Time-resolved RNA SHAPE chemistry: quantitative RNA structure analysis in one-second snapshots and at single-nucleotide resolution

RNA SHAPE chemistry exploits the discovery that conformationally dynamic nucleotides preferentially adopt conformations 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 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 °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 sec. Sites of 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 sec snapshots in time-resolved kinetic studies for RNAs of arbitrary length and complexity in a straightforward and concise experiment