RNaseH cleavage protection experiments for oligonucleotide targeting in RNA- protein complexes
Oligonucleotide-targeted RNase II digestion assay is a method to analyze RNA protein complexes (RNP, ribonucleoprotein particles), both for crude extracts and purified samples, and is very effective in analyzing the structure of the structural domains of RNP. This experiment is from "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
RNaseH cleavage protection experiments for oligonucleotide targeting in RNA-protein complexes
Principle
Oligonucleotide-targeted RNase II digestion experiments are a method for analyzing RNA protein complexes (RNP, ribonucleoprotein particles), both for crude extracts and purified samples, and are very effective for analyzing the structure of the structural domains of RNP.
Materials and Instruments
Protease K AMV Reverse transcriptase Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Buffer DNase Ⅰ DNase Ⅰ buffer Phenol Chloroform solution Sodium acetate RNasin E.coli RNase H TBE buffer Ammonium persulfate solution Urea gel spotting buffer Transfer buffer SSPE buffer Nucleotide solution Agarose Acrylamide RNP gel spotting buffer
Denaturing polyacrylamide gel electrophoresis Electrotransfer equipment Nylon membrane
All buffers and solutions must be free of RNAase contamination.
1. RNP preparation and DEAE chromatography
(1) Buffer D: 20 mmol/L HEPES (pH 8.0), 100 mmol/L KCl, 0.2 mmol/L EDTA, 20% glycerol, autoclaved. Add DTT to 1 mmol/L before use. add phenylmethylsulfonyl fluoride (PMSF) to 0.1 mmol/L.
(2) DEAE-Sepharose CL-6B ( Pharmacia Corporation, Piscaraway, NJ, USA).
(3) Buffer D100 : 20 mmol/L HEPES (pH 8.0), 100 mmol/L KCl, 10 mmol/L MgCl2, 20% glycerol, autoclaved. Add DTT to 1 mmol/L and PMSF to 0.1 mmol/L before use.
(4) Buffer D100: Same as Buffer D100, but with a KCl concentration of 400 mmol/L.
2. RNA preparation
(1) Proteinase K (PK): Prepare 10 mg/ml stock solution in dH2O and store at -20°C.
(2) 2X PK buffer: 20 mmol/L Tris-HCl (pH 7.5), 300 mmol/L NaCl, 25 mmol/L EDTA, 2% (m/V) SDS.
(3) RNase-free DNase Ⅰ ( 10 U/μl, Boehringer Mannheim Company, Indianapolis, IN, USA).
(4) 10X DNase Ⅰ buffer: 100 mmol/L Tris-HCl (pH 7.5), 50 mmol/L MgCl2.
(5) Phenol: chloroform solution: equal volumes of phenol and chloroform were mixed and equilibrated first with 100 mmol/L Tris-HCl (pH 7.5 ), and then with TE [ 10 mmol/L Tris-HCl ( pH 7.5 ), 1 mmol/L EDTA ] buffer.
(6) 3 mol/L sodium acetate (pH 7.0 ), autoclaved.
3. RNase H protection assay for oligonucleotide targeting
(1) DNA sequence complementary to the target RNA sequence, 12-20 nt long.
(2) RNasin ( 40 U/μl, Promega Corporation, Madison, WI, USA ).
(3) E.coli RNase H (1 U/μl, Bochringer Mannheim).
4. Northern blotting and denaturing polyacrylamide gel electrophoresis
(1) TBE buffer: 90 mmol/L Tris-borate, 2 mmol/L EDTA.
(2) Acrylamide-urea solution for denaturing polyacrylamide gel electrophoresis: prepare a solution containing 0% and 20% acrylamide (20:1 acrylamide:methacrylamide ) with 50% urea using TBE buffer, filter, and store in a brown bottle at room temperature.
(3) Prepare a 10% ammonium persulfate solution with dH2O.
(4) N,N,N',N',Tetramethylethylenediamine (TEMED).
(5) Urea gel spotting buffer: 50% urea (m/V), 1X TBE buffer, 0.05% bromophenol blue (m/V), 0.05% (m/V) xylene cyan.
(6) Transfer buffer: 10 mmol/L Tris-acetate (pH 7.8), 5 mmol/L NaAc, 0.5 mmol/L EDTA.
(7) Electrotransfer equipment (Biorad Corporation, Hercules, CA, USA ).
