Monitoring the flipping of RT by single-molecule FRET assay

Microscope quartz slides (G. Finkenbeiner, Inc.) and cover slips were passivated with PEG as described (33). Surface passivated slides, together with cover slips, were stored under inert conditions at −80°C until used.

Flow cells were assembled by sandwiching double-sided tape between a cover slip and a quartz slide. Typically the volume of the flow cell was ≤10 μl. A total of 100 μl of buffer A (50 mM Tris-HCl, pH 8.0, 50 mM NaCl and 6 mM MgCl₂) was applied through the flow cell before applying a 0.2 mg/ml streptavidin in buffer A for 1 min. Excess streptavidin was removed with 100 μl of buffer A before incubating a 50 pM biotin–DNA substrate. Finally, the flow cell was washed with 100 μl of buffer A to remove unbound DNA. Fluorescence measurements were performed in an imaging buffer consisting of 50 mM Tris-HCl, pH 8.0, 6 mM MgCl₂, 20 nM of RT-Cy3, 2 mM trolox, 0.2% triton-X100 and an oxygen scavenging system (0.3 mg/ml glucose oxidase and 40 μg/ml catalase with 5% (w/v) glucose as a substrate). Additionally, PEG 8K was used as a crowding agent and NaCl was used to vary the salt concentration. Where needed, the concentrations of NaCl and PEG 8K are mentioned.