 50 μL of Bead-solution buffer B (100 mM NaCl, 0.1% Triton X-100).

[0139] The magnetic beads were resuspended in 50 μL of blocking buffer (1% BSA, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.5% Triton X-100) and sealed on a rotating rack at 4°C for 1 hour.

[0140] 6. Magnetic beads capture RBP-RNA complex

[0141] After sealing, the magnetic beads were resuspended in 200 μL of blocking buffer, mixed, and added to the fragmented cell lysis buffer. The centrifuge tubes were then placed on a rotating rack at 4°C and incubated for 2 hours or overnight.

[0142] Remove the centrifuge tubes from the rotating rack, centrifuge briefly, and then place them on a magnetic rack to stand for 5 minutes until the solution becomes clear. Discard the supernatant and wash the magnetic beads twice with 500 μL of high-salt washing buffer (1M NaCl, 10mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 200 U / ml RNase inhibitor). Then wash the magnetic beads twice again with 500 μL of washing buffer (150mM NaCl, 10mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 200 U / ml RNase inhibitor).

[0143] 7. RNA fragmentation

[0144] Resuspend the magnetic beads in 500 μL MNase buffer (5 mM CaCl2, 10 mM Tris-HCl (pH 7.5)). Add 5 μL of Micrococcal Nuclease (NEB: MO247S) diluted 1:100 with MNase buffer to a final concentration of approximately 1:50,000. Place the solution in a 37°C metal bath and shake at 1000 rpm for 30 minutes. Add 0.2 μL of SuperRNase Inhibitor (Ambion, AM2694). Cool on ice for 3 minutes. Place the centrifuge tube on a magnetic rack and let stand for 5 minutes until the solution becomes clear. Aspirate the supernatant. Wash the magnetic beads twice with 500 μL washing buffer + EDTA (150 mM NaCl, 10 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 200 U / ml RNase inhibitor, 5 mM EDTA). Then wash with 500 μL PNK. The magnetic beads were washed twice with buffer (10mM MgCl2, 50mM Tris-HCl (pH 7.5), 2mM DTT, 0.5% Triton X-100, 200U / ml RNase inhibitor).

[0145] 8. T4 Polynucleotide kinase terminal phosphorylation and labeling

[0146] Prepare a T4 PNK reaction system: 100 μL of each sample (10 μL 10×T4 PNK buffer (NEB), 3 μL T4 polynucleotide kinase (NEB, MO201S), 200 U / ml RNase inhibitor), resuspend the magnetic beads, place in a 37°C metal bath and shake at 1000 rpm for 30 minutes, then place on ice for 3 minutes to cool. Place the centrifuge tube on a magnetic rack and let stand for 5 minutes until the solution becomes clear. Aspirate the supernatant. Wash the magnetic beads twice with 500 μL PNK buffer (10 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.5% Triton X-100, 200 U / ml RNase inhibitor). For the last wash, aspirate 50 μL of the resuspended beads and aliquot into new 1.5 ml Eppendorf tubes. Use ThermoPierce... TM The RNA 3' end biotinylation kit (Thermo, 20160) biotin-labels the 3' end of RNA in RBP-RNA complexes bound to magnetic beads for detection.

[0147] 9. Proteinase K digestion and RNA extraction

[0148] Prepare 200 μL of PK buffer (100 mM Tris-Cl (pH 7.5), 50 mM NaCl, 10 mM EDTA, 1% SDS, 190 μg / mL Pronase K). Resuspend the magnetic beads in 200 μL of PK buffer, mix thoroughly, and place on a metal bath at 55 °C and 1100 rpm for 1 hour for digestion.

[0149] Add 200 μL of RNA extraction buffer (phenol:chloroform:isoamyl alcohol = 25:24:1) to the digested centrifuge tube, vortex thoroughly, and place on ice for 15 minutes for extraction. Centrifuge the tube at 13300 rpm for 15 minutes at 4°C. After centrifugation, carefully remove the centrifuge tube and place it on a magnetic rack. Transfer the supernatant (approximately 200 μL) to a new 1.5 mL low-adsorption centrifuge tube and label it with Pulldown-RNA.

