Investigating the interplay between RNA structural dynamics and RNA chemical probing experiments

Small molecule chemical probes that covalently bond atoms of flexible nucleotides are widely employed in RNA structure determination. Atomistic molecular dynamic (MD) simulations recently suggested that RNA binding by chemical probes can be cooperative, leading to measured reactivities that differ from expected trends as probe concentration is increased. Here, we use selective 2 hydroxyl acylation analyzed by primer extension (SHAPE) and dimethyl sulfate (DMS) chemical probing to explore whether chemical probes influence RNA structural ensembles or nucleotide reactivities. We find that as the concentration of a probe increases, some nucleotides' modification rates increase nonlinearly or change unexpectedly. Importantly, we observe that some base-paired nucleotides that are unreactive at one probe concentration can become reactive at another. We show that this effect often corresponds with a shift in the modification rate of the complementary nucleotide, and we believe that this effect can be harnessed as evidence of pairing interactions. Additionally, we carry out one-dimensional nuclear magnetic resonance (NMR) spectroscopy phase-modulated chemical exchange (CLEANEX-PM) experiments to reveal that SHAPE-reactive base-paired nucleotides exhibit high imino proton exchange rates. Our results show that chemical probes structural influence on RNA structural ensembles and nucleotide modification rates can be measured to detect base pairs and that reactive paired nucleotides have high imino proton exchange rates.