Psoralen-mediated photocrosslinking reaction to study the interaction between RNA and protein molecules experimentally
An RNA-protein molecular complex analysis method based on the photochemical reaction of psoralen enables the analysis of the topological conformation of RNA-protein complexes under physiological conditions in the relative absence of data. This experiment was derived from "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Psoralen-mediated photocrosslinking reaction to study the interaction between RNA and protein molecules experimentally
Materials and Instruments
Proteinase K Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Denaturing gel electrophoresis buffer Non-denaturing gel electrophoresis buffer Binding buffer Hydrolysis buffer Acrylamide storage buffer RNA elution buffer Tetramethylethylenediamine Ammonium persulfate AMV Reverse transcriptase and reaction buffer Sodium phosphate 8-hydroxypsoralen 1 3-diiodopropane Potassium carbonate Acetone Petroleum ether Ethyl acetate Sodium sulfate Silica gel Aqueous trifluoroacetic acid Dimethylformamide
Polyacrylamide gel electrophoresis Photochemical reaction device Sep-pak C18 film Rotary vacuum dryer Sigmacote X-ray film
1. Polyacrylamide gel electrophoresis device.
2. Photochemical reaction device (360 nm long wavelength): Rayons RPR-100 photochemical reaction device (The Southern New England Ultraviolet Company, Branford, USA) is recommended. Other UV sources (e.g., Stratalinker) can also be used. In addition, long wavelength portable UV lamps (e.g., Model UVGL-25, San Gabriel, CA, USA) are also suitable for cross-linking, but require longer irradiation intervals.
3. Sep-pak C18 film, purchased from Waters ( MiIford, MA, USA).
4. rotary vacuum dryer.
5. Sigmacote (available from Sigma, St. Louis, MO, USA; a Sigma silylation reagent).
6. denaturing gel electrophoresis buffer (TBE): 0.09% mol/L Tris-boric acid, 0.002 mol/L EDTA. 5X TBE storage solution was prepared by dissolving 54 g of Tris and 27.5 g of boric acid in 900 ml of double-distilled water, and then adding 20 ml of 0.5 mmol/L EDTA (pH 8.0) to a volume of 1 L. The solution was stored under autoclave.
7. non-denaturing gel electrophoresis buffer: TBE buffer containing 0.1% Triton X-100.
8. 1X binding buffer: 50 mmol/L Tris-HCl (pH 7.5), 20 mmol/L KCl, and 0.1% Triton X-100.
9. 10X Hydrolysis Buffer: equal volumes of 0.5 mol/L Na2CO3 and 0.5 mol/L NaHCO3 (pH 9.2).
10. Nase T1, RNase T1 buffer: 16 mmol/L sodium citrate (pH 5.0 ), 0.8 mmol/L EDTA, 0.5 mg/mL tRNA, 3.5 mol/L urea.
11. Nase B cereos, Nase B cereos buffer: 16 mmol/L sodium citrate (pH 5.0 ), 0.8 mmol/L EDTA, 0.5 mg/ml tRNA.
12. Electrophoresis sample buffer: 9 mol/L urea, 1 mmol/L EDTA, 0.1% bromophenol blue or 98% formamide, 1 mmol/L EDTA, 0.1% bromophenol blue; non-denaturing electrophoresis sample buffer: 50 mmol/L Tris-HCl (pH 7.5), 20 mmol/L KCl, 0.1% Triton X-100, 40% ( m/V) glycerol, 0.1% ( m/V) Triton X-100, 0.1% ( m/V) Triton X-100, 0.5% ( m/V) Triton X-100, 0.1% ( m/V) Triton X-100, 0.1% ( m/V) Triton X-100. 40% ( m/V) glycerol, 0.1% bromophenol blue.
13. 40% (m/V) Acrylamide Storage Solution: 380 g of acrylamide, 20 g of methylenebisacrylamide, volume to 1 L, filter and store at 4 °C. Denaturing gel working solution contains 7 mol/L urea and 1X TBE; non-denaturing gel working solution contains 0.1% Triton X-100 and 1X TBE.
14. RNA elution buffer: TBE buffer (pH 7.2) (pH adjusted with 0.1 mol/L HCl).
15. tetramethylethylenediamine (TEMED) and ammonium persulfate.
16. psoralen-modified peptide or protein molecules, 5' or 3' end-labeled 32P RNA.
17. AMV reverse transcriptase and reaction buffer (purchased from Promega).
18. X-ray film, cassette and image processing equipment for radiographic autoradiography.
19. Proteinase K and reaction buffer: 10 mmol/L Tris-HCI (pH 7.8), 5 mmol/L EDTA, and 0.5% SDS.
20. 8-hydroxypsoralen, 1,3-diiodopropane, potassium carbonate, acetone, petroleum ether, ethyl acetate, sodium sulfate, silica gel (purchased from Aldrich).
