RNA footprinting and modification interference analysis experiments
RNA footprinting and modification interference analysis experiments
RNA footprinting and modification interference analysis are techniques used to study RNA-protein interactions.The principles of RNA sequencing and its structural analysis are the theoretical basis of RNA footprinting assays, which are based on the use of base-specific reagents to cleave RNA ends labeled with radioisotopes under denaturing, semi-denaturing, or natural conditions. RNA sequencing and its structural analysis are the theoretical basis of RNA footprinting assays, which are based on the use of base-specific reagents to cleave RNA labeled with radioisotopes under denaturing, semi-denaturing or natural conditions. This experiment is based on the "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
RNA footprinting and modification interference analysis experiments
Principle
RNA footprinting and modification interference analysis are techniques used to study RNA-protein interactions.The principles of RNA sequencing and its structural analysis are the theoretical basis of RNA footprinting assays, which are based on the use of base-specific reagents to cleave RNA ends labeled with radioisotopes under denaturing, semi-denaturing, or natural conditions. RNA sequencing and its structural analysis principles are the theoretical basis of RNA footprinting assays, which are based on the use of base-specific reagents under denaturing, semi-denaturing, or natural conditions to cleave RNAs labeled with radioisotopes at their ends.
Materials and Instruments
Protease K NTPαS complex Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
RNA enzyme inhibitors RNase T1 RNase V1 RNase T2 Aniline Citric acid Ethanol Ferric ammonium sulfate Sodium ascorbate EDTA NaAc Thiourea Diethyl pyrocarbonate N-Ethyl-N-nitrosourea Dimethyl sulfate Hydrazine anhydrous
Silanized Centrifuge Tubes
1. RNAase inhibitors ( RNasin et al.).
2. RNase T1.
3. RNase B.cereus ( Bc ).
4. RNase V1.
5. RNase T2.
6. protease K.
7. redistilled phenol, phenol:chloroform (1:1 volume ratio) and chloroform.
8. aniline.
9. NTPαS complex (New England Nuclear-Dupont).
10. 0.5 ml and 1.5 ml silanized centrifuge tubes.
11. 0.1 mol/L citric acid.
12. Ethanol.
13. 0.4 mol/L ammonium iron sulfate.
14. 2 mol/L sodium ascorbate.
15. 0.8 mmol/L EDTA ( pH 8.0 ).
16. Fe-EDTA.
17. 0.6% H2O2 (V/V).
18. 0.3 mol/L NaAc (pH 3.8).
19. 20 mmol/L thiourea (thiourea ).
20. diethyl pyrocarbonate (DEPC ).
21. N-ethyl-N-nitrosourea (Handle with care, strongly carcinogenic).
22. Dimethyl sulfate ( DMS ).
23. 3 mol/L NaAc (pH 4.5).
24. 200 mmol/L NaBH4.
25. anhydrous hydrazine.
26. Pure double-distilled aniline, revaporized under nitrogen prior to use and stored at -20°C under nitrogen protected from light.
27. 1-butanol.
28. 1% (m/V) sodium dodecyl sulfate (SDS ).
29. DEPC-treated H2O: 1 ml DEPC added to 1 L of water, left overnight with vigorous shaking, autoclaved.
30. 10X RNA-Protein Binding Buffer: varies according to the proteins analyzed, see notes for details.
31. calf liver tRNA: incubate at 37°C with 50 μg/ml proteinase K for 2 h, extract, ethanol precipitate, redissolve to a final concentration of 10 mg/ml.
32. 1.5X Gel Sampling Buffer: 10 mol/L urea, 1.5X TBE, 0.015% (m/V) bromophenol blue, 0.015% (m/V) xylene cyanine, 1% SDS, filtered to remove bacteria and stored at -20°C.
33. 10X Hydroxyl Buffer: 50 mmol/L NaHCO3/Na2CO3 (pH 9.2), 1 mmol/L EDTA (pH 8.0), filtered for sterilization and stored at -20°C.
34. 2X extraction buffer: 50 mmol/L Tris-HCl (pH 7.4), 300 mmol/L NaCl, 0.05 % NP-40, 10 μg/ml tRNA, 1 % SDS, filtered for sterilization and stored at -20 °C.
