DNA Origami Tension Sensors (DOTS) for Single-Molecule Force Measurements at Fluid Intermembrane Junctions

A key event in triggering adaptive immunity is the binding of a T cell receptor (TCR) to its antigen at the T cell–target cell interface. Mechanical forces are critical for TCR–antigen interactions, where piconewton (pN) forces modulate immune responses. A major challenge in studying these interactions is quantifying forces at the single-molecule scale, as T cells can activate in response to just 1–10 antigen molecules. To address this, we developed single-molecule DNA origami tension sensors (smDOTS) for high-resolution force mapping. Our design includes spectral fingerprint density reporters, multiple quenchers for extended force dynamics monitoring, and tunable cholesterol anchors for controlled mobility. We report unprecedented measurements of TCR–antigen forces at fluid membranes, detecting forces with magnitudes of 8 to 19 pN, and tracking ligand translocation. Multiplexing enabled the simultaneous imaging of sensors with different force thresholds. This approach could further reveal bond lifetimes and force dynamics, deepening our understanding of TCR-mediated signaling.