21. 0.1 mol/L sodium phosphate (pH 7.4 ).
22. 10% aqueous trifluoroacetic acid.
23. Dimethylformamide.
II. Methods of operation
1. Synthesis of psoralen by reaction with cysteine
i.e. Synthesis of compound No. 1: 8-[ ( 3-iodopropyl-1 )oxy]psor-alen].
(1) 8-Hydroxypsoralen (0.176 g, 1 mmol ), 1,3 -diiodopropane ( 1.15 ml, 2.96 g, 10 mmol ) and potassium carbonate (1.38 g, 10 mmol ) were dissolved in 15 ml of acetone in a 100 ml footed flask.
(2) The mixture was shaken at room temperature for 10 h. The reaction was examined by thin-bond chromatography (Expanding agent: petroleum ether: ethyl acetate, 4:1 ). The reaction is complete without starting material.
(3) Vacuum concentration of the dried reactants.
(4) Dissolve the dried product in 30 ml of water and extract with 20 ml of ethyl acetate three times.
(5) Wash the extracted product with 30 ml of water, and then wash the extracted product once with 10 ml of saturated sodium chloride.
(6) Add about 2 g of sodium sulfate, and leave at room temperature for 4~6 h.
(7) Chromatograph the reaction product with silica gel, and elute the product with petroleum ether: ethyl acetate (4 : 1 ). The purified compound No. 1 (0.31 g, 85% yield) was a light yellow solid.
2. Psoralen and protein binding
(1) Insertion of active cysteines into specific sites of proteins by targeted mutagenesis or solid-phase synthesis of peptides.
(2) After purification and characterization of the protein, the protein (1 nmol) was dissolved in 100 μl of dimethylformamide along with the previously prepared Compound 1 (10 nmol).
(3) Add 50 μl of 0.1 mol/L sodium phosphate buffer, pH 7.4, and adjust the pH to 7.0.
(4) Leave for 16 h at room temperature.
(5) Add about 10 μl of 10% trifluoroacetic acid aqueous solution to the reaction solution to adjust the pH of the solution to 4.0~5.0.
(6) Extract the unreacted psoralens with 0.5 ml of chloroform and repeat 3 times.
(7) Wash the organic phase with 0.2 ml of 1% aqueous trifluoroacetic acid for three times to recover the dissolved peptide.
(8) Combine the aqueous phases and concentrate the total volume to about 200 μl in a rotary vacuum concentrator.
(9) Purify the psoralen-bound protein by TIPLC (using Zorbax 300 SB-C8 column or other column material suitable for the protein under study). The yield of the resulting psoralen-bound protein is typically about 80%.
3. Photocrosslinking
(1) Small Volume Crosslinking Reaction
A denaturing polyacrylamide gel (containing 1X TBE and 7 mol/L urea) was prepared prior to the cross-linking reaction.
The prepared gel was pre-electrophoresed for 3~4 h at 30W power.
The 32P-labeled RNA was dissolved in 20 μl of binding buffer (final concentration of about 2.5 μmol/L) and heated at 85℃ for 3 min to denature the RNA, and then allowed to cool to room temperature to refold the RNA.
Take 2 μl of RNA, 2 μl of 5X binding buffer, 1 μl of 0.1 μg/μl yeast tRNA, 1~5 μl of double-distilled water, and then add psoralen-conjugated protein to a final volume of 10 μl (the amount of protein added can be analyzed by gel electrophoresis to select the appropriate RNA/protein ratio).
The reaction solution was mixed and ice-bathed for 20 min. A small clean glass plate was scrubbed with 95% ethanol and siliconized with Sigmacote for better recovery of crosslinked samples, and the plate was set on ice.
The prepared samples were transferred to the plate and irradiated with UV light (360 nm) for 10 min to 20 min (the optimal irradiation time should be determined from the time-performance curve).
The irradiated samples were transferred to a new tube, and the remaining sample spots on the plate were rinsed with 10 μl of sample buffer and added to the tube.
10 μl of sample buffer was added to the irradiated samples, and 1 μl of 20 μg/μl yeast tRNA was added to all samples to compete for RNA-protein binding.
All samples were heated at 90°C for 3 min and electrophoresed for 2-4 h at 30W power until the front of bromophenol blue was 5 cm from the bottom of the gel.
The gel was removed and wrapped in plastic wrap and autoradiographed at -80℃.
(2) Preparation of bulk crosslinks
A large number of cross-linking reactions are required to identify cross-linking sites on the RNA molecule. The cross-linking reactions described above can be expanded 100-fold or even larger, while the final volume can be reduced to half its original size. When cross-linking by UV irradiation, it is best to spot no more than 20 μl of sample on the plate to ensure efficient cross-linking and ease of handling.
