阅读说明：本技术 一种检测体内rna与dna及蛋白互作的试剂盒及其使用方法 (Kit for detecting interaction of RNA, DNA and protein in vivo and use method thereof ) 是由 不公告发明人 于 2020-08-12 设计创作，主要内容包括：本发明公开了一种检测体内RNA、DNA、蛋白三者互作的试剂盒及其使用方法,包括末端脱氧核糖核苷酸转移酶,生物素标记的dCTP,阳性探针及阴性探针,细胞裂解液,链霉亲和素磁珠,杂交液,洗液,蛋白酶K及其缓冲液,无RNA酶水,DNA洗脱液,蛋白洗脱液。本发明利用生物素标记的DNA探针抓取特异RNA,并随之获取与RNA互作的蛋白与DNA。首先用交联剂将体内互作的RNA、DNA和蛋白交联在一起,再利用与RNA序列互补且标记生物素的DNA探针与交联后的裂解液孵育,之后用链霉亲和素磁珠捕获RNA、DNA、蛋白互作复合物,最后分别洗脱RNA、蛋白以及DNA。(The invention discloses a kit for detecting the interaction of RNA, DNA and protein in vivo and a using method thereof. The invention uses a DNA probe marked by biotin to grab specific RNA and then obtains protein and DNA interacted with the RNA. Firstly, cross-linking RNA, DNA and protein interacted in vivo by using a cross-linking agent, then incubating the cross-linked lysate by using a DNA probe which is complementary to an RNA sequence and is marked with biotin, capturing an RNA, DNA and protein interaction complex by using streptavidin magnetic beads, and finally eluting the RNA, protein and DNA respectively.)

1. A kit for detecting the interaction of RNA, DNA and protein in vivo is characterized by comprising terminal deoxyribonucleotide transferase, dCTP labeled by biotin, a positive probe, a negative probe, cell lysate, streptavidin magnetic beads, hybridization solution, proteinase K and a buffer solution thereof, washing solution, RNA-free enzyme water, DNA eluent and protein eluent.

2. The kit for detecting the interaction between RNA, DNA and protein according to claim 1, wherein the positive probe is a TERC probe, the negative probe is a LacZ probe, and the probe sequence is as follows:

(1) nucleotide sequence of TERC Probe 1 5'-CAGGCCCACCCTCCGCAACC-3'

(2) Nucleotide sequence of TERC Probe 2 5'-GCAAAAGCACGGCGCCTACG-3'

(3) Nucleotide sequence of TERC Probe 3 5'-CTCTAGAATGAACGGTGGAA-3'

(4) Nucleotide sequence of TERC Probe 4 5'-GCCTCCAGGCGGGGTTCGGG-3'

(5) Nucleotide sequence of TERC probe 5 5'-GGCTGACAGAGCCCAACTCT-3'

(6) Nucleotide sequence of LacZ probe 1: 5'-GCCACATATCCTGATCTTCC-3'

(7) Nucleotide sequence of LacZ probe 2: 5'-TCATCGATAATTTCACCGCC-3'

(8) Nucleotide sequence of LacZ probe 3: 5'-GAAGCAGAACAACTTTAACG-3'

(9) Nucleotide sequence of LacZ probe 4: 5'-GTATCGCTGGATCAAATCTG-3'

(10) Nucleotide sequence of LacZ probe 5: 5'-GCTGATCCTTTGCGAATACG-3' are provided.

3. The kit for detecting the interaction of RNA, DNA and protein in vivo as claimed in claim 1, wherein the formulation of the lysis solution comprises the following raw materials:

1～1.5％(W/V)SDS

Tris-Cl 50～100mmol/L

EDTA 10～15mmol/L

PMSF 0.8～1.2mmol/L；

the pH value of the Tris-Cl is 7.0-7.5.

4. The kit for detecting the interaction of RNA, DNA and protein in vivo as claimed in claim 1, wherein the formulation of said hybridization solution comprises the following raw materials:

1～1.5％(W/V)SDS

15～20％(V/V)formamide

NaCl 500～750mmol/L

Tris-Cl 50～100mmol/L

EDTA 1～2mmol/L

PMSF 0.8～1.2mmol/L

the pH value of the Tris-Cl is 7.0-7.5.

5. The kit for detecting the interaction of RNA, DNA and protein in vivo as claimed in claim 1, wherein the formulation of the proteinase K buffer comprises the following raw materials:

0.3～0.8％(W/V)SDS

NaCl 100～150mmol/L

Tris-Cl 10～20mmol/L

EDTA 1～2mmol/L

the PH value of Tris-Cl is in two ranges, and the PH value of Tris-Cl is 7.0-7.5 when RNA is extracted; when extracting DNA, the PH value of Tris-Cl is 8.0-8.5.

6. The kit for detecting the interaction of RNA, DNA and protein in vivo as claimed in claim 1, wherein the formula of said washing solution comprises the following raw materials:

0.3～0.8％(W/V)SDS

NaCl 300～400mmol/L

30-40 mmol/L sodium citrate.

7. The kit for detecting RNA-DNA-protein interaction in vivo according to claim 1, wherein the formulation of the DNA eluent comprises the following raw materials:

0.8～1.2％(W/V)SDS

NaHCO3 50～80mmol/L。

8. the kit for detecting RNA, DNA and protein interaction according to claim 1, wherein the formulation of the protein eluent comprises the following raw materials:

220～280mmol/L Tris-HCl；

8.5～11.5％(W/V)SDS；

0.4-0.6% (W/V) bromophenol blue;

45-55% (V/V) glycerol;

4-6% (W/V) beta-mercaptoethanol;

the pH value of the Tris-HCL ranges from 6.6 to 7.0.

