Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

37-POS Board 19 SINGLE-MOLECULE FLUORESCENCE IMAGING AT MICROMOLAR CONCENTRATIONS WITHOUT NANOPHOTONIC HELP Riley C Gentry 4,5 ; Katherine Leon Hernandez 1 ; Ruben L Gonzalez, Jr. 2,3 ; Colin D Kinz Thompson 1 ; 3 Columbia University, Department of Physiology & Cellular Biophysics, New York, NY, USA 4 The Rockefeller University, Laboratory of Molecular Biophysics, New York, NY, USA 5 Columbia University, Department of Biological Sciences, New York, NY, USA Nature leverages weak, reversible interactions to drive biological processes such as biomolecular recognition. Single-molecule techniques, such as single-molecule fluorescence resonance energy transfer (smFRET), are particularly well-suited for studying such processes, because they enable direct observations of low-probability events. Unfortunately, an effect known as the concentration barrier prevents many smFRET experiments from being performed at physiologically relevant concentrations unless complex nanophotonic devices, such as zero mode waveguides, are employed. Here, we report a simple, generalized approach for conducting smFRET experiments at micromolar concentrations using a modified surface functionalization protocol that significantly mitigates nonspecific adsorption of biomolecules. With this strategy, we performed smFRET experiments with acceptor fluorophore-labeled biomolecules at up to 8 micromolar concentration in solution without using nanophotonic devices. Together, this advance enabled us to characterize the binding kinetics and thermodynamics of two systems: 8 mer oligonucleotide DNA hybridization at room temperature, and eukaryotic translation initiation factor eIF4A binding to messenger RNA–a system with a micromolar equilibrium dissociation constant. By pushing back against the concentration barrier, our approach opens the door to countless new investigations of weakly interacting biomolecular events using smFRET experiments. 1 Rutgers University-Newark, Chemistry, Newark, NJ, USA 2 Columbia University, Chemistry, New York, NY, USA

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