Flashbody: A Next Generation Fluobody with Fluorescence Intensity Enhanced by Antigen Binding.

Fluorescent probes are valuable tools for visualizing the spatiotemporal dynamics of molecules in living cells. Here we developed a genetically encoded antibody probe with antigen-dependent fluorescence intensity called "Flashbody". We first created a fusion of EGFP to the single chain variable region fragment (scFv) of antibody against seven amino acids of the bone Gla protein C-terminus (BGPC7) called BGP Fluobody, which successfully showed the intracellular localization of BGPC7-tagged protein. To generate BGP Flashbody, circularly permuted GFP was inserted in between two variable region fragments, and the linkers were optimized, resulting in fluorescence intensity increase of 300% upon binding with BGPC7 in a dose-dependent manner. Live-cell imaging using BGP Flashbody showed that BGPC7 fused with cell penetrating peptide was able to enter through the plasma membrane by forming a nucleation zone, while it penetrated the nuclear membrane with different mechanism. The construction of Flashbody will be possible for a range of antibody fragments and opens up new possibilities for visualizing a myriad of molecules of interest.

[1]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[2]  Yamamura Ken-ichi,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector , 1991 .

[3]  J. Takagi,et al.  A multipurpose fusion tag derived from an unstructured and hyperacidic region of the amyloid precursor protein , 2013, Protein science : a publication of the Protein Society.

[4]  J. Wagner,et al.  Generation of an intrabody‐based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells , 2014, Journal of molecular recognition : JMR.

[5]  A. Cattaneo,et al.  Redox State of Single Chain Fv Fragments Targeted to the Endoplasmic Reticulum, Cytosol and Mitochondria , 1995, Bio/Technology.

[6]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[7]  Ryoji Abe,et al.  Ultra Q-bodies: quench-based antibody probes that utilize dye-dye interactions with enhanced antigen-dependent fluorescence , 2014, Scientific Reports.

[8]  V. Papadopoulos,et al.  Printed in U.S.A. Copyright © 1998 by The Endocrine Society Peripheral-Type Benzodiazepine Receptor Function in Cholesterol Transport. Identification of a Putative Cholesterol Recognition/Interaction Amino Acid Sequence and Consensus Pattern* , 2022 .

[9]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[10]  D. Desplancq,et al.  The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells. , 2013, Experimental cell research.

[11]  Ryoji Abe,et al.  Insight into the Working Mechanism of Quenchbody: Transition of the Dye around Antibody Variable Region That Fluoresces upon Antigen Binding. , 2016, Bioconjugate chemistry.

[12]  C. Legrand,et al.  Mitochondria-targeted cpYFP: pH or superoxide sensor? , 2012, The Journal of general physiology.

[13]  Rainer Fischer,et al.  A Comprehensive Model for the Cellular Uptake of Cationic Cell‐penetrating Peptides , 2007, Traffic.

[14]  Bence Ölveczky,et al.  Rapid Diffusion of Green Fluorescent Protein in the Mitochondrial Matrix , 1998, The Journal of cell biology.

[15]  Takahiro Hohsaka,et al.  "Quenchbodies": quench-based antibody probes that show antigen-dependent fluorescence. , 2011, Journal of the American Chemical Society.

[16]  R. Tsien,et al.  Circular permutation and receptor insertion within green fluorescent proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Lourdes M. Aleman,et al.  A single-chain antibody/epitope system for functional analysis of protein-protein interactions. , 2002, Biochemistry.

[18]  R. Bird,et al.  Single chain antibody variable regions. , 1991, Trends in biotechnology.

[19]  Rebecca L Rich,et al.  Higher-throughput, label-free, real-time molecular interaction analysis. , 2007, Analytical biochemistry.

[20]  T. Tsuboi,et al.  Generation of a cGMP Indicator with an Expanded Dynamic Range by Optimization of Amino Acid Linkers between a Fluorescent Protein and PDE5α. , 2017, ACS sensors.

[21]  Kevin W Eliceiri,et al.  NIH Image to ImageJ: 25 years of image analysis , 2012, Nature Methods.

[22]  S. Futaki,et al.  Arginine-rich Peptides , 2001, The Journal of Biological Chemistry.

[23]  A. Schots,et al.  Fluobodies: green fluorescent single-chain Fv fusion proteins. , 1999, Journal of immunological methods.

[24]  S. Futaki,et al.  Cell-surface accumulation of flock house virus-derived peptide leads to efficient internalization via macropinocytosis. , 2009, Molecular therapy : the journal of the American Society of Gene Therapy.

[25]  G. Gloor,et al.  Expression of Single Chain Antibodies (ScFvs) for c‐myc Oncoprotein in Recombinant Escherichiacoli Membranes by Using the Ice‐Nucleation Protein of Pseudomonassyringae , 2000, Biotechnology progress.

[26]  Satoshi Arai,et al.  Genetically-Encoded Yellow Fluorescent cAMP Indicator with an Expanded Dynamic Range for Dual-Color Imaging , 2014, PloS one.

[27]  Jim Berg,et al.  A genetically encoded fluorescent reporter of ATP/ADP ratio , 2008, Nature Methods.

[28]  Atsushi Miyawaki,et al.  Molecular spies for bioimaging--fluorescent protein-based probes. , 2015, Molecular cell.

[29]  D. Morgan,et al.  Analysis of intracellular protein function by antibody injection. , 1988, Immunology today.

[30]  H. Sondermann,et al.  Structural basis for calcium sensing by GCaMP2. , 2008, Structure.

[31]  H. Ueda,et al.  Q-Bodies from Recombinant Single-Chain Fv Fragment with Better Yield and Expanded Palette of Fluorophores , 2016 .