Bidirectional photoriboswitch for translational regulation in mammalian cells

Yaxin Hu; Cheuk Yin Li; Lala Fu; Yiang Sun; Mingji Zhang; Tak Marx Yau; Chuxiao Xiong; Peng Shi; Yi Kuang

doi:10.1016/j.isci.2026.117671

. 2026 Sep 26;29(10):117671. doi: 10.1016/j.isci.2026.117671

Bidirectional photoriboswitch for translational regulation in mammalian cells

Yaxin Hu ^1,³, Cheuk Yin Li ^1,³, Lala Fu ¹, Yiang Sun ¹, Mingji Zhang ¹, Tak Marx Yau ¹, Chuxiao Xiong ², Peng Shi ², Yi Kuang ^1,^4,^∗

PMCID: PMC13639096 PMID: 42840230

Summary

Photoriboswitches are invaluable tools for synthetic biology because they provide direct, precise, and on-demand control of translation. While most existing photoriboswitches are predominantly one-directional, this study presents a bidirectional photoriboswitch platform capable of mimicking the orthogonal RNA-binding protein-mediated up-/downregulation of multiple mRNAs. Inspired by intein-based split protein reconstitution and light-induced protein dimerization, we first engineered a Light-OFF system that uses photodimerization to block intein reconstitution. We then validated that this Light-OFF system can function orthogonally with a Light-ON system that reconstitutes a different split intein pair under light. Together, a bidirectional photoriboswitch is generated, which senses light to orthogonally reconstitute RNA-binding proteins to tune the translation of their respective target mRNAs. We have demonstrated that the photoriboswitch produces bidirectional translation regulation. Importantly, the functional modules of the photoriboswitch can be easily altered to adapt for different purposes, allowing the bidirectional photoriboswitch to serve as a versatile tool for translation regulation.

Keywords: photoriboswitch, RNA-binding protein, bidirectional regulation, photodimerization

Graphical abstract

Highlights

•
Light-OFF riboswitch using photodimerization to block intein reconstitution
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Bidirectional photoriboswitch mimicking RBP-mediated regulation of multiple mRNAs
•
Orthogonal translational control in mammalian cells using a single light source

Cell biology; Functional aspects of cell biology; Molecular biology; Molecular mechanism of gene regulation

Introduction

In the field of synthetic biology, complex biological systems that mimic living cells are designed using circuits and devices with programmable and orthogonal regulation.¹^,²^,³ The advancement of synthetic biology in recent years has accelerated innovations in gene therapies,⁴^,⁵^,⁶ biosensors,⁷^,⁸ sustainable production,⁹^,¹⁰ and fundamental biological research.¹¹^,¹² Although various gene regulatory approaches in nature have been adapted by synthetic biology, the use of light to control gene expression has garnered increasing attention.¹³^,¹⁴ Photoregulation has several advantages, including accurate spatial control,¹⁵^,¹⁶ on-demand instant on/off switching of light,¹⁷^,¹⁸ and non-invasive administration.¹⁹^,²⁰ Additionally, as photoregulated gene expression enables high precision,²¹^,²² unintended effects on other genes or other locations are largely minimized,²³ promising safer therapeutic applications.²⁴^,²⁵

The photoregulation of gene expression in synthetic biology is typically achieved using photosensitive proteins or chemicals in combination with protein- or chemical-responsive DNA to form an optogenetic circuit.²⁶^,²⁷^,²⁸ Upon stimulation by the correct wavelength of light, the conformational change of the protein or chemical either turns on or turns off transcription, tuning the level of downstream protein production.²⁶^,²⁷^,²⁸ For example, photocleavable blockades on the promoter region enable the occurrence of transcription only under specific wavelengths of light, enabling dual-light-induced orthogonal transcription activation.²⁹ Photo-dimerization between a DNA-binding protein and a translation regulator can selectively turn on or turn off target gene translation.³⁰ These optogenetic circuits have been widely used to map or control neural activities mimicking natural neuron translation regulation, with great potential in curing neurological disorders, such as epilepsy and various neural degeneration diseases.³¹^,³²^,³³