(8) Nylon membranes: good results were obtained using either uncharged (Amersham Company, Little Chalfont, Buckinghamshire, UK) or positively charged nylon membranes (Boehringer Mannheim Company).
(9) 3 MM chromatography paper (Whatman Company, Clifton, NJ, USA ).
(10) 20X SSPE buffer: 3.6 mol/L NaCl, 200 mmol/L Na2HPO4 (pH 7.7), 2 mmol/L EDTA.
(11) Denhardt's solution (100X): 2% ( m/V) BSA component V; 2% (m/V) Ficoll 400; 2% (m/V) polyvinylpyrrolidone.
(12) 10% SDS (m/V) stock solution.
(13) Pre-hybridization solution: 5X SSPE buffer, 5X Denhardt's solution, 0.1% SDS, 50 μg/ml tRNA.
(14) 32P-labeled oligonucleotides (1 ng/μl, 1~5X107 cpm/μg DNA), labeled with T4 polynucleotide kinase and [ λ-32P ] ATP at the 5' end.
5. Primer extension
(1) AMV reverse transcriptase (5~10 U/μl, Promega), including 5X reaction buffer.
(2) Nucleotide solution containing dATP, dCTP, dGTP, dTTP (all at a concentration of 10 mmol/L).
(3) 32P-labeled DNA oligonucleotide fragments.
6. Gel electrophoresis of RNP in the natural state.
(1) Agarose.
(2) 20% acrylamide (80:1 acrylamide:methylene bisacrylamide) solution.
(3) RNP gel spotting buffer: 0.3X TBE buffer, 80% glycerol, 0.05% (m/V) bromophenol blue (m/V), 0.05% (m/V) xylene cyanide.
(4) 1.5 mm spacer and small comb for vertical plate gel.
II. Test Methods
1. Preparation of RNP
RNP can be prepared either in whole cells, cytoplasm or nucleus. although RNase H cutting assays can be successfully performed in crude extracts, the accuracy of these results needs to be further verified because the same RNA can be present in different RNPs or in different conformations, and also the degradation of RNP and RNase H cutting products is a serious problem when performing the assays in crude extracts. is a serious problem. These potential problems can be avoided to some extent by purification of RNP by DEAE chromatography, which enriches the spliceosome snRNP, but this process adds additional conditions to RNA-protein interactions, e.g., many RNPs are compressed into tightly packed and stable complexes when eluted with high salts, which affects their analysis.
All steps of DEAE chromatography must be carried out at 4°C.
(1) Prepare a 5 ml DEAE-Sepharose column for the graded separation of 50 ml of crude extract dialyzed through Buffer D. Wash the column with water and ethanol. Wash the column with water and ethanol, then wash the column with a small amount of Buffer D100. Load the column with a 1:1 suspension of D100 in DEAE-Sepharose, preferably with a peristaltic pump, and wash the column with 5 ml of Buffer D100.
(2) Adjust the concentration of MgCl2 in the crude extract to 10 mmol/L. Slowly add the crude extract into the chromatographic column, taking care not to let the column run dry, and wash the column with 50 ml of buffer D100 .
(3) Switch to buffer D100 to elute RNP, collect 15 tubes of eluent, 1 ml per tube.
(4) Determine the protein peak by analyzing the snRNP peaks in the fractions (usually between fractions 5 and 10), collect the appropriate portion, freeze in liquid nitrogen, and store at -70 °C.
2. RNA preparation
To determine whether the protection of RNA in the RNase H protection assay is due to a protein or RNA conformation that prevents the enzyme from approaching, the RNase H cutting assay must be controlled with the same ionic strength as the RNase H cutting assay that removes the protein followed by the RNA. The amount of RNA used for the extract and control reactions should be equal, and the amount for the following steps can be increased or decreased as required.
(1) Mix 100 μl of extract or RNP preparation with 100 μl of 2X Proteinase K buffer (protein-rich extract becomes slightly turbid at this point), add Proteinase K to a final concentration of 0.2 mg/ml, and incubate the reaction solution at 50°C for 1 h. The reaction solution should be incubated with the same ionic strength as the RNase H cutting assay.
(2) Phenol:chloroform extraction: add equal volumes of phenol:chloroform, shake for 10 s, centrifuge at 16,000 g for 2 min, transfer the supernatant to a new centrifuge tube, and repeat the extraction if there is a white protein layer between the two liquid surfaces.
