CN108427000A

CN108427000A - A kind of method and kit of capture nucleic acid binding protein

Info

Publication number: CN108427000A
Application number: CN201710080808.9A
Authority: CN
Inventors: 张必良; 米格尔·埃斯特班; 鲍习琛; 郭亨彭; 尹梦回; 王玮; 克雷格·梅洛
Original assignee: Guangzhou Ribobio Co ltd
Current assignee: Guangzhou Ribobio Co ltd
Priority date: 2017-02-15
Filing date: 2017-02-15
Publication date: 2018-08-21
Anticipated expiration: 2037-02-15
Also published as: CN108427000B

Abstract

The present invention relates to the method for capture RBP and corresponding kits, and the method includes nucleoside analog A is added in the biological sample system that can synthesize RNA；Photoactivation is handled, and makes RNA that photo-crosslinking occur with albumen；Cell pyrolysis liquid is added, the first molecule B is added；Obtain the content of biological sample system；Content is contacted with the carrier D with mark molecule C；Carrier of separating obtains RNA RBP compounds.Kit of the present invention can capture to more wide spectrum RNA and its RBP, and the limitation of nonpolyA RBP can not be obtained by solving the prior art.Can efficiently concentrating RBP, and specificity it is good, non-RBP albumen will not be captured.

Description

A kind of method and kit of capture nucleic acid binding protein

Technical field

The invention belongs to biotechnologies, particularly relate to a kind of method and reagent of capture nucleic acid binding protein Box.

Background technology

NonpolyA-RNA (including circRNAs, ppRNAs, eRNAs, tsRNAs etc.) and a variety of physiological and biochemical procedure phases It closes, such as transcriptional control and mRNA translations.NonpolyA-RNA usually with rna binding protein (RNA-binding Proteins, RBPs) form that forms albumen composition plays a role, and RBP participates in RNA montages, poly-adenosine, sequence are compiled Volume, rna transport, the various aspects for maintaining the RNA such as the stabilization and degradation of RNA, intracellular targeting and translational control metabolism.For research The interaction group of nonpolyA-RNA and its RBPs detach and identify the research that nonpolyA-RNA and its RBPs is indispensable Method, it would be desirable to be able to efficiently separate the technological means of nonpolyA-RNA and its RBPs.And existing RBPs isolation technics can not be real Existing effect obtains the purpose of nonpolyA-RBPs, such as Oligo (dT) technology (Castello, A.et al.Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.Cell 149,1393- 1406(2012))。

Invention content

An object of the present invention is to provide a kind of method of new capture RBP, and this method is created in the method for RICK methods Newly and improved.The method of the invention has important value in the interaction group of research RNA and RBP, while advantageous In finding new RBP, the function of new RBP and its regulatory mechanism, pathogenic mechanism research, drug targets screening can be widely applied to Equal fields.

Realize that above-mentioned purpose technical solution is as follows.

A method of capture RBP and/or RNA is mainly included the following steps that：

1) nucleoside analog with dipolarophile group is added in the biological sample system that can synthesize RNA；

2) photoactivation is handled, and makes RNA that photo-crosslinking occur with albumen；

3) the click-reaction liquid for the first mark molecule for containing 1,3-, bis- dipole groups is added, by click-reaction by first Mark molecule is covalently attached with the RNA mixed with nucleoside analog, so that RNA is marked by the first mark molecule, the click-reaction is Huisgen triazoles under cuprous ion catalysis are reacted, and include catalyst in click-reaction liquid, the catalyst is preferably monovalence Cu⁺Or Vitamin C and Cu²⁺Combination；

4) be added cell pyrolysis liquid, until sample solution clarify, the cell pyrolysis liquid include 0.15M-1M Li salt and Detergent, the detergent are preferably the detergent containing lithium ion of 0.1-1%；

5) be added and be connected with the second mark molecule of carrier, the second mark molecule can with the first mark molecule association reaction, By the association reaction of the second mark molecule and the first mark molecule, RNA- albumen compositions will be formed by and be coupled to On the carrier of two mark molecules；

6) separation obtains the RNA- albumen compositions on carrier；

7) separation obtains the albumen (RBP) and/or RNA in RNA- albumen compositions.

Being added in the click-reaction liquid in one of the embodiments, has the protein protective agent, the protein protective agent to be Aminoguanidine and/or THPTA.

