Multiple probes are required to explore and control the rugged energy landscape of RNA hairpins

Brownian dynamics simulations, as a function of temperature (T) and force (f) show that RNA hairpins form by multiple pathways thus revealing the rugged nature of the free-energy landscape. While low dimensional free-energy profiles can account for some aspects of thermodynamics of hairpin formation they cannot account for the observed pathway diversity during the refolding process. Thus, a single free-energy surface cannot be used to infer the experimentally observed multistate kinetics in hairpin formation in nucleic acids. The profound differences between the kinetics of folding upon f- and T-quench is due to the slow rate of loop nucleation when the search for the native conformation commences from stretched conformation as is the case upon f-quench.