Kinetic Dissection of Proton-Coupled Conformational Transitions in Nucleic Acids by Integrating pH-Dependent NMR and Chemical Modifications

Proton-coupled conformational transitions play fundamental roles in nucleic acid recognition, catalysis, and folding, yet the kinetic mechanisms underlying these multistep protonation reactions remain unknown. Here, we present an approach to resolve the dominant kinetic pathway and rate-limiting step, which combines NMR chemical exchange measurements with chemical perturbations that shift pK_a or modulate conformational equilibria. Applying the approach to three nucleic acid systems, we find the microscopic protonation step to be a diffusion-limited proton transfer reaction (k_prot ∼ 10¹¹ M^-1 s^-1), 2 orders of magnitude faster than diffusion-limited ligand-binding. For an A⁺-C mismatch in duplex DNA, protonation was the rate-limiting step occurring after the conformational change at a diffusion-limited k_on ∼ 10¹¹ M^-1 s^-1 via conformational selection of the wobble conformation, which forms rapidly and in significant abundance in the neutral ensemble. In RNA, the A-C wobble was sparsely populated in the neutral ensemble. The apparent k_on was 2 orders of magnitude slower, and the reaction followed an induced-fit mechanism, where the unpaired adenine was initially protonated, followed by rate-limiting intrahelical flipping. The apparent k_on was 5 orders of magnitude slower for the protonated G(syn)-C⁺ Hoogsteen conformation in duplex DNA in which cytosine protonation was rate-limiting occurring after the conformational change via conformational selection of an energetically disfavored G(syn)-C intermediate. These kinetic models quantitatively predicted the impact of pH shifts and chemical modifications on reaction kinetics. Our findings reveal how differences in nucleic acid conformational ensembles can drive diverse kinetic responses to pH changes and chemical modifications, even in binding reactions involving the simplest ligand: the proton.