Osmium tetroxide as a probe of RNA structure

Abstract

Structured RNAs have a central role in cellular function. The capability of structured RNAs to adopt fixed architectural structures or undergo dynamic conformational changes contributes to their diverse role in the regulation of gene expression. Although numerous biophysical and biochemical tools have been developed to study structured RNAs, there is a continuing need for the development of new methods for the investigation of RNA structures, especially methods that allow RNA structure to be studied in solution close to its native cellular conditions. Here we use osmium tetroxide (OsO₄) as a chemical probe of RNA structure. In this method, we have used fluorescence-based sequencing technologies to detect OsO₄ modified RNA. We characterized the requirements for OsO₄ modification of RNA by investigating three known structured RNAs: the M-box, glycine riboswitch RNAs, and tRNA^asp. Our results show that OsO₄ predominantly modifies RNA at uracils that are conformationally exposed on the surface of the RNA. We also show that changes in OsO₄ reactivity at flexible positions in the RNA correlate with ligand-driven conformational changes in the RNA structure. Osmium tetroxide modification of RNA will provide insights into the structural features of RNAs that are relevant to their underlying biological functions.