Force-Dependent Folding Kinetics of Single Molecules with Multiple Intermediates and Pathways

[Image: see text] Most single-molecule studies derive the kinetic rates of native, intermediate, and unfolded states from equilibrium hopping experiments. Here, we apply the Kramers kinetic diffusive model to derive the force-dependent kinetic rates of intermediate states from nonequilibrium pulling experiments. From the kinetic rates, we also extract the force-dependent kinetic barriers and the equilibrium folding energies. We apply our method to DNA hairpins with multiple folding pathways and intermediates. The experimental results agree with theoretical predictions. Furthermore, the proposed nonequilibrium single-molecule approach permits us to characterize kinetic and thermodynamic properties of native, unfolded, and intermediate states that cannot be derived from equilibrium hopping experiments.