Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

23-POS Board 11 GETVNA: A COMPLEMENTARY APPROACH TO SMFRET FOR PROTEIN AND DNA DYNAMICS Jakob Hartmann 1 ; Lars Richter 1 ; Alan Szalai 2 ; Giovanni Ferrari 1 ; Merve-Zeynep Kesici 1 ; Bosong Ji 1 ; Deep Sekhar Biswas 1 ; Izabela Kaminska 1 ; Ingrid Teßmer 3 ; Andrés M. Vera 1 ; Tim Schröder 1 ; Philip Tinnefeld 1 ; 1 Ludwig-Maximilians-Universität München, Department of Chemistry and Center for NanoScience, Munich, Germany 2 Consejo Nacional de Investigaciones Científicas y Técnicas, Centro de Investigaciones en Bionanociencias, Buenos Aires, Argentina 3 University of Würzburg, Rudolf Virchow Center, Würzburg, Germany Single-molecule FRET has been the workhorse of dynamic structural biology for three decades, enabling real-time monitoring of distances between two-point emitters under physiological conditions. Two-dimensional materials such as graphene can also act as quenchers, with energy transfer to the planar acceptor following a d ⁻⁴ distance dependence. Unlike organic dyes, graphene exhibits no photophysics or bleaching, enabling distance measurements with Ångström precision in the 8-25 nm regime. [1] We discovered that double-stranded DNA carrying a long single-stranded overhang stands vertically on graphene. This approach, which we term Graphene Energy Transfer with vertical Nucleic Acids (GETvNA), provides a defined orientation relative to the acceptor plane. [2] While the dsDNA height follows the expectation of a worm-like chain model, the rapid fluctuations of the polymer remain unresolved. In my talk, I tackle these challenges by monitoring fast dye–graphene distance fluctuations using shrinking-gate fluorescence correlation spectroscopy (sg-FCS), [3] which reveals intensity fluctuations correlated with changes in fluorescence lifetime. We observe sub-microsecond bending fluctuations of the biopolymer, modulated by ionic strength and local defects, such as gaps and mismatches. Beyond the structural dynamics of the Biopolymer itself, GETvNA serves as a scaffold for monitoring DNA-protein interactions. We achieve single-base pair resolution by tracking a dye labeled O ⁶ -alkylguanine DNA alkyltransferase (AGT) on the vNA construct. Further, we resolved dynamic DNA bending by endonuclease IV at abasic sites when only one dye label at the vNA construct is required. [2] All in all, GETvNA is a powerful platform for monitoring Brownian polymer dynamics under different conditions, as well as protein-DNA interactions, with sub-nanometer precision. [1] A. Ghosh, et al., Nat. Photonics 2019, 13, 860. [2] A. M. Szalai, G. Ferrari, L. Richter, et al., Nature methods 2025, 22, 135. [3] T. Schröder, et al., PNAS 2023, 120, e2211896120. [4] L. Richter, J. Hartmann, et al., bioRxiv 2026, 2026.06.06.730580.

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