  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.
