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-probe binding can be cooperative, leading to measured reactivities that differ from expected trends as probe concentrations are varied. Here, we use selective 2-hydroxyl acylation analyzed by primer extension (SHAPE), dimethyl sulfate (DMS) chemical probing, and nuclear magnetic resonance (NMR) spectroscopy to explore the relationship between RNA structural dynamics and chemical probe reactivity. Our NMR chemical exchange experiments revealed that SHAPE-reactive base-paired nucleotides exhibit high imino proton exchange rates. Additionally, we find that as the concentration of a probe increases, some nucleotides’ modification rates shift unexpectedly. For instance, some base-paired nucleotides that are unreactive at one probe concentration become reactive at another, often corresponding with a shift in the modification rate of the complementary nucleotide. We believe this effect can be harnessed to infer pairing interactions. Lastly, our results suggest that the overmodification of an RNA can impact its conformational dynamics, leading to modulations in the structural ensembles representing the RNA’s fold. Our findings suggest an intricate interplay between RNA conformational dynamics and chemical probing reactivity.