Next-generation chemical-genetic fluorescent markers for advanced biological imaging

Pushing the limit of biological imaging. Deciphering the complex mechanisms controlling cells and organisms requires effective imaging systems and fluorescent probes to observe and quantify biomolecules in real time with high spatiotemporal resolution. A common strategy for imaging proteins is to fuse them to peptide or protein sequences that provide fluorescence, such as autofluorescent proteins. Although the last decade’s advances in imaging have led (and will lead) to new discoveries in biology, there are still extraordinary opportunities for basic and clinical research in developing new fluo2fluorescent markers for further advancing imaging capabilities. The most useful techniques will enable to interrogate quantitatively and comprehensively living systems at the molecular, cellular and network levels. We thus aim at developing next generation
fluorescent markers for pushing the limits of biological imaging.

Coupling Chemistry and Biology. Our approach uses exogenously applied small synthetic fluorescent probes to label proteins. Selectivity is ensured through fusion to a genetic tag that binds selectively the tailored fluorescent molecules. The modular nature of such an approach enables one to tune the synthetic part by molecular engineering, in order to address biological questions with the molecular diversity offered by modern chemistry. To avoid unspecific background in cells and achieve high imaging contrast, we used fluorescent probes that display no fluorescence until labeling occurs. Such probes are often called fluorogenic probes to highlight their ability to generate fluorescence upon interaction with their target. Our chemical-genetic fluorescent markers are thus composed of a genetically encoded protein tag that forms fluorescent complexes with small organic fluorogenic chromophores (also called fluorogens). Such fluorogenic labeling allows selective background-free imaging even in presence of an excess of free fluorogen, opening great prospects for imaging in complex samples such as tissues and whole organisms.Figure1

We recently developed an inducible chemical-genetic fluorescent marker named FAST. FAST is a small protein tag that binds and switches on the fluorescence of hydroxybenzylidene rhodanine (HBR) analogs. FAST was evolved from the photoactive yellow protein (PYP). FAST proved to be highly effective to fluorescently label proteins in living cells and multicellular organisms. This unique marker has enabled the development of innovative labeling protocols, and opens unprecedented perspectives for addressing current challenges in imaging, in particular in multiplexing, super-resolution microscopy and biosensing.