35. 2X Hydroxyl termination/extraction buffer: 50 mmol/L Tris-HCl (pH 7.4), 300 mmol/L NaCl, 0.05% NP-40, 10 μg/ml tRNA, 1% SDS, 20 mmol/L thiourea, filtered for sterilization and stored at -20 °C.
36. freshly saturated N-ethyl-N-nimxsoure (ENU) solution: add excess ENU (careful handling, strongly carcinogenic) to 100 μl ethanol, centrifuge to remove undissolved ENU, and use the supernatant as a base modification reagent.
37. 10X ENU modification buffer: 500 mmol/L HEPES-KOH (pH 8.0), 10 mmol/L EDTA, filtered and sterilized and stored at -20 °C.
38. I2 Stock Solution: 10 mmol/L in ethanol.
39. 2X dimethyl sulfate extraction buffer: 50 mmoI/L Tris-HCl (pH 7.4), 300 mmol/L NaCl, 0.05% NP-40, 10 μg/ml tRNA, 1% SDS, 125 mmol/L mercaptoethanol, filtered for sterilization and stored at -20℃.
40. hydrazine/0.5 mmoI/L NaCl and hydrazine/3 mmol/L NaCl: NaCl was prepared by dissolving in anhydrous hydrazine to the appropriate concentration.
II. Methods of Operation
To successfully perform RNA-protein interaction footprinting and modification analysis experiments, it is necessary to first establish the conditions for specific RNA-protein interactions and understand their binding characteristics. Nuclease footprinting typically yields low-resolution information on RNA-protein complexes, and nucleases are significantly active under a wider range of conditions, including physiological conditions, whereas chemical modification reagents require more stringent conditions in some cases. However, nuclease is relatively large and its activity may be affected by neighboring bases, and chemical probes are much smaller than nuclease, approaching the size of solvents, and therefore provide finer-grained information on RNA-protein interactions. In order to characterize the full range of RNA-protein interactions, the best means is to first use nuclease to localize the interaction site, and then use chemical probes and modification interferences to investigate the mode of interaction. The best way to characterize RNA-protein interactions is to first use nucleases to locate the interaction site, and then use chemical probes and modification interference analysis to investigate the mode of interaction.
For modification interference studies, it is important to have a reliable method to isolate RNA-protein complexes from RNA-protein binding reaction solutions. Gel mobility shift analysis, filter bonding, and immunoprecipitation are commonly used to isolate RNA-protein complexes, and the key is to not disrupt the binding between RNA and protein. This experiment requires the use of gel-purified RNA labeled at one end only, and end labeling is suitable for RNA molecules with less than 200 nucleotides or within 200 nucleotides of the interacting region, whereas for RNA molecules with more than 200 nucleotides or with more than 200 nucleotides of the interacting region, it is preferable to identify the cleavage site by using a primer extension with a reverse-transcriptase enzyme.
1. RNA enzyme footprinting
RNA enzyme footprinting gives a low-resolution map of the RNA regions interacting with the protein. Before performing the footprinting reaction, the concentration of nuclease is determined so that only about one out of 10 RNA molecules is cleaved, which can be accomplished by applying different enzyme concentrations and different cleavage times in the RNA-protein binding buffer. A range of site-specific RNA enzymes are now available for RNA footprinting, e.g. for viral-associated (VA) RNA1 and double-stranded RNA-dependent protein kinase (PKR) footprinting assays, RNase T1 ( 0.00012 U/5 μl reaction system), T2 ( 0.003 U/5 μl reaction system) and Bc ( 0.25 U/5 μl reaction system). T2 (0.003 U/5 μl reaction system) and Bc (0.25 U/5 μl reaction system) are used to map single-stranded regions, and RNase V1 (0.006 U/5 μl reaction system) is used to map structural regions, and is often used because it recognizes short helices formed by four to five or more nucleotides. Approximately 50-100,000 counts/reaction (as determined by the Cerenkov counting method) of 5' or 3' end-labeled RNA are sufficient to perform several gel electrophoresis and keep the autoradiography time short. 
(1) For each of the nucleases used in the experiments, the following reactions were performed: a control without RNase was set up, and a -series of concentrations was used for each enzyme, and the experiments were performed in the presence of MgCl2 ( in the natural state) or in the absence of MgCl2 (in the semidenatured state), with the exception of RNase V1, for which the presence of Mg2+ was necessary for the enzyme to exert its activity.
