Figure 4
From: Prokaryotic Argonaute from Archaeoglobus fulgidus interacts with DNA as a homodimer
Single-molecule studies of AfAgo-DNA interactions in solution. (a) A schematic overview of the single-molecule assay. Left, free DNA; right, WT AfAgo-DNA (blue and green circles) complex in a looped state. (b) Fluorescence intensity trace with 1 ms time bin of 25 pM DNA with 2 nM AfAgo. Red: inverted acceptor fluorescence upon donor excitation, green: donor fluorescence upon donor excitation. (c) Left—E-S histogram of DNA alone. The top and side axes contain, respectively, one-dimensional E (proximity ratio) and S (donor/acceptor stoichiometry) histograms of all bursts. Denoted are areas representing donor-only DNA, acceptor-only DNA, and dual-labelled DNA. Right—E-S histogram of DNA with 2 nM AfAgo. The one-dimensional E histogram on top is derived from bursts with S = 0.2–0.9, designated by horizontal lines in the E-S histogram. The red curve is a two-Gaussian fit to the data that gave positions of the Gaussian maxima on the E-axis (0.13 ± 0.01 and 0.39 ± 0.02). (d) Left—dependence of the ratio of looped and unlooped DNA molecules (parameter K) on WT AfAgo concentration (open circles). Right—the dependence of K on the AfAgoΔ concentration (open circles). Red diamonds in both graphs represent the competition experiment performed with 1.2 nM WT AfAgo and 0.6 nM AfAgoΔ. All data points are average values of three measurements ± 1 standard deviation.
