Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Tuesday Speaker Abstracts

THE DARK SIDE OF FRET: SELF-QUENCHING DNA PROBES ENABLE HOUR LONG SMFRET AND LIVE-CELL RNA TRACKING Mirjam Kümmerlin 1,2 ; Alison Farrar 1,2 ; Piers Turner 1,2 ; Hafez El-Syyed 1,2 ; Edward Wheeler 1,2 ; Achillefs Kapanidis 1,2 ; 1 University of Oxford, Physics, Oxford, United Kingdom 2 Kavli Institute for Nanoscience Discovery, Oxford, United Kingdom FRET is best known as a molecular ruler, measuring distances through fluorescence emission. Here, we reverse the perspective and utilise the principles that govern FRET to programme the emission of short ssDNA probes. When unbound, terminal fluorophore–quencher pairs interact to suppress emission, upon hybridisation to a specific target sequence, the signal is recovered. Balancing quenching and de-quenching is critical to facilitate both good background suppression and high signal levels. The resulting fluorogenic ssDNAs support high probe concentrations (up to 10µM), fast exchange kinetics, and washing-free operation while maintaining signal specificity. Treating the fluorophore–quencher pair akin to a FRET pair makes this fluorogenic response rationally tunable, so that the probe design can flexibly be adapted to various experimental regimes: In REFRESH-FRET, continuously exchanging fluorogenic labels circumvent photobleaching and extend single-molecule FRET measurements to one hour at 100ms time resolution, spanning five orders of magnitude in timescale within a single experiment, while reproducing the conformational dynamics of covalently labelled references. When taken up by live E. coli, fluorogenic ssDNA probes can hybridise directly to native 16S rRNA (the catalytic core of the ribosome). The small size of the probes (<10kDa) and their chemical similarity to the target minimise perturbation, while bright and photostable organic fluorophores yield trajectories comprising several hundred localisations with ≈ 25nm precision. Extended observations ( ≈ 10min) cover several rounds of translation (5-20s) and reveal a rich landscape of translational heterogeneity reporting on diverse modes of native protein synthesis. We could establish fluorogenic ssDNA probes as a programmable and adaptable labelling strategy that pushes the boundaries of single-molecule fluorescence. Most excitingly, they extend these measurements into native biology, enabling direct observation of molecular processes in their full cellular complexity.

26