Researchers at the University of Toronto and the University of Saskatchewan have engineered a compact, far-red light-responsive protein pair that could redefine how scientists switch molecules on and off inside living cells. The new system, described in Nature Methods, pairs a 17-kilodalton monomeric biliverdin-binding photoreceptor called FenixS with a 6-kilodalton selective binder named Ash1. Together they form a light-controlled heterodimer with negligible binding in the dark and a binding affinity that increases more than 1,200-fold upon illumination at 700 nanometers, producing what the developers describe as an ultralow-background core for optogenetic applications.
Optogenetics relies on genetically encoded photoreceptor proteins that change shape or binding behavior when struck by specific wavelengths of light. In mammalian cells, the workhorse tools for red-light control have long been phytochrome-based photoreceptors. These bacterial and plant proteins respond to red and far-red light, wavelengths that penetrate tissue more deeply and scatter less than the blue and ultraviolet light used by earlier systems such as LOV-domain tools like iLID, LOVTRAP and cryptochrome-based pairs. But phytochromes come with significant baggage. At roughly 70 kilodaltons, they are large proteins that function as dimers, forcing any fused protein of interest into dimeric configurations that may perturb its natural behavior.
The limitations of existing red-light systems extend beyond sheer size. Background binding in the OFF state—unwanted interaction between the photoreceptor and its partner when the light is off—can trigger spurious signaling that confounds experiments. Binding affinities in the lit state are often modest, and the dynamic range between OFF and ON states can be narrow. Binding partners, whether naturally occurring phytochrome-interacting factors or engineered domains, can be bulky and interact in complicated ways. For researchers trying to recruit a signaling protein to a membrane, assemble a transcription factor, or nucleate a biomolecular condensate on demand, these imperfections translate into noisy data and ambiguous biology.
The Toronto team, led by Giang N. T. Le in G. Andrew Woolley’s laboratory in the Department of Chemistry, together with Maruti Uppalapati at the University of Saskatchewan, attacked the problem at its foundation: the photoreceptor core itself. Using structure-based design combined with directed evolution, they engineered FenixS from a cyanobacteriochrome-derived scaffold that binds biliverdin, a linear tetrapyrrole chromophore that mammals produce naturally. This choice matters enormously for practical applications. Because biliverdin is endogenously present in mammalian cells and tissues, FenixS-based tools can in principle work without supplementing cultures or animals with exogenous chromophore—a persistent complication for many bilin-binding optogenetic systems.
FenixS evolved from earlier fluorescent protein engineering efforts, including the miRFPnano family of small near-infrared fluorescent proteins, which established that compact biliverdin-binding domains can be made bright, monomeric and stable. The team screened variants for fluorescence, biliverdin incorporation efficiency and photoswitchability across rounds of bacterial selection, using fluorescence imaging of agar plate colonies to track progressive gains in on-state brightness and in the contrast between illuminated and dark-adapted states. The resulting FenixS photoreceptor is monomeric, roughly a quarter the mass of a phytochrome, and undergoes reversible photoconversion between a dark-adapted Pr state and a red-light-generated Pb state when illuminated at 700 nanometers, with blue or green light driving the reverse reaction.
The second half of the pair, Ash1, was discovered through phage display selection against the illuminated state of FenixS. Phage display allows researchers to sift through billions of variant peptides and select those that bind a target under specified conditions—here, selecting binders that recognize the red-light state while counter-selecting against the dark state. Iterative rounds of selection produced Ash1, a 6-kilodalton helical binder with high affinity for photoactivated FenixS and, critically, virtually no binding to the dark-state receptor. The dramatic affinity swing between the two states is what gives the system its headline figure: a greater than 1,200-fold increase in binding upon 700-nanometer illumination, translating into an exceptionally large dynamic range for light-controlled assembly.
To demonstrate versatility, the researchers deployed the FenixS–Ash1 pair across several optogenetic architectures. In vitro, FenixS-coated beads recruited mCherry-tagged Ash1 from solution under red light and released it under blue light, cycling repeatedly with quantifiable fluorescence changes. In HeLa cells, mitochondria-targeted Ash1 recruited cytosolic FenixS to the organelle surface upon red-light illumination at 680 nanometers, with the interaction reversing under green illumination. The team also built a red-light version of the Corelets condensate system, in which light-controlled recruitment drives liquid-liquid phase separation, forming intracellular droplets on demand—a capability increasingly sought for studying biomolecular condensates, which organize cellular biochemistry and have been implicated in transcription, stress responses and disease.
Perhaps the most consequential demonstration is a transcription control tool built on the pair. The researchers fused the components to a split transcription factor architecture in which red light brings together the pieces needed to activate gene expression in mammalian cells. In head-to-head comparisons with established red-light optogenetic tools, the FenixS–Ash1-based system controlled gene expression robustly and did so without adding biliverdin to the culture medium, confirming that endogenous chromophore levels suffice. Low background activation in the dark state—a direct payoff of Ash1’s OFF-state selectivity—means that illuminated and non-illuminated cells can be compared cleanly, an essential property for perturbation experiments, developmental studies and any application where even a small leak of activity would be biologically meaningful.
The far-red operating range of the system adds further strategic value. Far-red and near-infrared light penetrate biological tissue more deeply than shorter wavelengths and generate less phototoxicity, making red-shifted optogenetics attractive for organoids, thick tissue slices and ultimately living animals. Previous red-light tools such as the bacterial phytochrome-based BphP1–PpsR2 system and the red/far-red reversible toggle switches built on plant phytochromes demonstrated the promise of this spectral window, but the bulk and dimeric architecture of phytochromes constrained their design flexibility. A monomeric, 17-kilodalton receptor changes the calculus: fusion proteins remain closer to their native size and oligomeric state, and the small Ash1 binder imposes minimal steric load when appended to effectors.