The protein protective agent is the amino of the THPTA and 0.5-1.5mM of 0.3-1.5mM in one of the embodiments, Guanidine.

The cell pyrolysis liquid includes the LiCl and detergent of 0.45M-0.5M in one of the embodiments, described Detergent is preferably the LiDS of 0.5-1% (0.5-1g LiDS are dissolved in 100ml water).

The nascent RNA be the RNA newly transcribed, newly synthesized RNA or after nucleotide analog is added, one timing The RNA of interior synthesis, including polyA-RNA and nonpolyA-RNA.

The nucleoside analog is a kind of compound with similar nucleotide structure, and the nucleoside analog can be closed in RNA At when be inserted into RNA chains in, and its carry dipolarophile group.

The dipolarophile group is alkynyl, alkenyl or halogen in one of the embodiments, it is highly preferred that the alkynyl For acetenyl, propinyl, cyclic alkyne.

The nucleoside analog with dipolarophile group includes EU, EC, EG, EA, BrU, 4SU, 6SG or combinations thereof, excellent It is selected as EU.

1, the 3-, bis- dipole groups are preferably azido, oxidation itrile group, dizaomethyl, nitro, nitrile amido etc..

The light of the step 2) is UV254, UV365 or combinations thereof, preferably UV254.

Second mark molecule is combined into covalent bond or Non-covalent binding with the first mark molecule, preferably covalently In conjunction with.

The method that the step 6) separation obtains albumen (RBP) or RNA in RNA- albumen compositions include enzymatic treatment method, Filter membrane absorption method, electrophoresis or antibody combined techniques.

First mark molecule includes biotin, digoxin, quantum dot, gold particle, nano particle or antibody, preferably Biotin.

Second mark molecule include Streptavidin or identification digoxin, quantum dot or gold particle antibody, preferably For Streptavidin.

The carrier includes magnetic bead or sepharose 4B.

It is a further object of the present invention to provide a kind of kits of capture RBP and/or RNA.

Realize that the technical solution of above-mentioned purpose is as follows.

A kind of kit of capture RBP and/or RNA, mainly includes：

1) nucleoside analog of dipolarophile group is carried；2) contain the first mark molecule of 1,3-, bis- dipole groups；3) band There is the carrier of the second mark molecule, and with the first mark molecule association reaction can occur for the second mark molecule；4) click-reaction is urged Agent, the catalyst are preferably monovalence Cu⁺Or Vitamin C and Cu²⁺Combination；5) cell pyrolysis liquid.

Further include having 6) protein protective agent in one of the embodiments, the protein protective agent be aminoguanidine and/or THPTA。

The nucleoside analog is preferably the EU of 0.05-0.45mM.

First mark molecule is preferably the biotin of the azido group modification of 0.1-1mM.

The concentration of the copper ion is preferably 0.2-0.5mM, and the compound of the copper ion is preferably copper sulphate, protobromide The concentration of copper, the vitamin C is preferably 2-10mM.

The protein protective agent is guanidine derivatives and/or THPTA, and the guanidine derivatives are preferably 0.5-1.5mM ammonia Base guanidine, the THPTA concentration is preferably 0.1-2.0mM, more preferably the amino of the THPTA of 0.3-1.5mM and 0.5-1.5mM Guanidine.The protein protective agent is not destroyed for protected protein, while THPTA also has the function of catalytic group sustained release, can make For the stabilizer of catalyst.

The cell pyrolysis liquid includes the salt and detergent of high concentration, and salt is preferably the Li salt of 0.15M-1M, and detergent is excellent It is selected as the detergent containing lithium ion of 0.1-1%.

The method of capture RBP of the present invention, by mixing nucleoside analog in nascent RNA, such as 5- acetylene uracils (EU), using ultraviolet induction RNA- protein cross, by capturing the RNA- albumen compositions of biotin labeling, after realizing Continuous separation and identification to nascent RNA and its RBP, the especially separation to nonpolyA-RBP and nonpolyA-RNA and mirror It is fixed.

