Fluorogen-Activating Proteins: Next-Generation Fluorescence Probes for Biological Research

Abstract

Since their discovery, fluorescent probes have found widespread use in biological research. Over time, multiple next-generation probes increased the fluorescence catalog by offering novel capabilities of detection that have been previously difficult or lacking with conventional probes. One of such probes is called a fluorogen-activating protein (FAP). These are bimodular sensors, composed of a singlechain antibody that exhibits high-affinity and selectivity for small-molecule fluorogens. Because fluorogens are inherently nonfluorescent unless sterically restricted, upon the formation of the noncovalent FAP-fluorogen complex the fluorogen module emits fluorescence when excited by light. More interestingly, these bimodular sensors permit improvement of their biophysical properties. For instance, the fluorescence spectra and environmental sensing capabilities of fluorogens may be altered by the method of chemical modification at the fluorogen structural level. Also, optimizations of the single-chain antibody scaffold, via amino acid substitutions at the selectivity regions, may improve the detection brightness and affinities of fluorogens; this may also improve the biophysical stability of FAPs in different cellular environments. Additionally, when utilized as biological discovery probes, FAP biosensors exhibit functional activity as genetic fusion tags with cellular proteins; this results in high fluorescent sensitivities of cell surface and intracellular targets. Also, FAPs allow the monitoring of cellular traffic of surface receptors by fluorescence methods of real-time color switching, or signal onset and offset. They find application as biological probes integrated into biomaterials, or as soluble affinity reagents for whole live animal studies. Overall, this noncovalent activation of fluorogen particles results in advanced strategies of fluorescence detection.

Protein Delivery: If Your GFP (or Other Small Protein) Is in the Cytosol, It Will Also Be in the Nucleus

“…Fluorescent proteins provide a straightforward and effective readout to evaluating cytosolic access. The integral fluorescence of these proteins eliminates artifacts arising from intracellular separation of the fluorophore from the protein cargo. , Green fluorescent protein (GFP) is a robust and versatile model cargo that can passively diffuse from the cytosol into the nucleus through pores in the nuclear membrane . Nuclear fluorescence of GFP thus provides definitive evidence of cytosolic access, making it a versatile tool for evaluating intracellular protein delivery (Figure ).…”

Visualizing and Manipulating Biological Processes by Using HaloTag and SNAP‐Tag Technologies

“…The utility of FAPs is well reviewed elsewhere. [36] Alternatively, several groups have devised unique strategies for labelling of HaloTagand SNAP-tag using fluorogenic probes to remove the requisite washing step. Typical fluorogenic probes are designed for fluorescent response to small molecule species through chemical reaction to activate fluorescence.…”

The Cutting Edge of Disease Modeling: Synergy of Induced Pluripotent Stem Cell Technology and Genetically Encoded Biosensors

“…Finally, there are some biosensors that combine organic dyes and a genetically encoded protein (or peptide) (Figure 4C): the tetracysteine/biomarsenic system and its variants (FlAsH and ReAsH), based on high-affinity interactions between trivalent arsenic compounds and a short peptide sequence containing pairs of closely spaced thiols [63]; fluorogen-activated proteins (FAPs) obtained from single-chain antibodies capable of binding an organic dye [64,65]; SNAP-tag, Halo-tag, and CLIP-tag consisting of a small protein (such as hAGT and halogendehalogenase) that mediates covalent binding between a genetically encoded target and a fluorophore [66][67][68].…”