Engineering the mechanosensitivity of single DNA molecules via high-throughput microfluidic force spectroscopy

Single-molecule force spectroscopy (SMFS) is a powerful tool to measure how biomolecules respond to mechanical force, but limited sequence throughput constrains its potential. Here, we present a single-molecule, multiplexed, microfluidic force spectroscopy (SM ³ FS) assay that uses parallelized microfluidics to measure up to 80 sequence variants per experiment. Using SM ³ FS, we stretched, overstretched, or unzipped 241 different DNA structures and generated 131,847 single-molecule traces from a quarter-million observations. High sequence throughput allowed us to identify DNA structures that are kinetically stable yet mechanically fragile (F _rupture < 3 pN at 0.5 pN s ^-1 ), revealing how mechanosensitivity can arise as an intrinsic property of multivalent systems. By enabling systematic sequence-function mapping under force, SM ³ FS opens a path to high-throughput nonequilibrium studies of biomolecules.