Photo-activated nucleotide analog-mediated RNA-protein cross-linking experiments
Photochemical cross-linking technology is an effective means to study the interaction between RNA and protein in nuclear enameled egg complexes. The basic principle is that without any chemical modification of the RNA-protein natural complexes in the short wavelength ultraviolet (254 nm) radiation, the nucleotides in the RNA and amino acids in the protein will produce photochemical reactions, transformed into an active state, and then form intermolecular covalent crosslinks. This experiment is from "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Light-activated nucleotide analog-mediated RNA-protein cross-linking experiments
Principle
Photochemical cross-linking technology is an effective means to study the interaction between RNA and protein in nuclear enameled egg complexes. The basic principle is that without any chemical modification of RNA - protein natural complexes in the short wavelength ultraviolet (254 nm) irradiation, RNA nucleotides and amino acids in proteins will produce photochemical reactions, transformed into an active state, and then form intermolecular covalent crosslinks.
Materials and Instruments
FPLC purified nucleotides Sephadex G-50 Move -Materials and equipment For more product details, please visit Aladdin Scientific website.
Buffer A Glycerol Acetylated Bovine Serum Albumin Buffer B Buffer C Heparin Buffer F Phenol Chloroform Isoamyl Alcohol DNaseI without RNase Azidobenzoyl Bromide Buffer D Buffer C TBE Buffer Protein Sampling Buffer SDS Denaturing Polyacrylamide Gel Transfer Buffer Silver Dye Reagent
Ultraviolet light source number Centrifuge tube X-ray film Sensitization screen Cellulose nitrate membrane Electrotransfer mounts
1. full-length nucleotide analog-containing RNA synthesis
All buffer preparations should be made with distilled and sterilized deionized water
(1) Buffer A: 20 mmol/L Tris-HAc (pH 8.0), 10 mmol/L Mg(Ac )2, 50 mmol/L potassium glutamate.
(2) 5% (V/V) glycerol, 40 mmoI/L Na2EDTA (pH 8.0), 40 μg/mL acetylated bovine serum albumin, stored at -20℃.
(3) Buffer B: 1.5 mol/L NH4Ac, 37.5 mmol/L Na2EDTA (pH 8.0), 50 μg/ml RNase-free tRNA, stored at 20℃.
(4) Buffer C (2X): 8 mol/L urea, 0.1% (m/V) xylene cyan, 0.1% (m/V) bromophenol blue.
(5) FPLC purified nucleotides, ATP, CTP, GTP, UTP, stored at -80°C.
(6) 5-SH-UTP, 5-SH-CTP, 5-APAS-UTP and 5-APAS-CTP were prepared as a 1-3 mol/L aqueous solution and stored at -80°C, protected from light.
(7) T7 phage RNA polymerase or E. coli RNA polymerase.
(8) The working concentration of [ α-32P ] GTP ranges from 105 cpm/pmol to 107 cpm/pmol .
(9) 1 mol/L NH4Ac was prepared in anhydrous ethanol and stored at room temperature.
(10) Heparin: 0.5 mg/ml aqueous solution, stored at -20 °C.
(11) Buffer F: 40 mmol/L Tris-HCl (pH 7.6), 15 mmol/L MgCl2, 10 mmol/L β-mercaptoethanol, acetylated bovine haematocrit.
(12) Phenol: chloroform: isoamyl alcohol (25:24:1 ).
(13) 80% (V/V) ethanol.
(14) RQ1 RNase-free DNase I ( Promega Corporation).
(15) UV light source: Spectroline model XX-15B (Spectroline).
(16) 1 ml centrifuge tube.
2. RNA Synthesis of Nucleotide Analogs with Photocrosslinking Activity Doped on the Molecular Surface
(1) 100 mmol/L azidophenacyl bromide (APB, Sigma) was dissolved in dimethyl sulfate.
(2) Sephadex G-50, DNA grade.
(3) Buffer D: 30 mmol/L Tris-HCl ( pH 7.0), 10 mmol/L KCl, 0.5 mmol/L Na2EDTA ( pH 8.0).
3. Cross-linking of RNA and protein
(1) Buffer C (2X): 8 mol/L urea, 0.1% (m/V) xylene cyan, 0.1% (m/V) bromophenol blue.
(2) 10X TBE buffer: 0.89 moI/L Tris base, 0.89 mol/L boric acid, 20 mmol/L EDTA, stored at room temperature.
(3) X-ray film.
(4) Sensitized screen.
(5) RNase T1 digestion buffer: 25 mmol/L sodium citrate, 1 mmol/L Na2EDTA (pH 8.0), stored at -20 °C.
(6) RNase T1.
(7) 2X Protein Sampling Buffer: 60 mmol/L Tris-HCl (pH 8.0 ), 60 mmol/L DTT, 3.4% (m/V) SDS, 17% (V/V) glycerol, 0.02% (m/V) bromophenol blue; DTT was added before use.
(8) SDS denaturing polyacrylamide gel.
(9) Transfer buffer: 25 mmol/L Tris, 192 mmol/L glycine, 20% (V/V) methanol, stored at 4℃.
(10) Cellulose nitrate membrane.
(11) Electrotransfer mount.
(12) Silver dyeing reagent: 2% (m/V) sodium citrate, 0.8% (m/V) ferrous sulfate, 0.1% (m/V) silver nitrate, stored at 4 °C.
