Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Friday Speaker Abstracts

COMBINING SINGLE-MOLECULE FRET AND GRAPHENE ENERGY TRANSFER TO RESOLVE PROMOTER-DEPENDENT DNA DEFORMATION BY TBP Deep S Biswas 1 ; Fabio Morella 1 ; Sophia Sebina 1 ; Don C Lamb 1 ; Evelyn Ploetz 1 ; Philip Tinnefeld 1 ; 1 Ludwig-Maximilians-Universität München, Chemistry and Center for NanoScience, Munich, Germany The TATA-binding protein (TBP) is a central component of the transcription preinitiation complex. Upon binding to the minor groove of promoter DNA, TBP induces local melting, kinking, and bending of the double helix. Although TBP preferentially recognizes the consensus TATA box, it also binds divergent promoter sites,[1] whose composition strongly influences binding affinity, kinetics, and conformational landscape of the resulting TBP-DNA complex. We compared TBP binding to three promoter sequences:[2] the adenovirus major late promoter AdML (K d = 48 nM), the archaeal SSV promoter (930 nM), and the histone H2B promoter (4.2 nM). These representative promotors span nearly three orders of magnitude in affinity, enabling us to dissect how a canonical TATA box, a low-affinity TATA-like site, and a promoter containing multiple potential binding motifs differ in their TBP-induced structural response.We combined TIRF microscopy and PIE-MFD-based single-molecule spectroscopy to measure single-pair FRET between fluorophores flanking the TBP-binding region. FRET reported on promoter compaction and resolved distinct conformational subpopulations. To obtain complementary structural information, we applied graphene energy transfer with vertical nucleic acids, GETvNA.[3] In this approach, DNA is oriented approximately perpendicular to a graphene surface, and the fluorescence lifetime of a dye attached to the 5’ end reports on the axial fluorophore–graphene distance. Upon TBP binding, DNA kinking reduces this distance, allowing GETvNA to detect sequence-specific structural changes. AdML produces comparatively narrow populations consistent with a single binding geometry, whereas SSV exhibited slightly broader distributions, indicative of increased structural heterogeneity. In contrast, the H2B promoter displays multiple conformational states and more complex kinetics. Sequence variants of H2B support the presence of overlapping or competing TBP-binding motifs, explaining its high apparent affinity and heterogeneous structural response.[4]1. Ravarani, D. et al. 2016 Nat. Commun. 7, 10417. 2. Gietl, A. et al. 2014 Nucleic Acids Res. 42, 6219-6231. 3. Szalai, A. et al. 2025 Nat. Methods. 22, 135-144. 4. Heiss, G. et al. 2019 Nucleic Acids Res. 47, 2793-2806.

58