Fig. 5: R-loop dynamics on locking-deficient targets containing two internal mismatches.

a Sketch of the DNA construct with mismatched bases shown in red and the possible R-loop states: the unbound state (\(U\)), two intermediate R-loops (\(I\) before the first and \({I}^{*}\) before the second mismatch) and the almost full but unlocked R-loop (\({F}^{*}\)). Sequential transitions between these states can be described by six different rates. Example trajectory of the transitions between the different states measured for a C:C mismatches. b Trajectories and histograms of the DNA length recorded for targets containing C:C mismatches with varying proximities at a torque of −6.7 pN nm (light blue). Four-state and three-state (in case \(I\) and \({I}^{*}\) states were indistinguishable) approximations of the trajectories are shown as dark lines. The central panel shows enlarged views of the regions between the dashed lines. Solid lines in the histograms represent Gaussian fits experimental histograms, while horizontal dashed lines indicate the average DNA length of each state. Bars represent theoretically predicted occupancies using the parameters shown in Supplementary Table 1. c Model of free energy landscapes for R-loop formation for the 11–17 (top) and the 14–17 (bottom) double mismatch targets together with the normalized occupancies of the \(I\) and \({I}^{*}\) states obtained from the model. Source data are provided as a Source Data file.