9. The method of using the kit for in vivo RNA-DNA and protein interaction according to any one of claims 1 to 8, comprising the steps of:

preparation of S1 Probe: first, a DNA probe sequence was synthesized based on the RNA sequence design, and biotin-labeled dCTP was labeled to the 3' end of the DNA by terminal deoxynucleotidyl transferase.

S2 cell crosslinking: cells were cross-linked with formaldehyde or glutaraldehyde.

S3 cell lysis: cell lysis was performed by adding cell lysis solution.

S4 ultrasonic fragmentation of chromatin: breaking chromatin by ultrasound, wherein the fragment length range is 200-500 bp:

incubation of S5 probe with lysate: and (3) incubating the probe prepared by the S1 with the lysate after the ultrasonic treatment.

S6 magnetic bead incubation: the streptavidin magnetic beads were incubated with the complex in S5, RNA, and DNA and protein bound thereto were immobilized on the streptavidin magnetic beads by a biotin-labeled DNA probe, and unbound substances were washed away with a washing solution.

And (3) obtaining an S7 product: products include DNA, RNA and proteins.

10. The method of using the kit for detecting RNA-DNA interaction and protein interaction in vivo according to claim 9, wherein the steps S1-S7 are as follows:

preparation of S1 Probe: then, 10ul of 5 Xterminal transferase buffer, 10pmol of linear DNA, 100pmol of biotin-labeled dCTP, and 40U of terminal deoxyribonucleotide transferase were added in this order, and water was added to 50ul, the reaction was carried out at 37 ℃ for 15 to 30 minutes, and the reaction mixture was heated at 70 ℃ for 10 minutes.

S2 cell crosslinking: cells were crosslinked for 10 minutes by adding 1% formaldehyde or glutaraldehyde, and crosslinked for 5 minutes by adding glycine to a final concentration of 125nmol/L, and washed 2 times with PBS.

S3 cell lysis: 1mL of cell lysate was added to every 3-5 × 107 cells and lysed for 10 min on ice.

S4 ultrasonic fragmentation of chromatin: and (3) transferring the cells into an ultrasonic tube, setting the ultrasonic to be on for 30 seconds and off for 30 seconds, carrying out ultrasonic for 15-20 cycles, taking 10ul of the lysate after ultrasonic treatment, adding 80 mu L of proteinase K buffer solution (pH is 8.0) and 10 mu L of proteinase K, and incubating at 65 ℃ for 1-3 hours. DNA was extracted and fragment size was checked in 1% agarose.

Incubation of S5 probe with lysate: 10. mu.L of input was taken as RNA, 10. mu.L of input was taken as DNA, and 10. mu.L of input was taken as protein. 1mL of chromatin was transferred to a 15mL centrifuge tube, 2mL of hybridization solution was added, and 100pmol of probe from S1 was added. Incubating at 37 ℃ for 4-5 hours.

S6 magnetic bead incubation: washing streptavidin magnetic beads with 500 mu L of cell lysate, adding 100 mu L of magnetic beads into each sample, incubating at 37 ℃ for 30-60 minutes, centrifuging at 3000rpm for 2 minutes to remove supernatant, adding 1mL of washing solution to wash the magnetic beads, centrifuging to remove supernatant, and repeatedly washing for 5 times

And (3) obtaining an S7 product: RNA acquisition 85. mu.L of proteinase K buffer (pH7.0) was added to input RNA. 100ul of magnetic beads were resuspended in proteinase K buffer (pH 7.0). 5 μ L of Proteinase K was added to each tube, incubated at 50 ℃ for 45min, RNA extracted using RNA extraction kit or phenol chloroform, and RNA eluted using 15 μ L of RNase-free water. Obtaining DNA: to input DNA was added 85. mu.L of proteinase K buffer (pH 8.0). 100ul of magnetic beads were resuspended in proteinase K buffer (pH 8.0). 5 mu L of LProteinease K is added into each tube, the tubes are incubated for 45min at 50 ℃, DNA is extracted by a DNA extraction kit or a phenol chloroform method, and the DNA is eluted by 15 mu L of LDNA eluent. Obtaining protein: and directly adding 15 mu L of protein eluent to the input protein and the magnetic beads to elute the protein.

Technical Field

The invention relates to the field of molecular biology, in particular to a kit for detecting in-vivo RNA (ribonucleic acid), DNA (deoxyribonucleic acid) and protein interaction and a using method thereof

Background content

The non-coding RNA participates in the regulation of cell processes such as cell proliferation, migration, EMT transformation, stem cell differentiation, cell apoptosis and the like, and is an important regulation factor of organisms. The non-coding RNA mainly comprises lncRNA, circRNA, microRNA, eRNA and the like, and the functions of the RNA in cytoplasm are studied very deeply. In recent years, studies have shown that a large number of non-coding RNAs, particularly lncRNA and ernna, regulate cellular processes by participating in epigenetic processes.

