BPS2026 – Expanding the reach of FRET: Quantitative quenching for probing DNA allostery and sub-3 nm distance determination

Single-molecule Förster resonance energy transfer (smFRET) is widely used to monitor biomolecular conformations in the 3–10 nm range. However, the method is inherently less-sensitive to distance changes below 3 nm, such as those postulated between adjacent DNA bases during allostery. To address this limitation, we introduce quantitative quenching FRET (qqFRET), a method based on distance-dependent quenching between common dye pairs including Cy3B, ATTO550, Alexa546, Atto647N, and Alexa647. Molecular dynamics simulations indicate that quenching arises from intermolecular dye-dye interactions, and we developed a predictive model based on the overlap of dye-accessible volumes mapped onto biomolecular structures. This interaction produces a continuous, quantifiable signal sensitive to sub-3 nm changes, enabling resolution beyond the Förster radius. Using DNA duplexes as nanometer-scale rulers, we designed constructs where the acceptor dye position differs by a single nucleotide (3.4 Å) in either the 3′ or 5′ direction. These subtle changes yield distinct quenching levels, validating qqFRET as a high-resolution, orientation-sensitive tool. Importantly, quenching is not binary but continuous, allowing nuanced distance measurements without specialized instrumentation. Complementary spectral shift and fluorescence anisotropy measurements characterized dye environments and rotational mobility, providing insight into orientation and local interactions. We then applied qqFRET to a biologically relevant system: ComK transcription factor and its cooperative binding to a promoter region in Bacillus subtilis. ComK binds to two promoter “boxes,” inducing long-range allosteric communication. The quenching signal shifts in a manner undetectable by conventional smFRET, aligning with the expected ∼3 Å change and suggesting alterations in groove width and tension propagation in the spacer region. This study establishes qqFRET as a powerful extension of smFRET, enabling high-resolution insight into short-range DNA dynamics, cooperative protein-DNA interactions, and structural transitions inaccessible with traditional smFRET.