The method of new capture RBP of the present invention, is the capture by will click on Chemical activator in RBP, basic herein On establish suitable for RBP capture methodology system.Cell pyrolysis liquid (such as 500mM LiCl, the 0.5%LiDS) packet used Salt containing higher concentration and detergent, reaching thoroughly lytic cell, the effect of increase RNA- Protein Separations effectively to go Except the interaction of the indirect RNA and albumen that are mediated between nonspecific RNA and protein binding or albumen and albumen. In addition, RNA- albumen compositions can be stable in the presence of in the detergent and high salt conditions of ionic.

Protein protectant of the present invention can inhibit to click hydroascorbic acid salt and protein side in chemical reaction Side reaction between chain, while cupric by-product hydrolysis biomolecule can also be prevented.Catalyst stabilizer THPTA is added, Has the function of catalytic group sustained release.

In general, the method and corresponding kit of new capture RBP of the present invention has the following advantages：1) more RNA and its RBP is captured to wide spectrum, including obtains nonpolyA-RNA and nonpolyA-RBP, solving the prior art can not obtain The limitation of nonpolyA-RBP.2) the RNA types captured are more, and the nonpolyA-RNA abundance of capture is high；It 3) can efficiently concentrating RBP, especially nonpolyA-RBP；More than 300 kinds of RBP that is completely new, not identified by the prior art is captured simultaneously；And specificity It is good, non-RBP albumen will not be captured；4) method compatibility and reproducible is suitable for different cell lines, different condition；5) high pass Amount, can be used for the screening or verification of large-scale RBP；6) method is easy, speed is fast, and required initial biological sample is few.

Description of the drawings

The schematic diagram of the click chemistry reaction of Fig. 1, biotin labeling；

Fig. 2, RBP silver staining testing result schematic diagram；

Fig. 3, RBP Western testing result schematic diagrames；

Fig. 4, protein spectrum data results schematic diagram；

Fig. 5, albumen Venn diagram analysis result schematic diagram；

Fig. 6, CLIP detect RBP result schematic diagrams；

Fig. 7, RNA Concentration Testing result schematic diagram；

Fig. 8, RNA distribution of lengths result schematic diagram；

Fig. 9, RNA floristic analysing result schematic diagram；

Figure 10, circular rna analysis result schematic diagram；

Figure 11, ppRNA analysis result schematic diagram；

Figure 12, eRNA analysis result schematic diagram；

The horizontal testing result schematic diagram of Figure 13, rna expression；

Figure 14, argentation detect albumen result schematic diagram.

Specific implementation mode

To facilitate the understanding of the present invention, below with reference to relevant drawings to invention is more fully described.In attached drawing Give presently preferred embodiments of the present invention.But the present invention can realize in many different forms, however it is not limited to this paper institutes The embodiment of description.Keep the understanding to the disclosure more thorough on the contrary, purpose of providing these embodiments is Comprehensively.

Unless otherwise defined, all of technologies and scientific terms used here by the article and belong to the technical field of the present invention The normally understood meaning of technical staff is identical.Used term is intended merely to description tool in the description of the invention herein The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases Any and all combinations of the Listed Items of pass.

1.RICK：Nascent RNA InteractomeUsing Click Reaction, refer to and utilize nucleotide analog The method (being reacted referring to the click chemistry of biotin labeling in Fig. 1) of incorporation and click chemistry Reaction Separation rna binding protein.

2. nascent RNA：The RNA newly transcribed, newly synthesized RNA after nucleotide analog is added, are closed within a certain period of time At RNA, these RNA have incorporation nucleotide analog (such as EU).

3.junction reads：Refer to the site that two different exons combine when analyzing circular rna, herein The binding site for refering in particular to exon flip Trim, such as the combination of exon 5 and exon 4 (5 be 5 ' ends, and 4 be 3 ' ends).

4.Back splicing：Flip Trim, the exon big from sequence are cut after leaning on to the forward exon that sorts It cuts.

5.TR(Traveling Ratio)：For many genes in transcription, transcription 50nt or so will appear a termination State, therefore can find that the signal in this region is significantly more than the regions genebody in sequencing procedure, and TR is exactly Refer to the ratio of the two position signals.

6.nonpolyA-RBP:The special rna binding protein tended in conjunction with non-polyA-RNA.

7. nucleoside analog (nucleoside analogues), one kind has the compound of similar ribonucleotide structure, The nucleoside analog can be inserted into when RNA is synthesized in RNA chains, and it carries dipolarophile group.

