title: A single-chain-variable-fragment fluorescence biosensor activates fluorogens from dissimilar chemical families.
abstract: Current advancements in biological protein discovery utilize bi-partite methods of fluorescence detection where chromophore and scaffold are uncoupled. One such technology, called fluorogen-activating proteins (FAPs), consists of single-chain-variable-fragments (scFvs) selected against small organic molecules (fluorogens) that are non-fluorescent in solution, but highly fluorescent when bound to the scFv. In unusual circumstances a scFv may activate similar fluorogens from a single chemical family. In this report we identified a scFv biosensor with fluorescence activity against multiple fluorogens from two structurally dissimilar families. In-vitro analysis revealed highly selective scFv-ligand interactions at sub-micromolar ranges. Additionally, each scFv-fluorogen complex possesses unique excitation and emission spectra, which allows broader detection limits from the biosensor. Further analysis indicated that ligand activation, regardless of chemical family, occurs at a common scFv binding region that proves flexible, yet selective for fluorogen binding. As a protein reporter at the surface of mammalian cells, the scFv revealed bright signal detection and minimal background. Additionally, when tagged to a G-protein-coupled receptor, we observed agonist dependent signaling leading to protein traffic from cell surface to endosomes via multi-color fluorescence tracking. In summary, this report unveils a noncanonical scFv biosensor with properties of high ligand affinity and multi-channel fluorescence detection, which consequently offers expanded opportunities for cellular protein discovery.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Gallo
authors.forename: Sophia
authors.initials: S
authors.lastname: Wienbar
authors.forename: Avin C
authors.initials: AC
authors.lastname: Snyder
authors.forename: Kalin V
authors.initials: KV
authors.lastname: Vasilev
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Armitage
authors.affiliation: Carnegie Mellon University, 4400 Fifth Ave., MI 257, Pittsburgh, Pennsylvania, 15213, USA. jarvik@cmu.edu.
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Jarvik
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D057127:Single-Chain Antibodies