Fig. 6: Real-time observation of heteroduplex unwinding and riboswitch folding in VF assays.

a Individual real-time trajectory (left), overlay of all WT ZTP riboswitch folding trajectories (center, n = 56), and histograms of all trajectories at 0, 2, and 8 s after PIFE (right) using VF with Rep-X in the presence of 1 mM ZMP. The PIFE peaks were fit to a Gaussian (blue) to determine their centers for synchronizing trajectories. b Individual real-time trajectory (left), overlay of all WT ZTP riboswitch trajectories (center, n = 24), and histograms of all trajectories at 0, 2, and 8 s after PIFE (right) using VF with Rep-X in the absence of ZMP. The relative unwinding position is indicated by a red line, based on the experimentally determined Rep-X unwinding rate and the location of U32 on the template. Blue circles indicate time points corresponding to the unwinding and release of helices P1 and P2, helices P1-P4, and terminator helix PT. c Real-time trajectories of terminator mutant 81-83 A in the absence of ZMP using VF with Rep-X, showing an individual trajectory (left), all trajectories overlaid (center, n = 15), and histograms of all trajectories at 0, 2, and 8 s after PIFE (right). The time duration between ticks is 5 s. Histogram probabilities are on a log scale, with ticks of 0.1, 1, and 10 percent.