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

Abstract

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 (SM3FS) assay that uses parallelized microfluidics to measure up to 80 sequence variants per experiment. Using SM3FS, 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 (Frupture < 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, SM3FS opens a path to high-throughput nonequilibrium studies of biomolecules.