Epigenetics refers to the genetic variation in gene expression without alteration of the DNA sequence. Mainly comprises DNA methylation, histone modification such as methylation and acetylation, chromatin reconstruction and the like. In eukaryotes, the most clearly studied epigenetic regulatory process is the stem cell differentiation process. In the process, three core regulatory factors, namely Nanog, Sox2 and Oct2, are regulated and controlled by transcription factors, and then an activation compound or a inhibition compound is recruited, so that genes related to sternness are activated or inhibited, and the differentiation of stem cells is regulated and controlled. Many studies have shown that this regulatory mechanism is also suitable for the study of various diseases such as cancer. In recent years. Non-coding RNA is extensively studied as an emerging epigenetic regulatory molecule. Research results show that the non-coding RNA can be directly wound on the transcription factor or used as a bracket to regulate the transcription factor, so that histone and related silencing or activating compound are regulated and controlled, and the transcription of genes is further regulated and controlled.

The interaction of non-coding RNA with DNA and protein is the key to research on the participation of RNA in apparent regulation. The prior art separates the interaction of the three into two parts, firstly researches the interaction condition of RNA and protein through RIP or RNA pull down, then researches the interaction of DNA and protein through ChIP, and finally carries out biological statistics comparison to find the protein interacted by the three parts. The method can not unify experimental conditions, can not intuitively prove the interaction of the three, can only infer the interaction through experimental experiments, and has high false positive. Many experiments have no positive control and negative control to evaluate the accuracy of the experiment, and the repeatability of the experiment is poor.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to overcome the defects of the prior art and provide a kit for simply and conveniently detecting the interaction of RNA, DNA and protein with good specificity, economy and integrity and a using method thereof.

The purpose of the invention is realized by the following technical scheme:

a kit for detecting combined interaction of RNA, DNA and protein in vivo and a using method thereof comprise terminal deoxyribonucleotide transferase, biotin-labeled dUTP, a positive probe, a negative probe, cell lysate, streptavidin magnetic beads, hybridization solution, washing solution, proteinase K and buffer solution thereof, RNase-free water, DNA eluent and protein eluent. The invention uses a DNA probe marked by biotin to grab specific RNA and then obtains protein and DNA interacted with the RNA. Firstly, cross-linking RNA, DNA and protein interacted in vivo by using a cross-linking agent, then incubating the cross-linked lysate by using a DNA probe which is complementary to an RNA sequence and is marked with biotin, capturing an RNA, DNA and protein interaction complex by using streptavidin magnetic beads, and finally eluting the RNA, protein and DNA respectively.

Further, the kit for detecting the interaction of RNA, DNA and protein is characterized in that the positive probe is a TERC probe, the negative probe is a LacZ probe, and the probe sequence is as follows:

(1) nucleotide sequence of TERC Probe 1 5'-CAGGCCCACCCTCCGCAACC-3'

(2) Nucleotide sequence of TERC Probe 2 5'-GCAAAAGCACGGCGCCTACG-3'

(3) Nucleotide sequence of TERC Probe 3 5'-CTCTAGAATGAACGGTGGAA-3'

(4) Nucleotide sequence of TERC Probe 4 5'-GCCTCCAGGCGGGGTTCGGG-3'

(5) Nucleotide sequence of TERC probe 5 5'-GGCTGACAGAGCCCAACTCT-3'

(6) Nucleotide sequence of LacZ probe 1: 5'-GCCACATATCCTGATCTTCC-3'

(7) Nucleotide sequence of LacZ probe 2: 5'-TCATCGATAATTTCACCGCC-3'

(8) Nucleotide sequence of LacZ probe 3: 5'-GAAGCAGAACAACTTTAACG-3'

(9) Nucleotide sequence of LacZ probe 4: 5'-GTATCGCTGGATCAAATCTG-3'

(10) Nucleotide sequence of LacZ probe 5: 5'-GCTGATCCTTTGCGAATACG-3'

The TERC probe sequence is an optimized sequence, the sequence is complementary with a telomerase sequence, the intervals of all the sequences are uniform, the intervals are all between 80 and 100bp, and the GC content of the probe is all between 45 and 55 percent. LacZ is a prokaryote sequence, the probe sequence of the lacZ is also strictly optimized, a middle front end sequence of the lacZ sequence is selected to design a probe, the probe interval is between 200 and 300bp, and the GC content of the probe is between 45 and 55 percent. The number of the probes is 5, which is strictly determined by us, and the capture efficiency is highest when the number of the probes is 5.

The formula of the cell lysate comprises the following raw materials:

1～1.5％(W/V)SDS

Tris-Cl 50～100mmol/L

EDTA 10～15mmol/L

PMSF 0.8～1.2mmol/L；

the pH value of the Tris-Cl is 7.0-7.5.

Wherein, SDS is an ionic strong detergent, can crack cell membrane, SDS about 1% can crack cell membrane and nuclear membrane rapidly; Tris-HCl is used as a stable buffer solution, can maintain a stable pH value, has a pH value of 7.2-7.5, is consistent with a normal pH value (about 7.4) of cells, and keeps normal forms of DNA and protein; EDTA is EDTA, which can neutralize Mg in cells²⁺、Mn²⁺、Zn²⁺The activity of various enzymes in cells is inhibited by the complexation of divalent metal ions; PMSF is a phosphatase inhibitor, mainly inhibiting the activity of phosphatase.

The formula of the hybridization solution comprises the following raw materials:

1～1.5％(W/V)SDS

15～20％(V/V)formamide

NaCl 500～750mmol/L

Tris-Cl 50～100mmol/L

EDTA 1～2mmol/L

PMSF 0.8～1.2mmol/L

the pH value of the Tris-Cl is 7.0-7.5.