Although controlling transcription is vital for biological systems, the regulation of translation is equally essential for gene expression in nature.³⁴^,³⁵ Translation regulation offers a direct and immediate response to changes in protein production, enabling cells to adapt rapidly to stress and stimulation.³⁵ In particular, cells often use RNA-binding proteins (RBPs) to concurrently down- and upregulate different mRNAs during vital processes, including cell differentiation³⁶^,³⁷^,³⁸ and tumor progression.³⁹ For example, during epithelial-mesenchymal transition, RBFOX2 is enabled while ESRP1 is disabled, resulting in the collective upregulation of mesenchymal-related mRNAs and downregulation of epithelial-related mRNAs.³⁶^,³⁷ Photosensitive translation regulatory circuits (also known as photoriboswitches) that mimic natural translation regulation have garnered attention as counterparts of optogenetic circuits in the last decade.⁴⁰^,⁴¹^,⁴²^,⁴³^,⁴⁴^,⁴⁵^,⁴⁶ For example, photolabile and photoreversible ligands uncage mRNA to turn on translation under light⁴⁷^,⁴⁸; the light-oxygen-voltage (LOV) photoreceptor binds to mRNA under light to sequester translation⁴⁹; and photo-caged or photo-induced protein dimerization controls the localization of translation regulator proteins on mRNA.²³ However, although some approaches are based on RBPs, most current photoriboswitches are one-directional, which sense light to turn on translation.²³ To mimic natural translation regulation, there is a need of orthogonally operating bidirectional photoriboswitches that use a single light input to enable and disable different RBPs for achieving differential and complex translational regulation.⁵⁰

In this study, we first designed a light-inhibited split intein system using light-sensitive protein pairs, in which the conjugation of the split extein is blocked by light. Such a light-inhibited split intein system can work orthogonally with existing light-induced split intein systems to differentially control the conjugation of functional exteins. Based on well-established model RBPs that are known to suppress translation when bound onto an aptamer on the 5′ UTR,⁵¹^,⁵² we used protein structure simulation to guide the engineering of split RBPs, which only bind to their designated aptamers after reconstitution. The combination of these two designs offers a bidirectional photoriboswitch platform that can use a single light source to selectively and irreversibly reconstitute RBPs, affecting the translation of the target mRNA bearing the matching aptamer. As various light-sensitive protein pairs have been established⁴⁹^,⁵³^,⁵⁴ and different RBP-aptamer pairs have been validated in mammalian cells,²³^,²⁶ the bidirectional photoriboswitch platform can be easily engineered to sense different wavelengths of light to tune RBP-aptamer interaction, mediating translational regulation by design. Therefore, this study presents a useful tool to expand the current toolkit of photoriboswitches, facilitating the further engineering of versatile photoriboswitches that offer bidirectional translation regulation of synthetic and natural mRNAs.

Results

Evaluation of light-induced protein dimerization and split intein pairs

To build a Light-OFF split intein system that inhibits intein under light, we first explored two protein pairs that dimerize under light: the Magnet protein pair⁵³ (pMag and nMag) and cryptochrome 2 (CRY2) and N-terminal cryptochrome-interacting basic-helix-loop-helix 1 (CIBN) pair.⁵⁴ Both protein pairs are relatively small in size, independent from exogenous cofactors, and exhibit fast dimerization kinetics upon irradiation with blue light.⁵³^,⁵⁴ To evaluate the light-induced dimerization efficiency of the protein pairs in the cell, we fused fluorescent proteins with non-overlapping spectra to each of the two proteins in the pair and used a well-studied plasma membrane tag LCK from lymphocyte-specific protein tyrosine kinase⁵⁵ to dock one protein onto the cell membrane (denoted as PM-pMag-mCherry and nMag-monomeric near-infrared fluorescent protein [miRFP]; PM-CIBN-enhanced green fluorescent protein [EGFP] and CRY2-mCherry) (Figure 1A). mRNAs encoding these protein pairs were co-transfected to HEK293T cells along with a reference mRNA encoding membrane-tagged enhanced blue fluorescent protein (EBFP; PM-EBFP). As shown in Figure 1B, the miRFP signal from nMag-miRFP is mostly distributed in the cytosol under dark conditions. After 30 s of blue-light irradiation (488 nm, 3 mW cm⁻²),⁵³ more than 80% of the miRFP signal was present on the cell membrane. A representative single cell was selected for analyzing the co-localization of the fluorescent signal through intensity profiling across the cell⁵⁶^,⁵⁷ (along the plotted line; Figure 1C). Figures 1D and 1E show that the miRFP signal co-localized only with the mCherry signal under light. Both the drastic membrane enrichment of the miRFP signal and strong co-localization between the miRFP and mCherry signals under light confirmed efficient light-induced pMag-nMag dimerization. However, the CRY2-CIBN pair only exhibited partial membrane enrichment and weak mCherry and EGFP signal co-localization under light (Figures S1A–S1D). Therefore, the pMag-nMag pair was selected for the following experiments.

