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Fluorescence-activating and absorption-shifting tag

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FAST (Fluorescence-Activating and absorption-Shifting Tag) is a small, genetically-encoded, protein tag which allows for fluorescence reporting of proteins of interest. Unlike natural fluorescent proteins and derivates such as GFP or mCherry, FAST is not fluorescent by itself. It can bind selectively a fluorogenic chromophore derived from 4-hydroxybenzylidene rhodanine (HBR), which is itself non fluorescent unless bound. Once bound, the pair of molecules goes through a unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, hence providing high labeling selectivity. The FAST-fluorogen reporting system can be used in fluorescence microscopy, flow cytometry and any other fluorometric method to explore the living world: biosensors, protein trafficking.

FAST, a small 14 kDa protein, was engineered from the photoactive yellow protein (PYP) by directed evolution. It was reported for the first time in 2016 by researchers from Ecole normale supérieure de Paris.[1]

Mechanism[edit]

FAST pertains to a chemical-genetic strategy for specific labeling of proteins. A peptide domain, called "tag", is genetically encoded to be bound to a protein of interest (by combination of their respective genes by means of transfection or infection). This tag is the anchor for a synthetic fluorescent probe to be further added.[2] Such chemical-genetic approach was already implemented besides natural fluorescent proteins such as GFP or their derivatives such as mCherry in several systems already widely used:

  • since 2003, SNAP-tag, a bi-component reporting system consisting of a 19 kDa peptide derived from a human enzyme, O6-methylguanine-ADN methyltransferase, evolved to form covalent bonds with fluorescent O6-benzylguanine derivatives; SNAP-tag was later evolved into an orthogonal tag, CLIP-tag;
  • since 2008, HaloTag, a bi-component reporting system consisting of a 33 kDa peptide derived from a bacterial enzyme, a haloalkane deshalogenase, which can specifically bind functional halogenated synthetic ligands, most often fluorescent for cell imaging (e.g., Coumarine, Oregon Green, Alexa Fluor 488, diAcFAM, TMR).
Reversible binding between FAST and a fluorogene

Several versions of FAST have been described differing by a small number of mutations, e.g., FAST1 (a.k.a. Y-FAST), FAST2 (a.k.a. iFAST), or a dimer, td-FAST.[3] Also, a complementation split version for monitoring protein-protein interactions was developed, splitFAST.[4] A number of plasmids displaying FAST or splitFAST genes are available at Addgene.[5]

Applications[edit]

The FAST-fluorogen reporting system is used in fluorescence microscopy, flow cytometry and any other fluorometric methods to explore the living world: biosensors, protein trafficking. FAST has been reported for dynamic imaging of biofilms thanks to its unique capacity of fluoresceing in low-oxygen conditions.[6] For the same reason it allows for imaging and FACSing anaerobes, such as Clostridium, used for biomass fermentation like the ABE fermentation.[7] FAST has also been reported for super-resolution microscopy of living cells.[8]

A number of fluorogens were developed for FAST and its derivates by The Twinkle Factory, varying by their emission wavelength, their brightness and their tag affinity. Some are non permeant, i.e., they can't go through cell membranes, hence specifically labeling membrane proteins or extracellular proteins, allowing for, e.g., monitoring trafficking from synthesis until excretion.[9]

References[edit]

  1. ^ Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M.; Specht, Christian G.; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine (2015-12-28). "Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo". Proceedings of the National Academy of Sciences. 113 (3): 497–502. doi:10.1073/pnas.1513094113. ISSN 0027-8424. PMC 4725535. PMID 26711992.
  2. ^ Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M.; Specht, Christian G.; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine (2016-01-19). "Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo". Proceedings of the National Academy of Sciences. 113 (3): 497–502. doi:10.1073/pnas.1513094113. ISSN 0027-8424. PMC 4725535. PMID 26711992.
  3. ^ Tebo, Alison G.; Pimenta, Frederico M.; Zhang, Yu; Gautier, Arnaud (2018-10-02). "Improved Chemical-Genetic Fluorescent Markers for Live Cell Microscopy". Biochemistry. 57 (39): 5648–5653. doi:10.1021/acs.biochem.8b00649. ISSN 0006-2960. PMID 30204425.
  4. ^ Tebo, Alison G.; Gautier, Arnaud (2019-08-14). "Author Correction: A split fluorescent reporter with rapid and reversible complementation". Nature Communications. 10 (1): 3730. Bibcode:2019NatCo..10.3730T. doi:10.1038/s41467-019-11689-6. ISSN 2041-1723. PMID 31413330.
  5. ^ "Addgene: Arnaud Gautier Lab Plasmids". www.addgene.org. Retrieved 2019-11-25.
  6. ^ Monmeyran, Amaury; Thomen, Philippe; Jonquière, Hugo; Sureau, Franck; Li, Chenge; Plamont, Marie-Aude; Douarche, Carine; Casella, Jean-François; Gautier, Arnaud; Henry, Nelly (2018-07-09). "The inducible chemical-genetic fluorescent marker FAST outperforms classical fluorescent proteins in the quantitative reporting of bacterial biofilm dynamics". Scientific Reports. 8 (1): 10336. Bibcode:2018NatSR...810336M. doi:10.1038/s41598-018-28643-z. ISSN 2045-2322.
  7. ^ Charubin, Kamil; Bennett, R. Kyle; Fast, Alan G.; Papoutsakis, Eleftherios T. (Nov 2018). "Engineering Clostridium organisms as microbial cell-factories: challenges & opportunities". Metabolic Engineering. 50: 173–191. doi:10.1016/j.ymben.2018.07.012. ISSN 1096-7176.
  8. ^ Venkatachalapathy, Muthukumaran; Belapurkar, Vivek; Jose, Mini; Gautier, Arnaud; Nair, Deepak (2019). "Live cell super resolution imaging by radial fluctuations using fluorogen binding tags". Nanoscale. 11 (8): 3626–3632. doi:10.1039/c8nr07809b. ISSN 2040-3364.
  9. ^ Li, Chenge; Mourton, Aurélien; Plamont, Marie-Aude; Rodrigues, Vanessa; Aujard, Isabelle; Volovitch, Michel; Le Saux, Thomas; Perez, Franck; Vriz, Sophie; Jullien, Ludovic; Joliot, Alain (2018-06-20). "Fluorogenic Probing of Membrane Protein Trafficking". Bioconjugate Chemistry. 29 (6): 1823–1828. doi:10.1021/acs.bioconjchem.8b00180. ISSN 1043-1802.


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