(2) For each reaction, add 1 μl of 10X binding buffer, 1 μl of 10 mg/ml calf liver tRNA, end-labeled RNA, 1 μl of diluted RNase, and DEPC-H2O to a final reaction volume of 5 μl. Incubate at 37 ℃ for 15 min, and then add 1 μl of 10 mg/mg of tRNA and 10 μl of 1.5X Gel Dispensing Buffer.
(3) Digest with 10X RNase T1 at 68℃ (denaturing condition) for 5 min, so that each G is cleaved to produce a sequencing ladder band.
(4) Generate a hydrolysis ladder in one-base increments using strong base cleavage: 1 μl 10X hydroxyl buffer, 1 μl 10 mg/ml calf liver tRNA, end-labeled RNA, add DEPC-H2O to a final volume of 10 μl, incubate at 90 ℃ for 2 min, add 1 μl 10 mg/ml calf liver tRNA, 10 μl 0.1 moI/L citrate and The reaction was terminated by adding 1 μl of 10 mg/ml calf liver tRNA, 10 μl of 0.1 moI/L citric acid and 24 μl of 1.5X gel spotting buffer.
(5) Separate the products by denaturing polyacrylamide gel electrophoresis. For long RNA, several electrophoreses with different concentrations and different times may be required.
(6) Labeled RNA can be detected directly by radioautography or phosphor screen. For radiographic autoradiography, the gel is placed on an X-ray film, wrapped in plastic film, and placed in a dark box at -80°C for radiographic autoradiography. For phosphorimaging, the gel is placed on 3 M Whatman paper, wrapped in plastic film, vacuum-dried, and phosphorimaged at room temperature.
(7) Determine the conditions of enzymatic digestion so that 90%~95% of the added RNA will not be cleaved, which is beneficial for the next footprinting experiment.
(8) Remove the protein fraction from the reaction system prior to gel analysis. Dilute the reaction solution with DEPC-H2O to 200 μl, add 200 μl of 2X extraction buffer to isolate the RNA again, add an equal volume of phenol: chloroform, shake for a few minutes, mix well, and centrifuge the reaction solution in a small plastic centrifuge at the highest speed for 10 min ( about 16,000 g) to collect the upper aqueous phase. Add an equal volume of chloroform and extract again as described above, precipitate the RNA with 2.5 times the volume of ethanol, place on dry ice/ethanol for 10-20 min, centrifuge at 4°C for 15 min at 16,000 g, wash the precipitate with pre-cooled 70% ethanol, and re-dissolve the precipitate with 5 μl of water and 10 μl of 1.5X Spotting Buffer.
(9) The enzyme reaction solution without protein should be extracted and precipitated again at the same time, and the same amount should be kept in the sample for analysis, including the protein-RNA complex that was not digested by nuclease after extraction as a control. 
2. Footprinting and interference analysis using reagents specific to the ribosomal backbone chain
Footprinting with chemical probes is based on the same principles as footprinting with nuclease, except that the relatively small size of the chemical probes gives a higher degree of accuracy at the interaction site.
(1) Hydroxyl
The hydroxyl group has a neutral charge and is the smallest substance used for chemical footprinting. It has a short half-life and usually cleaves the 1' or 4' position of the ribose in a sequence-independent manner. Hydroxyl has the same effect on both double-stranded and single-stranded RNAs, however, the high level of structure involved in the backbone strand of the ribose will have an effect on the efficiency of the cleavage. The cleavage reaction is gentle and can be performed in a wide range of buffers, salts, temperatures and pH conditions. As with previous experiments, the conditions of cleavage need to be determined in the binding buffer prior to performing footprinting experiments on RNA-protein complexes.
① Prepare filtered and sterilized 0.4 mol/L ammonium iron sulfate, 2 mol/L sodium ascorbate, 0.8 mmol/L EDTA pH 8.0, and 0.6% H2O2. 0.4 mol/L ammonium sulfate was diluted to 0.4 mmol/L, then mixed with an equal volume of 0.8 mmol/L EDTA, diluted 2 mol/L sodium ascorbate to 20 mmol/ L, then mixed with an equal volume of Fe-EDTA mixture and an equal volume of 0.6% H2O2.
