Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

39-POS Board 19 A DYNAMIC DNA ORIGAMI DEVICE FOR CONTINUOUS SENSING VIA SINGLE MOLECULE FRET Line M Lund 1 ; Emily Tsang 1 ; Victoria Birkedal 1 ; Kurt V Gothelf 1 ; 1 Aarhus University, Chemistry, Aarhus, Denmark Continuous, real-time monitoring of molecular targets requires sensors that report reversibly rather than only once. Inspired by the motion of macroscopic machines, we designed a DNA origami nanodevice for programmable, continuous nanoscale sensing. The DNA origami offers nanometre-precise positioning of donor and acceptor fluorophores, making it an ideal scaffold for ratiometric FRET readouts. The device features a rotating top bundle that reversibly switches between distinct conformational states through toehold-mediated strand displacement. Each transition repositions the fluorophore pair, producing well-defined changes in fluorescence intensity and FRET efficiency that report the state of the device, while competing strands return it to its original configuration for repeated sensing cycles. Using ensemble fluorescence spectroscopy together with single-molecule microscopy, we demonstrated dual- and multiplexed sensing of nucleic acid targets. Single-molecule FRET measurements at the Aarhus Single Molecule Fluorescence (ASiMoF) infrastructure resolved individual devices and revealed real time dynamics and state transitions hidden in ensemble averages. Tracking FRET efficiency over time on immobilised structures let us distinguish discrete states, follow reversible switching, and measure the kinetics of target-driven transitions at the single-molecule level. A complementary dual-mode strategy combining FRET with fluorescence quenching extended the approach to multiplexed detection of several targets in parallel. Together, these results establish a versatile framework for the rational design of dynamic DNA nanodevices and underline single-molecule FRET as a powerful tool for resolving their motion. We anticipate this approach will enable reusable, continuously operating biosensors for a broad range of molecular analytes.

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