Light-Driven Switch Lets Scientists Turn Genes On and Off in the Same Cell

Synthetic biologists have long dreamed of controlling gene expression with the same elegance with which nature does it: precisely, reversibly, and in response to cues that can be applied and removed at will. A team at The Hong Kong University of Science and Technology has now taken a significant step toward that goal by building a bidirectional photoriboswitch, a light-controlled device that can simultaneously raise the production of one protein and lower the production of another inside the same mammalian cell. The work, published in iScience, addresses a stubborn gap in the optogenetics toolkit, where most light-responsive translation controls have been one-directional, able only to switch protein synthesis on when illuminated.

The appeal of using light as a regulatory input is easy to understand. Unlike drugs or small molecules, light can be applied instantly, confined to a specific region, and tuned in intensity without chemically perturbing the cell. Optogenetic circuits built from photosensitive proteins have transformed neuroscience, allowing researchers to map and manipulate neural activity with millisecond precision. But most of these circuits operate at the level of transcription, controlling whether a gene is read into messenger RNA in the first place. Translation, the step in which ribosomes convert mRNA into protein, offers an even more direct and immediate handle on protein levels, and it is the step that cells themselves regulate most heavily during stress, differentiation, and disease.

In living cells, that translational control is largely exerted by RNA-binding proteins, or RBPs, which latch onto specific sequences in messenger RNAs and either block or boost their translation. During epithelial-mesenchymal transition, for example, the RNA-binding protein RBFOX2 is enabled while ESRP1 is disabled, collectively raising the output of mesenchymal-related mRNAs and suppressing epithelial-related ones. Mimicking this kind of opposing, coordinated regulation with a synthetic device has been the missing piece. The HKUST team, led by Yi Kuang, set out to build a platform in which a single light input would activate one RNA-binding protein while simultaneously preventing the formation of another, allowing two different target mRNAs to be regulated in opposite directions at once.

The core of the design is a clever inversion of a standard optogenetic trick. The researchers began with a split intein, a pair of protein fragments derived from the DnaE intein of the cyanobacterium Nostoc punctiforme that spontaneously stitch themselves together and splice out, fusing whatever proteins are attached to their ends. Normally, light-induced dimerization is used to bring protein fragments together. Here, the team did the opposite: they fused one half of the intein to the blue-light dimerizing protein pMag, and the other half to its partner nMag, but inserted a rigid synthetic alpha-helical spacer of about 39.7 angstroms between the intein fragment and pMag. In the dark, the intein halves find each other and splice normally. Under blue light, pMag and nMag dimerize and physically drag the intein halves apart, holding them beyond the reach of the spacer and blocking splicing altogether.

The team validated the concept in HEK293T cells using fluorescent reporters. When cells expressing the two fusion proteins were kept in the dark, the fluorescent signals swapped localization, confirming that the intein had spliced and conjugated the proteins as intended. Western blot analysis sharpened the picture: dark-incubated cells showed a dominant band at 18.3 kilodaltons, the expected spliced product, accounting for 67.6 percent of the signal, while light-exposed cells showed a dominant band at 36.4 kilodaltons, the unspliced precursor, at 71.4 percent. Removing the spacer abolished the light response entirely, with splicing proceeding under both conditions, proving that the rigid helix was the element converting photodimerization into inhibition. The chosen light intensity of roughly 3.75 milliwatts per square centimeter caused no measurable loss of cell viability.

Crucially, the Light-OFF system proved orthogonal to an existing Light-ON intein system based on the light-oxygen-voltage (LOV) sensing domain, which assembles a different engineered intein pair, NpuM, under the same blue light. When both systems were placed in the same cells, one driving reconstitution of split sfGFP under light and the other driving reconstitution of split mCherry in the dark, the two fluorescent outputs cleanly inverted between conditions. sfGFP signal under light reached levels about 4.5-fold higher than in the dark, while mCherry in the dark was roughly twice the light-condition level. The researchers also showed that the Light-OFF mCherry output could be cycled: switching light and dark every 24 hours over five days made the mCherry signal repeatedly appear and disappear without harming the cells.

