CN108427000B - Method and kit for capturing nucleic acid binding protein - Google Patents

Abstract

The present invention relates to a method for capturing RBP and a corresponding kit, the method comprises adding a nucleoside analogue A into a biological sample system capable of synthesizing RNA; carrying out light activation treatment to enable the RNA and the protein to be subjected to light cross-linking; adding cell lysate and first molecule B; obtaining contents of a biological sample system; contacting the content with a carrier D with a marker molecule C; separating the carrier to obtain the RNA-RBP complex. The kit can capture RNA and RBP thereof more broadly, and solves the limitation that the prior art can not obtain nonpolyA-RBP. Can efficiently enrich RBP, has good specificity, and can not capture non-RBP protein.

Description

Method and kit for capturing nucleic acid binding protein

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method and a kit for capturing nucleic acid binding protein.

Background

NonpolyA-RNA (including circRNAs, ppRNAs, eRNAs, tsRNAs, etc.) is involved in various physiological and biochemical processes such as transcriptional regulation, mRNA translation, etc. nonpolyA-RNA generally functions in the form of protein complexes with RNA-binding proteins (RBPs) involved in various aspects of RNA metabolism such as RNA splicing, polyadenylation, sequence editing, RNA transport, maintenance of RNA stability and degradation, intracellular localization, and translational regulation. In order to research the interactomics of the nonpolyA-RNA and the RBPs thereof, the separation and identification of the nonpolyA-RNA and the RBPs thereof are indispensable research methods, and technical means capable of effectively separating the nonpolyA-RNA and the RBPs thereof are needed. However, the existing RBPs separation technology can not achieve the purpose of effectively obtaining non-enol A-RBPs, such as oligo (dT) technology (Castello, A.et al.Insights inter RNA biology from an atlas of mammalian mRNA-binding proteins. cell 149,1393-1406 (2012)).

Disclosure of Invention

It is an object of the present invention to provide a novel method for capturing RBPs, which is innovative and improved in the method of RICK method. The method has important value in researching the RNA and RBP interactomics, is beneficial to discovering new RBP, functions and regulation mechanism of the new RBP, and can be widely applied to the fields of disease mechanism research, drug target screening and the like.

The technical scheme for achieving the purpose is as follows.

A method for capturing RBP and/or RNA mainly comprises the following steps:

1) adding nucleoside analogues with dipole-philic groups into a biological sample system capable of synthesizing RNA;

2) carrying out light activation treatment to enable the RNA and the protein to be subjected to light cross-linking;

3) adding a click reaction solution containing a first marker molecule of a 1, 3-dipolar group, covalently connecting the first marker molecule with RNA doped with nucleoside analogue through click reaction to make the RNA be marked by the first marker molecule, wherein the click reaction is Huisgen triazole reaction under catalysis of cuprous ions, and the click reaction solution contains a catalyst which is preferably monovalent Cu⁺Or Vitamin C and Cu²⁺A combination of (1);

4) adding cell lysate to clarify the sample solution, wherein the cell lysate comprises 0.15M-1M Li salt and a detergent, and the detergent is preferably 0.1-1% detergent containing lithium ions;

5) adding a second marker molecule connected with a carrier, wherein the second marker molecule can be combined and reacted with the first marker molecule, and coupling the formed RNA-protein complex to the carrier with the second marker molecule through the combination and reaction of the second marker molecule and the first marker molecule;

6) separating to obtain RNA-protein complex on the carrier;

7) separating to obtain protein (RBP) and/or RNA in the RNA-protein complex.

In one embodiment, a protein protecting agent is added into the click reaction solution, and the protein protecting agent is aminoguanidine and/or THPTA.

In one embodiment, the protein protectant is 0.3-1.5mM THPTA and 0.5-1.5mM aminoguanidine.

In one embodiment, the cell lysate comprises 0.45M to 0.5M LiCl and a detergent, preferably 0.5 to 1% LiDS (0.5 to 1g LiDS in 100ml water).

