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.