The preparation of electrophoresis gel is as described above and also requires pre-electrophoresis.
After UV irradiation, precipitate RNA and RNA crosslinks with 1/10 volume of 3 mol/L NaAc (pH 5.2) and 2.5 times the volume of anhydrous ethanol at -80°C for 30 min.
The sample was centrifuged at 16000 g for 20 min to remove the supernatant, washed with a small amount of 75% ethanol, and centrifuged again to remove the supernatant. Evacuate under vacuum.
Resuspend the product in sample buffer, add yeast tRNA, heat at 90°C for 3 min, and electrophoresis the sample, while adding end-labeled RNA as a control in the adjacent well.
After disassembling the gel, wrap it with plastic wrap, and radiate autoradiography at room temperature (short time).
The RNA and cross-links are excised from the control autoradiography, mashed, eluted in RNA elution buffer, and recovered by ethanol precipitation in a model.
4. Determination of cross-linking sites
Cross-linking sites on RNA can be determined by performing incomplete RNase digestion and alkaline hydrolysis of purified RNA crosslinks. The size of the fragments can be determined by comparison with oligonucleotides of known sequence and length (RNA digested with RNasc T1 and RNase B. cereus).
For large amounts or structurally unstable RNAs that are not amenable to alkali hydrolysis, the precise sites on the RNA involved in cross-linking can be found by performing primer extension analysis on purified RNA cross-links. The cDNA copy synthesized by reverse transcription using RNA as a template terminates at the modification or cross-linking site of the RNA, and the termination site of reverse transcription can be found by comparing it with the control sequence.
(1) Determination of RNA cross-linking site by incomplete alkaline hydrolysis reaction
Add 32P-labeled RNA into two clean centrifuge tubes, labeled No.1 and No.2; add RNA-protein cross-links into the third centrifuge tube, labeled No.3.
Add 1 μl of 10X RNase buffer, 1 μl of 0.1 U/μl of RNase T1 or RNase B. cereus to tube No.1, add double-distilled water to 10 μl, and incubate for 6~12 min at 55℃. This reaction was a fragment size control for the hydrolysis reaction.
Add 1 μl of 10X hydrolysis buffer to the other two tubes, then add double-distilled water to 10 μl and incubate at 85°C for 8 min to obtain the alkaline hydrolyzed fragments of RNA.
Sampling buffer was added to all three tubes, and the tubes were placed on ice and heated at 95℃ for 2 min, then sampled and electrophoresed with denaturing gel (the gel had been pre-electrophoresed at 30W for 4 h) until all the bands were sufficiently separated.
Radiographic autoradiography was performed.
(2) Determination of RNA cross-linking sites by reverse transcription (primer extension analysis) method
The amount of RNA or RNA-protein crosslinker used in this method is usually 0.1-1 pmol (depending on the activity of the 32P-labeled DNA primer).
Digestion of RNA-protein cross-links with Proteinase K produces RNA-protein cross-links with peptides containing only a small number of amino acids: resuspend the RNA-protein cross-links purified from the gel with 50 μl of Proteinase K Reaction Buffer, add 1 μl of Proteinase K Stock Solution (2.5 mg/ml ), and react for 30 min at 37℃.
The RNA-protein cross-links were precipitated with ethanol (as before), and the RNA products were purified by electrophoresis of denaturing gel (as before).
Add 4 pmol of uncrosslinked RNA, 4 pmol of 32P-labeled DNA primer, 2 μl of 5X reverse transcription buffer, and 10 μl of double-distilled water to the reaction tube. Add digested RNA-protein cross-links, 1 pmol of 32P-labeled DNA primer, 0.5 μl of 5X reverse transcription buffer, and 2.5 μl of double-distilled water to the reaction tube. Anneal at 75°C for 3 min, cool gradually to room temperature, and collect the products by centrifugation.
Label five reaction tubes with G, A, C, U and X. Add 3 μl of nucleotide mixture and 1 μl of 5X reverse transcription buffer to each of the five reaction tubes, add 2.5 μl of uncrosslinked RNA mixture from step (3) to each of the five reaction tubes G, A, C, U, and add the RNA-protein cross-linking mixture from step (3) to the X reaction tube.
Add 1 μl ( 5 U/μl ) AMV reverse transcriptase to each reaction tube to start the extension reaction.
The reaction was incubated at room temperature for 10 min and at 42℃ for 50 min.
The reaction was terminated by adding 12 μl of sampling buffer to each reaction tube and heated at 95℃ for 5 min for denaturing gel electrophoresis.
Radiation autoradiography.