② The RNA was first digested in the absence of protein to determine the conditions under which the appropriate cutting ladder bands were obtained, and the efficiency of the cutting was adjusted by controlling the amount of the ion/EDTA/H2O2 mixture added; the optimal conditions for VA RNA1 were an equal amount of the digestion solution added to the binding reaction solution and incubation for 2 min at room temperature.
(iii) Dilute the reaction solution with DEPC-H2O to 200 μl, then quickly add an equal volume of 2X extraction buffer containing 20 mmol/L thiourea (hydroxyl-eliminator) to terminate the reaction, and then re-recover the RNA by using the method in step (8) of 1 "RNA enzymatic footprinting analysis".
④ After establishing the digestion conditions, compare the cleavage of the RNA-protein complex with that of unbound RNA and analyze the results obtained using the method described previously.
(2) N-ethyl-N-nitrosourea
N-ethyl-N-nitrosourea (ENU ) is a mild alkylating reagent that causes phosphoric acid on the backbone chain to form propyl phosphates, and these modifications can be recognized and cleaved by mild base reagents.The appropriate modification conditions were established by measuring unbound RNA to determine the reaction time and the amount of ENU used.Although this modification is not site-specific, and the high level structures involved in the RNA backbone Although this modification is not site-specific and the higher structures involved in the RNA backbone may have an effect on the reaction, ENU is able to be used for footprinting and modification interference analysis assays.
① Add 2 μl of 10X binding buffer, 1 μl of 10 mg/ml calf liver tRNA, end-labeled RNA, or RNA-protein complex, 5 μl of freshly prepared saturated ENU, add water to 20 μl, and incubate for 30 min at 37°C. Dilute to 200 μl with water, add an equal volume of phenol: chloroform, shake for a few minutes to mix, and then centrifuge for 10 min (~16,000 g) in a minicentrifuge at maximum speed. The RNA was precipitated with 2.5 times the volume of ethanol, incubated in dry ice/ethanol for 10-20 min, centrifuged at 16,000 g for 15 min at 4°C, and the precipitate was washed with 70% ethanol pre-cooled on ice. At the same time, 5 μl of ethanol was used as a blank control instead of ENU.
② Re-dissolve the RNA precipitate in 200 μl of 0.3 mol/L NaAc at pH 3.8, then add 600 μl of ethanol, incubate in dry ice/ethanol for 10-20 min, centrifuge at 16000 g for 15 min at 4℃, and wash the precipitate with ice-cooled 70% ethanol.
(iii) Freshly dissolve the RNA into 10 μl of 100 mmol/L Tris-HCl (pH 9.0), incubate at 50 ℃ for 5 min, precipitate the RNA in ethanol and analyze by denaturing gel electrophoresis.
④ If RNA modification is to be analyzed for interference beforehand, use 10X ENU modification buffer in step (1) and incubate at 80°C for 2 min.
(3) Phosphorothioate-containing transcripts
The Sp diastereoisomer of NTPαS is a substrate for phage RNA polymerase and is able to be incorporated into synthesized RNA like normal NTP. Phosphorothioate is sensitive to and cleaved by iodine/ethanol and is suitable for footprinting experiments because iodine is uncharged, very small, reacts quickly, and has no effect on electrophoretic mobility. It is therefore suitable for footprinting experiments. This can be used as an alternative to ENU modification, both to speed up the reaction time and to interpret the data without having to sequence the RNAase and degrade the ladder bands.
① Synthesize the phosphorothioate-containing transcripts, end-label and purify, and additionally perform a separate transcription reaction for each nucleotide and 0.2 mmol/L (5%) NTPαS, i.e., for an AαS-containing transcript to contain AαS, it must contain all 4 NTPs, but also perform a separate transcription reaction with 0.2 mmol/L ATPαS.
(ii) Unbound RNA and RNA-protein complexes were incubated with 1 mmol/L I2 for 2 min at room temperature for cleavage of phosphorothioate-based closures, while an equal amount of ethanol was used as a control reaction.
