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

A Severino, A M Monge, P Rissone and F Ritort

The remarkable accuracy and versatility of single-molecule techniques make new measurements that are not feasible in bulk assays possible. 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.

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