Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

43-POS Board 21 SPANNING THE CONCENTRATION RANGE OF INTERACTING BIOLOGICAL PARTNERS Fabio Morella 1 ; Don C Lamb 1 ; 1 Ludwig-Maximillians-Universität, Pharmazie und Chemie, München, Germany Biomolecular interactions can be investigated by single-molecule fluorescence techniques, such as fluorescence correlation spectroscopy (FCS). Key challenges include the detection of sub picomolar concentrations for biosensing purposes and the characterization of biological complexes. However, sub-picomolar concentrations are challenging to detect due to a severely reduced signal-to-background ratio. Fluorescence lifetime correlation spectroscopy (FLCS) takes advantage of the lifetime information, providing a means to overcome the concentration limitation by removing scattering and background components in FCS, enabling the detection of biomarkers at low concentration. So far we have successfully applied FLCS technique on a freely diffusing Atto488 solution and the lowest concentration achieved thus far has been 10 pM. Beyond detecting low concentrations, however, fully characterizing biological systems also requires resolving complexes beyond binary interactions. FCS already offers a way to perform pairwise cross-correlation to probe the formation of binary complexes. Nevertheless, two-color fluorescence cross-correlation spectroscopy (FCCS) is limited to the investigation of two-species complexes. Therefore, the enhancement of FCCS to a 3-color fluorescence correlation spectroscopy (3-color FCS) would set the stage for the investigation of the quantity and binding state of the interacting species in ternary complexes. For instance, those formed in PROTAC mediated targeted protein degradation, where a target protein, a bifunctional degrader, and an E3 ubiquitin ligase must simultaneously interact. Nonetheless, 3-color FCS is currently computationally demanding. After we will have overcome such a challenge, we propose combining FLCS and 3-color FCS to enable the investigation of a wider range of biological interactions, from systems at sub-picomolar concentrations to complexes involving multiple binding partners.

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