Understanding cooperativity effects in RNA structure probing experiments.

Abstract

Small molecule chemical probes, which form covalent bonds with atoms of flexible nucleotides, are commonly used to gain insight into the structure adopted by an RNA. In this work, we use selective 2’ hydroxyl acylation analyzed by primer extension (SHAPE) and nuclear magnetic resonance (NMR) spectroscopy experiments to explore whether the structure of an RNA is modulated by chemical probe binding events. We find that as the concentration of a chemical probe increases, modified nucleotides locally modulate the RNA structure, resulting in the increase or decrease of SHAPE reactivity in surrounding nucleotides. This cooperative effect is dependent on both chemical probe concentration and size. Critically, this effect impacts the accuracy of predicted RNA secondary structures when guided by chemical probing experiments. Altogether, our work highlights the importance of optimizing the conditions of chemical probing experiments to obtain secondary structural information about an RNA.