Wherein, SDS is an ionic strong detergent, which can crack cell membrane, and about 1% SDS can crack cell membrane rapidly; formamide is deionized formamide, which can form hydrogen bonds with nucleic acid to denature DNA or RNA, 15-20% of deionized formamide can keep free RNA in a denatured state, and DNA keeps a double-stranded structure to better combine a probe and RNA. Tris-HCl is used as a buffer solution, can maintain a stable pH value, has a pH value of 7.2-7.5, is consistent with a normal pH value (about 7.4) of cells, and keeps normal forms of DNA and protein; nacl is used as an electrolyte, 500-750 mmol/L of Nacl is in a high-salt state and high-concentration Na⁺Can neutralize the electric property on the nucleic acid and promote the base complementary pairing action, thereby promoting the probe and the probeBinding of RNA; EDTA is EDTA, which can neutralize Mg in cells²⁺、Fe²⁺、Mn²⁺、Zn²⁺The activity of various enzymes in cells is inhibited by the complexation of divalent metal ions; PMSF is a phosphatase inhibitor, mainly inhibiting the activity of phosphatase.

The formula of the proteinase K buffer solution comprises the following raw materials:

0.3～0.8％(W/V)SDS

NaCl 100～150mmol/L

Tris-Cl 10～20mmol/L

EDTA 1～2mmol/L

the PH value of Tris-Cl is in two ranges, the PH value of Tris-Cl is 6.5-7.0 when RNA is extracted, and the PH value of Tris-Cl is 8.0-8.5 when DNA is extracted.

Wherein, SDS is an ionic strong detergent, and about 0.5% SDS can denature protein, thereby enhancing the degradation capability of proteinase K; the NaCl is used as an electrolyte, 100-150 mmol/L of NaCl maintains the normal osmotic pressure of cells; Tris-HCl is used as a buffer solution, the pH can be adjusted, RNA is single-stranded nucleic acid, the pH is neutral when extraction is carried out, DNA is double-stranded nucleic acid, and the pH is alkaline when extraction is carried out; EDTA is EDTA, which can chelate Mg in the system²⁺、Fe²⁺、Mn²⁺、Zn²⁺Divalent metal ions are added to enhance the activity of proteinase K.

The formula of the lotion comprises the following raw materials:

0.3～0.8％(W/V)SDS

NaCl 300～400mmol/L

30-40 mmol/L sodium citrate

Wherein, SDS is an ionic strong detergent, and about 0.5% SDS can denature protein and better remove protein impurities. The Nacl is used as an electrolyte, 300-500 mmol/L Nacl is higher than the osmotic pressure of cells, and is a high-salt state, high-concentration and high-concentration Na⁺Can neutralize the electrical property on the nucleic acid, make the nucleic acid in a hydrophobic state, and can better separate the nucleic acid from other impurities during elution. Sodium citrate can complex Mg²⁺、Ca²⁺、Fe²⁺Of equal metalThe ion has higher solubility, is a strong alkali and weak acid salt, is a good buffer reagent, and maintains the stability of the system PH.

The formula of the DNA eluent comprises the following raw materials:

0.8～1.2％(W/V)SDS

NaHCO3 50～80mmol/L

among them, SDS is an ionic strong detergent, and 1% SDS can reduce the adsorption of DNA on the wall of the centrifuge tube. NaHCO3 is a strong base weak acid salt, can maintain the alkalescence of the system, and is a good buffer reagent, 50-80 mmol/L Na⁺The concentration protects the DNA from degradation.

The formula of the protein eluent comprises the following raw materials:

220～280mmol/L Tris-HCl；

8.5～11.5％(W/V)SDS；

0.4-0.6% (W/V) bromophenol blue;

45-55% (V/V) glycerol;

4-6% (W/V) beta-mercaptoethanol;

the pH value of the Tris-HCL ranges from 6.6 to 7.0.

Wherein, SDS of about 10% can denature protein, and make protein carry load evenly, bromophenol blue, point out the position of the protein band while electrophoresing; the glycerol can increase the viscosity of the solution, and the liquid is easier to precipitate after the sample is loaded; about 5% of beta-mercaptoethanol can denature protein; the protein is negatively charged, and the pH value of the solution is kept to be slightly acidic, so that the pH value of the Tris-HCL is adjusted to be 6.6-7.0.

The use method of the simple and convenient kit for detecting the interaction of RNA, DNA and protein comprises the following steps:

preparation of S1 Biotin-labeled DNA Probe: first, a DNA probe sequence was synthesized based on the RNA sequence design, and biotin-labeled dCTP was labeled to the 3' end of the DNA by terminal deoxynucleotidyl transferase.

S2 cell cross-linking, and cross-linking the cell with formaldehyde or glutaraldehyde.

S3 cell lysis: cell lysis was performed by adding cell lysis solution.

S4 ultrasonic fragmentation of chromatin: breaking chromatin by ultrasound, wherein the fragment length range is 200-500 bp:

incubation of S5 probe with lysate: and (3) incubating the probe prepared by the S1 with the lysate after the ultrasonic treatment.

S6 magnetic bead incubation: the streptavidin magnetic beads were incubated with the complex in S5, RNA, and DNA and protein bound thereto were immobilized on the streptavidin magnetic beads by a biotin-labeled DNA probe, and unbound substances were washed away with a washing solution.

And (3) obtaining an S7 product: products include DNA, RNA and proteins.

The specific steps of step S1 are: then, 10ul of 5 Xterminal transferase buffer, 10pmol of linear DNA, 100pmol of biotin-labeled dCTP, and 40U of terminal deoxyribonucleotide transferase were added in this order, and water was added to 50ul, the reaction was carried out at 37 ℃ for 15 to 30 minutes, and the reaction mixture was heated at 70 ℃ for 10 minutes.

