Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Friday Speaker Abstracts

BREAKING THE PHOTOBLEACHING LIMIT IN SINGLE-MOLECULE FRET BY DYECYCLING Sonja Schmid ; 1 University of Basel, Department of Chemistry, Basel, Switzerland 2 Swiss Nanoscience Institute, Basel, Switzerland With its unique spatio-temporal resolution at the single-molecule level, Förster resonance energy transfer (FRET) is an established approach to study conformational dynamics of proteins, DNA, and RNA, to understand biomolecular function beyond 3D structures. However, early photobleaching is a key limitation restricting the achievable information gain and the application range of single-molecule FRET. Specifically, it limits single-molecule observation times at fast time resolution and high signal-to-noise ratio, and it hinders the study of inter- and intra molecular heterogeneities – a specific hallmark of single-molecule experiments. Here, we establish nanophotonic DyeCycling, which bypasses photobleaching by reversible fluorophore binding and efficient background suppression using zero-mode waveguides. Nanoscale conformational changes are sensitively detected from milliseconds up to the hour range, enabling per-molecule kinetic analyses without ensemble averaging. As a result, slow kinetic regime changes within a single molecule are clearly resolved, and also static heterogeneity and outliers are confidently identified across the measured ensemble. We demonstrate the versatility of DyeCycling using DNA and protein systems, paving the way towards single-molecule FRET beyond photobleaching. For the future, we anticipate that the hour-long direct observations of biomolecular dynamics gained by DyeCycling will reveal previously missed effects and facilitate the elucidation of biomolecular systems with multiple states and rates.

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