The nascent RNA is newly transcribed RNA, newly synthesized RNA, or RNA synthesized within a certain time after the addition of nucleotide analogs, including polyA-RNA and nonpolyA-RNA.

The nucleoside analogue is a compound with a similar nucleoside structure, can be inserted into an RNA chain during RNA synthesis, and has a dipole-philic group.

In one embodiment, the dipole-philic group is an alkynyl group, an alkenyl group, or a halogen, and more preferably, the alkynyl group is an ethynyl group, a propynyl group, or a cyclic alkynyl group.

The nucleoside analogs with a dipole-philic group include EU, EC, EG, EA, BrU, 4SU, 6SG, or combinations thereof, preferably EU.

The 1, 3-dipolar group is preferably an azide group, an oxonitrile group, a diazomethyl group, a nitro group, a nitrilamine group or the like.

The light of step 2) is UV254, UV365 or a combination thereof, preferably UV 254.

The binding of the second label molecule to the first label molecule is covalently bound or non-covalently bound, preferably covalently bound.

The method for separating and obtaining the protein (RBP) or RNA in the RNA-protein complex in the step 6) comprises an enzyme treatment method, a filter membrane adsorption method, an electrophoresis method or an antibody binding method.

The first labeling molecule comprises biotin, digoxigenin, quantum dots, gold particles, nanoparticles or antibodies, preferably biotin.

The second labeling molecule comprises streptavidin or an antibody recognizing digoxin, quantum dots or gold particles, preferably streptavidin.

The carrier comprises magnetic beads or agarose beads.

Another object of the present invention is to provide a kit for capturing RBP and/or RNA.

The technical scheme for achieving the purpose is as follows.

A kit for capturing RBP and/or RNA mainly comprises:

1) nucleoside analogs with a dipole-philic group; 2) a first labeling molecule comprising a 1, 3-dipolar group; 3) a carrier with a second labeling molecule, wherein the second labeling molecule can perform a binding reaction with the first labeling molecule; 4) a click reaction catalyst, preferably monovalent Cu⁺Or Vitamin C and Cu²⁺A combination of (1); 5) cell lysate.

In one embodiment, the protein protective agent also comprises 6) protein protective agent, and the protein protective agent is aminoguanidine and/or THPTA.

The nucleoside analogue is preferably EU at 0.05-0.45 mM.

The first labeling molecule is preferably 0.1-1mM azide group-modified biotin.

The concentration of the copper ions is preferably 0.2-0.5mM, the compound of the copper ions is preferably copper sulfate and cuprous bromide, and the concentration of the vitamin C is preferably 2-10 mM.

The protein protective agent is guanidine derivatives and/or THPTA, the guanidine derivatives are preferably 0.5-1.5mM aminoguanidine, and the THPTA concentration is preferably 0.1-2.0mM, more preferably 0.3-1.5mM THPTA and 0.5-1.5mM aminoguanidine. The protein protective agent is used for protecting protein from being damaged, and the THPTA also has the function of catalytic group slow release and can be used as a stabilizer of a catalyst.

The cell lysate comprises high concentrations of salt, preferably 0.15M-1M Li salt, and detergent, preferably 0.1-1% lithium ion-containing detergent.

According to the method for capturing RBP, nucleoside analogues such as 5-Ethynyluracil (EU) are doped into the nascent RNA, the ultraviolet is utilized to induce RNA-protein crosslinking, and biotin-labeled RNA-protein complexes are captured, so that the subsequent separation and identification of the nascent RNA and the RBP thereof, particularly the separation and identification of nonpolyA-RBP and nonpolyA-RNA are realized.

The novel RBP capturing method is a methodology system which is suitable for RBP capturing and is established on the basis of applying click chemistry to RBP capturing. The cell lysate used (e.g., 500mM LiCl, 0.5% LiDS) contains higher concentrations of salts and detergents to achieve the effect of completely lysing the cells, increasing RNA-protein separation, and effectively removing non-specific RNA and protein binding or indirect RNA and protein interactions mediated between proteins. In addition, the RNA-protein complex is stable in ionic detergents and high salt conditions.