The specific steps of step S2 are: cells were added with 1% formaldehyde or glutaraldehyde, cross-linked with shaking for 10 minutes, added with glycine to a final concentration of 125nmol/L, terminated for 5 minutes, and washed 2 times with PBS.

In the step S2, if the final product is DNA or RNA, formaldehyde is used as a crosslinking agent, and if the final product is protein, glutaraldehyde is used as a crosslinking agent, and the formaldehyde can crosslink nucleic acid proteins and the like together by a crosslinking reaction,

but formaldehyde is easy to form a large cross-linked complex and is not suitable for protein cross-linking, glutaraldehyde can react with protein sulfydryl, hydroxyl, carboxyl and amino to alkylate the protein, and the large cross-linked complex cannot be formed and is suitable for protein cross-linking.

The specific steps of step S3 are: cells were collected, 1mL of cell lysate was added to every 3-5 × 107 cells, and lysed for 10 min on ice.

The specific steps of step S4 are: and (3) transferring the cells into an ultrasonic tube, setting the ultrasonic to be on for 30 seconds and off for 30 seconds, carrying out ultrasonic for 15-20 cycles, taking 10ul of the lysate after ultrasonic treatment, adding 80 mu L of proteinase K buffer solution (pH is 8.0) and 10 mu L of proteinase K, and incubating at 65 ℃ for 1-3 hours. DNA was extracted and fragment size was checked in 1% agarose.

In step S4, if the sample is not a cell, but a bacterial, fungal or plant tissue, it is recommended to set the ultrasound gradient on a 5-cycle basis, increasing 5 cycles at a time, until the sample is sonicated to the appropriate fraction.

The specific steps of step S5 are: 10. mu.L of input was taken as RNA, 10. mu.L of input was taken as DNA, and 10. mu.L of input was taken as protein. 1mL of chromatin was transferred to a 15mL centrifuge tube, 2mL of hybridization solution was added, and 100pmol of probe from S1 was added. And carrying out rotary incubation at 37 ℃ for 4-5 hours.

The specific steps of step S6 are: washing streptavidin magnetic beads with 500 mu L of cell lysate, adding 100 mu L of magnetic beads into each sample, incubating at 37 ℃ for 30-60 minutes, centrifuging at 3000rpm for 2 minutes to remove supernatant, adding 1mL of washing solution to wash the magnetic beads, centrifuging to remove supernatant, and repeatedly washing for 5 times

The specific steps of step S7 are: RNA acquisition 85. mu.L of proteinase K buffer (pH7.0) was added to input RNA. Resuspend 100. mu.L of the magnetic beads with proteinase K buffer (pH 7.0). mu.L of protease K was added to each tube, incubated at 50 ℃ for 45min, RNA was extracted using an RNA extraction kit or phenol chloroform method, and RNA was eluted using 15. mu.L of RNase-free water. Obtaining DNA: to the input DNA 85ul proteinase K buffer (pH8.0) was added. Resuspend 100. mu.L of the magnetic beads with proteinase K buffer (pH 8.0). 5ul of Proteinase K was added to each tube, incubated at 50 ℃ for 45min, DNA extracted using a DNA extraction kit or phenol chloroform, and eluted with 15. mu. LDNA. Obtaining protein: and directly adding 15 mu L of protein eluent to the input protein and the magnetic beads to elute the protein.

In step S7, different products are obtained, with different extracts. Therefore, only one product can be obtained in one experiment, and if three different products are to be obtained simultaneously, three experiments are required to be performed simultaneously, and each experiment obtains one product.

In the simple and convenient kit for detecting the interaction of RNA, DNA and protein, a positive control probe is TERC, and a negative control probe is Lac Z; detecting positive RNA as TERC and negative RNA as GAPDH; detecting that the protein is positive as TEBP and the protein is negative as GAPDH; and detecting the telomere repetitive sequence as DNA positive and the Alu repetitive sequence as DNA negative.

Compared with the prior art, the invention has the following beneficial effects:

(1) the design of the probe is flexible, and a plurality of probes grab the same RNA, thereby greatly improving the grabbing efficiency.

(2) The probes can be labeled by the terminal transferase, so that a plurality of probes can be labeled simultaneously, the labeling efficiency is greatly improved, and the cost is saved.

(3) According to different purpose requirements, the optimal cross-linking agent is selected, and the reliability of the result is ensured to the maximum extent.

(4) Can accurately obtain endogenous and RNA interactive protein, and can truly reflect the interaction condition of RNA and protein in organisms compared with RNA pull down result

(5) The obtained DNA can be subjected to qPCR or sequencing, and the flexibility is high.

