Verification of the Crooks fluctuation theorem and recovery of RNA folding free energies

TL;DR

This paper experimentally verifies the Crooks Fluctuation Theorem using optical tweezers on RNA molecules and demonstrates its utility in accurately determining biomolecular folding free energies from nonequilibrium work measurements.

Contribution

It provides the first experimental verification of the Crooks Fluctuation Theorem in biomolecular systems and introduces a method to extract folding free energies from nonequilibrium data.

Findings

Verified Crooks Fluctuation Theorem for RNA unfolding and refolding.

Showed accurate free energy estimation from nonequilibrium work distributions.

Demonstrated applicability to small biomolecules under various conditions.

Abstract

The description of nonequilibrium processes in nano-sized objects, where the typical energies involved are a few times, is increasingly becoming central to disciplines as diverse as condensed-matter physics, materials science, and biophysics. Major recent developments towards a unified treatment of arbitrarily large fluctuations in small systems are described by fluctuation theorems that relate the probabilities of a system absorbing from or releasing to the bath a given amount of energy in a nonequilibrium process. Here we experimentally verify the Crooks Fluctuation Theorem (CFT) under weak and strong nonequilibrium conditions by using optical tweezers to measure the irreversible mechanical work during the unfolding and refolding of a small RNA hairpin and an RNA three-helix junction. We also show that the CFT provides a powerful way to obtain folding free energies in biomolecules by determining the crossing between the unfolding and refolding irreversible work distributions. The method makes it possible to obtain folding free energies in nonequilibrium processes that dissipate up to of the average total work exerted, thereby paving the way for reconstructing free energy landscapes along reaction coordinates in nonequilibrium single-molecule experiments.