Tiny Titans: Nanobodies as Powerful Tools for TR‐FRET Assay Development

Abstract

AbstractOwing to their exceptional sensitivity and specificity, assays based on time‐resolved Förster resonance energy transfer (TR‐FRET) have become increasingly popular in biomedical research. TR‐FRET assays are highly versatile and compatible with complex biological environments. However, the paucity of straightforward strategies for the selective installation of TR‐FRET donor fluorophores on target proteins, which is a critical requirement for most assay designs, has limited the potential and adaptation of this powerful technology. To address this issue, we have developed a versatile toolbox strategy based on CoraFluor‐1‐functionalized nanobodies. The target‐agnostic modular design simplifies the TR‐FRET donor‐labeling of epitope tags, fluorescent proteins and primary antibodies, including their use with endogenous proteins in lysates. Our strategy, which we extend to enable the seamless adaptation of split‐luciferase systems for TR‐FRET approaches, greatly facilitates the development and design of TR‐FRET assays for both target engagement studies and the quantification of proteins of interest with sub‐nanomolar sensitivity.