We subsequently evaluated two split intein pairs: the well-established fast self-splicing Nostoc punctiforme DnaE intein pair⁵⁸ (denoted as Npu_N and Npu_C) and its engineered version (denoted as NpuM_N and NpuM_C), which has little cross-reactivity with the Npu_N-Npu_C pair.⁵⁹ We also utilized the aforementioned LCK tag and two fluorescent proteins to evaluate the self-splicing performance. As shown in Figures 1F and S1F, when HEK293T cells were transfected with mRNA encoding PM-Npu_N-mCherry or Npu_C-EGFP, the mCherry signal was largely distributed on the cell membrane, whereas the EGFP signal was evenly distributed inside the cell. The co-transfection of both mRNAs resulted in a clear localization shift of both mCherry and EGFP signals in over 80% of the cells, confirming efficient Npu intein self-splicing that conjugates the EGFP onto the membrane tag and detaches the mCherry with the intein pair. A similar change in signal localization was also observed on cells co-transfected with NpuM_N- and NpuM_C-encoding mRNAs with a weaker efficiency (Figures S1E and S1F). Therefore, the Npu_N-Npu_C pair was selected for the Light-OFF split intein system.

Design and evaluation of the Light-OFF split intein system

To use pMag-nMag dimerization to physically hinder the assembly of the Npu_N-Npu_C pair, we utilized a synthetic rigid ⍺ helix structure⁶⁰ as a spacer. This structure is derived from an algorithm specialized for designing short ⍺ helix structures (global sequence optimization [SEQOPT] method).⁶⁰ The spacer is placed between Npu_N and pMag, whereas Npu_C and nMag are directly linked to each other through a flexible short linker (GGGGS)_3.⁶¹^,⁶² The protein structure⁶³ in Figure 2A shows that a spacer of 39.7 Å is sufficient to segregate the Npu_N-Npu_C pair when pMag-nMag dimerize under light, and the spacer does not interfere with the assembly of Npu_N-Npu_C in dark conditions. To validate this design, we conjugated two fluorescent proteins to the two fusion proteins, respectively, and further fused the LCK tag onto Npu_N (denoted as PM-Npu_N-Spacer-pMag-mCherry and nMag-Npu_C-miRFP). mRNAs encoding the two proteins were delivered to HEK293T cells individually or at a molar ratio of 1:1 (Figures 2B, 2C, and S2A–S2D). Cells transfected with one mRNA were incubated under regular culture conditions, and the mCherry or miRFP signal was expressed on the cell membrane or cytosol, respectively. Cells co-transfected with both mRNAs were incubated under dark or blue-light conditions. A black plastic block covered the cells to create the dark condition. An identical black plastic block decorated by light-emitting diode (LED) light strips was used to create the light condition (estimated total light intensity of 3.75 mW cm⁻²) (Figure S1G). The selected light intensity is sufficient for pMag-nMag dimerization (3 mW cm⁻²)⁵³ and does not cause significant loss in cell viability or transcription suppression (Figure S1H). Under dark conditions, the location of mCherry and miRFP signals swapped owing to Npu_N-Npu_C self-splicing (Figures 2B, 2C, and S2A–S2D). The miRFP signal clearly co-localized with the reference EBFP signal, wherein the membrane/cytosol ratio reached the same level as that of cells solely transfected with the mCherry signal. The membrane/cytosol ratio of mCherry also reached the same level as that of cells solely transfected with the miRFP signal. Under light condition, both the mCherry and miRFP signals were distributed on the cell membrane with similar membrane/cytosol ratios owing to pMag-nMag dimerization.

