Development of fluorogen activating proteins (FAPs) over the past two decades has led to the diverse set of over 160 publications from CMU and others as listed below. The unique capabilities of FAPs have facilitated a broad variety of in vivo studies in systems ranging from bacterial, yeast and mammalian cells to nematode, fly, zebrafish and mouse. The following examples illustrate key properties of FAPs that make them powerful tools for basic and biomedical research and drug discovery.

The above images from our seminal paper (ref. 1) establish the basic behavior of FAPs. FAPs were discovered by screening a library of yeast displayed scFvs (antibody fragments) against pegylated fluorogenic dyes (I). The salient characteristic of FAPs that bind pegylated fluorogens is that they generate intense surface-specific fluorescence that for our most useful fluorogens (malachite green, MG-2p; thiazole orange, TO1-2p) are respectively enhanced by thousands-fold. This behavior enables essentially instant fluorescence visualization of a surface-expressed FAP-fusion protein simply by adding a fluorogen to the cell culture without need of washing out excess dye. For cell surface-specific applications, FAPs have a marked advantage over fluorescent proteins (GFPs) which are compromised by internal signal generated by the biosynthetic/secretory pathway (II). Importantly, FAP technology eliminates the need for resource-intensive labeling with dye-conjugated antibodies (II).

If one substitutes the PEG on malachite green with an ester, the fluorogen becomes cell-permeant (III). In these mammalian cells, the permeant fluorogen additionally lights-up the FAP on nuclear envelope ribosomes, Golgi and various secretory vesicles. Intracellular labeling has been used in several studies of nuclear, mitochondrial and Golgi proteins. Inside/outside differential labeling has been especially useful for dynamically tracking the endocytosis/exocytosis of GPCRs and the CFTR ion channel in the context of drug screening and studying the mouse synaptic BK channel. Surface-specific FAP imaging is also useful for visualizing cell/cell contacts (IV).

The above chemical structures and fluorescence spectra (ref. 13) depict our first demonstration of the chemical modularity of FAP technology. FAP sensor functionality can readily be modified by conjugating different fluorescent dyes to a fluorogen via the (branched) linker. In this case, a series of Cy3 donors quantitatively amplify the fluorogenic emission of a MG FAP via fluorescence resonance energy transfer (FRET). pH sensitive Cy3 and Cy5 analogs respectively convert MG and TO1 FAPs into sensors that track internalization of cell surface receptors. Other two color bichromophore constructs create spectrally shifted FAPs that can be used for pulse-labeling. MG and TO1 chromophores separated by a defined linker length can be used as a molecular ruler to assess distances between contacting cells respectively displaying cognate FAPs.

FAPs have been used to fluorescently label IgG Fc domains by photochemical conjugation or by using FAPs fused to protein A/G. These FAP/IgG reagents possess useful binding specificity for labeling purposes. Following the chemical modularity paradigm, Biocognon seeks to extend the use of antibody-based reagents by discovering Nanobodies that when fused to FAPs can be used to specifically deliver toxin or radionuclide payloads that are coupled to PEG-fluorogen.

The above fluorogen/FAP schematic and associated cell images document our remarkable discovery that the MG fluorogen can be converted into an efficient FAP-dependent photoablation reagent (ref. 66). When this iodine-modified fluorogen becomes encapsulated within the cognate FAP, it generates cytotoxic reactive oxygen species (ROS) that rapidly kill the host cell. Cell-targeted cytoxicity is entirely dependent on addition of the modified fluorogen, and thus applications are not compromised by background cytoxicity, as is the case with ROS-generating fluorescent proteins. The MG-2I/FAP has been used in transgenic zebrafish to ablate cardiac cells at defined stages of fish development and to ablate mitochondria in cells to study the effects of oxidative stress on telomeres.

Tight encapsulation of unmodified MG by the FAP renders the fluorogen more resistant to photobleaching than fluorescent proteins or fluorescent dyes conjugated to proteins. Photobleaching resistance, together with its far-red emission at 664 nm, makes the MG/FAP complex an exquisitely sensitive reporter for applications where signal is limiting. These properties have also made this complex useful for single molecule and super-resolution microscopy.

FAP-based labeling has been used for flow cytometric and plate-based small molecule drug discovery screens that monitor protein trafficking. As cartooned above and described under Mission and Technology, Biocognon is developing a Nanobody screening platform that employs two FAPs to monitor displayed Nanobodies and secreted target antigens expressed in a single yeast cell (ref. 152). The mechanism of the biosensor is not detailed here. We have demonstrated that our yeast system robustly co-expresses Nanobody-FAP and antigen-FAP fusions encoded by a modular vector that is optimized for synthetic biology. Nanobodies and target antigens that specifically associate on the cell surface can be flow cytometrically screened and identified using next generation sequencing analysis of barcoded output.


3.  Evanko D. The new fluorescent probes on the block — Research Highlights. Nat Methods. 2008 Mar;5(3):218.

14.  Sklar L, Edwards B. High throughput flow cytometry for discovery at UNMCMD and the NIH Molecular Libraries Initiative. Drug Discovery World. 2010 Oct 4;