Atomic force microscopy and optical-tweezer techniques can be used to study the properties of systems as small as a single molecule. For example, the helical molecule ribonucleic acid (RNA), can be made to unfold and refold by mechanical stretching. Typically, the molecule is forced back and forth between the two configurations rather quickly, often too fast to stay in thermal equilibrium with its environment. So the extraction of useful thermodynamic information from such experiments seemed unlikely. However, in Nature this week, Delphine Collin and colleagues1 show that it is possible to learn something about this molecule’s equilibrium situation, and their work puts recent theoretical advances in non-equilibrium thermodynamics on firmer ground.

Classical thermodynamics describes systems that are in equilibrium, or at least very close to it — in the sense that the processes by which energy is transferred within the system happen sufficiently slowly as to waste no heat. But conceptual advances in recent years promise to yield thermodynamical information about systems on which work is performed far away from this reversibility-defining limit. The work expended along non-equilibrium trajectories can be related to equilibrium free energies by the Crooks fluctuation theorem2. In the case of a stretched RNA molecule, the theorem suggests that the amount of useful work that can be extracted by going from the folded to the unfolded state (which is its equilibrium free energy) can be determined by repeatedly measuring the force required to switch back and forth between the two states, even when energy is dissipated along the way.

Collin and colleagues1 have verified the Crooks fluctuation theorem in a series of experiments. From their data on the folding and unfolding of an RNA ‘hairpin’ molecule and an RNA three-helix junction, they determined free energies associated with RNA folding with amazing accuracy. In fact, the method is sensitive enough to reveal the difference in the folding free energies of RNA molecules whose composition — from four bases, adesine, guanine, cytosine and uracil — varies by only one out of 34 base pairs.