To learn more, you can find our new preprint here! 13/13

www.biorxiv.org/content/10.6...

This work wouldn’t be possible without the support of my thoughtful co-advisors @pollyfordyce.bsky.social and @dunnlab.bsky.social, their creative labs, generous funders (Stanford Bio-X, NSF, NIH, @czbiohub.bsky.social), @stanford-chemh.bsky.social, and my amazing co-authors! 12/13

So much of life is mechanical: T-cell activation, antibody maturation, pathogenesis, development, homeostasis, and more. They all rely on molecular force sensing. Expanding throughput is the first step for unlocking the potential of engineered molecular force responses. 11/13

Our results challenge the prevailing model that avidity necessarily leads to mechanically robust linkages; instead, we show how mechanosensitivity can arise as an intrinsic, nonequilibrium property of multivalency. 10/13

As it turns out, some of biology’s most stable yet sensitive mechanosensors, Notch and chromatin, are multivalent too. We speculate their multivalency provides sufficient stability to prevent spontaneous activation yet allows sensitive detection of small forces. 9/13

To decouple stability from strength, we connected short, unstable duplexes via flexible linkers. Given enough repeats, the multivalent construct could achieve avidity-driven thermodynamic stability yet rupture at low forces because each duplex remained mechanically weak. 8/13

In total, we measured two libraries encoding 241 DNA sequence variants. We quantified 131,847 single molecules from 249,148 observations, using only 10 microfluidic chips. Our most stable yet sensitive duplex ruptured at 2.7 pN. How? 7/13

With SM3FS, we sought to engineer DNA duplexes that were thermodynamically stable yet rapidly ruptured at physiologically low forces, ~ 3 pN (the pulling force of a single head of Myosin II). This challenge required designing and measuring many variants of DNA. 6/13

Using established DNA nanomechanics (e.g.stretching), we ensured that we (1) measured single molecules, (2) applied calibrated forces, and (3) had high-fidelity multiplexing. These experiments validated SM3FS as a high-throughput single-molecule technology. 5/13

We developed a single-molecule, multiplexed, microfluidic force spectroscopy (SM3FS) assay that arrays many flow chambers in one FOV. Additional multiplexing enabled library-scale experiments. 4/13

Our goal was straight-forward: expand the capabilities of single-molecule force spectroscopy to unlock high-throughput single-molecule engineering. 3/13

Biological systems are constantly sensing and responding to force. However, only a handful of molecular force responses have been characterized. Characterization is bottlenecked by sensitive yet laborious technologies. 2/13

First preprint of the @pollyfordyce.bsky.social and @dunnlab.bsky.social collaboration! We used high-throughput microfluidics for sequence-strength mapping at the single-molecule level. Our new tech allowed us to discover a fundamental nonequilibrium property of multivalent systems. 1/13