Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

14-POS

Board 8

EXPANDING THE REACH OF FRET: QUANTITATIVE QUENCHABLE FRET FOR SUB-3 NM STRUCTURAL MEASUREMENTS Timothy Craggs 1,5 ; Sophie E Fountain 1 ; Benjamin Ambrose 1 ; Eitan Lerner 2 ; Hagen Hofmann 4 ; Thorben Cordes 3 ; 1 University of Sheffield, School of Mathematical and Physical Sciences, Sheffield, United Kingdom 2 The Hebrew University of Jerusalem, Department of Biological Chemistry, Jerusalem, Israel 3 Technische Universität Dortmund, Department of Chemistry and Chemical Biology, Dortmund, Germany 4 Weizmann Institute of Science, Department of Chemical and Structural Biology, Rehovot, Israel 5 Exciting Instruments Ltd, Sheffield, United Kingdom Förster resonance energy transfer (FRET) is a cornerstone of biomolecular structural biology, but its sensitivity diminishes below ~3 nm, where FRET efficiency approaches saturation and many biologically important structural changes remain difficult to resolve. Here, we introduce quantitative quenchable FRET (qqFRET), a method that exploits short-range fluorophore quenching as a quantitative structural readout in this previously inaccessible distance regime. Using DNA molecular rulers, we show that quenching increases systematically with predicted donor–acceptor accessible-volume overlap across six commonly used fluorophore pairs. Molecular dynamics simulations and complementary photophysical measurements support a mechanism involving direct dye–dye association and enhanced non-radiative decay. Critically, qqFRET distinguishes constructs differing by only a single nucleotide, demonstrating sensitivity to sub-nanometre changes in molecular structure. We demonstrate two complementary biological applications of qqFRET. First, qqFRET resolves DNA-mediated allostery. Cooperative binding of the Bacillus subtilis transcription factor ComK produced large, position-dependent changes in quenching within the DNA spacer separating its binding sites. These changes closely matched structural predictions, revealing local DNA rearrangements associated with cooperative communication between distant protein-binding sites that are difficult to detect using conventional FRET. Second, qqFRET reports protein conformational dynamics. In maltose binding protein, ligand binding produced a clear quenching response corresponding to closure of the two protein domains, while retaining the expected ligand affinity and non-cooperative binding behaviour. Together, these applications establish qqFRET as a reporter of both short range nucleic-acid structural communication and protein motions.qqFRET therefore transforms fluorophore quenching from an unwanted photophysical artefact into a quantitative nanoscale ruler. Requiring only common fluorophores and standard smFRET instrumentation, qqFRET extends fluorescence-based structural measurements below 3 nm and provides a broadly accessible approach for investigating DNA allostery, protein conformational dynamics and other biomolecular rearrangements at near-atomic length scales.

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