ABSTRACT
Fluorescent proteins enable targeted visualization of biomolecules in living cells, but their maturation is oxygen-dependent and they are susceptible to aggregation and/or suffer from poor photophysical properties. Organic fluorophores are oxygen-independent with superior photophysical properties, but targeting biomolecules in vivo is challenging. Here, we introduce two oxygen-independent chemogenetic protein (OICP) tags that impart fluorogenicity and fluorescence lifetime enhancement to bound organic dyes. We present a photo- and physicochemical characterization of thirty fluorophores interacting with two OICPs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of the proteins. The binding specificity of the tags and the superior photophysical properties of organic fluorophores enable microscopy of living bacterial and eukaryotic cells. The exchange of photobleached dye for unbleached fluorophore enables prolonged live-cell imaging. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein-protein interactions, and microscopy applications under strictly oxygen-free conditions.
Competing Interest Statement
The authors have declared no competing interest.
ABBREVIATIONS
- BODIPY
- 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene;
- s.d.
- standard deviation;
- HOMO
- highest occupied molecular orbital;
- LUMO
- lowest unoccupied molecular orbital