Single‑Molecule Force and Fluorescence Imaging of DNA–Intercalator Interactions Using an Integrated Optical Tweezers–Confocal Microscope

Abstract

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