RNA SHAPE Analysis of Small RNAs and Riboswitches

We describe structural analysis of RNAs by SHAPE chemical probing. RNAs are treated with 1-methyl-7-nitroisatoic anhydride (1M7), a reagent that detects local nucleotides flexibility, and N-methylisatoic anhydride (NMIA) and 1-methyl-6-nitroisatoic anhydride (1M6), reagents which together detect higher-order and non-canonical interactions. Chemical adducts are detected as stops during reverse transcriptase-mediated primer extension. Probing information can be used to infer conformational changes and ligand binding, and to develop highly accurate models of RNA secondary structures