[0150] Add 1 μL of glycogen, 1 / 10 volume (20 μL) of 3M sodium acetate and 3 times the volume (600 μL) of anhydrous ethanol to the Pulldown-RNA tube, and precipitate at -80°C for 1 hour or overnight.

[0151] Remove the centrifuge tube after alcohol precipitation and centrifuge at 13300 rpm for 30 minutes at 4°C, discarding the supernatant. Add 1 mL of 75% ethanol to wash the precipitate, centrifuge at 13300 rpm for 10 minutes at 4°C, discarding the supernatant. Then centrifuge again at 13300 rpm for 2 minutes at 4°C to remove residual ethanol solution. Open the tube cap and place it in a clean bench to dry for about 5-10 minutes. Dissolve the RNA precipitate in 8 μL of water and store at -80°C.

[0152] 10. Library Construction

[0153] Pulldown RNA was processed according to the fragmented RNA, and a small RNA library was constructed according to the library construction instructions of the kit (Takara SMARTer StrandedsmRNA-Seq Kit). The library was then purified and fragments were screened using KAPA Pure DNA beads (Roche, KK8000), retaining fragments longer than 150 nt.

[0154] Example 3. Analysis of RNA-binding protein substrates

[0155] This embodiment employs a metabolic labeling route, utilizing the methionine analog L-azidohomoalanine (L-AHA) to incorporate into newly synthesized peptide chains during protein translation, thereby labeling newly synthesized proteins in living cells (see Appendix for the detailed flowchart of the metabolic labeling route). Figure 3 ).

[0156] The process and steps in this embodiment are the same as those in Embodiment 2, except that the reagents used in the following two steps are different:

[0157] 1. Prepare cells and label RNA with 5-ethynyl uridine.

[0158] As described in Example 2, after the cells have firmly adhered to the culture medium and grown stably, add 6 μL of 200 mM 5-ethynyl uridine and 12 μL of 200 mM L-AHA (L-AHA is a reagent used to label RBPs; it is added here because L-AHA is an amino acid analog that is utilized during protein translation and needs to be added at an earlier time so that the cells can absorb it and gradually add it to the peptide chain via ribosomes) to the culture medium according to the experimental requirements. After gently shaking to mix, continue to incubate at 37°C in a 5% CO2 incubator for the specified time. The specific labeling time depends on the experimental requirements and the type of RNA molecule to be labeled. This forms the first complex.

[0159] 2. Collect cells and label them with RBP.

[0160] As described in Example 2, the cell suspension was placed in a 15 mL centrifuge tube, and 50 μL of 1M N-ethylmaleimide (NEM, C6H7NO2) was added. After gentle mixing, the mixture was incubated at 37°C for 15 minutes. The centrifuge tube was gently inverted every 3-5 minutes to keep the cells suspended. The mixture was then centrifuged at 200×g at 37°C for 3 minutes. The supernatant was discarded, and the cell pellet was washed twice with 2 mL of 1×PBS. After each resuscitation by gentle pipetting, the pellet was centrifuged at 200×g at 37°C for 3 minutes. The supernatant was discarded, thus forming the second complex.

[0161] 3-10. Continue the experiment according to step 3-10 described in Example 2.

[0162] Example 4. Sequencing and Data Analysis

[0163] The successfully constructed library was sequenced using the HiSeq-PE150 sequencing platform for paired-end sequencing. The raw data obtained from sequencing was processed and analyzed using various bioinformatics software. First, the FASTX-Toolkit's fastx_clipper was used to remove sequencing adapter sequences from the raw sequencing data. fastq_quality_trimmer was used to filter reads based on base quality and length, removing reads shorter than 24 nt. Subsequently, after removing PCR repeats, poly A sequences, and barcode sequences from the reads, Trimmomatic software was used to remove shorter reads with a base quality below 20 and a length less than 35 nt, completing the initial quality control of the raw data reads. The preprocessed reads were then aligned to a reference genome sequence using bwa software, with the human hg19 version as the reference genome (parameters: -n 0.06 -q 20). After alignment, mutation peak calling was performed on all aligned specific reads using the tag2peak.pl algorithm in the CLIP Tool Kit (CTK). Subsequently, the CIMS and CITS models were used sequentially to determine the location of the RBP's clipping reaction on RNA by detecting sites of substitution and truncation during cDNA reverse transcription in library construction. When analyzing mutation sites using the CIMS and CITS models, default parameters were used, or parameters could be adjusted according to actual conditions (including FDR, tagNumber, mutationFreq, etc.). Extending the position on the genome by 10 nt to the left and right yielded the RBP target information. The bedtools tool was used to annotate the RBP target information, and the HOMER software was used for motif analysis of RBP-binding substrates to obtain characteristic information of the RBP-binding substrates.