(3) Precipitate RNA with 3 times volume of anhydrous ethanol, wash with 70% ethanol and dry the precipitate.
(4) Dissolve the precipitate with 89 μl of water, add 10 μl of DNase Ⅰ buffer and 1 μl of DNase Ⅰ, incubate at 37℃ for 30 min.
(5) Extract with equal volume of phenol: chloroform, add 1/10 volume of 3 mol/L NaAc (pH 7.0) and 3 times the volume of anhydrous ethanol for precipitation, wash with 70% ethanol and dry the precipitate.
(6) Dissolve the RNA precipitate with 100 μl of water and store at -20℃.
2. Oligonucleotide-targeted RNase H protection assay
Knowledge of the secondary structure of the RNA is important for the selection of DNA oligonucleotide hybridization strand targeting sequences, which are mostly located in single-stranded or circular regions. Although a 4-base pair annealed oligonucleotide is sufficient as a substrate for RNase H cleavage, an oligonucleotide of no less than 12 base pairs is usually required for efficient specific pairing, and it is desirable to compare and evaluate empirically obtained DNA oligonucleotide sequences targeting different sequences of RNA. Primer extension efficiency can be used to select or test oligonucleotides for their ability to bind directly to a specific RNA.
(1) Standard RNase protection assays are typically performed in 25 μl systems containing 15 μl of extract or RNP preparation, 40 mg/ml DNA oligonucleotide, 12 mmol/L HEPES (pH 8.0), 60 mmol/L KCl, 3 mmol/L MgCl2, 1 mmol/L DTT, 20 U RNasin, 1 U E.coli RNasin, 1 U E.coli RNasin, and 1 U E.coli RNasin. 1 U E.coli RNase H. Controls were performed using the same amount of RNA from the same source under the same ionic conditions. The reaction solution was incubated at 30 ℃ for 60 min.
(2) For the analysis of RNA fragments, RNA was prepared by adding 75 μl of redistilled water, 100 μl of 2X PK buffer, and 4 μl of Proteinase K to terminate the reaction, as described previously [steps (1)-(5) in 2 "Preparation of RNA"].
(3) If RNP fragments are analyzed, the reaction solution can be used directly in the following experiments.
4. Analysis of RNase H Cutting Products - RNA Fragments
Analysis of RNA fragments after RNase H cutting can be performed by Northern blot or primer extension.
(1) Northern blot
In RNA Northern blot analysis, the probes are generally chosen to be complementary fragments of DNA oligonucleotides, which can specifically detect very small RNA fragments. Therefore, in the following procedure, special hybridization and washing conditions are designed for oligonucleotide probes.
① Denaturing PAGE gel electrophoresis: mount a small vertical gel (17 cm X 18 cm) glass plate with a spacer thickness of 0.4 mm.
② 8% gel preparation: mix 10 ml of 20% acrylamide/50% urea and 15 ml of 0% acrylamide/50% urea storage solution, then add 200 μl of 10% APS and 20 μl of TEMED, mix well, and immediately fill the gel. After polymerization is complete (minimum 3 min required), pre-electrophoresis at 800V for 30 min with 1X TBE buffer.
(iii) The RNA precipitate was re-dissolved in urea spotting buffer and the samples were heated at 90°C for 1 min prior to sampling. The samples were briefly centrifuged to collect the samples from the bottom of all reaction tubes, and a suitable amount of the top sample was taken and electrophoresed with TBE buffer at 800V until the bromophenol blue reached the bottom of the gel.
④ Electrotransfer the RNA from the polyacrylamide gel to the nylon membrane, make a sandwich structure to ensure that the gel and the nylon membrane can be in close contact with each other, soak two sponge pads and three sheets of 3 MM filter paper in the transfer buffer, at the same time, equilibrate the gel and the nylon membrane in the transfer buffer, in a gripper to place the layers in the following order, the sponge pads, the two layers of 3 MM filter paper, the gel, and the nylon membrane, The layers were placed in a holder in the following order: sponge cushion, two layers of 3 MM filter paper, gel, nylon membrane, the second layer of 3 MM filter paper, sponge cushion, avoiding air bubbles between the layers when placing the layers, and after placing the layers, they were mounted on the electrotransfer device, and the transfer buffer was added.