The protein protective agent can inhibit side reaction between dehydroascorbate and a protein side chain in click chemistry reaction, and can prevent a byproduct of bivalent copper from hydrolyzing biological molecules. The catalyst stabilizer THPTA is added, and the function of catalytic group slow release is achieved.

In general, the novel method for capturing RBPs and the corresponding kit of the present invention have the following advantages: 1) the method can capture RNA and RBP thereof more broadly, including obtaining nonpolyA-RNA and nonpolyA-RBP, and solves the limitation that nonpolyA-RBP can not be obtained in the prior art. 2) The variety of the captured RNA is multiple, and the abundance of the captured nonpolyA-RNA is high; 3) the RBP can be efficiently enriched, in particular nonpolyA-RBP; more than 300 brand-new RBPs which are not identified by the prior art are captured simultaneously; the specificity is good, and non-RBP protein can not be captured; 4) the method has good compatibility and repeatability, and is suitable for different cell lines and different conditions; 5) high throughput, can be used for large-scale screening or validation of RBPs; 6) the method is simple and fast, and needs few initial biological samples.

Drawings

FIG. 1, schematic representation of a biotin-labeled click chemistry reaction;

FIG. 2 is a schematic diagram showing the result of RBP silver staining;

FIG. 3 is a schematic diagram showing the result of RBP Western detection;

FIG. 4 is a schematic diagram showing the results of protein mass spectrometry data analysis;

FIG. 5 is a schematic diagram showing the results of analysis of a protein Venturi diagram;

FIG. 6 is a schematic diagram showing the result of CLIP RBP detection;

FIG. 7 is a diagram showing the results of RNA concentration measurement;

FIG. 8 is a graph showing the results of RNA length distribution;

FIG. 9 is a diagram showing the results of RNA species analysis;

FIG. 10, schematic representation of the results of circular RNA analysis;

FIG. 11, schematic diagram showing the result of analysis of ppRNA;

FIG. 12, eRNA analysis results diagram;

FIG. 13 is a schematic diagram showing the results of detection of RNA expression levels;

FIG. 14 is a schematic diagram showing the protein detection results by silver staining.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

1, RICK: nascent RNA Interactive Click Reaction, refers to the use of nucleotide analogue incorporation and Click chemistry Reaction separation of RNA binding protein method (see figure 1 biotin labeled Click chemistry Reaction).

2. Nascent RNA: newly transcribed RNA, newly synthesized RNA, RNA synthesized after addition of nucleotide analogs for a certain period of time, these RNAs having incorporated nucleotide analogs (e.g., EU).

Connection reads: it refers to the binding site of two different exons, specifically the binding site of exon reverse splicing, such as the binding of exon 5 and exon 4 (5 is 5 'end and 4 is 3' end) when analyzing circular RNA.

Back meshing: reverse splicing, where splicing occurs from the top to the bottom of the large-sized exon.

Tr (tracking ratio): in many genes, transcription is terminated by about 50nt during transcription, so that signals in the region are found to be significantly larger than the genebody region during sequencing, and TR is the ratio of the signals at the two positions.

NonpolyA-RBP RNA-binding proteins specifically prone to binding to non-polyA-RNA.

7. Nucleoside analogues (nucleosides analogues), a class of compounds with a ribonucleoside-like structure, which can be inserted into the RNA strand during RNA synthesis and which carry a dipole-philic group.

8. The dipole-philic group is an alkynyl group, an alkenyl group or a halogen, more preferably, the alkynyl group is an ethynyl group, a propynyl group, a cyclic alkynyl group or the like.

1, 3-Didipolar groups are preferably: azido, oxonitrile, diazomethyl, nitrone, nitrilamine, and the like.

10. Click reaction: the Huisgen triazole reaction under the catalysis of cuprous ions can be completed at normal temperature and high yield in water or various organic solvents. Such as copper-catalyzed covalent reactions between azides and alkynes.

