A custom dual-trap optical tweezers setup built around an upright fluorescent microscope (AxioImager.Z1, Carl Zeiss, Oberkochen, Germany) was used as described previously [42 (link),52 (link)]. In brief, a ND:YVO4 1064 nm laser beam (Spectra-Physics, Mountain View, CA, USA) was split in two using a polarizing beam splitter cube and focused with an oil immersion lens (LOMO 100X, NA 1.32, St. Petersburg, Russia) to generate two orthogonally polarized optical traps. The x–y position of one of the traps was operated by the mirror mounted on a piezo platform (S-330.80L, Physik Instrumente, Karlsruhe, Germany). The images of the trapped beads were obtained with an EMCCD camera (Andor Technology, iXon Ultra 897, Belfast, UK) and further processed for real-time measurements of DNA-tether length and applied tension with 30 ms time resolution. Force–clamp and force–extension measurements were performed using custom software developed in LabView.
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