Studying T-cell force activation and DNA repair dynamics at the single-molecule level with optical tweezers and correlated fluorescence microscopy

DNA repair, the collection of highly regulated mechanisms by which a cell identifies and repairs DNA damage, remains one of the most essential processes of human life. Without DNA repair mechanisms cells lose the ability to transcribe important regions of their genome, resulting in harmful mutations, which could eventually jeopardize cellular wellbeing. Sources of DNA damage include double-stranded breaks and DNA intra- and interstrand crosslinks, which can ultimately become malignant tumors, leading to cancer. To study DNA repair, single-molecule studies have proven to greatly enhance understanding at the molecular level. However, it is often challenging to obtain adequate sensitivity and resolution, as well as biological conditions mimicking in vivo environments. The C-Trap™ system allows for realtime visualization of the interaction between DNA and DNA repair proteins, under biologically relevant conditions with high spatial and temporal resolution. The C-Trap™ is the only instrument that integrates optical tweezers, confocal/STED microscopy, and an advanced microfluidics system in a truly correlated manner. It enables live, simultaneous and correlative visualization and manipulation of molecular interactions with sub-picoNewton (pN) force resolution and a kilo-Hz (to mega-Hz) temporal resolution. Previously we have presented applications in: protein (un)folding and conformational changes; DNA-protein interactions and genome modifications; effects of mechanical stress on DNA/RNA structure; motility of cytoskeletal molecular motors; and protein droplet and aggregation dynamics. By culturing or adhering cells to the surface of the flow chamber, single-molecule cell-based studies can also be performed with the C-Trap. Cell-surface bound mechano-receptors can be activated by forming tethers with an optically trapped particle and then creating a controlled tension on the molecule. Correlated fluorescent imaging can elucidate internal protein cascades and other similar reactions to the applied force. Here, we present our experiments visualizing and quantifying the mechanisms of DNA repair and the force activation of T-cells using the C-Trap system. These experiments show that the technological advances in hybrid single-molecule methods can be turned into an easy-to-use and stable instrument that opens up new venues in many research areas. Citation Format: Ali Raja, Leif Anderson, Willem Peutz, Andrea Candelli, Gerrit Sitters. Studying T-cell force activation and DNA repair dynamics at the single-molecule level with optical tweezers and correlated fluorescence microscopy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1759.