Time-resolved RNA SHAPE chemistry: quantitative RNA structure analysis in one-second snapshots and at single-nucleotide resolution.
RNA selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry exploits the discovery that conformationally dynamic nucleotides preferentially adopt configurations that facilitate reaction between the 2'-OH group and a hydroxyl-selective electrophile, such as benzoyl cyanide (BzCN), to form a 2'-O-adduct. BzCN is ideally suited for quantitative, time-resolved analysis of RNA folding and ribonucleoprotein (RNP) assembly mechanisms because this reagent both reacts with flexible RNA nucleotides and also undergoes auto-inactivating hydrolysis with a half-life of 0.25 s at 37 degrees C. RNA folding is initiated by addition of Mg(2+) or protein, or other change in solution conditions, and nucleotide resolution structural images are obtained by adding aliquots of the evolving reaction to BzCN and then 'waiting' for 1 second. Sites of the 2'-O-adduct formation are subsequently scored as stops to primer extension using reverse transcriptase. This time-resolved SHAPE protocol makes it possible to obtain 1-second structural snapshots in time-resolved kinetic studies for RNAs of arbitrary length and complexity in a straightforward and concise experiment.
Mortimer
Stefanie A
SA
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, USA.
Weeks
Kevin M
KM
D000215:Acylation; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics*; D006900:Hydroxylation; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000032:analysis* / Q000378:metabolism; D012313:RNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043262:Ribonuclease P / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
D017931:DNA Primers; D009711:Nucleotides; D012313:RNA; D043262:Ribonuclease P