Fig. 2: smFRET observations of DNA unwinding by T4 helicase using LAGOON.

a, Representative donor (green), acceptor (red) and FRET time traces (out of n = 117 traces) showing the unwinding of a single DNA molecule upon continuous ATP uncaging with 4 W cm⁻² UV laser power density (purple). b, Representative FRET time traces showing the unwinding of individual DNA molecules at different ATP concentrations in a standard experiment (left, n = 775, 1,388, 145 and 441) or at different uncaging laser intensities in a LAGOON experiment with continuous uncaging (right, n = 85, 178, 153 and 303). c, Histograms of the ratio between Cy5 fluorescence intensities upon 532-nm and 638-nm excitation before and after unwinding in a standard experiment (left, n = 1,513) or before and after continuous uncaging for 1 minute with 0.75 W cm⁻² UV laser power density in three FOVs on the same slide in a LAGOON experiment with 10 mM DMNPE-ATP (right, n = 1,454, 1,327 and 1,106). The Cy5_532nm/Cy5_638nm ratio was chosen instead of FRET because the donor-labeled DNA strand dissociated upon unwinding, leading to the absence of any detectable FRET signal. d, DNA unwinding rates (mean ± s.e.m.) as a function of ATP concentration (n = 637, 775, 1,800, 1,388, 145, 441, 182, 222, 209 and 131) or uncaging laser power density in the presence of 2 mM DMNPE-ATP (n = 85, 141, 134, 169, 178, 252, 104, 94, 186, 153, 210, 305, 303, 229 and 85) upon continuous uncaging. The plot on the right represents an enlarged version of the region marked in yellow in the plot on the left, scaled to overlay the linear sections of two titration curves. e,f, High-throughput smFRET observations of DNA unwinding using LAGOON on a motorized objective-type TIRF setup. e, Representative FRET time traces showing the unwinding of individual DNA molecules during continuous uncaging with approximately 2.5 W cm⁻² 405-nm laser in the presence of 200 nM T4 helicase, 10 mM DMNPE-ATP and 10 U ml⁻¹ of hexokinase (out of n = 64,243 traces from 112 FOVs). f, Histogram of DNA unwinding rates (n = 158,523 unwinding events). The experiments (e,f) were carried out on a Nikon Eclipse Ti2-E motorized microscope equipped with a CFI Apochromat TIRF 60XC objective. a.u., arbitrary unit.