To confirm that self-splicing between the Npu_N and Npu_C pair occurred under dark condition, we added a FLAG tag sequence after nMag-Npu_C-miRFP (denoted as nMag-Npu_C-miRFP-FLAG) for western blot analysis (Figure S2E).⁶⁴^,⁶⁵ We extracted total cellular proteins from HEK293T cells co-transfected with mRNAs encoding nMag-Npu_C-miRFP-FLAG and PM-Npu_N-Spacer-pMag-mCherry. As a reference, total cellular proteins were also collected from HEK293T cells transfected with mRNAs encoding nMag-Npu_C-miRFP-FLAG or PM-miRFP-FLAG (the expected product after Npu_N-Npu_C splicing). Anti-FLAG-antibody staining revealed that intact nMag-Npu_C-miRFP-FLAG has a molecular weight of 36.4 kDa, while PM-miRFP-FLAG has a molecular weight of 18.3 kDa. Co-transfected cells incubated under dark condition exhibited a dominant band at 18.3 kDa (67.6% ± 2.3%), and co-transfected cells incubated under light condition exhibited a dominant band at 36.4 kDa (71.4% ± 0.8%). These data not only confirmed that splicing between the Npu_N-Npu_C pair occurred under dark condition to yield the expected protein product but also validated that the splicing was inhibited under the light condition. It is to note that unspliced proteins still exist under dark condition, which is likely due to the limited splicing efficiency of the DnaE intein.⁶⁶ Similarly, the limited cellular pMag-nMag dimerization efficiency leads to random pairing between Npu_N-Npu_C under light condition.⁵³ To validate that the inhibited intein splicing under light condition stems from the spacer, we removed the spacer sequence from PM-Npu_N-Spacer-pMag-mCherry (denoted as PM-Npu_N-pMag-mCherry). mRNAs encoding PM-Npu_N-pMag-mCherry and nMag-Npu_C-miRFP were co-transfected into HEK293T cells, and the cells were incubated under dark or light conditions. Figure S2F showed that under both dark and light conditions, the miRFP signal clearly co-localized with the reference EBFP, and the mCherry signal was mostly distributed in the cytosol. These data suggested that intein splicing occurred under both light/dark conditions. These results confirmed that the spacer design successfully inhibits split intein assembly under light without interfering with intein splicing in the dark.⁶⁷^,⁶⁸^,⁶⁹

Construction of the bidirectional light-sensing protein reconstitution system

Subsequently, we evaluated whether the Light-OFF split intein system can function orthogonally with the existing light-induced (Light-ON) intein system to control functional protein reconstitution (Figure 3A). Split mCherry⁷⁰ was used as the reporter protein for the Light-OFF split intein system (Light-OFF mCherry). The Light-ON system was based on the well-established LOV-sensing domain,⁴⁹ which also senses blue light to undergo conformational change and has been used to induce NpuM_N-NpuM_C assembly. Similarly, split sfGFP,⁷¹ which has a different split design from split mCherry,⁷⁰ was used as the reporter to form the Light-ON sfGFP system. mRNAs encoding the Light-ON sfGFP and Light-OFF mCherry were co-transfected into HEK293T cells at equal molar ratios. Figure 3B shows that the sfGFP signals were strong under light condition, while only a marginal sfGFP signal can be detected under dark condition (0.22-fold to that of the light condition). On the other hand, strong mCherry signals can only be observed under dark conditions, with only 0.46-fold of mCherry signals being detected under light condition. Although the efficiencies of both pMag-nMag dimerization and LOV domain conformational change are high,⁵³^,⁶³ random intermolecular assembly may still occur, resulting in the leakage of mCherry and sfGFP signals under light/dark conditions, respectively. Nonetheless, the merged sfGFP/mCherry images clearly depicted the intensity shift of the two signals between dark and light conditions, confirming that the Light-ON and Light-OFF intein systems functioned independently to control the reconstitution of functional proteins.

Additionally, we synthesized a plasmid carrying the Light-OFF mCherry design and examined prolonged light-controlled mCherry production in cells. As shown in Figure S3A, when cells cultured under dark condition overnight were moved to irradiated conditions for 16 h, most of the mCherry signals disappeared. Therefore, we switched the light or dark condition every 24 h and observed that the mCherry signal repeatedly appeared when cells were incubated in the dark and near-completely disappeared under light (Figure 3C). The light/dark cycle did not impact the viability of the cells (Figure S3B). Instead, the cells slightly outgrew the untreated cells, and the crowded condition reduced the overall protein production. Nonetheless, these results suggested that the Light-OFF split intein system can be applied for repeated or prolonged translation regulation.

