Improved chemical-genetic fluorescent markers for live cell microscopy

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Improved chemical-genetic fluorescent markers for live cell microscopy, Biochemistry2018, 57 (39), pp 5648–5653

 

The ubiquitous implementation of fluorescence microscopy to address biological questions has led to the development of a series of markers based on small molecules, proteins, and hybrid techniques. The discovery of the green fluorescent protein (GFP) and the subsequent expansion of the color palette of fluorescent proteins have revolutionized biological imaging due to being genetically encoded and their absolute labeling specificity. In parallel, a suite of small molecule probes with excellent brightness and photostability complements the toolbox of fluorescent markers. However, both approaches suffer from drawbacks —fluorescent proteins are large and have slow maturation times, while small molecule probes suffer from off-target labeling. Thus, hybrid techniques have sought to combine the advantages of the two approaches for live cell imaging, while circumventing their respective drawbacks.

 

 

Furthermore, the introduction of these mutations demonstrates that FAST could be further modified in efforts to expand the FAST system, such as to further improve the fluorescence performance and physicochemical properties, but also to expand the color palette or engineer new fluorogen specificity.

 

 

Résumé: 

Biochemistry2018, 57 (39), pp 5648–5653

 

Inducible chemical-genetic fluorescent markers are promising tools for live cell imaging requiring high spatiotemporal resolution and low background fluorescence. The Fluorescence-Activating and absorption Shifting Tag (FAST) was recently developed to form fluorescent molecular complexes with a family of small, synthetic fluorogenic chromophores (so-called fluorogens). Here, we use rational design to modify the binding pocket of the protein and screen for improved fluorescence performances with four different fluorogens. The introduction of a single mutation results in improvements in both quantum yield and dissociation constant with nearly all fluorogens tested. Our improved FAST (iFAST) allowed the generation of a tandem td-iFAST that forms green and red fluorescent reporters 1.6-fold and 2-fold brighter than EGFP and mCherry, respectively, while having comparable size.

 

 

Group: 
Chimie Biophysique
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Improved chemical-genetic fluorescent markers for live cell microscopy

 

Alison G. Tebo, Frederico M. Pimenta, Yu Zhang & Arnaud Gautier

 

PASTEUR, Département de Chimie, École Normale Supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France.

 

Biochemistry2018, 57 (39), pp 5648–5653

 

DOI : 10.1021/acs.biochem.8b00649