Characterization of the kinetic and thermodynamic landscape of RNA folding using a novel application of isothermal titration calorimetry

A novel isothermal titration calorimetry (ITC) method was applied to investigate RNA helical packing driven by the GAAA tetraloop–receptor interaction in magnesium and potassium solutions. Both the kinetics and thermodynamics were obtained in individual ITC experiments, and analysis of the kinetic data over a range of temperatures provided Arrhenius activation energies (ΔH(‡)) and Eyring transition state entropies (ΔS(‡)). The resulting rich dataset reveals strongly contrasting kinetic and thermodynamic profiles for this RNA folding system when stabilized by potassium versus magnesium. In potassium, association is highly exothermic (ΔH(25°C) = −41.6 ± 1.2 kcal/mol in 150 mM KCl) and the transition state is enthalpically barrierless (ΔH(‡) = −0.6 ± 0.5). These parameters are sigificantly positively shifted in magnesium (ΔH(25°C) = −20.5 ± 2.1 kcal/mol, ΔH(‡) = 7.3 ± 2.2 kcal/mol in 0.5 mM MgCl(2)). Mixed salt solutions approximating physiological conditions exhibit an intermediate thermodynamic character. The cation-dependent thermodynamic landscape may reflect either a salt-dependent unbound receptor conformation, or alternatively and more generally, it may reflect a small per-cation enthalpic penalty associated with folding-coupled magnesium uptake.