With the light-sensing machinery in hand, the team turned to the harder problem of engineering the RNA-binding proteins themselves. They chose two widely used model RBPs, the MS2 bacteriophage coat protein (MCP) and the PP7 coat protein (PCP), which bind distinct RNA aptamers and are workhorses for tracking and manipulating RNA in living cells. When bound to aptamers placed in the 5-prime untranslated region of a reporter mRNA, these proteins suppress translation. The challenge was that split inteins prefer particular amino acids at the splicing junction, and the flexible random-coil regions of MCP and PCP did not naturally offer suitable split sites. The researchers inserted cysteine residues at engineered positions and used AlphaFold 3 to simulate whether each candidate split pair would reconstitute into a structure resembling the original protein, screening candidates by template modeling score and root-mean-square deviation before ever testing them in cells.

The simulations paid off. Split MCP with a cysteine inserted at the 38/39 position and split PCP split at 36/37 proved the most potent, each reconstituting into functional repressors that bound their cognate aptamers and suppressed reporter expression. The team then assembled the full bidirectional photoriboswitch: light induces formation of PCP, which suppresses an EGFP reporter bearing the PP7 aptamer, while darkness allows formation of MCP, which suppresses an iRFP reporter bearing the MS2 aptamer. In cells carrying the full device, the EGFP-to-iRFP ratio shifted by more than 4.3-fold between light and dark conditions, and the platform performed similarly in HeLa cells, with a 6.7-fold shift, demonstrating that the effect is not confined to a single cell line.

Perhaps most strikingly, the platform could be flipped from repression to activation. Many natural RNA-binding proteins enhance translation rather than block it, and the team mimicked this by fusing the VPg translation-promoter motif, derived from a viral protein genome-linked factor, onto the split RBP fragments, while fitting the reporter mRNAs with a translation-deficient cap. Now, light-induced formation of PCP-VPg localized the enhancer onto the EGFP switch and boosted its output, while darkness enabled MCP-VPg to upregulate the iRFP switch. The light-to-dark ratio shift in this configuration exceeded 81.7-fold, a dramatic demonstration that the same architectural principle can be adapted to opposing modes of translational control simply by swapping the functional domain attached to the reconstituted protein.

The authors are candid about the current limitations. Blue light, the input used throughout the study, penetrates tissue poorly and can cause DNA damage, so extending the platform to red or near-infrared sensing pairs such as BphP1/QPAS1 will be essential for any move beyond cultured cells. The system also produces irreversible RBP formation, since intein splicing cannot be undone, although natural protein degradation and dilution during cell growth limit the duration of the effect and allow repeated light-dark cycles of regulation. And the demonstration so far regulates two model reporter mRNAs; extending the approach to multiple physiologically relevant transcripts remains future work. Even so, the design principles on display, using reversible photodimerization to gate irreversible protein assembly and using computational structure prediction to engineer split points where none naturally exist, offer a genuinely expandable foundation. As RNA-based therapeutics and synthetic mRNA circuits mature, tools that can dial protein production up and down with nothing more than light are likely to find eager users, from basic researchers dissecting gene regulation to engineers building cell therapies that respond to optical commands.

Subject of Research: A bidirectional light-controlled riboswitch for regulating translation of synthetic mRNAs in mammalian cells

Article Title: Bidirectional photoriboswitch for translational regulation in mammalian cells

Article References: Hu, Y., Li, C. Y., Fu, L., Sun, Y., Zhang, M., Yau, T. M., Xiong, C., Shi, P., & Kuang, Y. (2026). Bidirectional photoriboswitch for translational regulation in mammalian cells. iScience, 29(10), Article 117671. https://doi.org/10.1016/j.isci.2026.117671

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117671

Keywords: synthetic biology, optogenetics, photoriboswitch, translation regulation, RNA-binding proteins, split intein, mRNA, blue light, AlphaFold 3, mammalian cells, gene expression control, HKUST