11. Click reaction catalyst: divalent or monovalent copper ions or in combination with ascorbic acid, preferably copper sulphate, cuprous bromide, sodium ascorbate.

12. LiDS: and (3) dodecyl lithium sulfate.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The method or the kit can be used for capturing RBPs in organs, tissues or cells cultured in vivo or in vitro, wherein the cells are preferably stem cells, nematode cells, mouse cells, tumor cells and immune cells. The organism is preferably rat, mouse, nematode.

Example 1: capture of RBP (RNA binding protein)

1. Capturing RNA-protein complexes

1.1 EU-incorporating RNA

HeLa (obtained from ATCC) cells were cultured in a culture dish having a diameter of 100mm in 10mL of a high-sugar medium containing 10% FBS at 37 ℃ in 5% CO₂Culturing under the condition. When the cells were cultured to a density of 80%, EU was added to a final concentration of 0.25mM and the cells were incubated with EU for 16 hours to allow sufficient incorporation of EU into the intracellular newly synthesized RNA.

1.2 washing

The cells were washed twice with PBS buffer to remove residual media.

1.3 photocrosslinking

PBS was removed, and the culture dish containing the cells of step (2) was placed on ice at 254nm (0.4J/CM)²) Irradiating for 1min with ultraviolet lamp to make RBP and interacting RNA form covalent bond.

1.4 fixation of cells

The dish was removed from the UV lamp, 5mL of 90% ethanol was added to treat the cells, the cells were fixed by placing on ice for 15min, and washed 3 times with PBS.

1.5 permeabilizing cells

PBS was removed and 5mL of 0.5% Triton X-100 treated cells were added to the dish for 15min followed by 3 washes with PBS.

1.6RNA biotinylation

PBS was removed, and the cells obtained in step (5) were mixed with 10mL of a click reaction solution (see Table 1 for components)

Incubate the dishes for 3-30 minutes.

1.7 washing

The click reaction solution in step (6) was removed, and 10mL of 0.5% Triton X-100 wash solution (to which EDTA was added to a final concentration of 2 mM) was added to the petri dish, washed 3 times, and then washed 2 times with 10mL of PBS.

1.8 lysis of cells with lysis solution

2mL of cell lysate (see Table 1 for components) is added into the culture dish, after 15-30 minutes of lysis, the cell is scraped and collected to obtain a lysate, and the supernatant is collected by centrifugation.

1.9 preparation of magnetic beads

70uL of streptavidin magnetic beads (Thermo Fisher Scientific) were removed, adsorbed by a magnetic holder, washed 2 times with 300uL of cell lysate, and resuspended in 50uL of cell lysate.

1.10 magnetic bead Capture

And (3) adding the streptomycin avidin magnetic beads prepared in the step (9) into the supernatant obtained in the step (8), incubating for 3 hours by using a rotator, and capturing biotinylated RNA-protein complexes in the lysate.

1.11 washing of the beads to remove non-specific binding

Adsorbing the magnetic beads by a magnetic seat, standing for 2min, and removing lysate; adding 500uL lysine Buffer, washing for 10min by a rotary instrument, and removing a supernatant after magnetic bead adsorption; adding 500uL Buffer 1 (the components are shown in Table 1), washing for 10min by a rotary instrument, and removing a supernatant after magnetic bead adsorption; adding 500uL Buffer 2 (see the composition in Table 1), washing for 10min with a rotary instrument, removing supernatant after magnetic bead adsorption, adding 500uL Buffer 3 (see the composition in Table 1), and washing for 10min with a rotary instrument. At this point, RNA-protein complexes bound to the magnetic beads are obtained.

TABLE 1 reagent Table (working concentration)

2. Separating to obtain RBP and RNA

2.1RBP

2.1.1 obtaining RBP

The RNA-protein complex obtained in step 1 was resuspended in 50uL of the eluate (see Table 1 for composition), 1uL of 10mg/mL RNase A (Sigma) was added and treated at 37 ℃ for 1hr, followed by boiling in a boiling water bath for 5min, and the supernatant was aspirated.

