A SNAP-tag fluorogenic probe mimicking the chromophore of the red fluorescent protein Kaede.

Self-labelling protein tags with fluorogenic probes serve as great fluorescence imaging tools to understand key questions of protein dynamics and functions in living cells. In the present study, we report a SNAP-tag fluorogenic probe 4c mimicking the chromophore of the red fluorescent protein Kaede. The molecular rotor properties of 4c were utilized as a fluorogenic probe for SNAP-tag, such that conjugation with SNAPf protein leads to inhibition of twisted intramolecular charge transfer, triggering fluorogenecity. Upon conjugation with SNAPf, 4c exhibited approximately a 90-fold enhancement in fluorescence intensity with fast labelling kinetics (k2 = 15 000 M-1 s-1). Biochemical and spectroscopic studies confirmed that fluorescence requires formation of folded SNAPf·4c covalent conjugate between Cys 145 and 4c. Confocal microscopy and flow cytometry showed that 4c is capable of detecting SNAPf proteins or SNAPf fused with a protein of interest in living cells. This work provides a framework to develop the large family of FP chromophores into fluorogenic probes for self-labelling protein tags.

[1]  G. Ning,et al.  Modulation of Fluorescent Protein Chromophores To Detect Protein Aggregation with Turn-On Fluorescence. , 2018, Journal of the American Chemical Society.

[2]  K. Tan,et al.  S- Cis Diene Conformation: A New Bathochromic Shift Strategy for Near-Infrared Fluorescence Switchable Dye and the Imaging Applications. , 2018, Journal of the American Chemical Society.

[3]  W. Zhou,et al.  A naphthalimide-derived fluorogenic probe for SNAP-tag with a fast record labeling rate , 2017 .

[4]  K. Tan,et al.  Fluorogenic Protein Labeling Probes to Study the Morphological Interplay between PreLamin and Mature Lamin. , 2017, Bioconjugate chemistry.

[5]  Lu Miao,et al.  SNAP-tag fluorogenic probes for wash free protein labeling , 2017 .

[6]  Chunyang Lei,et al.  DNA mimics of red fluorescent proteins (RFP) based on G-quadruplex-confined synthetic RFP chromophores , 2017, Nucleic acids research.

[7]  Samie R. Jaffrey,et al.  A homodimer interface without base pairs in an RNA mimic of red fluorescent protein , 2017, Nature chemical biology.

[8]  Qinglong Qiao,et al.  A wash-free SNAP-tag fluorogenic probe based on the additive effects of quencher release and environmental sensitivity. , 2017, Chemical communications.

[9]  A. K. Boal,et al.  The Cation−π Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification , 2017, Biochemistry.

[10]  M. Rols,et al.  Conjugates of Benzoxazole and GFP Chromophore with Aggregation-Induced Enhanced Emission: Influence of the Chain Length on the Formation of Particles and on the Dye Uptake by Living Cells. , 2016, Small.

[11]  Alexander M. Spokoyny,et al.  Atomically precise organomimetic cluster nanomolecules assembled via perfluoroaryl-thiol SNAr chemistry , 2016, Nature chemistry.

[12]  M. Liptak,et al.  Suppression of Kasha's rule as a mechanism for fluorescent molecular rotors and aggregation-induced emission , 2016, Nature Chemistry.

[13]  S. Hell,et al.  Fluorogenic Probes for Multicolor Imaging in Living Cells. , 2016, Journal of the American Chemical Society.

[14]  S. Hell,et al.  Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super‐Resolution STED Microscopy in Living Cells , 2016, Angewandte Chemie.

[15]  K. Johnsson,et al.  Imaging and manipulating proteins in live cells through covalent labeling. , 2015, Nature chemical biology.

[16]  K. Tan,et al.  Protein sensing in living cells by molecular rotor-based fluorescence-switchable chemical probes , 2015, Chemical science.

[17]  Mark D. Smith,et al.  Mimic of the green fluorescent protein β-barrel: photophysics and dynamics of confined chromophores defined by a rigid porous scaffold. , 2015, Journal of the American Chemical Society.

