A small cationic probe highly selective for RNA tertiary structure

RNA is unique among the large biomolecules due to the intersection of its distinctive electrostatic properties, created by the permanent charge at each phosphate group, coupled with its ability to form diverse three-dimensional tertiary structures. We demonstrate that at rare, but distinctive, sites tertiary folding creates electronegative pockets that preferentially react with a small positively-charged chemical probe, trimethyloxonium (TMO), which alkylates RNA. TMO performs the same chemistry with RNA as the classical uncharged probe, dimethyl sulfate (DMS). Sites preferentially reactive with the TMO cation, relative to the uncharged DMS, are detected efficiently by massively parallel sequencing, at low read depths. These motifs, which we call T-sites, reflect preferential interaction of TMO at an electronegative pocket created by juxtaposition of a reactive nucleobase with non-bridging oxygen atoms, and specifically map to the centers of higher-order structural interactions and functional cores in model RNAs. T-site probing is a facile robust strategy, poised to create broad opportunities to detect and analyze RNA tertiary structures transcriptome-wide. Overall design: In-vitro transcribed TPP riboswitch aptamer domain, Group II intron catalytic domain, and RNase P catalytic domain constructs with structure cassettes, either native sequence or various mutants, were refolded and structure was probed with TMO, DMS, or a non-reactive control. Naked RNA extracted from dengue virions was also chemically probed. Modified RNA was reverse transcribed under MaP conditions using SuperScript II. cDNA sequencing libraries were prepared and sequenced on an Illumina MiSeq instrument.