Revealing hidden protonated conformational states in RNA dynamic ensembles

Identifying protonated states within RNA ensembles, quantifying their pKas, and elucidating the kinetic mechanisms by which they form is essential for understanding protonation-coupled biochemical reactions and how RNAs sense and adapt to pH fluctuations. However, detecting protonated states is challenging when they are short-lived and lowly populated. Here, using pH-dependent NMR chemical exchange, kinetic solvent isotope effects, and mutation, we show that a low-populated (0.4% at pH 6.4) conformational state of HIV-1 TAR RNA is coupled to protonation of a C⁺-C mismatch. Despite an intrinsic pKa of ~7.1, the energetic penalty to form this alternative conformation depressed the apparent pKa to ∼4.0, below the pH range typically probed experimentally. Substituting C-C with a G-C base pair abolished the pH-dependence of these dynamics, confirming C-C as the protonation site. This hidden protonated state competes with a more abundant conformation harboring a C-A⁺ mismatch, producing a non-monotonic ensemble response to pH. Both transitions follow an induced-fit mechanism, in which solvent-exposed nucleobases are rapidly protonated followed by slower changes in secondary structure. These findings reveal a general mechanism for protonation-coupled conformational switching in RNA and provide a framework for dissecting sparsely populated protonated states and their multi-protonation-state dynamics.