[0164] The analysis results of the sequencing data are shown below. Figure 4 . Figure 4 A represents the distribution of RNA types corresponding to the target sequences of the reporter RNA-binding protein PTBP1 captured using the RCLICK crosslinking method described in this application. 293T cells were divided into two groups and cultured at 37℃ (Ctrl) and 43℃ (HS) for 30 minutes respectively before harvesting to investigate the effects of heat shock treatment on the target sequences, localization, and function of PTBP1. It was found that the target sequences corresponded mostly to mRNA, with a smaller portion being lincRNA and precursor transcripts, consistent with the currently known biological functions of PTBP1 (RNA processing and splicing regulation). Figure 4 B and Figure 4C shows the location distribution characteristics of the PTBP1 target sequence in mRNA. It can be observed that PTBP1 mainly binds to the intron region of mRNA, while in the exon region, it is mainly enriched in the 3'UTR, with a lower proportion binding to the CDS region and very little binding to the 5'UTR. Figure 4 D shows the PTBP1 binding motif on RNA obtained from RCLICK-Seq data. This result is completely consistent with previously reported PTBP1 binding motifs (UUUC). Furthermore, in the PTBP1 motif statistics, this UC-rich motif ranks 1st, and the P-value also demonstrates its high significance. Figure 4 E represents the target peak distribution of PTBP1 on the same chromosome obtained from different repeat data of RCLICK-Seq. The consistency of IGVtrack in different repeat data proves that the RCLICK-Seq technique of this application has good reproducibility.

[0165] Example 5. Comparison of efficiency and specificity between UV crosslinking and chemical crosslinking

[0166] To compare the RNA capture efficiency of the method in this application with that of existing technologies (UV crosslinking), and to compare the RNA capture efficiency of the known RNA-binding protein PTBP1 with that of the known non-RNA-binding protein EGFP (as a negative control), the following experiments were designed and conducted:

[0167] Cells from the same batch cultured in vitro were divided into three groups (i.e., experimental groups one, two, and three), and experiments were conducted according to the procedures described in steps 1-3 of Example 2; however, the treatment of each experimental group was slightly different, specifically in the following aspects:

[0168] (1) The first experimental group was irradiated with 365nm ultraviolet light after washing with PBS (i.e., using ultraviolet crosslinking commonly used in existing technology, without crosslinking through CLICK reaction), and was denoted as UV-CLIP treatment group (U).

[0169] (2) The second experimental group did not add 5-EU, but only added NHS-azide for chemical labeling, and then cross-linked by CLICK reaction, which was denoted as CLICK-non-cross-linked group (N);

[0170] (3) The third experimental group was labeled with 5-EU 16 hours before the start of the experimental treatment and chemically labeled with NHS-azide. Then, cross-linking was carried out using CLICK reaction, and it was designated as CLICK-cross-linking group (C).

