Highly-parallel, microfluidics-based force spectroscopy on single motor proteins

Optical trapping experiments have provided crucial insight into the operation of molecular motors. However, these experiments are mostly limited to one measurement at a time. Here, we describe an alternative, highly-parallel, microfluidics-based method that allows for rapid data collection. We applied tunable hydrodynamic forces to stepping kinesin-1 motors via DNA-tethered beads and utilized a large field-of-view to simultaneously track the velocities, run lengths and interaction times of hundreds of individual kinesin-1 molecules under varying resisting and assisting loads. Importantly, the 16-μm long DNA tethers between the motors and the beads significantly reduced the vertical component of the applied force. Consequently, forces were predominantly exerted in the direction of motor movement, rather than away from the microtubule surface as unavoidable in conventional optical tweezers experiments. Our approach is readily applicable to other motors and constitutes a new methodology for parallelized single-molecule force studies.