CAREER: High-throughput multi-axial tension-inducing DNA origami device for investigating mechanosensitive signaling pathways and protein structures under defined tension

Minuscule mechanical forces within us regulate the functions of mechanosensitive proteins, which subsequently control how cells act and react. By studying integrins, the tiny proteins that serve as cellular anchors and sensors for the mechanical cues from their environments, new rules governing how cells translate mechanical forces they feel into decisions regarding their behavior can be uncovered. This insight will advance the understanding of living systems and has enormous potential in understanding diseases like cancer, where the stickiness and mechanical properties of cells play key roles. Beyond its scientific merit, the research's impact will stretch beyond the laboratory. An innovative, hands-on course that immerses undergraduate students in Biological Physics research, fostering the next wave of scientists, will be designed. Additionally, artists will help create science-inspired cartoons, making complex scientific concepts accessible and thrilling for children of all ages. This project will develop a novel approach to understanding the mechanotransduction pathways and structural biology of integrin proteins, a critical factor for cell adhesion and signaling. At the heart of this project is the Multi-Axial Entropic Spring Tweezers along Ring-shaped Origami (MAESTRO), a low-cost, high-throughput technology for applying controlled mechanical tensions to individual integrins from multiple directions. MAESTRO leverages the entropic elasticity of single-stranded DNA to generate mechanical tension, the exquisite positional control of DNA origami to control the directions of applied tensions, and device miniaturization to reduce cost and increase throughput. To validate the performance of MAESTRO, the tension-inducing tool will be benchmarked by assessing the kinetics of Holliday junctions under mechanical tension. Measurements will be compared with published data from optical tweezer experiments and theoretical predictions derived from the Boltzmann distribution. Advanced single-molecule methods, including confocal single-molecule Fluorescence Resonance Energy Transfer (smFRET) and total internal reflection microscopy, will be used to dissect the intricate signaling of integrins in response to these multi-directional mechanical stimuli. Finally, cryo-electron microscopy will be used to visualize the tension-induced state changes of integrins, offering structural insights into their signaling roles. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.