Nanophotonic DyeCycling delivers single-molecule FRET beyond photobleaching to identify heterogenous dynamics in DNA and protein systems
- 1University of Basel
Abstract
With its unique spatiotemporal resolution at the single-molecule level, Forster resonance energy transfer (FRET) is a powerful tool for investigating biomolecular conformational dynamics and function. However, early photobleaching remains a key limitation restricting the achievable observation time and therefore the information gain and application range of single-molecule FRET. Here, we establish nanophotonic DyeCycling, which overcomes photobleaching by reversible fluorophore binding and efficient background suppression using zero-mode waveguides. Nanoscale conformational changes are sensitively resolved from milliseconds up to the hour range, enabling robust kinetic analyses at the single-molecule level. As a result, outlier molecules reflecting static heterogeneity and time-dependent kinetics within individual molecules reflecting dynamic heterogeneity are reliably resolved. The versatility of DyeCycling is demonstrated using DNA and protein systems. Altogether, nanophotonic DyeCycling extends smFRET observations beyond the conventional photobleaching limit, revealing previously inaccessible kinetic effects and providing deeper insight into biomolecular systems with multiple states and rates.
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BibTeX
@misc{vermeer2026nanophotonic,
title = {{Nanophotonic DyeCycling delivers single-molecule FRET beyond photobleaching to identify heterogenous dynamics in DNA and protein systems}},
author = {B. Vermeer and D. H. Shin and A. Vogel and F. Zundel and S. Caneva and S. Schmid},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.23.753919},
url = {https://www.biorxiv.org/content/10.64898/2026.09.23.753919v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Nanophotonic DyeCycling delivers single-molecule FRET beyond photobleaching to identify heterogenous dynamics in DNA and protein systems
AU - B. Vermeer
AU - D. H. Shin
AU - A. Vogel
AU - F. Zundel
AU - S. Caneva
AU - S. Schmid
PY - 2026
DA - 2026/09/23
PB - bioRxiv
DO - 10.64898/2026.09.23.753919
UR - https://www.biorxiv.org/content/10.64898/2026.09.23.753919v1
AB - With its unique spatiotemporal resolution at the single-molecule level, Forster resonance energy transfer (FRET) is a powerful tool for investigating biomolecular conformational dynamics and function. However, early photobleaching remains a key limitation restricting the achievable observation time and therefore the information gain and application range of single-molecule FRET. Here, we establish nanophotonic DyeCycling, which overcomes photobleaching by reversible fluorophore binding and efficient background suppression using zero-mode waveguides. Nanoscale conformational changes are sensitively resolved from milliseconds up to the hour range, enabling robust kinetic analyses at the single-molecule level. As a result, outlier molecules reflecting static heterogeneity and time-dependent kinetics within individual molecules reflecting dynamic heterogeneity are reliably resolved. The versatility of DyeCycling is demonstrated using DNA and protein systems. Altogether, nanophotonic DyeCycling extends smFRET observations beyond the conventional photobleaching limit, revealing previously inaccessible kinetic effects and providing deeper insight into biomolecular systems with multiple states and rates.
ER -