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 <sup>3</sup> FS) assay that uses parallelized microfluidics to measure up to 80 sequence variants per experiment. Using SM <sup>3</sup> 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 <sub>rupture</sub> < 3 pN at 0.5 pN s <sup>-1</sup> ), revealing how mechanosensitivity can arise as an intrinsic property of multivalent systems. By enabling systematic sequence-function mapping under force, SM <sup>3</sup> FS opens a path to high-throughput nonequilibrium studies of biomolecules.
Authors
- Jennifer E. Ortiz-Cárdenas
- Magnus S. Bauer (ORCID: https://orcid.org/0000-0003-1357-2852)
- Matthew DeJong (ORCID: https://orcid.org/0000-0002-0589-124X)
- Yujia Bian
- Polly M. Fordyce (ORCID: https://orcid.org/0000-0002-9505-0638)
- Bianca Figueroa
- Ashley R. Dunn
- Lillian Brixi
- Ananya Pant
- Eduardo Posadas-Barrera
Institutions
- Cardiovascular Institute of the South (US)
- Chan Zuckerberg Initiative (United States) (US)
- Stanford Medicine (US)
- Stanford University (US)
Publication Details
- Journal
- bioRxiv (Cold Spring Harbor Laboratory)
- Published
- 2026-02-25
- DOI
- https://doi.org/10.64898/2026.02.24.707783
- Primary Topic
- Force Microscopy Techniques and Applications
- Type
- article
- Field-Weighted Citation Impact
- 0.00