[18]  Grigory S. Filonov,et al.  Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution , 2014, Journal of the American Chemical Society.

[19]  D. Ekiert,et al.  De Novo-Designed Enzymes as Small-Molecule-Regulated Fluorescence Imaging Tags and Fluorescent Reporters , 2014, Journal of the American Chemical Society.

[20]  V. Subramaniam,et al.  Evaluation of fluorophores to label SNAP-tag fused proteins for multicolor single-molecule tracking microscopy in live cells. , 2014, Biophysical journal.

[21]  Hsin-Yun Hsu,et al.  A rapid SNAP-tag fluorogenic probe based on an environment-sensitive fluorophore for no-wash live cell imaging. , 2014, ACS chemical biology.

[22]  L. Reymond,et al.  A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red. , 2014, ACS chemical biology.

[23]  Y. Hori,et al.  Small-molecule-based protein-labeling technology in live cell studies: probe-design concepts and applications. , 2014, Accounts of chemical research.

[24]  Y. Hori,et al.  Protein labeling with fluorogenic probes for no-wash live-cell imaging of proteins. , 2013, Current opinion in chemical biology.

[25]  M. Grabolle,et al.  Relative and absolute determination of fluorescence quantum yields of transparent samples , 2013, Nature Protocols.

[26]  Suliana Manley,et al.  A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. , 2013, Nature chemistry.

[27]  Vladislav V Verkhusha,et al.  Chromophore transformations in red fluorescent proteins. , 2012, Chemical reviews.

[28]  Chaoran Jing,et al.  Chemical tags for labeling proteins inside living cells. , 2011, Accounts of chemical research.

[29]  S. Jaffrey,et al.  RNA Mimics of Green Fluorescent Protein , 2011, Science.

[30]  Ming-Qun Xu,et al.  Development of SNAP-Tag Fluorogenic Probes for Wash-Free Fluorescence Imaging , 2011, Chembiochem : a European journal of chemical biology.

[31]  D. Trono,et al.  Measuring in vivo protein half-life. , 2011, Chemistry & biology.

[32]  H. Okuno,et al.  Real-time measurements of protein dynamics using fluorescence activation-coupled protein labeling method. , 2011, Journal of the American Chemical Society.

[33]  Young‐Tae Chang,et al.  Recapture of GFP chromophore fluorescence in a protein host. , 2011, ACS combinatorial science.

[34]  L. M. Tolbert,et al.  Steric and electronic effects in capsule-confined green fluorescent protein chromophores. , 2011, Journal of the American Chemical Society.

[35]  Kai Johnsson,et al.  How to obtain labeled proteins and what to do with them. , 2010, Current opinion in biotechnology.

[36]  Y. Hori,et al.  Covalent protein labeling based on noncatalytic beta-lactamase and a designed FRET substrate. , 2009, Journal of the American Chemical Society.

[37]  Atsushi Miyawaki,et al.  Monitoring cellular movement in vivo with photoconvertible fluorescence protein “Kaede” transgenic mice , 2008, Proceedings of the National Academy of Sciences.

[38]  Marjeta Urh,et al.  HaloTag: a novel protein labeling technology for cell imaging and protein analysis. , 2008, ACS chemical biology.

[39]  Kai Johnsson,et al.  An engineered protein tag for multiprotein labeling in living cells. , 2008, Chemistry & biology.

[40]  M. Sheetz,et al.  In vivo protein labeling with trimethoprim conjugates: a flexible chemical tag , 2005, Nature Methods.

[41]  M. Howarth,et al.  Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase , 2005, Nature Methods.

[42]  G. Nolan,et al.  A general approach for chemical labeling and rapid, spatially controlled protein inactivation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[43]  A. Miyawaki,et al.  An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[44]  R Y Tsien,et al.  Specific covalent labeling of recombinant protein molecules inside live cells. , 1998, Science.

[45]  H. Vogel,et al.  A general method for the covalent labeling of fusion proteins with small molecules in vivo , 2003, Nature Biotechnology.