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Determination of thermodynamics and kinetics of RNA reactions by force.

Tinoco I, T X Li P, Bustamante C

Department of Chemistry, University of California, Berkeley, CA, USA.

1. Introduction 22. Instrumentation 42.1 Instruments to study mechanical properties of RNA 42.1.1 AFM 42.1.2 Magnetic tweezers 42.1.3 Optical tweezers 62.2 Optical trap instrumentation 62.3 Calibrations 82.3.1 Calibration of trap stiffness 82.3.2 Calibration of force 92.3.3 Calibration of distance 102.4 Types of experiments 102.4.1 Force-ramp 102.4.2 Force-clamp or constant-force experiments 112.4.3 Extension-clamp or constant extension experiments 112.4.4 Force-jump, Force-drop 122.4.5 Passive mode 123. Thermodynamics 123.1 Reversibility 123.2 Gibbs free energy 133.2.1 Stretching free energy 143.2.1.1 Rigid molecules 143.2.1.2 Compliant or flexible molecules 153.2.2 Free energy of a reversible unfolding transition 153.2.3 Free energy of unfolding at zero force 163.2.4 Free energy of an irreversible unfolding transition 163.2.4.1 Jarzynski's method 173.2.4.2 Crooks fluctuation theorem 194. Kinetics 214.1 Measuring rate constants 214.1.1 Hopping 214.1.2 Force-jump, Force-drop 234.1.3 Force-ramp 244.1.4 Instrumental effects 264.2 Kinetic mechanisms 274.2.1 Free-energy landscapes 274.2.2 Kinetics of unfolding 295. Relating force-measured data to other measurements 305.1 Thermodynamics 305.2 Kinetics 336. Acknowledgements 337. References 34Single-molecule methods have made it possible to apply force to an individual RNA molecule. Two beads are attached to the RNA; one is on a micropipette, the other is in a laser trap. The force on the RNA and the distance between the beads are measured. Force can change the equilibrium and the rate of any reaction in which the product has a different extension from the reactant. This review describes use of laser tweezers to measure thermodynamics and kinetics of unfolding/refolding RNA. For a reversible reaction the work directly provides the free energy; for irreversible reactions the free energy is obtained from the distribution of work values. The rate constants for the folding and unfolding reactions can be measured by several methods. The effect of pulling rate on the distribution of force-unfolding values leads to rate constants for unfolding. Hopping of the RNA between folded and unfolded states at constant force provides both unfolding and folding rates. Force-jumps and force-drops, similar to the temperature jump method, provide direct measurement of reaction rates over a wide range of forces. The advantages of applying force and using single-molecule methods are discussed. These methods, for example, allow reactions to be studied in non-denaturing solvents at physiological temperatures; they also simplify analysis of kinetic mechanisms because only one intermediate at a time is present. Unfolding of RNA in biological cells by helicases, or ribosomes, has similarities to unfolding by force.

Published 16 October 2006 in Q Rev Biophys.
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