by Luc Lenglet, R&D Manager, Twinkle Bioscience SAS (The Twinkle Factory)
Genetically encoded fluorescent labels are central to modern live-cell microscopy, but the two workhorses—fluorescent proteins and self-labeling enzyme tags—share a trait: once attached, the label stays attached. A different family of reporters gets around this, using small fluorogenic dyes that light up only when bound to their protein partner and can be removed back. This article examines three—FAST, splitFAST and CATCHFIRE—and where their reversible, low-background chemistry fits alongside established tags, such as SNAP-tag and HaloTag.
From GFP to CATCHFIRE: a short history
It all started with a discovery that reshaped biology studies: the green fluorescent protein (GFP). Engineered over the years, GFP and numerous derivate fluorescent proteins have invaded all laboratories. Then, chemical biologists have exercised unrestrained creativity.
Self-labeling enzyme tags were the next step, attaching synthetic dyes to dark protein tags on demand. SNAP-tag (2003), derived from human O6-alkylguanine-DNA alkyltransferase, forms a covalent bond with benzylguanine substrates1; its companion CLIP-tag (2008) reacts with benzylcytosine, so two proteins can be labeled orthogonally in one cell2. HaloTag arrived the same year, an engineered bacterial dehalogenase capturing chloroalkane ligands quickly3. All three decoupled protein tag from dye, giving access to bright fluorophores such as the far-red silicon-rhodamines. Later on, fluorogenic ligangs allowed for lowered background and reduced wash-away steps.
A third idea materialized in 2016: instead of attaching a dye covalently, reversibly bind a small molecule, the TFFluorogen, that stays dark until it docks in the protein’s pocket. This is the principle behind FAST (Fluorescence-Activating and absorption-Shifting Tag)4, which grew over the years into a family: splitFAST (2019), a reversible interaction reporter5, and CATCHFIRE (2023), a self-reporting, reversible, proximity switch6. Unbound fluorogen stays dark, so background is low, and reversible binding lets the fluorescence be switched on and off at will.
How FAST works
FAST (Fluorescence-Activating and absorption-Shifting Tag) is a 14-kDa monomeric protein engineered from the photoactive yellow protein of Halorhodospira halophila4. It is about half the size of GFP. Its cognate fluorogens are hydroxybenzylidene-rhodanine (HBR) derivatives. Free in solution, fluorogens dissipate energy by bond rotation and stay almost dark. When one binds the FAST pocket, two things happen: the chromophore is held rigid, raising its quantum yield, and deprotonated to a phenolate form, red-shifting its absorption4. Only bound fluorogen absorbs at that shifted wavelength, so exciting there reports the bound population alone, the “absorption-shifting” effect behind the system’s selectivity and low background (Figure 1).
Figure 1: The FAST fluorogen-activating tag. A fluorogenic ligand, dark in solution, becomes strongly fluorescent on reversible, non-covalent binding to the FAST protein, hence labeling the tagged protein of interest.
Several useful properties follow. With no chromophore maturation step and high deliverability of the small ligand, labeling is almost instant: fluorescence appears the moment fluorogen and tag meet, which suits kinetics monitoring from the very protein translation, and fast events. It needs no oxygen, so FAST works in anaerobic and hypoxic settings where fluorescent proteins such as GFP derivatives mature poorly to not at all. Brightness tracks fluorogen concentration, so the signal is tunable. Washing the fluorogen out extinguishes it within seconds, enabling re-labelling. It extends to flow cytometry and microplate assays.
This oxygen-independence and the straightforward penetration of small fluorogens through microorganisms’ wall has made FAST a workhorse in strict anaerobes and low-oxygen niches. Reported uses include:
- FAST-equipping a variety of strictly anaerobic bacteria (where oxygen-dependent GFP fails) to expedite metabolic engineering, e.g., Clostridium acetobutylicum, up to thermophilic methanogenic archaea, e.g., Methanococcus maripaludis;
- deciphering virulence and secretion mechanisms in pathogenic Clostridia: Clostridioides difficile, Clostridium perfringens;
- live imaging usually-non-anaerobic bacteria exposed to hypoxic conditions, e.g., in biofilms (E. coli, Bacillus thuringiensis) or in the gut and in tumors (E. coli).
None of these reporters replaces the others; increasingly, the point is that they work together. A striking example is provided by Papoutsakis’ lab in 2020 establishing FAST, HaloTag and SNAP-tag in Clostridium acetobutylicum and C. ljungdahlii, and resolving FAST from HaloTag and from SNAP-tag in mixed cultures8. Three orthogonal reporters in a strict anaerobe is not a curiosity: it is what makes real-time subpopulation tracking possible in synthetic cocultures and microbiomes, where the alternative has been cumbersome PCR.
Watching interactions come and go: splitFAST
splitFAST continues on this reversible theme5. FAST is divided into two moieties fused to two proteins of interest. Both moieties, alone, almost don’t associate, but when both proteins of interest interact, FAST fragments reassemble and, upon fluorogen addition, fluoresce. Both the reassembly and the fluorogen binding are dynamic, so the signal tracks an interaction as it forms and dissolves, within seconds. That suits transient or oscillating interactions, such as signaling dynamics and cyclical assemblies, which an irreversible trap would blur.
splitFAST has been widely used to detect and quantify dynamic organelle contact sites (mitochondria–ER, lipid-droplet–organelle) with minimal perturbation. Also, a tripartite version that fluoresces only when three proteins assemble has been developed to resolve ternary complexes.
Controlling and seeing proximity at once: CATCHFIRE
Chemically induced dimerization (CID) systems let researchers force two proteins together on cue, to reposition proteins, recruit enzymes or trigger a pathway; FKBP–FRB–rapamycin is the textbook example. Most are non-fluorescent, so confirming dimerization needs a separate reporter, and many reverse slowly.
Figure 2: The CATCHFIRE chemically induced dimerization system. Adding a fluorogenic “match” molecule simultaneously assembles the FIREmate and FIREtag domains and switches on fluorescence, hence a reversible, self-reporting readout of induced proximity.
CATCHFIRE (Chemically Assisted Tethering of CHimera by Fluorogenic Induced REcognition) merges the actuator and the readout6. Its two small domains (an 11-residue peptide and a ~114-residue partner) come from the FAST scaffold and dimerize upon the addition of a fluorogenic “match” molecule hence inducing the proximity of fused proteins of interest. When the ternary complex forms, it turns about 100-fold brighter. The induced proximity is hence self-reporting (Figure 2). Association and dissociation each take tens of seconds, and removing the match reverses the assembly, so the interaction can be toggled repeatedly. Matches of different colors, and a non-fluorescent variant for actuation without a readout, add flexibility.
CATCHFIRE applications are now building up rapidly: from forcing the tethering of mitochondria to the microtubule motor KIF17, to reversible switches reassembling split enzymes (luciferases, proteases, recombinases), and an inducible gene-expression system.
Outlook
In July 2026, Tran and colleagues reported NovoTags in Science: protein tags, computationally designed from scratch, that bind bright synthetic Janelia Fluor dyes with nanomolar affinity and mutual orthogonality across the green, orange and far-red range9. The same study added dye-induced dimerization and a split, inducible variant, echoes of CATCHFIRE and splitFAST though non-fluorogenic. Whether such designer tags complement or replace today’s evolved and fluorogenic systems, time will tell.