  Experimental variables of optical tweezers instrumentation that affect RNA
folding/unfolding kinetics were investigated. A model RNA hairpin, P5ab, was
attached to two micron-sized beads through hybrid RNA/DNA handles; one bead was
trapped by dual-beam lasers and the other was held by a micropipette. Several
experimental variables were changed while measuring the unfolding/refolding
kinetics, including handle lengths, trap stiffness, and modes of force applied
to the molecule. In constant-force mode where the tension applied to the RNA
was maintained through feedback control, the measured rate coefficients varied
within 40% when the handle lengths were changed by 10 fold (1.1 to 10.2 Kbp);
they increased by two- to three-fold when the trap stiffness was lowered to one
third (from 0.1 to 0.035 pN/nm). In the passive mode, without feedback control
and where the force applied to the RNA varied in response to the end-to-end
distance change of the tether, the RNA hopped between a high-force folded-state
and a low-force unfolded-state. In this mode, the rates increased up to
two-fold with longer handles or softer traps. Overall, the measured rates
remained with the same order-of-magnitude over the wide range of conditions
studied. In the companion paper (1), we analyze how the measured kinetics
parameters differ from the intrinsic molecular rates of the RNA, and thus how
to obtain the molecular rates.
