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Force Unfolding Kinetics of RNA using Optical Tweezers. II. Modeling Experiments

M. Manosas , J.-D. Wen ^*, P. T. X. Li ^*, S. B. Smith , C. Bustamante ^*  , I. Tinoco, Jr. ^* and F. Ritort  ^¶

^* Department of Chemistry, Department of Physics, and Department of Molecular and Cell biology and Howard Hughes Medical Institute, University of California at Berkeley, Berkeley, California; Department de Fisica Fonamental, Universitat de Barcelona, Barcelona, Spain; and ^¶ CIBER de Bioingenieria, Biomateriales i Nanomedicina, Instituto de Sanidad Carlos III, Madrid, Spain

Correspondence: Address reprint requests to I. Tinoco, Tel.: 510-526-3817; E-mail: intinoco{at}lbl.gov.

By exerting mechanical force, it is possible to unfold/refold RNA molecules one at a time. In a small range of forces, an RNA molecule can hop between the folded and the unfolded state with force-dependent kinetic rates. Here, we introduce a mesoscopic model to analyze the hopping kinetics of RNA hairpins in an optical tweezers setup. The model includes different elements of the experimental setup (beads, handles, and RNA sequence) and limitations of the instrument (time lag of the force-feedback mechanism and finite bandwidth of data acquisition). We investigated the influence of the instrument on the measured hopping rates. Results from the model are in good agreement with the experiments reported in the companion article. The comparison between theory and experiments allowed us to infer the values of the intrinsic molecular rates of the RNA hairpin alone and to search for the optimal experimental conditions to do the measurements. We conclude that the longest handles and softest traps that allow detection of the folding/unfolding signal (handles 5–10 Kbp and traps 0.03 pN/nm) represent the best conditions to obtain the intrinsic molecular rates. The methodology and rationale presented here can be applied to other experimental setups and other molecules.

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Force Unfolding Kinetics of RNA Using Optical Tweezers. I. Effects of Experimental Variables on Measured Results

Jin-Der Wen, Maria Manosas, Pan T. X. Li, Steven B. Smith, Carlos Bustamante, Felix Ritort, and Ignacio Tinoco, Jr.
Biophys. J. 2007 92: 2996-3009. [Abstract] [Full Text]  

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				J.-D. Wen, M. Manosas, P. T. X. Li, S. B. Smith, C. Bustamante, F. Ritort, and I. Tinoco Jr. Force Unfolding Kinetics of RNA Using Optical Tweezers. I. Effects of Experimental Variables on Measured Results Biophys. J., May 1, 2007; 92(9): 2996 - 3009. [Abstract] [Full Text] [PDF]