Multiplexed microfluidics for massively parallel single-molecule force spectroscopy
The study of mechanosensitive biomolecules is limited primarily to highly informative but low-throughput single-molecule force spectroscopy assays. Here, we present a spatially multiplexed, microfluidic, single-molecule force spectroscopy (SM3FS) assay that combines bead-based flow assays with a programmable microfluidic device to dramatically increase the throughput of force spectroscopy measurements. To validate our approach, we measured the force-extension curves of a set of 15 DNA molecules in parallel, measuring a total of >10,000 individual DNA tethers at 20-nm and sub-pN resolutions. We next applied SM3FS to systematically investigate the mechanical stability of a library of 700 designed DNA duplexes resulting in hundreds of thousands of single-molecule rupture measurements. These data reveal that avidity, while enhancing equilibrium stability, does not necessarily enhance mechanical stability. Instead, avidity can generate jagged free energy landscapes with remarkable tension sensitivity. Ongoing work aims to apply SM3FS to explore and define the mechanobiome, the set of mechanically regulated interactions within and between living cells.
Dr. Alexander R. “Alex” Dunn is a Professor in the Department of Chemical Engineering at Stanford University. He received his Ph.D. from the California Institute of Technology in 2003, where he studied the catalytic mechanism of selective C–H bond oxidation by cytochrome P450 enzymes under the direction of Harry Gray. After postdoctoral work with James Spudich in biochemistry at Stanford, he joined the faculty and now leads research investigating how molecular-scale mechanical information and protein asymmetries are translated into the physical form and function of cells, tissues and organs. His work leverages cutting-edge microscopy, single-molecule biophysics and modeling to unravel how cells sense and respond to mechanical stimuli, with implications for stem cell biology, tissue engineering and mechanobiology. His contributions have been recognized by a number of prestigious awards including the HHMI Faculty Scholar Award, the NIH Director’s New Innovator Award, and the Burroughs Wellcome Career Award.