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bioRxiv · 10.1101/2025.09.14.675960

Massively Parallel Bead-Free Force Spectroscopy with Fluorescence

Yasunaga, A. B.·Riopel, R.·Bakker, D. t. R.·Raghu, D.·Yang, M.·Li, I. T. S.
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Abstract

Single-molecule force spectroscopy (SMFS) has transformed our understanding of biomolecular mechanics. However, current high-throughput implementations rely on beads to apply force, introducing size and surface chemistry variability, requiring per-bead calibration, and are prone to multi-tether artifacts. Long handles further complicate measurements by convolving target conformational changes with handle stretching. We introduce Tether Force Spectroscopy (TFS), a bead-free SMFS platform where a single DNA tether acts as both the force applicator and internal calibrator. In TFS, shear flow acting on identical DNA tethers applies piconewton-scale forces directly to surface-anchored molecules whose conformational dynamics are simultaneously monitored by single-molecule fluorescence. This guarantees single-tether results with uniform, internally calibrated forces and is inherently compatible with single-molecule fluorescence. We achieved high-resolution, high-throughput measurements across hundreds of molecules, enabling both force-extension and rupture experiments without specialized instrumentation. The combination of simplicity and simultaneous force-fluorescence capability makes TFS broadly accessible for correlating structure and function in diverse biomolecular systems.

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Yasunaga, A. B., Riopel, R., Bakker, D. t. R., Raghu, D., Yang, M., Li, I. T. S.. 2025-09-17. Massively Parallel Bead-Free Force Spectroscopy with Fluorescence. https://doi.org/10.1101/2025.09.14.675960

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