8. dipolarophile group is alkynyl, alkenyl or halogen, it is highly preferred that the alkynyl is acetenyl, propinyl, cyclic annular alkynes Base etc..

9. 1,3- bis- dipole group is preferably：Azido, oxidation itrile group, dizaomethyl, nitrone base, nitrile amido etc..

10. click-reaction：The lower Huisgen triazoles of cuprous ion catalysis are reacted, reaction can at normal temperatures, water or a variety of have High yield is completed in solvent.As copper catalysis azide and alkynes between covalent reaction.

11, click-reaction catalyst：The copper ion of divalent or monovalence or the combination with ascorbic acid, preferably sulfuric acid copper, bromine Change cuprous, sodium ascorbate.

12、LiDS：Lithium dodecyl sulfate.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The method or kit of the present invention can be used for capturing in organism or in the organ of in vitro culture, tissue or cell Cell described in RBP is preferably stem cell, elegans cell, mouse cell, tumour cell, immunocyte.The biological preferably rat, Mouse, nematode.

Embodiment 1：The capture of RBP (rna binding protein matter)

1. capturing RNA- albumen compositions

1.1EU mixes RNA

Cell (is obtained) from ATCC using the culture dish culture HeLa of diameter 100mm, culture solution is 10mL containing 10%FBS's High glucose medium, in 37 DEG C, 5%CO₂Under the conditions of cultivate.When cell culture to density is 80%, it is added final concentration of 0.25mM EU, cell are incubated 16 hours altogether with EU, and EU is made fully to be incorporated into the newly synthesized RNA of intracellular.

1.2 washing

PBS buffer solution washs cell twice, with the remaining culture medium of removal.

1.3 photo-crosslinking

PBS is removed, the culture dish of the cell containing step (2) is placed on ice, 254nm (0.4J/CM²) ultraviolet lamp shine 1min is penetrated, the RBP and RNA of interaction is made to form covalent bond.

1.4 fix cell

Culture dish is taken out under from ultraviolet lamp, 90% alcohol treatment cells of 5mL are added, and places 15min on ice with fixed thin Born of the same parents, PBS are washed 3 times.

1.5 permeabilized cells

PBS is removed, the Triton X-100 processing cells 15min of 5mL 0.5%, subsequent PBS washings 3 are added in culture dish It is secondary.

1.6RNA biotinylation

PBS is removed, by the click-reaction liquid of cell and 10mL in step (5) (ingredient is shown in Table 1)

It is incubated 3-30 minutes in culture dish.

1.7 washing

Click-reaction liquid in removal step (6), then washing for 10mL 0.5%Triton X-100 is added in culture dish Liquid (EDTA that final concentration of 2mM is wherein added) is washed, after washing 3 times, 10mL PBS is added and wash 2 times.

1.8 lysate lytic cells

Cell pyrolysis liquid 2mL (component is shown in Table 1) is added in culture dish, after cracking 15-30 minutes, cell scraper, which collects to obtain, to be split Product is solved, supernatant is collected by centrifugation.

1.9 prepare magnetic bead

The Streptavidin MagneSphere (Thermo Fisher Scientific) of 70uL is taken out, 300uL is added in magnetic support absorption Cell pyrolysis liquid washs 2 times, and magnetic bead is resuspended in 50uL cell pyrolysis liquids.

1.10 magnetic capture

The streptomysin avidin magnetic bead prepared in step (9) is added to the supernatant obtained in step (8), gyroscope is incubated 3hr captures biotinylated RNA- albumen compositions in pyrolysis product.

1.11 washing magnetic beads are to remove non-specific binding

Magnetic bead is adsorbed with magnetic support, stands 2min, removes lysate；500uL Lysis Buffer, gyroscope washing is added 10min removes supernatant after magnetic bead absorption；500uL Buffer 1 (ingredient is shown in Table 1) are added, gyroscope washs 10min, and magnetic bead is inhaled Attached removal supernatant；500uL Buffer 2 (ingredient is shown in Table 1) are added, gyroscope washs 10min, after magnetic bead absorption in removal Clearly, after 500uL Buffer 3 (ingredient is shown in Table 1) being added, it is placed in gyroscope washing 10min.It obtains and is attached on magnetic bead at this time RNA- albumen compositions.