(iii) Dilute the reaction solution with DEPC-H2O to 200 μl, add an equal volume of extraction buffer to isolate the RNA again, add an equal volume of phenol:chloroform, shake for a few minutes, mix well, and centrifuge at the highest speed for 10 min in a minicentrifuge (about 16,000 g), collect the upper aqueous phase, add an equal volume of chloroform to extract again as described before, and precipitate the RNA with 2.5-fold the volume of ethanol, and incubate the reaction in dry ice/ethanol. The RNA was precipitated with 2.5 times the volume of ethanol, incubated in dry ice/ethanol for 10-20 min, centrifuged at 16,000 g for 15 min at 4°C, washed with 70% ethanol pre-cooled on ice, and re-dissolved in 5 μl of water and 10 μl of 1.5X spotting buffer.
④ In this experiment, the cleavage for specific NTPαS allowed the end-bound RNA to form sequencing ladder bands, which made the analysis of the gel easier.
3. Footprinting and interference analysis experiments using base-specific reagents
2 The method described in "Footprinting and Interference Analysis Experiments with Reagents Specific to the Ribosomal Backbone Chain" allows for the study of proteins that interact with the RNA backbone; however, other portions of the RNA, such as bases, can also modulate specific RNA-protein interactions, and a number of chemistries are currently available to perform experiments to obtain information about these information about the bases.
(1) Diethyl pyrocarbonate
Diethyl pyrocarbonate (DEPC ) carboxyethylates the N-7 position of purines (A>>G), a modification that can be inhibited by base stacking and regulated by divalent ions in solution. The concentration of the probe is first titrated, and the conditions and procedure described here are suitable for VA RNA1.
Add 2 μl of 10X binding buffer, 1 μl of 10 mg/ml calf liver tRNA, end-labeled unbound RNA or protein RNA complex, 2 μl of DEPC, add water to 20 μl and incubate for 10 min at 37°C. Dilute with water to 200 μl, add an equal volume of phenol: chloroform, shake for a few minutes to mix, and then centrifuge for 10 min in a minicentrifuge at maximum speed (~16000 g). (~16000 g), collect the upper aqueous phase, add an equal volume of chloroform as described above and extract again, precipitate the RNA with 2.5 times the volume of ethanol, incubate in dry ice/ethanol for 10-20 min, centrifuge at 16000 g for 15 min at 4°C, and then wash the precipitate with 70% ethanol pre-cooled on ice.
② If you want to generate DEPC sequencing ladder bands or pre-modify the RNA used for interference analysis, mix 2 μl of DEPC, end-labeled RNA, 1 μl of 10 mg/ml calf liver tRNA, 3.3 μl of 3 mol/L NaAc (pH 4.5), and 1 μl of 200 mmol/L EDTA (pH 8.0) with water to a final volume of 200 μl, and then add water to the final volume of 200 μl. Incubate at 90°C for 2 min, add 25 μl of 3 mol/L NaAc (pH 4.5) and 750 μl ethanol, and recover and precipitate the RNA.
(iii) RNA precipitated in step (1) or (2) was re-dissolved in 200 μl of 0.3 mol/L NaAc (pH 3.8), and 600 μl of ethanol was added to precipitate the RNA, which can be used for interferences analysis, cleavage of modification sites or denaturing gel electrophoresis.
(2) Dimethyl sulfate
Dimethyl sulfate (DMS) is able to N-methylate guanine (N7 ), cytosine (N3 ), and adenine (N1 ), and only the G and C modifications can be detected by the base cleavage method described by Krol and Carbon. the G-N7 reaction can be inhibited by base stacking, while the C-N3 reaction can be inhibited by base-pairing, and the conditions here are suitable for RV RNA1.
① Add 20 μl of 10X binding buffer, 1 μl of 10 mg/ml calf liver tRNA, end-labeled unbound RNA or protein and complex, add water to 200 μl, and incubate for 10 min at 37 ℃ with 0.5 μl of DMS.
② Recover the RNA using 2X DMS extraction buffer containing 125 mmol/L β-mercaptoethanol as described in step (8) of the "RNA enzymatic footprinting analysis" in 1.
