Forced-unfolding and force-quench refolding of RNA hairpins

Nanomanipulation of individual RNA molecules, using laser optical tweezers, has made it possible to infer the major features of their energy landscape. Time-dependent mechanical unfolding trajectories, measured at a constant stretching force (f(S)) of simple RNA structures (hairpins and three-helix junctions) sandwiched between RNA/DNA hybrid handles show that they unfold in a reversible all-or-none manner. To provide a molecular interpretation of the experiments we use a general coarse-grained off-lattice Gō-like model, in which each nucleotide is represented using three interaction sites. Using the coarse-grained model we have explored forced-unfolding of RNA hairpin as a function of f(S) and the loading rate (r(f)). The simulations and theoretical analysis have been done both with and without the handles that are explicitly modeled by semiflexible polymer chains. The mechanisms and timescales for denaturation by temperature jump and mechanical unfolding are vastly different. The directed perturbation of the native state by f(S) results in a sequential unfolding of the hairpin starting from their ends, whereas thermal denaturation occurs stochastically. From the dependence of the unfolding rates on r(f) and f(S) we show that the position of the unfolding transition state is not a constant but moves dramatically as either r(f) or f(S) is changed. The transition-state movements are interpreted by adopting the Hammond postulate for forced-unfolding. Forced-unfolding simulations of RNA, with handles attached to the two ends, show that the value of the unfolding force increases (especially at high pulling speeds) as the length of the handles increases. The pathways for refolding of RNA from stretched initial conformation, upon quenching f(S) to the quench force f(Q), are highly heterogeneous. The refolding times, upon force-quench, are at least an order-of-magnitude greater than those obtained by temperature-quench. The long f(Q)-dependent refolding times starting from fully stretched states are analyzed using a model that accounts for the microscopic steps in the rate-limiting step, which involves the trans to gauche transitions of the dihedral angles in the GAAA tetraloop. The simulations with explicit molecular model for the handles show that the dynamics of force-quench refolding is strongly dependent on the interplay of their contour length and persistence length and the RNA persistence length. Using the generality of our results, we also make a number of precise experimentally testable predictions.