⑤ Even level the sandwich, dry the nylon membrane in air for 10 min, cross-link the RNA and the membrane using a UV cross-linker (312 nm), with the RNA side facing the light source, and irradiate the membrane for 10 min at the maximum intensity, and then dry the membrane completely in air.
⑥ Pre-hybridization with hybridization solution was performed at 37℃ for not less than 2 h. Then 200 ng of 32P-labeled oligonucleotide probe was added and hybridization was performed at 37℃ overnight.
(vii) Wash the membrane twice with 5X SSPE, 0.1% SDS solution, and then twice with 2X SSPE, 0.1% SDS solution, each time at 37 ℃ for 15 min.
(8) Wrap the wet nylon film completely with plastic film and place it in a dark box for X-ray film development.
(2) Primer extension
① Reaction: Dissolve RNA precipitate with 13.5 μl of dH2O, add 4 μl of 5X reverse transcription buffer and 1 μl of 32P-labeled oligonucleotide probe (1 ng/μl), incubate at 70°C for 5 min, and then incubate on ice for 5 min.
② Transcription reaction: Add 1 μl of 10 mmol/L dNTP and 0.5 μl of reverse transcriptase to 18.5 μl of annealing reaction solution and incubate at 42°C for 45 min.
Incubate at 42°C for 45 min. ③ Add 2 μl of 3 mol/L NaAc (pH 7.0) and 60 μl of anhydrous ethanol to precipitate the nucleic acid, dry the precipitate, dissolve the precipitate with urea spotting buffer, and then separate the nucleic acid by denaturing PAGE electrophoresis.
④ Dry the gel and develop the X-ray film.
5. Analysis of RNase H cut product-RNP fragments
If site-specific cleavage occurs, analysis of the RNP fragments produced by RNase H cleavage provides insight into the structure of the structural domains, i.e., which part of the protein binds to which RNP sub-structural domain. Tests to isolate specific RNPs from different RNPs and RNAs include glycerol gradient centrifugation, CsCI density gradient centrifugation, and RNP nondenaturing gel electrophoresis, and RNP RNA can be detected by Northern blot or primer extension as described above. The procedure for non-denaturing gel electrophoresis analysis is given below.
(1) Mount a small vertical gel glass plate with a 1.5 mm spacer.
(2) Prepare acrylamide solution: 12.6 ml 20% acrylamide (acrylamide: methylene bisacrylamide 80:1), 14.4 ml 50% glycerol, 2.2 ml 10X TBE buffer, 5.8 ml water, and 0.96 ml 10% APS are mixed well.
(3) Add 0.36 g of agarose to 36 ml of water, melt in the microwave, and wait until the temperature of the agarose has dropped to approximately 60 °C before adding it to the acrylamide solution.
(4) Add 35 μl of TEMED and mix well. Pour the gel immediately and allow the gel to coalesce for at least 1 h. The gel should not be too thick.
(5) A standard oligonucleotide-targeted RNase H cutting reaction solution plus 5 μl of RNP spotting buffer can be used directly as electrophoresis samples. For comparison, dilute one portion of the reaction solution with 75 μl of water, and prepare the RNA by the method described previously [Steps (1)~(3) in 2 "Preparation of RNA"] by dissolving the RNA in 10 μl of RNA buffer and then adding the RNA to 10 μl of RNA buffer. Dissolve RNA in 10 μl of water, add 15 μl of Buffer D and 5 μl of RNP Spotting Buffer.
(6) Pre-electrophoresis with 0.3X TBE at 60V for 30 min at room temperature.
(7) Spot sample and electrophoresis at 175V (17 V/cm) for at least 4~5 h until the bromophenol blue dye reaches the bottom of the gel. Longer gels may be required due to variations in RNP fragment size. The gel temperature should be constant; elevated temperatures may cause denaturation of the RNP.
(8) Electrotransfer the RNA from the RNP from the gel to the nylon membrane. The transfer should be carried out for 5 h at a constant pressure of 60 V at 4°C. It is important to make sure that the temperature of the transfer buffer does not rise, and to use a condensation system if necessary. After transfer, the nylon membrane was processed as described previously [ 4 "Analysis of RNA fragments by RNase H cutting products" (1) "Northern blot" steps ④~7].