Sensitivity Of Dna-hairpins Dynamics To The Mechanism Of Force Feedback Probed Using A Surface-coupled Passive Force Clamp

Abstract

Optical-trapping experiments have yielded new insight into the mechanical behavior of individual biomolecules. A common experimental assay consists of an enzyme or nucleic acid molecule attached to a cover slip at one end and to a small polystyrene bead at the other. The bead is captured and held under tension with an optical trap. Active feedback maintains constant force, often called a force clamp, to increase measurement precision. Yet, active feedback is inherently bandwidth limited. This limited bandwidth leads to significant fluctuations in force that are particularly pronounced for rapid, large changes in molecule extension (e.g. DNA hairpin unfolding). A novel, passive force clamp circumvents this limitation by pulling the bead to a non-linear region of the trap where ktrap = 0. To date, this passive force clamp has required a specialized dual optical trapping apparatus where one trap measures position and the other measures force. Here, we demonstrate a passive force clamp achieved with a single trap in a surface-coupled assay using a previously characterized DNA hairpin. In an active force clamp, rapid back-and-forth transition between open and closed hairpins states were observed within the update period of the active force clamp (2 or 10 ms) as well as a change in the long term dynamics. By using a passive force clamp, these spurious transitions were eliminated and the correct dynamics measured. By analyzing the fluctuations in the bead position in conjunction with the known elasticity of DNA, we simultaneously measured force and position in a single-beam, passive force clamp. Thus, the benefits of the passive force clamp are now available to the widely used surface-coupled optical trapping assays.