Single‑Molecule Force and Fluorescence Imaging of DNA-Intercalator Interactions Using an Integrated Optical...

Single-molecule techniques enable direct measurement of DNA mechanics while simultaneously visualizing ligand-associated fluorescence. This protocol describes DNA-tethered bead preparation, flow-cell passivation, power-spectrum trap calibration, confocal alignment, kymograph acquisition, and uniform analysis of force-extension curves with an extensible worm-like chain (eWLC) model. Representative data include synchronized raw force and fluorescence trajectories during a variable-force acquisition and force-extension measurements at 0, 50, 100, and 400 nM SYTOX Orange. The tested range did not define a saturation plateau; therefore, no dissociation constant (K_d) or kinetic rate is reported. The workflow can be adapted to other DNA-binding ligands after appropriate calibration and control experiments. A typical experimental session requires 4-6 h. The representative same-session dataset comprised 0, 50, 100, and 400 nM SYTOX Orange. After numerical and visual quality control, 5/6, 4/5, 5/5, and 6/6 force-extension records were retained, respectively. Mean ± SD eWLC contour lengths were 16.386 ± 0.130, 16.067 ± 0.075, 16.752 ± 0.219, and 18.916 ± 0.573 µm, respectively. The response did not reach a demonstrable plateau within this concentration range and was therefore not fitted to obtain K_d. © 2026 Wiley Periodicals LLC. Basic Protocol 1: DNA‑tether formation and single‑molecule force measurement Basic Protocol 2: Confocal alignment and kymograph acquisition for intercalation dynamics Support Protocol 1: Preparation of passivated flow cell and oxygen scavenging buffers Alternate Protocol 1: Using PCA/PCD oxygen scavenging system instead of glucose oxidase/catalase.