(6) Compared with the method of separately purchasing synthesized terminal marker DNA, magnetic beads and reagents, the method is more economical and can be industrially produced;

(7) and (3) completeness: comprises a marking component, an enrichment component, a positive control probe, a negative control probe and a corresponding nucleic acid detection primer;

sequence listing

Nucleotide sequence of TERC Probe 1 5'-CAGGCCCACCCTCCGCAACC-3'

Nucleotide sequence of TERC Probe 2 5'-GCAAAAGCACGGCGCCTACG-3'

Nucleotide sequence of TERC Probe 3 5'-CTCTAGAATGAACGGTGGAA-3'

Nucleotide sequence of TERC Probe 4 5'-GCCTCCAGGCGGGGTTCGGG-3'

Nucleotide sequence of TERC probe 5 5'-GGCTGACAGAGCCCAACTCT-3'

Nucleotide sequence of LacZ probe 1: 5'-GCCACATATCCTGATCTTCC-3'

Nucleotide sequence of LacZ probe 2: 5'-TCATCGATAATTTCACCGCC-3'

Nucleotide sequence of LacZ probe 3: 5'-GAAGCAGAACAACTTTAACG-3'

Nucleotide sequence of LacZ probe 4: 5'-GTATCGCTGGATCAAATCTG-3'

Nucleotide sequence of LacZ probe 5: 5'-GCTGATCCTTTGCGAATACG-3'

Telomerase qPCR detection primers:

F：TGTCTAACCCTAACTGAGAAGG

R:CTCTAGAATGAACGGTGGAA

GAPDH detection primer:

F:CCAGAAGACTGTGGATGGCC

R:CATGCCAGTGAGCTTCCC

the sequence of the dotblot probe for detecting the telomere repeat sequence is as follows:

TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG

the dotblot probe sequence for detecting Alu repeat sequence is:

CACTTTGGGAGGCCGAGGCGGGCGGATCAC

drawings

FIG. 1 is a schematic and flow diagram of the present invention;

FIG. 2 is a graph showing the detection of RNA acquisition efficiency in the present invention

FIG. 3 is a comparison of the silver staining pattern of the present invention and the conventional RNA pull down result

FIG. 4 is a graph showing the detection of the protein of the present invention

FIG. 5 is a diagram showing DNA acquisition detection according to the present invention

Detailed Description

The present invention will be further described in detail with reference to the following specific examples, which are not intended to limit the invention in any manner.

Example 1

A kit for detecting combined interaction of RNA, DNA and protein in vivo and a using method thereof comprise terminal deoxyribonucleotide transferase, biotin-labeled dUTP, a positive probe, a negative probe, cell lysate, streptavidin magnetic beads, hybridization solution, washing solution, proteinase K and buffer solution thereof, RNase-free water, DNA eluent and protein eluent.

The positive probe is a TERC probe, the negative probe is a LacZ probe, and the probe sequence is as follows:

(1) nucleotide sequence of TERC Probe 1 5'-CAGGCCCACCCTCCGCAACC-3'

(2) Nucleotide sequence of TERC Probe 2 5'-GCAAAAGCACGGCGCCTACG-3'

(3) Nucleotide sequence of TERC Probe 3 5'-CTCTAGAATGAACGGTGGAA-3'

(4) Nucleotide sequence of TERC Probe 4 5'-GCCTCCAGGCGGGGTTCGGG-3'

(5) Nucleotide sequence of TERC probe 5 5'-GGCTGACAGAGCCCAACTCT-3'

(6) Nucleotide sequence of LacZ probe 1: 5'-GCCACATATCCTGATCTTCC-3'

(7) Nucleotide sequence of LacZ probe 2: 5'-TCATCGATAATTTCACCGCC-3'

(8) Nucleotide sequence of LacZ probe 3: 5'-GAAGCAGAACAACTTTAACG-3'

(9) Nucleotide sequence of LacZ probe 4: 5'-GTATCGCTGGATCAAATCTG-3'

(10) Nucleotide sequence of LacZ probe 5: 5'-GCTGATCCTTTGCGAATACG-3'

The method comprises the following specific steps:

the S1 probe was prepared by adding 10ul of 5 Xterminal transferase buffer, 10pmol of linear DNA, 100pmol of biotin-labeled dCTP, and 40U of terminal deoxyribonucleotide transferase sequentially to 50ul of water, reacting at 37 ℃ for 15 to 30 minutes, and heating at 70 ℃ for 10 minutes.

S2 cell crosslinking, adding 1% formaldehyde or glutaraldehyde into the cells, crosslinking for 10 min, adding 125nmol/L glycine, terminating crosslinking for 5min, and washing with PBS 2 times.

S3: cell collection: 1mL of cell lysate was added to every 3-5 × 107 cells and lysed for 10 min on ice.

S4 chromatin sonication: and (3) transferring the cells into an ultrasonic tube, setting the ultrasonic to be on for 30 seconds and off for 30 seconds, carrying out ultrasonic for 15-20 cycles, taking 10ul of the lysate after ultrasonic treatment, adding 80 mu L of proteinase K buffer solution (pH is 8.0) and 10 mu L of proteinase K, and incubating at 65 ℃ for 1-3 hours. DNA was extracted and fragment size was checked in 1% agarose.

S5: and (3) probe incubation: in the lysate after sonication, 10. mu.L of input as RNA was taken, 1mL of chromatin was transferred to a 15mL centrifuge tube, 2mL of hybridization solution was added, and 100pmol of probe prepared from S1 was added. Incubating at 37 ℃ for 4-5 hours.

S6, magnetic bead incubation: washing streptavidin magnetic beads with 500 mu L of cell lysate, adding 100 mu L of magnetic beads into each sample, incubating at 37 ℃ for 30-60 minutes, centrifuging at 3000rpm for 2 minutes to remove supernatant, adding 1mL of washing solution to wash the magnetic beads, centrifuging to remove supernatant, and repeatedly washing for 5 times

S7 extraction of RNA 85ul proteinase K buffer (pH7.0) was added to the input RNA. 100ul of magnetic beads were resuspended in proteinase K buffer (pH 7.0). 5ul of protease K was added to each tube, incubated at 50 ℃ for 45min, RNA was extracted using an RNA extraction kit or phenol chloroform method, and RNA was eluted with 15ul of RNase-free water.

