Innovations in the Field of Fluorogen-Activating Proteins: Engineering scFv Biosensors as Universal Affinity Reagents, Multi-Specific Activation for Multi-Color Applications on Live Cells, and the Generation of scFv Multimer Assemblies for Advanced Fluorescence Detection

Current advancements in biological protein discovery utilize bi-partite methods of fluorescence detection where chromophore and scaffold are uncoupled. An innovative biosensor platform utilizes single-chain-variable-fragments (scFvs) selected against fluorogenic molecules, where fluorogens emit fluorescence only when bound to the scFv. In order to by-pass current FAP technology methods of
reporter tagging – a process that is time and labor intensive – here I report different universal affinity reagent approaches based on FAP fusions with IgG-binding proteins (Protein-A or Protein-G) or biotin binding proteins (streptavidin or avidin). Fluorescence results using various techniques indicate minimal background and high target specificity for exogenous and endogenous proteins in mammalian cells. Additionally, FAP-based affinity reagents provide enhanced properties of detection previously absent
using conventional affinity systems: (1) signal wavelengths (excitation and emission) determined by the particular fluorogen chosen, (2) real-time user controlled fluorescence onset and termination, (3) signal wavelength substitution while performing live analysis, and (4) enhanced resistance to photobleaching. In unusual circumstances a scFv may activate multiple fluorogens. In this work I present two
different multi-specific scFvs with sub-micromolar affinities. Furthermore, each scFv-fluorogen complex
possesses unique excitation and emission spectra, which allows broader detection limits from each
biosensor. Accordingly, one may perform advanced fluorescence detection at the surface of mammalian
cells because the scFv-ligand complex forms via non-covalent interactions. More specifically, fluorogen
addition or removal from the cellular medium results in modular signal (onset versus offset). Likewise,
simultaneous labelling with different fluorogens permits multi-color detection or real-time color-switch. Overall, the strength of affinity interactions between scFv and cognate ligands determines the scope of signal manipulations – removal, co-labelling, or exchange. On a different approach, here I also investigate the cell surface display of tandem scFvs for enhanced fluorescence detection. By analyzing different combinations of scFv-dimers, I correlated scFv location (cell membrane distal versus proximal) and selection with biosensor cell surface activities. For scFv combinations where absence or poor cell surface signals occurred, the likely mechanisms include protein misfolding (intramolecular or intermolecular interactions) and secretory-pathway mediated degradation. Further in-vitro work determined scFv monomer stability to associate with live-cell tandem scFv activities, and explains the biases for scFv location and selections. When utilized as protein
reporters in mammalian cells, one may perform advanced detection strategies such as multi-color vesicular traffic, fluorescence signal amplification, and Förster-resonance energy transfer between the fluorophores.