Fig. 2
From: Single molecule high-throughput footprinting of small and large DNA ligands
Binding selectivity, kinetics, and thermodynamics of Echinomycin. a The FC protocol in presence of Echinomycin at 300 nM is repeatedly applied to unzip H0 DNA hairpin and detect blocking events, shown in red, blue, and green. b Histogram of molecular extension in phase 2 converted to base-pairs, for the bead in a, shows three peaks. The histogram is fitted to Gaussian functions centered around the three binding sites. Results shown correspond to 135 cycles. c Distribution of blocking times, τ, for the three binding sites at F test = 17 pN for the bead in a. Error bars are inversely proportional to the square root of the number of points for each bin. d Schematics of ligand unbinding described as a Kramer Bell-Evans activated process. e Logarithm of the unbinding rate, k = 1/<τ> as a function of the applied force, F test, for the three binding sites. The k and <τ> values are computed as the average over 10 beads. Error bars are the s.e.m. The result of the linear fits are also shown. (Inset) Average blocking time, <τ>, as a function of F test for the three binding sites. f Equilibrium extension trace for H0 in the presence of Echinomycin at force F hop = 15.5 pN, showing hopping between the three partially unzipped configurations blocked at the Echinomycin binding sites. The three configurations are represented at the right side of the panel. g Schematics of the free energy landscape of the hairpin in presence of Echinomycin that shows three minima corresponding to the three partially unzipped hairpin configurations (upper panel) and histogram of the molecular extension shown in f, presenting three peaks, that is fitted to Gaussian functions (lower panel). The relative weights of the Gaussian are used to compute the probabilities of the different configurations and to estimate the Echinomycin binding energy (Methods and Supplementary Table 3)