1 reagent table of table (working concentration)

2. separation obtains RBP and RNA

2.1RBP

2.1.1 RBP is obtained

The RNA- albumen compositions that step 1 obtains are resuspended in the eluent (ingredient is shown in Table 1) of 50uL, 1uL is added 37 DEG C of 10mg/mL RNase A (Sigma companies) handle 1hr, and then boiling water bath boils 5min, draw supernatant.

2.1.1RBP detection

(1) silver staining detects

After SDS-PAGE electrophoretic separation, fixer (50% methanol and 5% acetic acid) is handled 40 minutes, 50% methanol and double Moisture time washing is steamed, distilled water washs after 0.02% sodium thiosulfate is handled 1 minute, 0.1%AgNO₃Dark processing 20 minutes Distilled water washs afterwards, and 2% sodium carbonate and 0.04% formaldehyde colour developing is added, and 5% acetic acid terminates reaction.

Fig. 2 shows the albumen obtained using silver staining method detection RICK methods.The Input of different condition has clearly item Band, and the sample being only added in Pull-down after EU crosslinkings obtains clear band, is not crosslinked group and RNA enzyme processing group is equal There is no the enrichment of rna binding protein.Experimental result illustrates that RICK methods are capable of the enrichment rna binding protein of differential high efficient.(note： Crosslinking indicates 365nm UV photo-crosslinkings 1 minute；RNase is RNase A, RNA and albumen can be kept compound with degradation of rna Object dissociates；EU is uracil analogues."+" indicates exist, and "-" is removal, and Input is full cell pyrolysis liquid, Pull-down tables Show and the rna binding protein to get off is enriched with from Input by RICK methods, the following drawings is identical.)

(2) western blot

Albumen loading, PAGE gel electrophoresis will be on the protein delivery to pvdf membrane after electrophoretic separation (Millipore), it is incubated overnight at 4 DEG C with primary antibody after (solution containing 5% milk of TBST configurations) closing 1hr in confining liquid It educates, after TBST washings, 4 DEG C of the secondary antibody that film and HRP are conjugated is incubated 2 hours, TBST washings, ECL plus (Amersham) inspections It surveys.

The result shows that 4 kinds of RBP, POLR2A (RNA polymerase subunit), DDX5 (RNA helicase), HNRNPK is (inhomogenous Ribonucleoprotein k), PTBP1 (poly pyrimidine channel Binding Protein 1) are captured by RICK methods, and non-RBP albumin As CTIN (is held Family's gene protein), TUBULIN (housekeeping gene albumen) will not be captured, it was demonstrated that RICK method specific enrichment rna binding proteins. As a result referring to Fig. 3.

(3) LC-MS/MS is detected

A detection methods

After 50mM TCEP and 200mM MNTS reduction albumen, 70% ethyl alcohol, the TEAB washings by several times of 8M urea and 0.25M. Again with being digested overnight albumen at 1 37 DEG C of μ g/ μ L trypsase, 3000 highly effective liquid phase chromatographic systems of Ultimate (Dionex, USA) peptide fragment mixture to be used to detach.(PhenomenexGemini-NX 3u C after upper prop₁₈11OA chromatographic columns) 95% it is molten Liquid A (20mM ammonium acetate solutions, 2MNaOH, pH 10.0) is washed, solution B (20mM HCOONH₄, 2M NaOH, 80%CAN, PH 10.0) binary linear gradient (15%-50%) elute 45 minutes to detach peptide fragment, flow velocity 0.2mL/min.UV detectors are set It is scheduled on 214/280nm, it is per minute to collect once, after the group lease making traditional vacuum drying of separation, the detection of nanometer reversed-phase liquid chromatography It is spare.Q ExactiveTM HF mixing quadrupole rods mass spectrometer systems (ThermoFisher Scientific) analyze eluent, according to Rely property acquisition mode.Mass Spectrometer Method condition:Mass range 400-1,250m/z, high resolution model (>30,000), highly sensitive Pattern (resolution ratio>15000) data are recorded, protein Pilot softwares extract data.