2.1.1RBP detection

(1) Silver staining detection

After SDS-PAGE, the resulting gel was treated with a fixed solution (50% methanol and 5% acetic acid) for 40 minutes, washed with 50% methanol and double distilled water in portions, treated with 0.02% sodium thiosulfate for 1 minute and then washed with double distilled water, and 0.1% AgNO₃Washing with double distilled water after dark treatment for 20 min, adding 2% carbonic acidSodium and 0.04% formaldehyde developed and 5% acetic acid stopped the reaction.

FIG. 2 shows the detection of proteins obtained by the RICK method using the silver staining method. While Input under different conditions has clear bands, only samples added with EU crosslinking in Pull-down have clear bands, and neither the non-crosslinking group nor the RNase treatment group have enrichment of RNA binding protein. The experimental result shows that the RICK method can specifically and efficiently enrich the RNA binding protein. (Note: Cross-linking means 365nm UV-Crosslinking for 1 minute; RNase is RNase A which degrades RNA and dissociates RNA and protein complexes; EU is uracil analogue "+" indicates the presence, "-" indicates the absence, Input is whole cell lysate, Pull-down indicates RNA-binding protein enriched from Input by RICK method, the same figures are as follows.)

(2) Western blot

The proteins were loaded, subjected to SDS-PAGE gel electrophoresis, and the electrophoretically separated proteins were transferred to a PVDF membrane (Millipore), blocked in a blocking solution (5% milk-containing solution prepared in TBST) for 1hr, incubated with primary antibody at 4 ℃ overnight, washed in TBST, incubated with HRP-conjugated secondary antibody at 4 ℃ for 2 hours, washed in TBST, and detected by ECL plus (Amersham).

The results show that 4 RBPs, POLR2A (RNA polymerase subunit), DDX5(RNA helicase), HNRNPK (heterogeneous ribonucleoprotein k), PTBP1 (polypyrimidine channel binding protein 1) are captured by the RICK method, and non-RBP proteins ACTIN (housekeeping gene protein) and TUBULIN (housekeeping gene protein) are not captured, and that the RICK method specifically enriches RNA binding protein. The results are shown in FIG. 3.

(3) LC-MS/MS detection

A detection method

After 50mM TCEP and 200mM MNTS reduction of protein, 70% ethanol, 8M urea and 0.25M TEAB washing. The protein was then digested with 1. mu.g/. mu.L trypsin overnight at 37 ℃ and used in separation of peptide fragment mixtures using the Ultimate 3000 high performance liquid chromatography system (Dionex, USA). After column loading (Phenomenex Gemini-NX 3u C₁₈11OA column) 95% solution a (20mM ammonium acetate in water, 2m naoh, pH 10.0) and solution B (20mM hcooonh)₄2M NaOH, 80% CAN, pH 10.0) binary Linear gradient(15% -50%) was eluted for 45 minutes to isolate the peptide fragment at a flow rate of 0.2 mL/min. The UV detector is set at 214/280nm, and the components are collected once per minute, and the separated components are dried by vacuum centrifugation and then detected by nanometer reversed phase liquid chromatography for later use. The eluate was analyzed by a Q ExactivetHF mixed quadrupole mass spectrometry system (ThermoFisher Scientific), dependent acquisition mode. Mass spectrum detection conditions of mass range 400->30,000), high sensitivity mode (resolution)>15000) Data are recorded and extracted by protein Pilot software.

