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

TL;DR

This study introduces a non-equilibrium single-molecule method using Kramers kinetic diffusive model to determine force-dependent folding kinetics, barriers, and energies of DNA hairpins with multiple intermediates, surpassing equilibrium methods.

Contribution

The paper presents a novel non-equilibrium approach to derive kinetic and thermodynamic properties of folding intermediates from pulling experiments, validated on DNA hairpins.

Findings

Experimental results agree with theoretical predictions.

Method characterizes properties not accessible via equilibrium hopping.

Applicable to complex folding pathways with multiple intermediates.

Abstract

Most single-molecule studies derive the kinetic rates of native, intermediate, and unfolded states from equilibrium hopping experiments. Here, we apply Kramers kinetic diffusive model to derive the force-dependent kinetic rates of intermediate states from non-equilibrium 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 non-equilibrium 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.