Efficient methods for determining folding free energies in single-molecule pulling experiments

TL;DR

This paper reviews methods for accurately estimating folding free energies in single-molecule pulling experiments, emphasizing elastic energy correction techniques using DNA and RNA hairpins with optical tweezers.

Contribution

It introduces optimized approaches for elastic energy estimation to improve free energy calculations in single-molecule force spectroscopy.

Findings

Enhanced accuracy in free energy estimation through elastic energy correction.

Methodology applicable to various force-spectroscopy techniques.

Validated approach using DNA and RNA hairpins as models.

Abstract

The remarkable accuracy and versatility of single-molecule techniques make possible new measurements that are not feasible in bulk assays. Among these, the precise estimation of folding free energies using fluctuation theorems in nonequilibrium pulling experiments has become a benchmark in modern biophysics. In practice, the use of fluctuation relations to determine free energies requires a thorough evaluation of the usually large energetic contributions caused by the elastic deformation of the different elements of the experimental setup (such as the optical trap, the molecular linkers and the stretched-unfolded polymer). We review and describe how to optimally estimate such elastic energy contributions to extract folding free energies, using DNA and RNA hairpins as model systems pulled by laser optical tweezers. The methodology is generally applicable to other force-spectroscopy techniques and molecular systems.