[0171] Subsequently, the first, second, and third experimental groups were simultaneously subjected to immunoprecipitation under the same conditions to enrich specific RBP proteins and their bound RNA fragments. Differences in the amount of enriched RNA fragments were compared by fluorescent staining after electrophoresis, and library construction and sequencing were performed to compare the differences in conserved motifs and motif distribution of the enriched RNA fragments. Specifically, the experiments were first conducted according to steps 2-9 of Example 2. After the T4-PNK enzyme end treatment of the bound RNA fragments was completed, the RBP-RNA complex bound to the beads was biotin-labeled using the Thermo Nucleic Acid Detection Kit (Cat. No. 89880) to detect RNA abundance. After labeling, the beads were washed twice with PNK buffer, and 25 μL of 2×SDS loading buffer was added. The mixture was heated at 95°C for 5 minutes to obtain protein complex samples suitable for SDS-PAGE gel electrophoresis analysis. Two 8% acrylamide gels were prepared, with 5 μL of sample loaded per gel. The sample arrangement was as follows: Figure 5 Two gels were used for biotin exposure detection of RNA content and Western spectroscopy detection of protein levels, respectively. Electrophoresis was first performed at 90V for approximately 1.5 hours, followed by wet transfer at 75V for approximately 2 hours to transfer the electrophoretically separated RBP-RNA complexes from the two gels onto two PVDF membranes. One membrane was blocked, developed, washed, and exposed using the ThermoNucleic Acid Detection Kit (Cat. No. 89880) according to the standard protocol. The other membrane was blocked with TBST + 5% milk for 20 minutes, then incubated with Sigma Rabbit anti-Flag antibody (F7425) at a 1:1000 dilution at room temperature for 2 hours. It was washed three times with TBST buffer for 5 minutes each time, then incubated with Sigma Goat Anti-Rabbit IgG Antibody, Peroxidase Conjugated (AP132P) at a 1:5000 dilution at room temperature for 0.5 hours. After washing three times with TBST buffer, it was exposed with a suitable ECL chemiluminescence solution to obtain the Western chromatogram results.

[0172] The results of the crosslinking efficiency and specificity are shown in [the original text]. Figure 5 ,in, Figure 5 Lanes 1 and 2 of both A and B were EGFP negative controls, while lanes 3 and 4 were PTBP1 positive results. Figure 5 Lanes 1 and 3 in swim bladder A are the second experimental group, which are not cross-linked. Figure 5 Lanes 2 and 4 of swim bladder A are the third experimental group, with CLICK crosslinking. Figure 5Lanes B, 1 and 3, are the first experimental group, subjected to ultraviolet cross-linking; Figure 5 Lanes B, 2 and 4, are the third experimental group, with CLICK crosslinking. Figure 5 A study demonstrated that the RCLICK cross-linking reaction produces highly efficient and specific RBP-RNA cross-links. After cross-linking and immunoprecipitation enrichment, a small sample was used for biotin labeling of RNA to reveal the RNA signal. RCLICK cross-linking of the non-RNA-binding protein EGFP did not show any RNA binding signal, indicating the specificity of the cross-linking. However, RCLICK cross-linking of the RNA-binding protein PTBP1, compared to the non-cross-linked control group, showed a significant increase in the amount of bound RNA, demonstrating the effectiveness of the cross-linking. Figure 5 B controls RCLICK crosslinking and PAR-CLIP 365nm UV crosslinking demonstrate that although RCLICK crosslinking efficiency is slightly reduced, it also reduces non-specific crosslinking reactions. RCLICK crosslinking of the non-RNA-binding protein EGFP did not show any RNA binding signal, but UV crosslinking showed a weaker background signal.

[0173] The results of the cross-linking reagent concentration and cross-linking time are attached. Figure 6 , Figure 6 A represents the results of the cross-linking reaction time. The experimental results show that the efficiency is low in the 0-5 minute range, acceptable and similar in the 10-20 minute range, and good in the 20-40 minute range. However, excessive processing time may have strong cytotoxicity or even lead to cell death. Therefore, the cross-linking time should not exceed 20 minutes in practical applications. Figure 6 B represents the results of crosslinking reagent labeling time. The experimental results confirm that the labeling efficiency gradually increases from 0.5-1 hour, 1-2 hours, and 2-16 hours. Figure 6 C represents the results of the crosslinking reagent concentrations. The experimental results confirm that the 5-EU concentration of 0.1-0.2 mM and the L-AHA concentration of 0.2-0.4 mM are effective.

