FAST (''Fluorescence-Activating and absorption-Shifting Tag'') is a small, genetically-encoded,
protein tag
Protein tags are peptide sequences genetically grafted onto a recombinant protein. Tags are attached to proteins for various purposes. They can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or a ...
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.
__TOC__
Mechanism
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. Such chemical-genetic approach was already implemented besides natural fluorescent proteins such as
GFP GFP may refer to:
Organisations
* Gaelic Football Provence, a French Gaelic Athletic Association club
* Geheime Feldpolizei, the German secret military police during the Second World War
* French Group for the Study of Polymers and their Applicat ...
or their derivatives such as
mCherry
mCherry is a member of the mFruits family of monomeric red fluorescent proteins (mRFPs). As a RFP, mCherry was derived from DsRed of '' Discosoma'' sea anemones unlike green fluorescent proteins (GFPs) which are often derived from '' Aequoera vic ...
in several systems already widely used:
* since 2003,
SNAP-tag
SNAP-tag® is a self-labeling protein tag commercially available in various expression vectors. SNAP-tag is a 182 residues polypeptide (19.4 kDa) that can be fused to any protein of interest and further specifically and covalently tagged with a sui ...
, a bi-component reporting system consisting of a 19 kDa peptide derived from a human enzyme, ''O''
6-methylguanine-ADN methyltransferase, evolved to form covalent bonds with fluorescent ''O''
6-benzylguanine derivatives; SNAP-tag was later evolved into an orthogonal tag,
CLIP-tag
SNAP-tag® is a self-labeling protein tag commercially available in various expression vectors. SNAP-tag is a 182 residues polypeptide (19.4 kDa) that can be fused to any protein of interest and further specifically and covalently tagged with a sui ...
;
* since 2008,
HaloTag
HaloTag is a self-labeling protein tag. It is a 297 residue protein (33 kDa) derived from a bacterial enzyme, designed to covalently bind to a synthetic ligand. The bacterial enzyme can be fused to various proteins of interest. The synthetic ligand ...
, 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).
![FAST fr](https://upload.wikimedia.org/wikipedia/commons/4/4d/FAST_fr.png)
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. Also, a complementation split version for monitoring protein-protein interactions was developed, splitFAST. A number of plasmids displaying FAST or splitFAST genes are available at Addgene.
Applications
The FAST-fluorogen reporting system is used in
fluorescence microscopy
A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscop ...
,
flow cytometry
Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
In this process, a sample containing cells or particles is suspended in a fluid and injected into the flo ...
and any other fluorometric methods to explore the living world, including
biosensor
A biosensor is an analytical device, used for the detection of a chemical substance, that combines a biological component with a physicochemical detector.
The ''sensitive biological element'', e.g. tissue, microorganisms, organelles, cell rece ...
s and
protein trafficking
:''This article deals with protein targeting in eukaryotes unless specified otherwise.''
Protein targeting or protein sorting is the biological mechanism by which proteins are transported to their appropriate destinations within or outside the c ...
. FAST has been reported for dynamic imaging of biofilms because of its unique capacity of fluorescence in low-oxygen conditions. For the same reason it allows for imaging and FACSing anaerobes, such as ''
Clostridium'', used for
biomass fermentatio
Biomass is plant-based material used as a fuel for heat or electricity production. It can be in the form of wood, wood residues, energy crops, agricultural residues, and waste from industry, farms, and households. Some people use the terms bi ...
like the
ABE fermentation
Abe or ABE may refer to:
People and fictional characters
* Shinzo Abe (1954–2022), former Prime Minister of Japan
* Abe (given name), a list of people and fictional characters with the given name or nickname
* Abe (surname), a list of people an ...
. FAST has also been reported for
super-resolution microscopy
Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techni ...
of living cells.
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.
References
{{Reflist
Proteins
Biochemistry methods