Experimental setup and single-molecule FRET (smFRET) assay

A prism-type total internal reflection fluorescence (TIRF) setup, equipped with an Olympus IX-71 microscope and an Olympus 60×, 1.20 NA water objective, was utilized to perform smFRET measurements. Single molecule sensitivity was achieved by an Andor Ixon EMCCD camera (iXon DV 887-BI EMCCD, Andor Technology, CT, USA). A λ = 532 nm green laser (SpectraPhysics Excelsior) was used to excite the donor (Cy3) fluorophores. Emissions from donor and acceptor (Cy5) fluorophores were separated by a dichroic mirror and projected on two halves of a CCD screen.

The quartz slides and glass coverslips were extensively cleaned, including Piranha etching, and coated with polyethylene glycol (PEG). 1% of PEG molecules were tagged with biotin to provide attachment points for biotinylated DNA molecules, which bind to biotin-PEG via neutravidin. In addition, the surface was coated with 1 mg/ml casein (overnight incubation) or 1% (v/v) Tween-20 (15-minute incubation before adding neutravidin) to reduce non-specific binding of BLM to the surface. To provide adequate statistics for the analysis, 250–350 DNA molecules per imaging area (∼5 × 10³ μm²) was targeted as the ideal density in most measurements.

To improve the photostability of Cy3 and Cy5, an oxygen scavenging system (0.1 mg/ml glucose oxidase, 0.02 mg/ml catalase, 0.8 mg/ml glucose) was added to the imaging buffer that contains Tris–HCl (50mM, pH 7.5), 2 mM Trolox, 0.1 mg/ml bovine serum albumin (BSA), 150 mM KCl and 2 mM MgCl₂. Depending on the experiment, the imaging buffer also contained SM ligands, BLM, and ATP at desired concentrations. 1 mM dithiothreitol (DTT) was included in imaging buffer for measurements that included BLM. Before introducing BLM into a sample chamber, proper GQ folding was checked via smFRET histogram. We immediately started recording either short movies (15 frames) or long movies (1500–3000 frames) after introducing BLM into the sample chamber. The laser power and frame integration time (50 ms in Figure ​Figure11 and 200 ms in Figure ​Figure2)2) were adjusted depending on the goal of the measurement. All smFRET measurements were performed at room temperature, 23°C.