Performing reverse transcription on the obtained RNA, and then performing qPCR detection, wherein the method specifically comprises the following steps:

and respectively carrying out reverse transcription on the obtained 15ul of IP group RNA and the obtained input group RNA by using a reverse transcription kit to obtain 20ul of cDNA, respectively taking 1ul of cDNA to carry out qPCR detection, and obtaining the capture efficiency of the probe.

FIG. 2 shows the results of qPCR, and it can be seen from FIG. 2 that the TERC probe has very high enrichment efficiency for TERC, which is about 3000 times that of the negative probe LacZ. Enrichment of LacZ negative probes was near negative. This indicates that the positive and negative probes designed by us are very effective.

Example 2

A kit for detecting combined interaction of RNA, DNA and protein in vivo and a using method thereof comprise terminal deoxyribonucleotide transferase, biotin-labeled dUTP, a positive probe, a negative probe, cell lysate, streptavidin magnetic beads, hybridization solution, washing solution, proteinase K and buffer solution thereof, RNase-free water, DNA eluent and protein eluent.

The positive probe is a TERC probe, the negative probe is a LacZ probe, and the probe sequence is as follows:

(1) nucleotide sequence of TERC Probe 1 5'-CAGGCCCACCCTCCGCAACC-3'

(2) Nucleotide sequence of TERC Probe 2 5'-GCAAAAGCACGGCGCCTACG-3'

(3) Nucleotide sequence of TERC Probe 3 5'-CTCTAGAATGAACGGTGGAA-3'

(4) Nucleotide sequence of TERC Probe 4 5'-GCCTCCAGGCGGGGTTCGGG-3'

(5) Nucleotide sequence of TERC probe 5 5'-GGCTGACAGAGCCCAACTCT-3'

(6) Nucleotide sequence of LacZ probe 1: 5'-GCCACATATCCTGATCTTCC-3'

(7) Nucleotide sequence of LacZ probe 2: 5'-TCATCGATAATTTCACCGCC-3'

(8) Nucleotide sequence of LacZ probe 3: 5'-GAAGCAGAACAACTTTAACG-3'

(9) Nucleotide sequence of LacZ probe 4: 5'-GTATCGCTGGATCAAATCTG-3'

(10) Nucleotide sequence of LacZ probe 5: 5'-GCTGATCCTTTGCGAATACG-3'

The method comprises the following specific steps:

the S1 probe was prepared by adding 10ul of 5 Xterminal transferase buffer, 10pmol of linear DNA, 100pmol of biotin-labeled dCTP, and 40U of terminal deoxyribonucleotide transferase sequentially to 50ul of water, reacting at 37 ℃ for 15 to 30 minutes, and heating at 70 ℃ for 10 minutes.

S2 cell crosslinking, adding 1% glutaraldehyde into the cells, crosslinking for 10 min, adding glycine with a final concentration of 125nmol/L, terminating crosslinking for 5min, and washing with PBS 2 times.

S3: cell collection: 1mL of cell lysate was added to every 3-5 × 107 cells and lysed for 10 min on ice.

S4 chromatin sonication: and (3) transferring the cells into an ultrasonic tube, setting the ultrasonic to be on for 30 seconds and off for 30 seconds, carrying out ultrasonic for 15-20 cycles, taking 10ul of the lysate after ultrasonic treatment, adding 80 mu L of proteinase K buffer solution (pH is 8.0) and 10 mu L of proteinase K, and incubating at 65 ℃ for 1-3 hours. DNA was extracted and fragment size was checked in 1% agarose.

S5: and (3) probe incubation: mu.L of input as protein was taken, 1mL of chromatin was transferred to a 15mL centrifuge tube, 2mL of hybridization solution was added, and 100pmol of probe from S1 was added. Incubating at 37 ℃ for 4-5 hours.

S6, magnetic bead incubation: washing streptavidin magnetic beads with 500 mu L of cell lysate, adding 100 mu L of magnetic beads into each sample, incubating at 37 ℃ for 30-60 minutes, centrifuging at 3000rpm for 2 minutes to remove supernatant, adding 1mL of washing solution to wash the magnetic beads, centrifuging to remove supernatant, and repeatedly washing for 5 times

S7 protein elution: 15ul of protein eluent is directly added into the input protein and the magnetic beads to elute the protein.

Carrying out silver staining, immunoblot detection and mass spectrometry on the obtained protein, and specifically comprising the following steps:

(1) silver staining: and (3) taking 1-3 ul of the eluted protein sample, performing polyacrylamide gel electrophoresis, separating the protein sample, performing silver staining on the polyacrylamide gel, displaying a protein band, and analyzing differential protein.

The results are shown in fig. 3, wherein a is the silver staining result of the invention, and B is the silver staining result of the RNA pull down experiment, and comparing the two results, it can be seen that the experimental result of the invention is more clear in protein band, the background of the negative probe is lower, and the experimental result is obviously superior to the traditional RNA pull down experimental result.

(2) Protein spot hybridization: spotting 3ul of the protein extract on a PVDF membrane, vacuum drying, blocking with 3% BSA for 10 min, adding primary antibody, incubating at room temperature for 2 hours, washing the membrane, adding secondary antibody, incubating at room temperature for 1 hour, washing the membrane, and adding hair liquid for exposure.