TABLE 2 chromatographic separation parameters

Mobile phase A	Mobile phase B	Flow rate of flow	Gradient of B phase elution
				0.1％FA	0.1％FA	300nL/min	5％-40％，70min
5％ACN	95％ACN

B Mass Spectrometry data analysis

LC-MS/MS detected 1,353 RBP candidates, and the proteins were classified into high-confidence (720, red) and low-confidence (633, blue) proteins by confidence assessment, and the 720 proteins identified by RICK method based on unique peptide fragment counts (FIG. 4). FIG. 4a shows that the correlation coefficients of the 3 experimental results are all greater than or equal to 0.828, which indicates that the method has high repeatability and high efficiency. Fig. 4b shows the mass spectrum experiment after background exclusion. The RICK panel enriched a number of authentic RNA binding proteins. Of the 720 proteins, 350 of them coincided with the protein captured by oligo (dT) method reported in the known literature (Castello, A.et al. instruments inter RNA biology from an atlas of mammalin mRNA-binding proteins. cell 149,1393-1406(2012)), and 370 (51.4%) were brand new, unreported RBPs specifically detected by RICK. Based on the significant difference in RNA species isolated by the two methods, it was concluded that the 370 protein was a candidate non-polyARBP (see FIG. 5 a). Comparison of RBP data in different cell lines showed that 307 proteins (. about.83.0%) of the 370 candidate non-polyA RBPs were specifically captured by RICK, and that 307 proteins were RBPs that were not identified by all methods (SerlC method, see Conrad, T.et al.Serial interaction capture of the human cell nucleus. Nat Commun7,11212 (2016)).

FIG. 4a repeatability and correlation analysis of the results of 3 experiments. The red letters indicate the proteome in the low confidence region and the bluish blue indicates the proteome with the interaction.

FIG. 4 b: in mass spectrum, comparing the unique peptide segments of the control group and the RICK experimental group, wherein red represents high-reliability protein, blue represents low-reliability protein, and black represents background protein.

Venturi FIG. 5a RNA-binding protein group obtained by RICK method in HeLa cells and RNA-binding protein group identified by oligo (dT) method were analyzed in comparison.

FIG. 5 b-analysis of RNA binding proteins identified by the RICK method and different cell lines including Huh7, HEK293T, K562.

(3) RBP binding Capacity analysis

From 370 candidate nonpolyA RBPs, 20 nonpolyA-RBPs were selected using the PAR-CLIP method and tested for their ability to bind to RNA (GFP was used as a negative control, CCAR2 and HNRNPK as positive controls). FIG. 6 shows that, after cross-linking, the protein with positive RNA binding capacity has a strong signal (RNA-protein), and the candidate nonpolyA-RBP can bind to RNA. Wherein SMARCC1, SMC1A, INTS9, CDK2, CDK4, CDK9, MCM2, MCM5 and MCM6 are newly discovered proteins. In FIG. 5, "-" indicates a sample which was not subjected to UV crosslinking treatment, and "+" indicates a sample which was subjected to UV crosslinking treatment at 365 nm. And detecting the protein expression quantity of the Anti-Flag at the same position.

2.2 RNA

2.2.1 obtaining RNA

Resuspending the RNA-protein complex obtained in step 1 in 200uL 1 PK buffer, adding 20uL 20mg/ml proteinase K (Merck), treating at 56 deg.C and 1000rpm for 1hr, and extracting RNA by phenol/chloroform method.

2.2.2 RNA detection

(1) Concentration and distribution detection

The Qubit 2.0 measures the RNA concentration (fig. 7), and the Agilent2200 analyzes the size and distribution of RNA fragments (fig. 8). As can be seen from FIG. 7, the RNA concentration was 13.8ng/uL, and the total amount was 179 ng. In fig. 7: EU-sample is negative control, EU + is experimental group sample, Input is positive control). FIG. 8 shows that the kurtosis of RNA obtained by RICK reaction is higher than that of 25nt standard lower, and the main enrichment region is 50nt to 6000nt RNA.

(2) RNA sequencing

Alignment of the sequencing data with the human hg38 genome using bowtie2 generated a whole genome sequence density profile.

