Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

19-POS Board 9 SYSTEMATIC EVALUATION OF BIO-ORTHOGONAL CHEMISTRIES AND FLUORESCENT DYES FOR SINGLE-MOLECULE FRET MEASUREMENTS IN NATIVE HUMAN PROTEINS Luis Gaiser 1 ; Andreas Schmidbauer 1 ; Gerti Beliu 2 ; Dina Grohmann 1 ; 1 University of Regensburg, Department for Microbiology, Regensburg, Germany 2 University of Regensburg, Department of Pharmacy, Regensburg, Germany Single-molecule FRET (smFRET) measurements are a powerful tool for investigating intra- and inter-molecular dynamics in biomolecules. Recently, we developed the SLAM-FRET approach that allows the site-specific labeling of native mammalian proteins for smFRET measurements based on bio-orthogonal chemistries. Although numerous fluorescent dyes and labeling reactions are available, a systematic comparison of their performance in smFRET experiments is lacking. This work is closing this gap by evaluating four coupling chemistries and a range of dye pairs using the model proteins Rbp4/7 of the human RNA polymerase II. Rpb4/7 forms a stable heterodimer, which allows consistent measurements of a single static FRET population. Labelling of Rpb4/7 is achieved by targeting either natural or unnatural amino acids that were site-specifically introduced. Reactions utilized include azido-phenylalanine with phosphine linked dyes through a Staudinger-Bertozzi ligation or with dibenzocyclooctyne-linked dyes through strain-promoted alkyne-azide cycloaddition (SPAAC), tetrazine-linked dyes that react with trans-cyclooct-2-en-L-lysine in an inverse electron-demand Diels-Alders (IEDDA) reaction and the reaction of cysteine residues with maleimide-linked dyes through a Michael addition. Rpb4/7 is purified from HEK cells by immobilization on FLAG beads through a FLAG-tag mutated into Rpb4 and directly labelled on the beads using SLAM-FRET. FRET measurements are carried out on an objective TIRF setup with the labelled Rpb4/7 being immobilized on a surface via antibodies. This approach allows the quantification of parameters like labelling efficiency, labelling specificity or FRET-pair yield – a pre-requisite to gather meaningful smFRET data. Moreover, these measurements enable the evaluation of how linker length, potential protein-dye interactions and dye properties influence the quality of FRET efficiency distributions. The best performing dye pairs, regardless of coupling chemistry, were found to be Cy3B/Cy5 and Dylight550/Dylight650, while Atto550/Atto647N performed the worst.

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