Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

51-POS Board 25 AN ALTERNATIVE DNA-FRET STANDARD Christian Rönnau 1 ; Carla Hollermann 1 ; Verena Hirschfeld 1 ; Christian G Hübner 1 ; 1 University of Luebeck, Institute of Physics, Lübeck, Germany Double-stranded DNA (dsDNA) serves as the gold standard for FRET-based spectroscopic rulers, enabling precise distance measurements through systematic variation of fluorophore positions along the helix. In the conventional approach, two complementary strands — each internally labeled with a donor or acceptor dye — are hybridized to form B-DNA. However, this approach requires a dedicated set of labeled strands for each target distance, which scales linearly with the number of separations probed. Here we present a method requiring only two labeled strands, regardless of the number of separations probed. This is achieved by incorporating two auxiliary single-stranded DNA strands for each sample. The resulting DNA FRET standards were characterized by fluorescence lifetime measurements and single-molecule FRET (smFRET) for several donor-acceptor fluorophore combinations. The results show well defined FRET populations across the full range of transfer efficiencies. Given its reduced synthesis demands and cost efficiency, this approach has the potential to become a widely adopted standard in FRET-based distance measurements.

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