RNA Unzipping and Force Measurements with a Dual Optical Trap

In order to mechanically unfold a single RNA molecule, an RNA/DNA hybrid construction is prepared which allows specific attachment to two micrometer-sized beads. A dual-beam optical trap thus holding the construct in solution captures the beads separately. Unfolding of a molecule is obtained by increasing the distance between the traps, one trap being slowly moved while the other is held fixed. Force is measured to sub-piconewton precision by back focal plane interferometry of the bead in the fixed trap. The experiment allows us to measure structure and base-sequence-dependent force signals. In this chapter, important technical aspects of this type of single-molecule force measurements are considered.