A small cationic probe for accurate, punctate discovery of RNA tertiary structure

RNA can fold to form complex three-dimensional tertiary structures, with large roles in RNA biology. However, accurate discovery of motifs that form true tertiary structures remains an unsolved challenge. Here we demonstrate that at rare but distinctive sites, RNA tertiary folding creates electronegative pockets that undergo selective chemistry with a small positively-charged chemical probe, trimethyloxonium (TMO). Sites preferentially reactive with the TMO cation, relative to uncharged dimethyl sulfate (DMS), termed T-sites, reflect preferential interaction of TMO at punctate electronegative pockets created by juxtaposition of a reactive nucleobase with non-bridging oxygen atoms in the RNA backbone. T-sites map specifically to centers of higher-order structural interactions and functional cores in diverse RNAs, including in specific states in conformational ensembles. Probing the 10,723-nt dengue virus RNA genome revealed exactly three T-sites, each of which is embedded within a complex RNA structure with direct roles in viral replication. T-site chemistry creates broad opportunities for high-confidence discovery and analysis of functional RNA tertiary structures across long and complex RNAs, including transcriptome-wide.