A single-chain-variable-fragment fluorescence biosensor activates fluorogens from dissimilar chemical families

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. © 2014 bentham science publishers.