RNA Construct Manipulation Using Optical Tweezers

The experimental setup for optical tweezers has been described previously (29 (link),30 (link)). More detailed description for an instrument similar to the one we used can be found in a recent report (31 (link)). Briefly, the two ends of an RNA construct were attached to two polystyrene beads (2.1 µm in diameter; Spherotech) coated with streptavidin and anti-digoxigenin antibodies, respectively. One bead was fixed on a micropipette and the other was trapped by laser beams, the position of which can be tuned to control the relative distance between these two beads. For force-ramp experiments, the trap was moved at a rate of 100 nm/s. For constant-force experiments, the position of the trap was feed-backed (with a frequency of 2 KHz) to maintain a preset force. The experiments were performed in a buffer containing 10 mM Tris–HCl, pH 7.0, 200 mM KCl and 20 mM MgCl₂, unless otherwise noted.