Fluorogen-activating proteins: beyond classical fluorescent proteins

Xu, Shengnan; Hu, Hai-Yu

Fluorescence imaging is one of the most powerful techniques to observe biomolecules in real-time with high spatial and temporal resolution, which reveals the fundamental insights into the production, localization, trafficking, and biological functions of biomolecules in living systems [1] [2] [3] [4] . As the biological objects are poorly fluorescent, fluorescent probes including fluorescent proteins and organic fluorescent dyes are essential molecular tools for bio-imaging 5, 6 . Among them, a diverse set of genetically encodable fluorescent biosensors have been designed to probe dynamic cellular events. These sensors that generally involve the incorporation of a fluorescent tag into a protein or a selected protein domain, have enabled researchers to track various components of intracellular signaling networks in real time within the native cellular environment. In the past several decades, two approaches have been developed to construct genetically encodable biosensors for live cell studies ( Fig. 1 ): 1) fluorescent protein-based reporters: chimeric genetic fusions of fluorescent proteins (e.g., GFP and its variants) with a protein (or RNA) domain 7 ; 2) fluorogen-based reporters: a genetically encodable tag binds a fluorogenic ligand (endogenously present or exogenously applied) and activates its fluorescence. As the fluorogenic chromophore is non-fluorescent by its own and becomes strongly fluorescent only upon binding its target, unspecific fluorescence background in cells remains minimal even in the presence of an excess of dye, thus ensuring high imaging contrast 8 . Labeling with fluorogenic probes can be covalent, relying on chemical or enzymatic reaction, or noncovalent, relying on binding equilibrium. In the past 20 years, great efforts have been dedicated to exploring covalence-based self-labeling tags, such as the commercially available SNAP-tag [9] [10] [11] , CLIP-tag 12 and Halo Tag [13] [14] [15] . Parallel to the development of covalent fluorogenic protein labeling strategies, methods based on the non-covalent interaction between a protein tag and a fluorogenic dye have emerged 16, 17 . Unlike the covalent labeling strategies, non-covalent labeling can be very fast since no covalent bond has to be created. Moreover, systems based on reversible non-covalent binding could provide an additional degree of control as fluorescence could also be switched off by washing away the fluorogenic ligand, given that the off-rate is fast enough. In this review, we describe the discovery of one of the noncovalence-based fluorogenic probes based on fluorogen activating proteins (FAPs), the design strategy of FAP fluorogens, the application of the FAP technology and the advances of FAP technology in protein labeling systems.