[0174] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof. SEQUENCE LISTING <110> Beijing Institute of Genomics, Chinese Academy of Sciences (National Center for Biotechnology Information) <120> A method for analyzing RNA-binding protein substrates <130> IDC220041 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 530 <212> PRT <213> artificial <220> <223> PTBP1 protein <400> 1 Met Asp Gly Ile Val Pro Asp Ile Ala Val Gly Thr Lys Arg Gly Ser 1 5 10 15 Asp Glu Leu Phe Ser Thr Cys Val Thr Asn Gly Pro Phe Ile Met Ser 20 25 30 Ser Asn Ser Ala Ser Ala Ala Asn Gly Asn Asp Ser Lys Lys Phe Lys 35 40 45 Gly Asp Ser Arg Ser Ala Gly Val Pro Ser Arg Val Ile His Ile Arg 50 55 60 Lys Leu Pro Ile Asp Val Thr Glu Gly Glu Val Ile Ser Leu Gly Leu 65 70 75 80 Pro Phe Gly Lys Val Thr Asn Leu Leu Met Leu Lys Gly Lys Asn Gln 85 90 95 Ala Phe Ile Glu Met Asn Thr Glu Glu Ala Ala Asn Thr Met Val Asn 100 105 110 Tyr Tyr Thr Ser Val Thr Pro Val Leu Arg Gly Gln Pro Ile Tyr Ile 115 120 125 Gln Phe Ser Asn His Lys Glu Leu Lys Thr Asp Ser Ser Pro Asn Gln 130 135 140 Ala Arg Ala Gln Ala Ala Leu Gln Ala Val Asn Ser Val Gln Ser Gly 145 150 155 160 Asn Leu Ala Leu Ala Ala Ser Ala Ala Ala Val Asp Ala Gly Met Ala 165 170 175 Met Ala Gly Gln Ser Pro Val Leu Arg Ile Ile Val Glu Asn Leu Phe 180 185 190 Tyr Pro Val Thr Leu Asp Val Leu His Gln Ile Phe Ser Lys Phe Gly 195 200 205 Thr Val Leu Lys Ile Ile Thr Phe Thr Lys Asn Asn Gln Phe Gln Ala 210 215 220 Leu Leu Gln Tyr Ala Asp Pro Val Ser Ala Gln His Ala Lys Leu Ser 225 230 235 240 Leu Asp Gly Gln Asn Ile Tyr Asn Ala Cys Cys Thr Leu Arg Ile Asp 245 250 255 Phe Ser Lys Leu Thr Ser Leu Asn Val Lys Tyr Asn Asn Asp Lys Ser 260 265 270 Arg Asp Tyr Thr Arg Pro Asp Leu Pro Ser Gly Asp Ser Gln Pro Ser 275 280 285 Leu Asp Gln Thr Met Ala Ala Ala Phe Gly Leu Ser Val Pro Asn Val 290 295 300 His Gly Ala Leu Ala Pro Leu Ala Ile Pro Ser Ala Ala Ala Ala Ala 305 310 315 320 Ala Ala Ala Gly Arg Ile Ala Ile Pro Gly Leu Ala Gly Ala Gly Asn 325 330 335 Ser Val Leu Leu Val Ser Asn Leu Asn Pro Glu Arg Val Thr Pro Gln 340 345 350 Serum Leu Phe Ile Leu Phe Gly Val Tyr Gly Asp Val Gln Arg Val Lys 355 360 365 Ile Leu Phe Asn Lys Lys Glu Asn Ala Leu Val Gln Met Ala Asp Gly 370 375 380 Asn Gln Ala Gln Leu Ala Met Ser His Leu Asn Gly His Lys Leu His 385 390 395 400 Gly Lys Pro Ile Arg Ile Thr Leu Ser Lys His Gln Asn Val Gln Leu 405 410 415 Pro Arg Glu Gly Gln Glu Asp Gln Gly Leu Thr Lys Asp Tyr Gly Asn 420 425 430 Ser Pro Leu His Arg Phe Lys Lys Pro Gly Ser Lys Asn Phe Gln Asn 435 440 445 Ile Phe Pro Pro Ser Ala Thr Leu His Leu Ser Asn Ile Pro Pro Ser 450 455 460 Val Ser Glu Asp Leu Lys Val Leu Phe Ser Ser Asn Gly Gly Val Val 465 470 475 480 Lys Gly Phe Lys Phe Phe Gln Lys Asp Arg Lys Met Ala Leu Ile Gln 485 490 495 Met Gly Ser Val Glu Glu Ala Val Gln Ala Leu Ile Asp Leu His Asn 500 505 510 His Asp Leu Gly Glu Asn His His Leu Arg Val Ser Phe Ser Lys Ser 515 520 525 Thr Ile 530 <210> 2 <211> 239 <212> PRT <213> artificial <220> <223> EGFP protein <400> 2 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235