The engineering pipeline itself may prove as influential as the tool. The combination of structure-guided scaffold selection, directed evolution of a photoreceptor, and phage-display counter-selection for state-specific binding offers a general recipe for building next-generation optogenetic cores. The Woolley and Uppalapati groups had previously reported optimized phage-display selection methods for heterodimerizing optogenetic tools, and the FenixS–Ash1 work shows that approach paying off at scale. The plasmids for the new system will be deposited with Addgene, lowering the barrier for laboratories worldwide to adopt it. If FenixS–Ash1 performs as robustly in other hands as the published characterizations suggest, red-light optogenetics may finally gain a small, clean, high-contrast switch to match the blue-light tools that launched the field—and open the door to precise, minimally invasive optical control of signaling, transcription and condensate dynamics deep inside living systems.
The choice of biliverdin as the chromophore deserves particular attention from a cell-biology standpoint. Biliverdin is the product of heme oxygenase activity in the heme degradation pathway, and it is continuously generated inside mammalian cells at concentrations that, while modest, appear sufficient to populate the binding pockets of engineered biliverdin-binding domains. This metabolic availability distinguishes FenixS from plant-derived phytochromes that prefer phycocyanobilin or related bilins not found in animal tissues, which historically forced experimenters to add costly chromophore analogs to culture media or to accept incomplete holoprotein assembly. The cyanobacteriochrome lineage from which FenixS descends is notable in this regard: these compact, single-domain members of the phytochrome superfamily retain the bilin-binding GAF domain while dispensing with the flanking domains that make canonical phytochromes so large, a structural economy that earlier work on near-infrared fluorescent proteins exploited to dramatic effect.
The spectral separation built into the system also opens practical possibilities for multiplexing. Because FenixS is driven to its binding-competent state by far-red light and reversed by blue or green illumination, it occupies a spectral window distinct from the blue-light tools that dominate the optogenetic toolkit. In principle, a cell could carry both a blue-light-controlled LOV-domain module and a far-red-controlled FenixS–Ash1 module, each addressing different targets with minimal crosstalk. The reversibility of the switch, with light of different colors pushing the receptor between states, further allows a single experiment to cycle recruitment on and off repeatedly, which is valuable for probing the kinetics of cellular responses rather than merely triggering a one-time event.
For researchers weighing adoption, the reported head-to-head transcription experiments provide a useful benchmark, since gene activation assays integrate every weakness of a photoreceptor core—dark-state leak, slow switching, incomplete chromophore incorporation—into a single readable output. The demonstration that endogenous biliverdin suffices removes a logistical hurdle that has discouraged many laboratories from adopting bilin-based systems, particularly for work in primary cells, organoids or animal models where chromophore delivery is awkward. Combined with the small size of both components, which reduces the risk of perturbing fused effector proteins, these properties position the system as a candidate for experiments ranging from single-cell transcriptional perturbation to light-controlled assembly of synthetic signaling complexes in contexts where tissue penetration and low phototoxicity are at a premium.
Subject of Research: Engineering of a small biliverdin-binding far-red light-responsive protein pair for ultralow-background optogenetic control in mammalian cells
Article Title: An ultralow-background far-red light-responsive optogenetic tool based on an engineered biliverdin-binding domain
Article References: Le, G. N. T., Pham, L. M. T., Xue, B., Uppalapati, M., & Woolley, G. A. (2026). An ultralow-background far-red light-responsive optogenetic tool based on an engineered biliverdin-binding domain. Nature Methods. https://doi.org/10.1038/s41592-026-03223-6
Image Credits: AI Generated
DOI: 10.1038/s41592-026-03223-6
Keywords: optogenetics, FenixS, Ash1, biliverdin, photoreceptor, directed evolution, phage display, red light, gene expression, protein engineering, mammalian cells, biomolecular condensates
Cite Scienmag News
APA
MLA
Chicago
Juliet Wilcox. (September 12, 2026). Tiny red-light switch FenixS–Ash1 delivers ultralow-background optogenetic control. Scienmag. https://scienmag.com/tiny-red-light-switch-fenixs-ash1-delivers-ultralow-background-optogenetic-control/
Juliet Wilcox. “Tiny red-light switch FenixS–Ash1 delivers ultralow-background optogenetic control.” Scienmag, 12 September 2026, https://scienmag.com/tiny-red-light-switch-fenixs-ash1-delivers-ultralow-background-optogenetic-control/. Accessed 12 September 2026.
Juliet Wilcox. “Tiny red-light switch FenixS–Ash1 delivers ultralow-background optogenetic control.” Scienmag. September 12, 2026. https://scienmag.com/tiny-red-light-switch-fenixs-ash1-delivers-ultralow-background-optogenetic-control/
Copy citation
Download RIS
Tags: Ash1biliverdinbiliverdin-binding photoreceptorsbiomolecular condensatesdirected evolutionfar-red light-responsive proteinsFenixSFenixS-Ash1 heterodimer systemgene expressiongenetically encoded photoreceptor proteinsimprovements over phytochrome-based toolslight-controlled molecule bindingmammalian cellsoptogenetic controloptogeneticsphage displayphotoreceptorProtein Engineeringred lightred-light control in mammalian cellssmall optogenetic switchestissue penetration of far-red lightultralow-background optogenetic systemsultralow-background optogenetics
