  Recent advances in non-equilibrium statistical mechanics and single molecule
technologies make it possible to extract free energy differences from
irreversible work measurements in pulling experiments. To date, free energy
recovery has been focused on native or equilibrium molecular states, whereas
free energy measurements of kinetic states (i.e. finite lifetime states that
are generated dynamically and are metastable) have remained unexplored. Kinetic
states can play an important role in various domains of physics, such as
nanotechnology or condensed matter physics. In biophysics, there are many
examples where they determine the fate of molecular reactions: protein and
peptide-nucleic acid binding, specific cation binding, antigen-antibody
interactions, transient states in enzymatic reactions or the formation of
transient intermediates and non-native structures in molecular folders. Here we
demonstrate that it is possible to obtain free energies of kinetic states by
applying extended fluctuation relations. This is shown by using optical
tweezers to mechanically unfold and refold DNA structures exhibiting
intermediate and misfolded kinetic states.
