Fig. 1: Using AuRBT to detect Cas12a binding and R-loop formation.

A Schematic of rotor bead tracking assay. A torsionally constrained DNA tether is stretched between a cover slip and magnetic bead. An evanescent field generated by TIR illuminates the rotor bead. Torque (τ) is measured from the difference between magnet position (θ) and the angular deflection of the rotor bead (Ψ), reporting on structural transitions in the target sequence. B R-loop formation event in the presence of dLbCas12a. Measured torque (above) and calculated change in equilibrium twist (below) as a function of total twist imposed by rotating magnets. Raw data are shown in black; 1 s averaged traces are shown in red. The change in equilibrium twist is shown in units of base pairs unwound, assuming that changes are due to unwinding of B-DNA. R-loop formation produces a jump in torque and a corresponding step in equilibrium twist. C Torque vs. twist for unwinding and rewinding cycles in the presence of dAsCas12a. Magnets were rotated to cycle between positive and negative twist, driving repeated R-loop formation and collapse events. Raw data for a single cycle of unwinding (black) and rewinding (gray) are shown with overlayed time-averaged (1 s) unwinding (red-orange) and rewinding (blue-green) traces from the same cycle and two additional magnet cycles. Vertical arrows indicate locations of R-loop formation at negative torque (pink) and collapse at positive torque (blue), respectively. Diagonal arrows show the direction of magnet rotation during unwinding (black) and rewinding (gray). D Example R-loop formation event for dAsCas12a with a fully-matched (FM) guide RNA (gRNA). Additional panels show zoomed-in regions. Yellow lines show idealizes traces generated by change-point analysis. Dashed magenta lines are at 5 bp, 17 bp, and 20 bp to guide the eye. E Example R-loop formation event for dLbCas12a with a FM sgRNA. Insets: From left to right, zoomed-in views of R-loop formation, hopping between distal R-loop states, and R-loop collapse.