a, Power spectrum showing the Brownian fluctuation in the angular position of the rotor attached to the anchor by 52 bp of dsDNA. Representative of 3 independent biological replicates. Red line shows the modified Lorentzian fit, as described in Supplementary Discussion, Supplementary Equation (1). This fit yields the torsional stiffness (κ) and hydrodynamic drag (γ). b, Dependence of the inverse of κ on the length of dsDNA segment between the rotor and origami. The slope of the linear fit yields the torsional stiffness per unit length of the dsDNA in the absence of an applied force (Supplementary Discussion, Supplementary Equation (3)), C = 200 ± 10 pN nm2 rad−1, which is consistent with previous measurements under zero force12. The inverse of the y-offset from this fit, κ other = 30 ± 8 pN nm rad−1 (Supplementary Discussion, Supplementary Equation (3)), represents the torsional stiffness of the remainder of the structure, which is the equivalent of about 20 bp of dsDNA. c, Dependence of the relaxation time, τ = γ/κ, on the length of the dsDNA segment between the rotor and anchor calculated using κ and γ derived from the power spectrum fit (Supplementary Discussion, Supplementary Equation (1)). Data in b, c are mean ± s.e.m. (n = 203, 195 and 133 (from left to right) rotor–anchor complexes from 3 independent biological replicates). d, Dependence of γ of the origami rotor on the viscosity of the buffer. The origami rotor was connected by a 92-bp dsDNA segment to the anchor. The different viscosities were achieved using 0%, 10% and 25% glycerol. Data are mean ± s.e.m. (n = 195, 210 and 150 (from left to right) rotor–anchor complexes from 3 independent biological replicates). e, Standard deviation of the angular positions of the rotor as a function of integration time. Black line shows the s.d. measured from a single rotor connected to the anchor with a 52-bp dsDNA segment tracked at 3 kHz after down-sampling to the indicated integration time. Representative of 3 independent biological replicates. Red and blue curves show predicted precision with and without (respectively) taking into account the contribution of localization uncertainty (Supplementary Discussion, Supplementary Equation (2)). κ = 7.8 pN nm rad−1, γ = 5.0 fN nm s and localization uncertainty per frame \({\sigma }_{L}^{2}\) = 0.038 rad2. κ and γ were derived from the measurements of multiple (n = 203) rotors with 52-bp dsDNA segment connecting the rotor to the anchor. \({\sigma }_{L}^{2}\) was estimated using the measurement uncertainty in radial position, and converted to an angular value using the radius of the circular trajectory. The crossing points of the top and bottom dashed lines with the s.d. versus integration time curve give the integration times required for detection of single-base-pair rotation (34.6°) with a signal-to-noise ratio of 1 and 3, respectively. f, Radial localization uncertainty (s.d.) during processive unwinding by RecBCD as a function of the average fluorescence signal intensity (mean) from individual rotors. Representative of 3 independent biological replicates. We apply a localization uncertainty threshold of 16 nm (0.1 pixel), shown in red, to select only trajectories with relatively high localization precision. All measurements were performed with 0% glycerol, except for d.