Evaluation of the bidirectional photoriboswitch for orthogonal translational regulation

To construct the bidirectional photoriboswitch for the orthogonal manipulation of RBPs, we utilized two model RBPs: MS2 bacteriophage coat protein⁵¹ (MCP) and PP7 bacteriophage coat protein⁷² (PCP). Both RBPs were chosen as they are the most widely used RBP tools for visualizing, tracking, and manipulating RNA molecules in living cells.⁵¹^,⁷²^,⁷³ We first searched for suitable split sites on the random coil fragments of both RBPs, as the flexible random coil region topologically favors structure assembly after reconstitution.⁷⁴ The Npu inteins prefer cysteine (Cys) at the +1 position and serine or threonine (Ser/Thr) at the +2 position of the splicing junction.⁷⁵^,⁷⁶ However, the random coil fragments of MCP only carry Ser/Thr, and neither Cys nor Ser/Thr can be found on PCP. Therefore, we engineered candidate split MCPs and PCPs, with Cys inserted before the Ser/Thr sites on MCP or onto the center of random coil fragments on PCP. For instance, as 39 aa of MCP represents Ser, MCP1-38 aa formed the candidate MCP_N fragment, and Cys followed by MCP39-129 aa formed the candidate MCP_C fragment (Cys-insertion site denoted as 38/39). AlphaFold 3⁷⁷ was used to simulate the reconstituted candidate MCPs and PCPs. The best simulated structure of each candidate was compared with the original RBP structures (Figures 4A and S3E). Both the template modeling (TM) score, which is used to assess the topological similarity,⁷⁸^,⁷⁹ and the root-mean-square deviation (RMSD) value, which indicates structural alignment based on the superposition of Cα atoms,⁸⁰ suggested that the candidate split-RBPs could reconstitute into structures that were similar to the original RBPs⁸¹^,⁸² (Figures S3C and S3D). We transfected cells with mRNAs encoding each pair of split intein-RBP candidates (i.e., MCP_N-Npu_N and Npu_C-MCP_C). Following the design of the RBP-sensing riboswitch in a previous study,⁸³ we also co-transfected the cells with MCP- or PCP-sensing riboswitches, which carry an MS2 or PP7 aptamer⁵¹^,⁵² that binds the respective RBPs on 5′ UTR to suppress the expression of EGFP. The level of EGFP signal repression indicated the efficiency of RBP reconstitution. Figures 4A and S3E show that all candidate split-RBP pairs successfully induced the reduction of reporter EGFP levels, with the Cys insertion sites at 38/39 (MCP) and 36/37 (PCP) being the most potent.

Finally, utilizing the selected split RBPs as described earlier, we constructed a model bidirectional photoriboswitch that uses Light-OFF and Light-ON intein systems to orthogonally reconstitute RBPs to control aptamer-bearing mRNAs. The scheme in Figure 4B shows the design of the model bidirectional photoriboswitch: light induces the formation of PCP, leading to the suppression of the EGFP signal of the reporter EGFP-switch via PCP-PP7 binding. The dark condition enables the formation of MCP, leading to the downregulation of reporter iRFP-switch via MCP-MS2 binding. In the control cells that were only transfected with the two reporter switches, the levels of the EGFP and iRFP signals were both high and at near-equal ratios (Figures 4C and 4D). When cells were transfected with the model photoriboswitch, strong iRFP or EGFP signals were observed only under the light or dark condition, respectively (logarithmic ratio at −0.40 and 0.23, respectively) (Figures 4C and 4D). The light/dark conditions changed the EGFP/iRFP ratio by more than 4.3-fold, confirming strong light-controlled orthogonal translation regulation based on RBP-induced translation suppression. Moreover, we also tested this model bidirectional photoriboswitch on another cell line. Figures S4A and S4B showed that light/dark conditions triggered a similar EGFP/iRFP ratio shift (6.7-fold) on HeLa cells, confirming the bidirectional translation regulation performance by the photoriboswitch. Importantly, as many RBPs in nature can enhance translation, we also studied whether our platform can mimic the regulation of enhancer RBPs. A translation promoter motif, viral protein genome (VPg)-linked, was fused onto the C-terminal fragments of split RBPs⁵¹^,⁸³; translational-deficient cap (G(5′)ppp(5′)A)⁵¹^,⁸⁴ was placed onto the reporter switches (Figure S4C). Light induces the formation of PCP-VPg, leading to the enhancement of the EGFP signal through PCP-PP7 aptamer-binding-mediated localization of VPg onto the EGFP switch. The dark condition enables the formation of MCP-VPg, leading to upregulation of iRFP through MCP-MS2 aptamer-induced localization of VPg onto the iRFP switch. On the control cells that were only transfected with the two reporter switches, the levels of both EGFP and iRFP were low and at near-equal ratios (Figures S4D and S4E). When cells were transfected with this model photoriboswitch, strong EGFP or iRFP signals were observed only under the light or dark condition, respectively (logarithmic ratio of 1.57 and −0.34, respectively) (Figures S4D and S4E). The light/dark condition changed the EGFP/iRFP ratio by more than 81.7-fold, confirming that our platform can be easily adapted to bidirectionally tune the function of different RBPs.

