Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

9-POS Board 5 PARALLEL STOPPED FLOW INTERROGATION OF DIVERSE BIOLOGICAL SYSTEMS AT THE SINGLE-MOLECULE SCALE Ryan Brady 1 ; Roman Kiselev 1 ; Arnab Modak 1 ; Francisco Aaron Cruz-Navarrete 1 ; Jose L Alejo 1 ; Daniel S Terry 1 ; Roger B Altman 1 ; Wesley B Asher 2,3 ; Jonathan A Javitch 2,3,4 ; Scott C Blanchard 1,5 ; 1 St. Jude Children's Research Hospital, Department of Structural Biology, Memphis, TN, USA 2 Columbia University, Department of Psychiatry, New York, NY, USA 3 New York State Psychiatric Institute, Division of Molecular Therapeutics, New York, NY, USA 4 Columbia University, Department of Molecular Pharmacology and Therapeutics, New York, NY, USA 5 St. Jude Children's Research Hospital, Department of Chemical Biology and Therapeutics, Memphis, TN, USA Single-molecule fluorescence resonance energy transfer (smFRET) has proven to be a uniquely powerful tool to study the function of a wide array of biomolecular systems by directly observing their underlying structural dynamics. Comparative smFRET studies, for example the comparison of a wild-type protein to one bearing disease-causing mutations, have conventionally required the consecutive measurement of each sample. Not only does this constrict experimental throughput, but it can also introduce experimental variances which obscure functional distinctions in systems with subtly different kinetic behavior. Here, we introduce Parallel Rapid Exchange (PRE) smFRET, which enables simultaneous measurements of a broad range of biomolecular systems under identical conditions. PRE-smFRET combines micropatterning with DNA-hybridization mediated immobilization to enable site-specific pull-down of DNA-barcoded molecules of interest from a complex mixture. In this way, multiple distinct samples can be simultaneously interrogated within one field-of-view, thereby enabling confident comparison of even subtle differences in behavior that could otherwise have been confounded with experiment to-experiment variances. This immobilization strategy is broadly applicable to a range of biomolecular systems including nucleic acids, proteins, and RNA-protein complexes. PRE smFRET is well-suited for both steady- and pre-steady-state measurements. A computer controlled fluidics system facilitates rapid exchange of solution during imaging, with complete solution exchange occurring within 60 ms, enabling highly reproducible studies of fast out-of equilibrium processes. We believe that this generalizable and scalable method will broaden the scope and reproducibility of quantitative single-molecule interrogations of biomolecular function, and have ourselves demonstrated the utility in a number of cases including; elucidating the timing of distinct conformational events underpinning β -arrestin1 activation, unmasking antibiotic-induced impacts on messenger RNA decoding fidelity, and showing conclusively that endogenously encoded ribosomal RNA sequence variation modulates antibiotic sensitivity.

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