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RNA Structure Determination Using Chemical Methods Mark Caprara Cold Spring Harb Protoc; doi: 10.1101/pdb.prot078485 Email Alerting Service Subject Categories

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Protocol

RNA Structure Determination Using Chemical Methods Mark Caprara

Information about the secondary structure of RNA is often useful when assessing the potential for certain RNAs to interact with proteins or when determining whether RNAs that are dissimilar in sequence can form the same structure. In this protocol we discuss chemical methods for RNA structure determination. These methods rely on the fact that certain reagents interact with RNA bases when they are single stranded, but do not react when the bases are involved in Watson–Crick base pairs. For example, dimethylsulfate (DMS) methylates the N1 position of adenosine, the N7 position of guanine, and the N3 position of cytosine only when these bases are in single-strand regions. Modifications of adenosine and cytosine create blocks to reverse transcriptase; accordingly, these modifications are detected as stops to primer extension. Modification of guanine does not create reverse transcriptase stops, but these modifications can be detected by cleavage of the modified RNA after borohydride reduction and aniline cleavage. Because DMS and other chemical reagents modify only single-stranded RNA, double-stranded regions are inferred by the lack of modification.

MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.

Reagents

Aniline (1 M; pH 4.5) To prepare 10 µL of this reagent, add 1 µL of aniline (11 M) and 7 µL of glacial acetic acid (17 M) to 3 µL of H2O. Make fresh for each use. Scale up amounts according to numbers of reactions.

Annealing buffer (2×) (20 mM Tris/80 mM KCl) To prepare 10 mL of this reagent, add 0.2 mL of 1 M Tris (pH 8.3) and 0.4 mL of 2 M KCl to 9.4 mL of H2O. Store indefinitely at 4˚C.

CMCT in H2O (cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate) (25 mg/mL) Diethylpyrocarbonate (DEPC; Sigma-Aldrich) Dimethylsulfate (DMS) (diluted 1:6 in 100% ethanol) EDTA (200 mM, pH 8.0) Ethanol (100%) Loading dye Adapted from RNA: A Laboratory Manual, by Donald C. Rio, Manuel Ares Jr, Gregory J. Hannon, and Timothy W. Nilsen. CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2013 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078485

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M. Caprara

Modification reaction buffer A (denaturing) Modification reaction buffer A (native) Modification reaction buffer B (denaturing) Modification reaction buffer B (native) Oligonucleotide primers (5′ -end-labeled; prepared using T4 polynucleotide kinase) Reverse transcriptase (M-MuLV, 200 units/μL or SuperScript II, 200 units/μL) Reverse transcription buffer (5×) RNA to be probed (0.2–1 µg) Sodium acetate (3 M, pH 5.2) Sodium borohydride (0.2 M) TBE electrophoresis buffer Tris-HCl (1 M, pH 8.3) tRNA carrier (E. coli 10 µg/μL) Equipment

Denaturing polyacrylamide gel electrophoresis system and power supply to run an 8% gel Dry ice Gel dryer Heat block (95˚C) Ice Microcentrifuge Phosphorimager or autoradiography materials SpeedVac Vortex mixer Water baths (set to temperatures to accommodate specific reactions) METHOD Here we present methods for modifying RNA with DMS (Steps 1–7), CMCT (Steps 8–12), and DEPC (Steps 13–18). The conditions described provide a starting point for concentrations of reagents and times of incubation. These should be titrated on denatured RNA (unfolded) to find the optimum modification conditions for individual substrates. The concentrations of the reagents should be adjusted so that they modify an RNA substrate only once. A good rule of thumb is to ensure that the total amount of modified or cleaved material in a sample is no >20% of the total amount of RNA. After optimizing conditions for each reagent, assemble two reactions, one in “native” buffer (folded RNA) and one in “denaturing” buffer (unfolded RNA). Avoid buffers that contain amines (such as Tris) because many of the reagents used here modify primary amines. The sites of modification are monitored by primer extension using a 5′ -endlabeled oligonucleotide (Steps 22–28). If RNAs are longer than 100 nucleotides, more than one primer may be needed (one every 100 nucleotides). The primer extension ladders from two samples are compared with each other and a residue is scored as protected if the band intensity of the “native” is

RNA structure determination using chemical methods.

Information about the secondary structure of RNA is often useful when assessing the potential for certain RNAs to interact with proteins or when deter...
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