Development of electromagnetic tweezers for single-molecule energetics of kinesin

Optical tweezers have enabled single-molecule measurements of kinesin mechanics and energetics but face challenges for <i>in vivo</i> applications due to photothermal effects and refractive-index-dependent force calibration. Here, we developed electromagnetic tweezers featuring horizontal magnetic field geometry that enables force application along the microtubule axis for translational motor measurements. We validated the platform by reproducing established kinesin mechanics: stall force of ~6 pN and, using the Harada-Sasa equality to quantify nonequilibrium dissipation, an estimated energy conversion efficiency of ~20%, both consistent with optical tweezers results. We further demonstrated that FDT violations increase with ATP concentration following Michaelis-Menten kinetics, consistent with kinesin's enzymatic turnover. These results establish electromagnetic tweezers as a complementary tool offering advantages in high-throughput measurements and environment-independent force calibration, providing a foundation for future applications where these capabilities are essential.