2 chromatographic isolation parameter of table

Mobile phase A	Mobile phase B	Flow velocity	B phase gradients
				0.1%FA	0.1%FA	300nL/min	5%-40%, 70min
5%ACN	95%ACN

B MASS SPECTRAL DATA ANALYSISs

LC-MS/MS detects 1, and albumen is divided into high confidence level by the candidate of 353 kinds of RBP by reliability assessment (720, red) and low confidence level (633, blue) albumen, counts, 720 eggs that RICK methods are identified according to unique peptide fragment (Fig. 4) in vain.Fig. 4 a show that the related coefficient of 3 experimental results is all higher than and illustrate that this method has very high repetition equal to 0.828 Property and high efficiency.Fig. 4 b show in Mass spectrometry experiments, after excluding background.RICK experimental groups are enriched a large amount of believable RNA and combine Albumen.In 720 albumen, wherein have 350 Oligo reported with known references (dT) methods capture albumen (Castello, A.et al.Insights into RNA biology from an atlas of mammalian mRNA-binding Proteins.Cell 149,1393-1406 (2012)) it coincides, and it is that RICK specific detections arrive to have 370 (51.4%) , the completely new RBP not being reported.Based on the significant difference of the isolated RNA types of two methods, may infer that above-mentioned 370 kinds of albumen are the non-polyARBP of candidate (see literary formula Fig. 5 a).RBP comparings in different cell lines show 370 kinds There is 307 kinds of protein (~83.0%) are special to be captured by RICK in candidate non-polyA RBP, this 307 albumen are all (SerlC methods are referring to document by the not certified RBP of method：Conrad,T.et al.Serial interactome capture of the human cell nucleus.Nat Commun7,11212(2016))。

Fig. 4 a:The repeatability and correlation analysis of 3 experimental results.Red word indicates the albumen in low trusted area Group, cyanic colours indicate the protein groups with interaction.

Fig. 4 b：Control group and the exclusive peptide fragment of RICK experimental groups compare in mass spectrum, and red indicates high confidence level albumen, blue Indicate that low confidence level albumen, black indicate background proteins.

Venn diagram 5a:The rna binding protein group that RICK methods obtain in HeLa cells is identified to obtain with Oligo (dT) method Rna binding protein group comparative analysis.

Literary formula Fig. 5 b:RICK methods include that Huh7, HEK293T, K562 identify the RNA combination eggs taken from different cell lines White analysis.

(3) RBP binding abilities are analyzed

Using PAR-CLIP methods, from 370 candidate nonpolyA RBP, 20 kinds of nonpolyA-RBP, test are selected The binding ability of itself and RNA (GFP is used as negative control, and CCAR2 and HNRNPK are as positive control).Fig. 6 shows crosslinking Afterwards, the albumen of the RNA binding abilities positive has strong signal (RNA-protein), candidate nonpolyA-RBP that can be tied with RNA It closes.Wherein SMARCC1, SMC1A, INTS9, CDK2, CDK4, CDK9, MCM2, MCM5, MCM6 are complete newfound albumen.Fig. 5 Middle "-" indicates to handle sample without UV crosslinking, and "+" is indicated by 365nm UV crosslinking treated sample.Anti-Flag It is detected for the expressing quantity of same position.

2.2 RNA

2.2.1 RNA is obtained

The RNA- protein complexes that step 1 obtains are resuspended in 200uL 1*PK buffer, 20uL is added 20mg/ml Proteinase Ks (Merck), after 56 DEG C of 1000rpm handle 1hr, phenol/chloroform method extracts RNA.

2.2.2 RNA is detected

(1) concentration is detected with distribution

Qubit 2.0 detects RNA concentration (Fig. 7), and Agilent2200 analyzes size and the distribution (Fig. 8) of RNA segments.From Fig. 7 is it is found that RNA a concentration of 13.8ng/uL, total amount 179ng.In Fig. 7：EU- samples are negative control, and EU+ is experimental group sample Product, Input are positive control).Fig. 8 shows to react the standard items lower of the kurtosis ratio 25nt of obtained RNA by RICK Height, main rich region are the RNA of 50nt to 6000nt.

(2) RNA is sequenced

The hg38 genome alignments of bowtie2 and people are used sequencing data, whole genome sequence density profile is generated.