A RNA species analysis

A plurality of RNAs are obtained by the method, as shown in the left of FIG. 9: ribosomal RNA (rRNA, 45.0%), messenger RNA (mRNA, 24.7%), mitochondrial ribosomal RNA (mtRNA, 5.5%), other RNA (24.8%); in particular, a variety of non-poly A-RNAs have been isolated, such as long non-coding RNAs (lncRNA), circular RNAs (circRNAs), enhancer RNAs (eRNAs), proximal promoter RNAs (ppRNAs), and the like. FIG. 9 right shows the ratio of RNA transcripts isolated by RICK method to respective types of RNA (e.g., micro RNA (miRNA), antisense RNA (antisense) etc.).

Identification of B candidate circRNAs (circular RNAs)

The original sequencing reads were pre-processed to filter out duplicates, adaptors, low quality sequences, etc., using high quality sequence to genome alignments (Tophat alignment software). As compared with oligo (dT) method, 6199 reverse splicing sites of circRNA were detected in RNA isolated by RICK method, whereas only 57 reverse splicing sites were detected by oligo (dT). 828 of 6199 reverse splicing sites can be aligned at circBase datasets; while the oligo (dT) method only has 2 alignments (FIG. 10). It was confirmed that the method successfully isolated circRNA and that the amount of enriched circRNA was significantly higher than the corresponding data in the oligo (dT) method.

In fig. 10: normalized junction reads number (light color), normalized circular RNA number (dark color)

C ppRNA (proximal promoter RNA) analysis

Studies have found that there is proximal promoter signal accumulation at the gene site where RNA polymerase II is halted. The density of peaks in the proximal promoter sequence density of RNA obtained by RICK method was high in transcripts with TR greater than 4 compared to the TR <4 gene, confirming that ppRNA was isolated and that no substantial proximal promoter signal was detected by Oligo method, see FIG. 11 c. FIG. 11d analysis shows that the RICK method is less peaked near PolyA in the TR <4 gene. The enrichment effect of the method in the near-end promoter RNA is proved to be better than that of an oligo (dT) method, and the method is closer to the real situation in cells.

D eRNA (enhancer RNA) analysis

The results of eRNA sequencing and bidirectional eRNAs sequence density comparison of the FANTOM5 database show that RNA separated by the RICK method has stronger enhancer signals which are obviously stronger than oligo (dT), and the eRNA is successfully captured by the method. See figure 12 for a detailed analysis. FIG. 12e shows that RNA captured by RICK method has higher peak density near sites known to be near all possible enhancers, compared to the RNA sequence density distribution of two active enhancers H3K27ac, H3K4me 1. FIG. 12f shows that RICK-seq data and eRNA database searches found about 6% of the eRNA identified, whereas the RNA detected by oligo (dT) method found a lower proportion of eRNA analysis.

(3) RT-qPCR RNA expression level detection

The RNA obtained by separation is reversely transcribed into cDNA by using a SuperScipt III kit (Thermo Fisher Scientific), quantitative determination is carried out by SYBR Green kit (Takara) RT-PCR, and ACTB is selected as an expression level of a standardized target gene of an internal reference.

FIG. 13: quantitative analysis of representative RNAs from multiple classes of RNA showed that the RICK method has advantages over oligo (dt) methods in isolating and enriching circular RNA, etrna, snRNA, rRNA, lincRNA, and nonproly (a) mRNAs, with significantly higher enrichment levels than the latter.

Example 2: dose optimization of protein protectants

Detecting RBP by silver staining method: the results are shown in FIG. 14. (see Table 3 for test conditions set up, see example 1 for further procedures.)

TABLE 3 protein protectant conditions

Protein protective agent	THPTA concentration	Aminoguanidine concentration
			Condition
1	0mM	1mM
			Condition
2	0.3mM	1mM
			Condition
3	0.9mM	1mM
			Condition
4	1.5mM	1mM
			Condition
5	0.9mM	0.5mM
			Condition
6	0.9mM	1mM
			Condition
7	0.9mM	1.5mM
			Condition
8	0.9mM	0mM

As shown in fig. 14, when THPTA is not added to the click reaction solution (condition 1), the protein bands are clearly seen to be dispersed and clear in silver staining detection, which proves that the protein is damaged during the click reaction; when THPTA ( conditions 2, 3 and 4) is added, clear bands can be seen in silver staining detection, and the effect is more obvious along with the increase of the concentration. In the absence of aminoguanidine (condition 8), the protein condition also appears in a diffuse, hazy morphology; when the protectant aminoguanidine ( conditions 5, 6, and 7) was added, a clear band was exhibited, and the band was more pronounced as the concentration of the protectant increased. The two experiments show that the protective agents THPTA and aminoguanidine can play an obvious protective effect on protein in the click chemistry reaction process.

