Poster abstracts

Poster number 39 submitted by Sophia Mitton-Fry

Disentangling ligand-induced shifts in probing chemistry from RNA structural rearrangements

Sophia R. Mitton-Fry (Chemical and Biological Engineering, Northwestern University ), Edric K. Choi (Chemical and Biological Engineering, Northwestern University), Julius B. Lucks (Chemical and Biological Engineering, Northwestern University)

Abstract:
Ligand-induced structural rearrangements are central to RNA riboswitch regulation, where binding at an aptamer domain propagates to an expression platform and ultimately modulates gene expression. Chemical probing is widely used to study these rearrangements through differential reactivity measurements, but conventional analyses are largely relative. Ad hoc normalization can reveal where structural changes occur but profile-dependent scaling obscures effect size and limits quantitative comparison across ligands, conditions, and model systems. Probing-Resolved Inference of Molecular Energetics (PRIME) provides a kinetic framework to deconvolute probe chemistry from intrinsic RNA energetics, creating an opportunity to measure ligand-induced structural effects on a unified energetic scale. Here, we extend this framework to RNA-ligand interactions using well-characterized tetrahydrofolate (THF), ZMP, and fluoride aptamers. Surprisingly, time-course DMS probing revealed that each ligand perturbs probe chemistry in distinct and nonuniform ways, producing apparent reactivity changes independent of bona fide RNA structural rearrangements. We further validated these ligand-dependent kinetic effects using independent time-resolved 1D NMR. Results reveal that additives in the probing environment can exert unpredictable, base-dependent effects on apparent chemical reactivities, potentially confounding interpretation of the underlying RNA folding response. Therefore, it is essential to explicitly deconvolve these chemical effects in order to quantitatively resolve how ligand binding reshapes local RNA energetics, propagates allosteric information, and modulates the local dynamics that underpin RNA function.

Keywords: Chemical Probing, Ligand Interactions, Structural Energetics