Figure 4.

Wash-off experiments using scavenger DNA. (a) DNA extension as a function of time (green) in a wash-off experiment performed at 12 pN (orange) using peptide-free buffer. The extension decreases with time due to the unbinding of Thiocoraline. However, upon increasing the force after 2 h, an exponential elongation is observed, showing that small amounts of ligand remained in the microfluidics chamber. This effect can be suppressed by transiently flushing a high concentration of scavenger DNA (purple). Normal timescale after 100 min. (b) Unzipping the DNA hairpin (6.8 kb) without ligand shows a characteristic quasi-reversible sawtooth pattern due to base-pair disruption (gray). In the presence of Thiocoraline, a series of force peaks is observed during pulling (dark blue) indicative of binding events. During refolding (light blue), kinetic states that temporarily block the hairpin rezipping are sometimes observed (*). (c) To test the wash-off method, we flushed the scavenger DNA in, and removed the bound ligands by unzipping and rezipping the hairpin (blue). If the hairpin is immediately unzipped again (red), very few binding events remain. (d) Consecutive unzipping cycles performed during the following 10 min do not show binding (cycles shifted upward for clarity), and only occasionally (<20%) individual binding events are observed (arrow).