A RNA floristic analysings

The isolated a variety of RNA of this method, as shown in the left sides Fig. 9：RRNA (rRNA, 45.0%), mRNA (mRNA, 24.7%), mt rRNA (mtRNA, 5.5%), other RNA (24.8%)；It has especially isolated a variety of Non- poly-A tail RNA (non polyA-RNA) such as long-chain non-coding RNA (lncRNA), circular rna (circRNA), increases Hadron RNA (eRNA), proximal promoter RNA (ppRNA) etc..The right sides Fig. 9 are shown, and the rna transcription sheet of RICK methods separation is each Ratio from the RNA of type shared by (such as Microrna (miRNA), antisense RNA (antisense) etc.).

B candidates circRNA (circular rna) is identified

Primitive sequencer reading is preprocessed with filtering removal repeated fragment, connector, low-quality sequence etc., uses high quality Sequence and genome alignment (comparisons software is Tophat).Compared with Oligo (dT) method, in the RNA of RICK methods separation The 6199 flip Trim sites circRNA are detected, and Oligo (dT) only detects 57 flip Trim sites.6199 In flip Trim site, 828 can arrive in circBase datasets comparisons；And Oligo (dT) method only has 2 comparisons and arrives (Figure 10).Confirm that this method has been successfully separated circRNA, and the circRNA quantity being enriched with is significantly higher than in Oligo (dT) method Corresponding data.

In Figure 10：Junction reads quantity (light color) after standardization, the circular rna quantity (dark color) after standardization

C ppRNA (proximal promoter RNA) are analyzed

Research finds at the gene location that rna plymerase ii pauses there is the accumulation of proximal promoter subsignal.With TR<4 base Because comparing, in transcripts of the TR more than 4, the RNA proximal promoter sequence densities peak value that RICK methods obtain is high, it was demonstrated that separation PpRNA is obtained, and Oligo methods do not detect any substantive proximal promoter subsignal, see Figure 11 c.Figure 11 d analyses are said It is illustrated in TR<RICK methods kurtosis is relatively low near PolyA in 4 gene.Prove the method for the invention in proximal promoter The concentration effect of RNA is better than Oligo (dT) method, closer to true situation in cell.

D eRNA (enhancer RNA) are analyzed

ERNA is sequenced and the result of FANTOM5 database two-way pumping station eRNAs sequence densities shows the separation of RICK methods There are stronger enhancing subsignals by RNA, and signal is significantly stronger than Oligo (dT), shows that this method successfully captures eRNA.Specifically Figure 12 is shown in analysis.Figure 12 e are shown and H3K27ac, and the RNA sequence Density Distribution of two active enhancers of H3K4me1 compares, RICK The RNA of method capture location proximates near known all possible enhancer have higher peak density.Figure 12 f are shown RICK-seq data find that there are about 6% eRNA to be accredited with eRNA database search, and are detected using Oligo (dT) method RNA in eRNA analyses find that ratio is relatively low.

(3) RT-qPCR rna expressions level detects

It is reversed using the RNA that SuperScipt III kits (Thermo Fisher Scientific) obtain separation Record is cDNA, and SYBR Green kits (Takara) RT-PCR is quantitative, and it is the table that internal reference standardizes target gene to select ACTB Up to level.

Figure 13：RICK methods are cyclic annular with enrichment in separation to be shown to the quantitative analysis of the representative RNA of multiple classification RNA On RNA, eRNA, snRNA, rRNA, lincRNA, nonpoly (A) mRNAs, opposite Oligo (dT) method has advantage, rich It catchments to put down and is significantly higher than the latter.

Embodiment 2：The injectivity optimizing of protein protective agent

Argentation detects RBP：Testing result is shown in Figure 14.(experimental condition setting is shown in Table 3, other steps are referring to embodiment 1.)

3 protein protective agent condition of table

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

If Figure 14 such as shows, in click-reaction liquid, when THPTA (condition 1) is not added, albumen can be apparent in silver staining detection See that protein band is in disperse shape, can not see clearly band, it was demonstrated that albumen is destroyed during click-reaction；When adding When entering THPTA (condition 2,3,4), it can be seen that clearly band in silver staining detection, and with the raising of concentration, effect is more Obviously.In no addition aminoguanidine (condition 8), the fuzzy form of disperse is equally presented in albumen condition；When addition protective agent amine When base guanidine (condition 5,6,7), then clearly band can be showed, and brighter also with protectant concentration increase band It is aobvious.The albumen in click chemistry reaction process can be played obviously by the two description of test protective agent THPTA and aminoguanidine Protecting effect.