II. Methods of operation
1. Full-length RNA synthesis with nucleotide analogs
(1) Add 100 nmol/L template DNA, 100 nmol/L E. coli RNA polymerase and buffer A (or T7 phage polymerase and buffer F) to 100 μl of the reaction system and incubate at 37℃ for 5 min.
(2) 避光加入 100 μmol/L ATP，10 μmol/L UTP 或 CTP，20 μmol/L [ α-32P ] GTP，150 μmol/L 5-APAS-UTP 或 5-APAS-CTP，以及加入浓度逐渐递增的 UTP 或 CTP ( 0 μmol/L，1 μmol/L，5 μmol/L， 10 μmol/L, 20 μmol/L) and incubated for 5 min.
(3) Add 1 μl of heparin and incubate at 37 ℃ for 10 min.
(4) Add RQ1 RNase-free DNase I (5U) and incubate at 37 ℃ for 10 min.
(5) Add 100 mmol/L MgCl2 and 20 μg of RNase-free tRNA.
(6) Add an equal volume (100 μl) of phenol: chloroform: isoamyl alcohol (25:24:1), mix well with shaking, and centrifuge at 14000 g for 5 min.
(7) Take the upper aqueous phase into a new centrifuge tube, add 3 times the volume (300 μl) of ethanol containing 1 mol/L NH4Ac, mix well, and centrifuge at 14000 g for 5 min in an ice bath for 10 min.
(8) Carefully remove the supernatant, wash the RNA with 100 μl of 80% (V/V) ethanol and centrifuge at 14000 g for 5 min.
(9) Carefully remove the supernatant and allow to dry.
(10) Dissolve RNA in 100 μl deionized water or buffer.
(11) Aliquot the reaction product and add half of it to a clean centrifuge tube, place the tube 1.5 cm from a UV (302 nm max) light source and irradiate for 2 mm at room temperature, and place the other half of the reaction product at room temperature and out of the light during irradiation.
(12) Add 100 mmol/L DTT to reduce the residual azide, and avoid light for at least 5 min (no need to avoid light for subsequent operations).
(13) Add an equal volume of 2X buffer C; incubate at 95°C for 2 min; cool in an ice bath; electrophoresis with 7 mol/L urea/poly(endocene amide) gel (gel concentration and length depend on the size of the RNA to be detected, and there is a correlation between the size of the RNA and the concentration of the gel, which is as follows: greater than 100 nt, 6%; 30~100 nt, 5%; less than 30 nt, 25%).
(14) Lay filter paper, gel, plastic wrap, X-ray film, and sensitized screen in a cassette; radiograph at -80°C overnight.
(15) Analyze the optimal UTP or CTP concentration from the autoradiography results.
(16) Perform RNA-protein cross-linking according to the method "RNA-Protein Cross-linking".
2. RNA Synthesis of Nucleotide Analogs Doped on Molecular Surfaces with Photocrosslinking Activity
(1) Prepare and purify RNA inserted into 5-SH-UTP or 5-SH-CTP according to steps (1) to (10) in Method "Full-length RNA synthesis with nucleotide analogs", and adjust pH 7.0.
(2) Add APB (dissolved in dimethyl sulfone) at a final concentration of 5 mmol/L in an environment protected from light.
(3) Incubate for 2 h at room temperature away from light.
(4) Preparation of Sephadex G-50 column: Weigh 7 g of Scphadex G-50, add 100 ml of deionized water and autoclave the suspension. Add 1 ml of Sephadex G-50 suspension to 1 ml of column, centrifuge at 3500 g for 3 min and repeat until the gel volume reaches 750 μl.
(5) Equilibrate the column with 200 μl of Buffer D. Centrifuge at 3500 g for 4 min and repeat three times, discarding the eluate.
(6) Add 200 μl of reaction solution, protected from light [the ratio of gel to sample solution (750:200) is critical].
(7) Centrifuge at 3000 g for 3 min.
(8) Collect the effluent and perform RNA-protein cross-linking according to the method "RNA-Protein Cross-linking".
3. RNA-protein cross-linking
(1) In RNA-protein cross-linking, use the optimal UTP and CTP concentrations optimized in the previous experiments. Repeat steps (1)~(9) in Method 1, resuspend the RNA in sterilized deionized water or a suitable buffer (according to the corresponding RNA or protein), and add a suitable concentration of the protein that may bind to the RNA.
(2) Aliquot the reaction product, add half of it to a clean centrifuge tube, place the tube at 1.5 cm from the UV light source and irradiate it for 2 min at room temperature (some binding requires specific reaction temperature), and place the other half of the reaction product at room temperature and away from light to serve as a negative control during the irradiation process.
(3) Add DTT to a final concentration of 100 mmol/L and leave for at least 5 min, protected from light (no need to protect from light in subsequent steps).
(4) Remove a small amount of sample from the unirradiated sample to identify the correct length of RNA used. [See step (13) in the method "Synthesis of full-length RNA containing nucleotide analogs"].
(5) Add an equal volume of RNase T1 digestion buffer and 10 U/μl RNase T1 to the remaining sample and incubate at 37°C for 10 min.
(6) Add an equal volume of 2X Protein Sampling Buffer, heat at 94°C for 2~3 min, and perform denaturing SDS-PAGE electrophoresis.
(7) Conventional sandwich electrotransfer method: 500 mA for 2 h.
(8) Remove the membrane and silver stain until a band is seen (about 5 min), then dry under air.
(9) X-ray film, radiographic autoradiography at -80°C until crosslinked proteins are visible (several days may be required for proteins with low crosslinking efficiency). 