Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Thursday Speaker Abstracts

QUANTITATIVE SMFRET OF LOW-AFFINITY COMPLEXES IN NANOHOLES Mariska Haas 1 ; Jerome Wenger 2 ; Jens Michaelis 1 ; 1 Ulm University, Institute of Biophysics, Ulm, Germany 2 Aix-Marseille University, CNRS, Centrale Med, Institut Fresnel, AMUTech, Marseille, France Single-molecule (sm) FRET measurements allow for quantitative distance measurements, thus making them a great tool for structural biology1–5. The advantage of smFRET is, that structural information of transient, low affinity complexes can also be obtained. However, measurements on diffusing complexes in a confocal microscope, are practically limited to high-affinity complexes. Metallic nanoholes (NHs) reduce the observation volume enabling sm measurements at higher concentrations. smFRET measurements of DNA and protein samples in NHs demonstrate the potential of this approach for studying biomolecular interactions. However, the proximity of the fluorophores to the metal alters the photophysics thus increasing the complexity for quantitative smFRET analysis.We present unpublished data, using DNA-based smFRET standards with defined donor-acceptor distances3, to systematically evaluate the effects of the NH environment on quantitative FRET measurements. Our data shows that the NH increases fluorescence-brightness and reduces donor fluorescence-lifetime, consistent with previous reports6. We show that, in addition to adapting standard analysis procedures to account for altered photon statistics, NH-induced changes in fluorophore photophysics requires calibration of the effective Förster radius (R0). Comparison of data from confocal illumination and from NHs revealed a reduced R0 for the NH. Using this experimentally determined R0, quantitative smFRET measurements can be performed, thus highlighting the importance of the calibration for reliable structural interpretation.We apply this quantitative approach using NHs to a biological application, where confocal measurements were unsuccessful, given the transient nature of the investigated complexes.1. Muschielok, A. et al., Nat Methods 5, 965–971 (2008).2. Nagy, J et al., J Chem Phys 148, 123308 (2018).3. Hellenkamp, B. et al., Nat Methods 15, 669–676 (2018).4. Lerner, E. et al., Elife 10, e60416 (2021).5. Agam, G. et al., Nat Methods 20, 523–535 (2023).6. Torres, J. de, et al., Chemphyschem 16, 782–788 (2015).

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