Figure 2.

Simultaneous measurements of FRET and torque. (a) A GC-repeat sequence was incorporated in a DNA tether. The magnetic bead was rotated to change the total twist (θ) in the DNA while using a rotor bead to measure the torque (τ) and a local FRET pair (green star, Cy3; orange star, Cy5) to monitor the B-Z structural transition. (b) The B-Z transition is observed via simultaneous jumps in torque (top, 250 Hz) and FRET efficiency (bottom, 5 Hz). Only the rewinding portion of the torque-twist curve is shown. Thick gray lines are fits to torque-twist data before and after the B-Z transition. (c) Combined torque and FRET measurements are shown for four different molecules. In the orange and magenta traces, one of the fluorophores bleaches before the structural transition in the SOI is observed. Data are block-averaged in 0.05 rotation (2 s) windows. (d) The FRET efficiency shows continuous variation with torque in both the B- and Z-DNA states of the SOI. Trend lines (black) were fit to the data from (c), after replotting on the torque-FRET plane. FRET changes at a rate of −0.5% (pN nm)⁻¹ when the SOI is in B-form (top) and at a rate of −1.3% (pN nm)⁻¹ when the SOI is in Z-form (bottom).