Fluorogen-activating single-chain antibodies for imaging cell surface proteins

  title={Fluorogen-activating single-chain antibodies for imaging cell surface proteins},
  author={Christopher Szent-Gyorgyi and Brigitte F. Schmidt and Yehuda Creeger and Gregory W. Fisher and Kelly L. Zakel and Sally A. Adler and James A. J. Fitzpatrick and Carol A. Woolford and Qi Yan and Kalin V Vasilev and Peter B. Berget and Marcel P. Bruchez and Jonathan W. Jarvik and Alan S Waggoner},
  journal={Nature Biotechnology},
Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and… 
Fluorogen activating proteins (FAPs) are genetically encoded tags made from single chain antibody fragments designed to bind fluorogens with high specificity and can be modified to provide flexibility in properties such as affinity, membrane permeability, spectra, and quantum yield.
Fluorogen-Activating Proteins: Next-Generation Fluorescence Probes for Biological Research.
  • E. Gallo
  • Biology, Chemistry
    Bioconjugate chemistry
  • 2019
Non-covalent activation of fluorogen particles results in advanced strategies of fluorescence detection, and bi-modular sensors composed of a single-chain antibody that exhibits high-affinity and selectivity for small-molecule fluorogens are used.
Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors
FAP/affibody coupling provides a new approach to construct compact and modular affinity probes that label endogenous proteins on living cells and can be used for studying rapid changes in receptor pools involved in trafficking.
Fluorogen-activating proteins: beyond classical fluorescent proteins
A variable light domain fluorogen activating protein homodimerizes to activate dimethylindole red.
It is reported here that both nuclear magnetic resonance and X-ray diffraction methods indicate the M8V(L) forms noncovalent, antiparallel homodimers that are the fluorogen activating species.
Blue fluorescent dye-protein complexes based on fluorogenic cyanine dyes and single chain antibody fragments.
This work expands the repertoire of genetically encodable scFv-dye pairs by selecting and characterizing a group of scFvs that activate fluorogenic violet-absorbing, blue-fluorescing cyanine dyes, based on oxazole and thiazole heterocycles.
Fluorogen-Activating scFv Biosensors Target Surface Markers on Live Cells Via Streptavidin or Single-Chain Avidin
A novel FAP reagent platform that offers a rapid and efficient approach for cell surface protein detection and indicates high target specificity, minimal background, and bright signal detection is presented.
Covalent live-cell labeling of proteins using a photoreactive fluorogen.
Developing Bright GFP-Like Fluorogens for Live-Cell Imaging with Nonpolar Protein-Chromophore Interactions.
A novel fluorogen directly engineered from green fluorescent protein (GFP) chromophore by a unique double-donor-one-acceptor strategy is reported, which exhibits an over 550-fold FE upon FAST binding and a high extinction coefficient.


A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure
A small fluorescent chelator whose membrane-impermeant complex with nontoxic Zn2+ ions binds tightly but reversibly to hexahistidine (His6) motifs on surface-exposed proteins, demonstrating externalization of STIM1 upon depletion of Ca2+ from the endoplasmic reticulum.
Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase
A highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes that accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction.
Receptor-mediated Targeting of Fluorescent Probes in Living Cells*
A strategy was developed to label specified sites in living cells with a wide selection of fluorescent or other probes and applied to study pH regulation in Golgi, providing direct evidence that resting Golgi pH is determined by balanced leak-pump kinetics rather than the inability of the H+/ATPase to pump against an electrochemical gradient.
The Fluorescent Toolbox for Assessing Protein Location and Function
The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy.
Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein
The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1, and three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.
Fluorescent proteins: maturation, photochemistry and photophysics.
  • S. Remington
  • Chemistry, Biology
    Current opinion in structural biology
  • 2006
Blue-fluorescent antibodies.
A principle and method are described in which antibodies can direct the outcome of photophysical and photochemical events that take place on excited-state potential energy surfaces and a step was taken toward the use of antibody-based photochemical sensors for diagnostic and clinical applications.