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Verification of the Crooks fluctuation theorem and recovery of RNA folding free energies


Atomic force microscopes and optical tweezers are widely used to probe the mechanical properties of individual molecules and molecular interactions, by exerting mechanical forces that induce transitions such as unfolding or dissociation. These transitions often occur under nonequilibrium conditions and are associated with hysteresis effects—features usually taken to preclude the extraction of equilibrium information from the experimental data. But fluctuation theorems1,2,3,4,5 allow us to relate the work along nonequilibrium trajectories to thermodynamic free-energy differences. They have been shown to be applicable to single-molecule force measurements6 and have already provided information on the folding free energy of a RNA hairpin7,8. Here we show that the Crooks fluctuation theorem9 can be used to determine folding free energies for folding and unfolding processes occurring in weak as well as strong nonequilibrium regimes, thereby providing a test of its validity under such conditions. We use optical tweezers10 to measure repeatedly the mechanical work associated with the unfolding and refolding of a small RNA hairpin11 and an RNA three-helix junction12. The resultant work distributions are then analysed according to the theorem and allow us to determine the difference in folding free energy between an RNA molecule and a mutant differing only by one base pair, and the thermodynamic stabilizing effect of magnesium ions on the RNA structure.

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Figure 1: Force–extension curves.
Figure 2: Test of the CFT using an RNA hairpin.
Figure 3: Free-energy recovery and test of the CFT for non-gaussian work distributions.
Figure 4: Use of CFT to extract the stabilizing contribution of Mg 2+ to the free energy of the S15 three-helix junction (wild type).


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We thank G. Hummer and A. Szabo for many discussions and G. E. Crooks, D. Chandler and J. Liphardt for a careful reading of the manuscript. F.R. was supported by the Spanish Research council and the Catalan Government (Distinció de la Generalitat). C.J. was supported by an NIH grant and the US Department of Energy. I.T. was supported by an NIH grant. C.B. was supported by the Howard Hughes Medical Institute and the David and Lucile Packard Foundation.

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Collin, D., Ritort, F., Jarzynski, C. et al. Verification of the Crooks fluctuation theorem and recovery of RNA folding free energies. Nature 437, 231–234 (2005).

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