Embodiment 3：The optimization of lysate

Under different lysate condition settings (table 4), other compositions are same as Example 1, compare the albumen of acquisition Relative concentration (the mutual comparison of albumen, is not the absolute concentration of itself, i.e., do not use standard items make standard curve into Row absolute quantitation), other steps are referring to embodiment 1.Test result shows that the lysate of the application can be more efficiently enriched with RBP。

4 lysate condition of table

Lysate ingredient	Albumen relative concentration (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

Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, it is all considered to be the range of this specification record.

Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.

Claims

1. a kind of method of capture RBP and/or RNA, which is characterized in that mainly include the following steps that：

3) the click-reaction liquid for the first mark molecule for containing 1,3-, bis- dipole groups is added, is marked first by click-reaction Molecule is covalently attached with the RNA mixed with nucleoside analog, and RNA is made to be marked by the first mark molecule, and the click-reaction is cuprous Huisgen triazoles under ionic catalysis are reacted, and include catalyst in the click-reaction liquid；

4) cell pyrolysis liquid is added, until sample solution is clarified, include in the cell pyrolysis liquid 0.15M-1M Li salt and The detergent containing lithium ion of 0.1-1%；

5) the second mark molecule for being connected with carrier is added, the second mark molecule can pass through with the first mark molecule association reaction The association reaction of second mark molecule and the first mark molecule will be formed by RNA- albumen compositions and be coupled to the second mark It scores on the carrier of son；

6) separation obtains the RNA- albumen compositions on carrier；

7) separation obtains the RBP and/or RNA in RNA- albumen compositions.

2. the method for capture RBP and/or RNA according to claim 1, which is characterized in that add in the click-reaction liquid Entering has protein protective agent, and the protein protective agent is aminoguanidine and/or THPTA.

3. the method for capture RBP and/or RNA according to claim 2, which is characterized in that the protein protective agent is The aminoguanidine of the THPTA and 0.5-1.5mM of 0.3-1.5mM.

4. the method for capturing RBP and/or RNA according to claim 1-3 any one of them, which is characterized in that the cell is split Solution liquid includes the LiDS of the LiCl and 0.5-1% of 0.45M-0.5M.

5. the method for capturing RBP and/or RNA according to claim 1-3 any one of them, which is characterized in that the dipolarophile Group is acetenyl；The nucleoside analog with dipolarophile group includes EU, EC, EG, EA or combinations thereof；1, the 3- bis- Dipole group is azido；The catalyst is monovalence Cu⁺Or Vitamin C and Cu²⁺Combination.

6. the method for capturing RBP and/or RNA according to claim 1-3 any one of them, which is characterized in that the step 2) Light be UV254, UV365 or combinations thereof.

7. the method for capturing RBP and/or RNA according to claim 1-3 any one of them, which is characterized in that first mark Attached bag of scoring includes biotin, digoxin, quantum dot, gold particle, nano particle or antibody；Second mark molecule includes identification The first mark molecule digoxin of Streptavidin or identification of first mark molecule biotin, the antibody of quantum dot or gold particle； The carrier includes magnetic bead or sepharose 4B.

8. a kind of kit of capture RBP and/or RNA, which is characterized in that mainly include：

1) nucleoside analog of dipolarophile group is carried；

2) contain the first mark molecule of 1,3-, bis- dipole groups；

3) carrier of the second mark molecule is carried, and with the first mark molecule association reaction can occur for the second mark molecule；

4) click-reaction catalyst, the catalyst are Cu⁺, or be Vitamin C and Cu²⁺Combination；

5) cell pyrolysis liquid includes the Li salt of 0.15M-1M and being gone containing lithium ion for 0.1-1% in the cell pyrolysis liquid Dirty agent.

9. the kit of capture RBP and/or RNA according to claim 8, which is characterized in that further include having 6) albumen guarantor Agent is protected, the protein protective agent is the aminoguanidine of the THPTA and 0.5-1.5mM of 0.3-1.5mM.

10. the kit of capture RBP and/or RNA according to claim 8, which is characterized in that the nucleoside analog is The EU of 0.05-0.45mM；The biotin that the azido group that first mark molecule is 0.1-1mM is modified；The copper ion The compound of a concentration of 0.2-0.5mM, the copper ion are copper sulphate, cuprous bromide, a concentration of 2- of the vitamin C 10mM。