Nanotracker

DNA constructs were incubated for 2 h with 2 μm silica beads (Bangs Laboratories, Inc), which were previously covalently functionalized with anti-digoxigenin F_ab fragments (Roche). The mixture was diluted in optical tweezers buffer (50 mM sodium phosphate buffer, 250 mM NaCl, 1% (w/v) d-glucose, pH 7.1) and mixed with streptavidin-coated 1 μm silica beads (Bangs Laboratories, Inc.). Measurements were carried out at room temperature in optical tweezers buffer after addition of an oxygen scavenger system (26 U/ml glucose oxidase, 17 000 U/ml catalase). DNA conjugates concentration was adjusted to only sparsely cover the beads leading mainly to single-tether formation. The beads were trapped in the foci of a dual beam optical tweezers platform (JPK NanoTracker) (9 (link)). Both trapped beads were brought into close proximity for tether formation. The beads were then separated with a constant velocity yielding force vs. extension traces. Trap stiffness was determined using a previously described calibration protocol (10 ). The effective trap stiffness was determined with an error of ∼10% and varied from 0.056 to 0.062 pN/nm. Data were acquired at a sampling rate of 50 kHz. The signals were corrected for crosstalk.