The results are shown in FIG. 4, where the positive probe was a TERC probe and the negative probe was a LacZ probe. The positive detection protein is TEBP telomerase binding protein, and the negative detection protein is GAPDH glyceraldehyde phosphate dehydrogenase. From the results in the figure, we can see that the TERC probe clearly captures the TEBP protein bound to TERC, while the negative probe LacZ clearly does not capture the TEBP protein, indicating that the efficiency and specificity of our probe for capturing the RNA-binding protein are very high.

Example 3

A kit for detecting combined interaction of RNA, DNA and protein in vivo and a using method thereof comprise terminal deoxyribonucleotide transferase, biotin-labeled dUTP, a positive probe, a negative probe, cell lysate, streptavidin magnetic beads, hybridization solution, washing solution, proteinase K and buffer solution thereof, RNase-free water, DNA eluent and protein eluent.

The positive probe is a TERC probe, the negative probe is a LacZ probe, and the probe sequence is as follows:

(1) nucleotide sequence of TERC Probe 1 5'-CAGGCCCACCCTCCGCAACC-3'

(2) Nucleotide sequence of TERC Probe 2 5'-GCAAAAGCACGGCGCCTACG-3'

(3) Nucleotide sequence of TERC Probe 3 5'-CTCTAGAATGAACGGTGGAA-3'

(4) Nucleotide sequence of TERC Probe 4 5'-GCCTCCAGGCGGGGTTCGGG-3'

(5) Nucleotide sequence of TERC probe 5 5'-GGCTGACAGAGCCCAACTCT-3'

(6) Nucleotide sequence of LacZ probe 1: 5'-GCCACATATCCTGATCTTCC-3'

(7) Nucleotide sequence of LacZ probe 2: 5'-TCATCGATAATTTCACCGCC-3'

(8) Nucleotide sequence of LacZ probe 3: 5'-GAAGCAGAACAACTTTAACG-3'

(9) Nucleotide sequence of LacZ probe 4: 5'-GTATCGCTGGATCAAATCTG-3'

(10) Nucleotide sequence of LacZ probe 5: 5'-GCTGATCCTTTGCGAATACG-3'

The method comprises the following specific steps:

the S1 probe was prepared by adding 10ul of 5 Xterminal transferase buffer, 10pmol of linear DNA, 100pmol of biotin-labeled dCTP, and 40U of terminal deoxyribonucleotide transferase sequentially to 50ul of water, reacting at 37 ℃ for 15 to 30 minutes, and heating at 70 ℃ for 10 minutes.

S2 cell crosslinking, adding 1% formaldehyde into the cells, crosslinking for 10 minutes, adding glycine with the final concentration of 125nmol/L, terminating crosslinking for 5 minutes, and washing with PBS for 2 times.

S3: cell collection: 1mL of cell lysate was added to every 3-5 × 107 cells and lysed for 10 min on ice.

S4 chromatin sonication: and (3) transferring the cells into an ultrasonic tube, setting the ultrasonic to be on for 30 seconds and off for 30 seconds, carrying out ultrasonic for 15-20 cycles, taking 10ul of the lysate after ultrasonic treatment, adding 80 mu L of proteinase K buffer solution (pH is 8.0) and 10 mu L of proteinase K, and incubating at 65 ℃ for 1-3 hours. DNA was extracted and fragment size was checked in 1% agarose.

S5: and (3) probe incubation: mu.L of input as DNA was taken, 1mL of chromatin was transferred to a 15mL centrifuge tube, 2mL of hybridization solution was added, and 100pmol of the probe prepared from S1 was added. Incubating at 37 ℃ for 4-5 hours.

S6, magnetic bead incubation: washing streptavidin magnetic beads with 500 mu L of cell lysate, adding 100 mu L of magnetic beads into each sample, incubating at 37 ℃ for 30-60 minutes, centrifuging at 3000rpm for 2 minutes to remove supernatant, adding 1mL of washing solution to wash the magnetic beads, centrifuging to remove supernatant, and repeatedly washing for 5 times

S7 extraction of DNA 85ul proteinase K buffer (pH8.0) was added to the input DNA. 100ul of magnetic beads were resuspended in proteinase K buffer (pH 8.0). 5ul of protein K was added to each tube, incubated at 50 ℃ for 45min, DNA was extracted using a DNA extraction kit or phenol chloroform method, and 15ul of DNA-free eluent was used to elute the DNA.

1ul of the obtained DNA is respectively taken to carry out dot hybridization detection or construct a DNA library.

DNA dot hybridization: dropping 1-3 ul of the obtained DNA on a nylon membrane with positive points, sealing the membrane with salmon sperm DNA for 10 minutes, washing the membrane, adding a probe, incubating at 37 ℃ for 1 hour, washing the membrane, adding streptavidin magnetic bead-HRP, incubating for 30 minutes, adding a luminescent solution, and exposing and developing.

FIG. 5 shows the results of the detection, wherein the positive probe is a TERC probe and the negative probe is a LacZ probe. The positive detection DNA is telomere repetitive sequence, and the negative detection DNA is Alu repetitive sequence. From the results in the figure, we can see that TERC probe clearly captures telomere repeat sequence binding to TERC, while negative probe LacZ clearly does not capture telomere repeat sequence, indicating that our probe has very high efficiency and specificity for capturing DNA binding to RNA.

Sequence listing

<110> Guangzhou Saichong Biotechnology Limited

<120> kit for detecting in vivo RNA, DNA and protein interaction and use method thereof

<130> 341357421

<140> 2020107989585

<141> 2020-08-12

<160> 10

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