The effect of force on thermodynamics and kinetics: unfolding single RNA molecules

Abstract

We have used laser tweezers to unfold single RNA molecules at room temperature and in physiological-type solvents. The forces necessary to unfold the RNAs are over the range 10-20 pN, forces that can be generated by cellular enzymes. The Gibbs free energy for the unfolding of TAR (transactivation-responsive) RNA from HIV was found to be increased after the addition of argininamide; the TAR hairpin was stabilized. The rate of unfolding was decreased and the rate of folding was increased by argininamide.

Figure 1. Force unfolding and refolding of TAR RNA

(a) Secondary structure of the TAR hairpin we studied. (b) A force–extension curve for the TAR hairpin in 100 mM KCl, 10 mM Hepes (pH 8.0) and 1 mM EDTA is shown. The loading rate was approx. 3 pN · s⁻¹; the amount of hysteresis and the distribution of forces at which the rips occur depend on the loading rate.

Figure 2. Force clamp kinetics

Upper panel: the time dependence of two successive force jumps and drops; lower panel: the corresponding changes in extension. Measurement of the lifetime of the unchanged molecule after each jump or drop in force yields the rate constants.

Figure 3. Base-10 logarithms of the rate constants for unfolding and refolding TAR RNA are plotted against force

Unfolding and refolding for TAR RNA in EDTA (pH 8) with 100 mM KCl (circles) or 100 mM NaCl (squares), and TAR RNA in NaCl buffer, but with 10 mM argininamide added. The temperature was 20°C.