Claims

1. A method for capturing RNA interacting with a target RBP, the method comprising: Step (I): Provide a cell sample and transfer a plasmid containing the target RBP into the cells; Step (II): Provide a first reagent having an alkyne group to the cell so that the first reagent having an alkyne group forms a first complex with the RNA in the cell; The first reagent is a terminal alkyne-substituted ribonucleotide analog that can be absorbed by cells and utilized by RNA polymerase II to be incorporated into nascent RNA; Step (III): Provide a second reagent having an azide (-N3, azido) group and contact it with the cells so that the second reagent having the azide group forms a second complex with the target RBP in the cells; The second reagent is an azide-modified reagent that can permeate the cell membrane and can be incorporated into the target RBP in living cells through covalent binding or metabolism. Step (IV): The first and second complexes within the cell are brought into contact to form a third complex under conditions that allow copper-catalyzed cycloaddition of alkynes and azide (CUAAC) without the use of UV crosslinking. Step (V): Capture of RNA interacting with the target RBP is achieved by capturing the third complex.

2. The method of claim 1, wherein the plasmid further comprises a first ligand.

3. The method of claim 2, wherein in step (V), a second ligand is provided, and the third complex is captured by the binding interaction between the first ligand and the second ligand, thereby achieving the capture of RNA interacting with the target RBP.

4. The method of claim 3, wherein the first ligand is biotin and the second ligand is streptavidin.

5. A method for analyzing RNA or RBP, the method comprising: Step (i): Capture the third complex by the method described in any one of claims 1-4; Step (ii): Sequencing the RNA in the third complex to obtain RNA information, thereby obtaining information about the target RBP; Optionally, prior to step (ii), the RNA in the third complex is treated to sequence the RNA in step (ii).

6. The method of claim 5, wherein prior to step (ii), an RNA library is constructed based on the RNA in the third complex.

7. The method according to any one of claims 1-6, wherein, The method has one or more of the following: (1) The first reagent contains 5-ethynyl uridine; (2) The second reagent comprises azide-succinimide (NHS-azide) and / or L-azide-high alanine (L-AHA); optionally, the second reagent further comprises N-ethylmaleimide and / or maleimide polyethylene glycol (Maleimide-PEG). (3) In step (IV), a reagent containing copper ions is provided; optionally, in step (IV), an accelerating agent, a reducing agent, or any combination thereof is also provided; (4) Before step (III), the cells are treated with Trypsin-EDTA.

8. The method of claim 7, wherein, The method has one or more of the following: (1) The second reagent contains NHS-azide and N-ethylmaleimide; (2) The second reagent contains L-AHA and N-ethylmaleimide; (3) In step (IV), a reagent containing copper ions, a reagent to accelerate the reaction, and a reducing agent are provided; (4) The reagent used to accelerate the reaction is selected from BTTAA, MBHTM, Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), aminoguanidine, or any combination thereof; (5) The reagent containing copper ions is selected from copper sulfate, copper tetra(acetonitrile)hexafluorophosphonate, copper acetate, and copper bromide; (6) The reducing agent is selected from sodium ascorbate, tris(2-carboxyethyl)phosphine, dithiothreitol, β-mercaptoethanol, or any combination thereof.

9. The method of claim 8, wherein in step (IV), the reagent containing copper ions and the reagent that accelerates the reaction are first brought into contact with the cells, and then the reducing agent is brought into contact with the cells.

10. The method of claim 8, wherein, The method has one or more of the following: (1) The reagents that accelerate the reaction are THPTA and aminoguanidine; (2) The reagent containing copper ions is copper sulfate; (3) The reducing agent is sodium ascorbate.

11. The method according to any one of claims 1-6, wherein, The plasmid contains one or more target RBPs, which are derived from the cell.

12. The method of claim 1, wherein the cell is a single cell or a group of cells.

13. The method of claim 1, wherein, The cells in question are mammalian cells.

14. The method of claim 1, wherein, The cells are human, mouse, or rat cells.

15. A library comprising the sequence of RNA captured by the method of any one of claims 1-14.

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