  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.
