Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results

TL;DR

This study investigates how various experimental variables in optical tweezers affect RNA unfolding and refolding kinetics, revealing that handle length, trap stiffness, and force mode influence measured rates within a consistent order of magnitude.

Contribution

It systematically quantifies the effects of handle length, trap stiffness, and force mode on RNA kinetics measurements using optical tweezers.

Findings

Measured rates vary within 40% with handle length changes.

Lower trap stiffness increases rates two- to three-fold.

Rates remain within the same order-of-magnitude across conditions.

Abstract

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.