Example 3: optimization of lysates

The relative concentrations of the proteins obtained (comparison of the proteins with each other, not their own absolute concentrations, i.e. no absolute quantification using a standard as a standard curve) were compared under different lysate conditions (table 4) for the other components as in example 1, and the other steps are referred to in example 1. Test results show that the lysate can be used for enriching RBP more efficiently.

TABLE 4 lysate conditions

Lysate component	Relative concentration of protein (mg/ml)
		150mM NaCl，0.1％SDS,0.5％NP40	0.873
150mM LiCl，0.1％LiDS	0.738
		500mM LiCl，0.1％LiDS	0.789
500mM LiCl，0.5％LiDS	0.904
		500Mm LiCl，1％LiDS	0.955

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (7)

1. A method for capturing RBP and/or RNA is characterized by mainly comprising the following steps:

1) adding nucleoside analogues with dipole-philic groups into a biological sample system capable of synthesizing RNA; the nucleoside analogs with a dipole-philic group include EU, EC, EG, EA, or combinations thereof;

2) carrying out light activation treatment to enable the RNA and the protein to be subjected to light cross-linking;

3) adding a click reaction solution containing a first marker molecule of a 1, 3-dipolar group, covalently connecting the first marker molecule with RNA doped with nucleoside analogues through a click reaction to make the RNA be marked by the first marker molecule, wherein the click reaction is a Huisgen triazole reaction under catalysis of cuprous ions, the click reaction solution comprises a catalyst and a protein protective agent, and the protein protective agent is aminoguanidine and THPTA; the 1, 3-dipolar group is azido;

4) adding cell lysis solution to clarify the sample solution, wherein the cell lysis solution comprises 0.15-1M of Li salt and 0.1-1% of detergent containing lithium ions;

5) adding a second marker molecule connected with a carrier, wherein the second marker molecule can be combined and reacted with the first marker molecule, and coupling the formed RNA-protein complex to the carrier with the second marker molecule through the combination and reaction of the second marker molecule and the first marker molecule;

6) separating to obtain RNA-protein complex on the carrier;

7) separating to obtain RBP and/or RNA in the RNA-protein complex.

2. The method for capturing RBP and/or RNA as claimed in claim 1, wherein said protein protectant is 0.3-1.5mM THPTA and 0.5-1.5mM aminoguanidine.

3. The method for capturing RBP and/or RNA as claimed in claim 2, wherein said protein protectant is 0.6mM THPTA and 1mM aminoguanidine.

4. A method for capturing RBPs and/or RNAs as in any of claims 1-3, wherein said cell lysate comprises LiCl at 0.45M to 0.5M and LiDS at 0.5 to 1%.

5. The method for trapping RBPs and/or RNAs as claimed in any one of claims 1 to 3, wherein said catalyst is monovalent Cu⁺Or Vitamin C and Cu²⁺Combinations of (a) and (b).

6. The method for capturing RBPs and/or RNAs as claimed in any one of claims 1 to 3, wherein the light of step 2) is UV254, UV365 or a combination thereof.

7. The method for capturing RBPs and/or RNAs as claimed in any one of claims 1 to 3, wherein said first labeling molecule comprises biotin, digoxigenin, a gold particle, a nanoparticle or an antibody; the second marker molecule comprises streptavidin which recognizes the biotin of the first marker molecule; the carrier comprises magnetic beads or agarose beads.

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