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

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 single-chain 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.