Direct Observation of Folding Energy Landscape of RNA Hairpin at Mechanical Loading Rates.

By applying a controlled mechanical load using optical tweezers, we measured the diffusive barrier crossing in a 49 nt long P5ab RNA hairpin. We find that in the free-energy landscape the barrier height (G‡) and transition distance (x‡) are dependent on the loading rate (r) along the pulling direction, x, as predicted by Bell. The barrier shifted toward the initial state, whereas ΔG‡ reduced significantly from 50 to 5 kT, as r increased from 0 to 32 pN/s. However, the equilibrium work (ΔG) during strand separation, as estimated by Crook's fluctuation theorem, remained unchanged at different rates. Previously, helix formation and denaturation have been described as two-state (F ↔ U) transitions for P5ab. Herein, we report three intermediate states I1, I, and I2 located at 4, 11, and 16 nm respectively, from the folded conformation. The intermediates were observed only when the hairpin was subjected to an optimal r, 7.6 pN/s. The results indicate that the complementary strands in P5ab can zip and unzip through complex routes, whereby mismatches act as checkpoints and often impose barriers. The study highlights the significance of loading rates in force-spectroscopy experiments that are increasingly being used to measure the folding properties of biomolecules.