DMS footprinting of structured RNAs and RNA-protein complexes.
We describe a protocol in which dimethyl sulfate (DMS) modification of the base-pairing faces of unpaired adenosine and cytidine nucleotides is used for structural analysis of RNAs and RNA-protein complexes (RNPs). The protocol is optimized for RNAs of small to moderate size (< or = 500 nt). The RNA or RNP is first exposed to DMS under conditions that promote formation of the folded structure or complex, as well as 'control' conditions that do not allow folding or complex formation. The positions and extents of modification are then determined by primer extension, polyacrylamide gel electrophoresis and quantitative analysis. From changes in the extent of modification upon folding or protein binding (appearance of a 'footprint'), it is possible to detect local changes in the secondary and tertiary structure of RNA, as well as the formation of RNA-protein contacts. This protocol takes 1.5-3 d to complete, depending on the type of analysis used.
Tijerina
Pilar
P
Department of Chemistry and Biochemistry and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
Mohr
Sabine
S
Russell
Rick
R
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D007438:Introns; D009690:Nucleic Acid Conformation; D023302:Protein Footprinting / Q000379:methods*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013463:Sulfuric Acid Esters / Q000737:chemistry*; D013768:Tetrahymena / Q000235:genetics
D015800:Protozoan Proteins; D016337:RNA, Catalytic; D016601:RNA-Binding Proteins; D013463:Sulfuric Acid Esters; D012313:RNA; C007482:dimethyl sulfate