(iii) To generate a sequencing ladder of DMS or pre-modified RNA suitable for interference analysis, mix 1 μl of DMS, end-labeled RNA, 1 μl of 10 mg/ml calf liver tRNA, 3.3 μl of 3 mol/L NaAc (pH 4.5), and 1 μl of 200 mmol/L EDTA (pH 8.0) in water to a final volume of 200 μl. Incubate at 90°C for 1 min.
Incubate at 90°C for 1 min. ④ Add 25 μl of 3 mol/L NaAc (pH 4.5) and 750 μl ethanol to recover and precipitate RNA.
⑤ The RNA precipitated in step (1) or (2) was re-dissolved in 200 μl of 0.3 mol/L NaAc (pH 3.8), and 600 μl of ethanol was added to precipitate the RNA. These modified RNAs can be used for interfering analysis or re-dissolved in 100 μl of water, divided into two portions, and dried in vacuum.
(vi) Redissolve one portion in 10 μl of 1 mol/L Tris-HCl, add 10 μl of freshly prepared 200 mmol/L NaBH4, incubate on ice for 30 min, avoiding light, and perform G-N7 cleavage.
(vii) Dissolve the other portion in 10 μl of ice-cooled 50% (V/V) anhydrous hydrazine and incubate on ice for 5 min for C-N3 cleavage.
(8) RNA precipitated in step (4) or (5) was re-dissolved in 200 μl of 0.3 mol/L NaAc (pH 3.8), and 600 μl of ethanol was added to precipitate the RNA, and the dissolution and precipitation was repeated, and the RNA was used to perform aniline cleavage.
(3) RNA modified by aniline cleavage
① Re-dissolve the RNA precipitate in 20 μl of 1 mol/L aniline (freshly diluted with 0.3 mol/L pH 3.8 NaAc) and incubate at 60 ℃ for 20 min, protected from light.
② Terminate the reaction by adding 1.4 ml of n-butanol, shaking briefly, and centrifuge in a microcentrifuge at the highest speed at room temperature (about 16,000 g), remove the n-butanol carefully, and re-dissolve the RNA precipitate in 150 μl of 1% (m/V) SDS, add 1.4 ml of n-butanol, shaking briefly, and precipitate the RNA as before.
(iii) Wash the precipitate with anhydrous ethanol, dry completely, and redissolve the RNA precipitate in 4 μl of DEPC-H2O and 8 μl of 1.5X gel spotting buffer, and the sample is ready for gel analysis. The labeled unmodified RNA should also be treated with aniline.
4. Modification Interference Analysis
Modification interference analysis using pre-modified RNA can identify important sites for protein interactions.DEPC, ENU, DMS-modified RNA and phosphorothioate-containing transcripts can be subjected to interference analysis. Chemical modification of RNA under denaturing conditions may result in modification of all sites, and the modified RNA is recovered and subjected to a binding reaction. the RNA-protein complex is isolated from the unbound RNA, and protein-RNA complexes are extracted as described earlier. These RNAs were cleaved at the modification sites, unmodified RNA was tested in parallel to generate a sequencing ladder control, unmodified RNA was also used as a control without specific binding, and the products were analyzed by denaturing gel electrophoresis and radioautography. The procedure for analyzing modifications of pyrimidine bases using hydrazine is given below.
Hydrazine removes pyrimidine bases for interference analysis, and depending on the conditions chosen, the reaction can be performed for U, C, or both, with the conditions here being suitable for VA RNA1.
1. Precipitate unlabeled RNA and 1 μl of 10 mg/ml calf liver tRNA.
2. To perform the U-specific reaction, redissolve the RNA in 10 μl of water, add 10 μl of anhydrous hydrazine and incubate the mixture on ice for 10 min.
3. Perform the U- and C-specific reactions as described above, except that the RNA precipitate is redissolved in 20 μl of freshly prepared anhydrous hydrazine/0.5 mol/L NaCl and incubated on ice for 30 min.
4. For the C-specific reaction, the RNA precipitate is redissolved in freshly prepared anhydrous hydrazine/3 mol/L NaCI and incubated on ice for 30 min.
5. The RNA precipitated in steps 2, 3 and 4 was redissolved in 200 μl of 0.3 mol/L NaAc (pH 3.8), 600 μl of ethanol was added to precipitate the RNA, and the precipitate was redissolved once, and recovered for the interference binding and aniline cleavage assays.