Discussion

In this study, we first engineered a Light-OFF split intein trans-splicing system. Although the photodimerization of protein is commonly used to bring protein fragments into close proximity,⁵³^,⁸⁵^,⁸⁶ we used photodimerization to spatially segregate the split intein by placing a rigid spacer between one of the photosensitizer-split intein fusions. The natural assembly between split inteins under dark conditions affords efficient intein trans-splicing. We demonstrated that the Light-OFF split intein system operates independently, with the existing Light-ON intein system offering bidirectional protein reconstitution. Importantly, we successfully demonstrated that the protein structure simulation can assist in the design of split RBPs that lack suitable split sites. Only when the split RBPs are reconstituted via intein into functional RBPs can they locate onto aptamers to tune translation. These two strategies together help realize a bidirectional photoriboswitch platform that mimics natural RBP-based translational regulation events that differentially control the protein production levels of different mRNAs.

Although the bidirectional photoriboswitch exhibits clear differential translation regulation, the current design still has several limitations. For example, we used blue light in this study, which has low tissue penetration and causes high levels of DNA damage.⁸⁷^,⁸⁸ Alternatively, infrared light-sensing protein pairs, such as BphP1/QPAS1,⁸⁹^,⁹⁰ can be utilized to further develop an infrared light-sensing bidirectional riboswitch. Another limitation is that in our demonstration, continuous light irradiation is used to maximize the output. Although light irradiation is a mild condition that did not cause noticeable changes in cell viability, further optimization of light irradiation should be explored to minimize side effects and shorten the regulatory cycle. Finally, in contrast to reversible photodimerization that offers a precise duration of the regulatory effect, our photoriboswitch design utilized reversible photo-sensing to generate irreversible split intein self-splicing, leading to irreversible RBP formation. Although transient regulation cannot be achieved by our system, the irreversibility of RBPs offers clear-cut regulation that may become advantageous for applications with strong regulatory effects, such as photo-computation and phototherapy.²³ Importantly, although RBP formation is irreversible, natural protein degradation and dilution of RBP during cell growth/proliferation limited the duration of the RBP effect. Thus, repeated light/dark administration can offer cycles of translational regulation. Considering that RBP-based translational regulation is a major translation regulatory approach in nature, the fundamental design principles of the bidirectional photoriboswitch presented in this work offer useful tools for controlling the translation of synthetic and natural mRNAs on demand, thereby broadening the toolkit and expanding the potential applications of photoriboswitches.

Limitations of the study

Despite the establishment of a bidirectional photoriboswitch platform for translational regulation in mammalian cells, this study has several limitations. First, the platform utilized blue-light-sensing proteins, which limited the application of the current platform to cultured cells as blue light has low tissue penetration. Further studies on red- or far-red-sensing proteins should be conducted to broaden the application of the platform. Second, two model mRNAs were orthogonally regulated using the current platform. In order to mimic the complex bidirectional translation regulation events, future studies on regulating multiple physiologically relevant mRNAs remain to be investigated.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yi Kuang (kekuang@ust.hk).

Materials availability

All the 5′ UTR and 3′ UTR sequences are shown in Tables S1 and S2, respectively. All the transfection conditions are shown in Table S3.

Data and code availability

•
Representative raw image data have been deposited at Mendeley Data at https://doi.org/10.17632/wsj22ttzvd.1 and are publicly available as of the date of publication. Other data reported in this paper will be shared by the lead contact upon request.
•
This paper does not report original code.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

We appreciate Prof. Pak Hang Peter Cheung at HKUST for providing HEK293T cell lines. We appreciate Prof. Ben Zhong Tang at HKUST for providing HeLa cell lines. We appreciate Prof. Hirohide Saito at Kyoto University for providing plasmids of EGFP and MCP. We appreciate Prof. Liting Duan at CUHK for providing plasmids of CIBN and CRY2. We thank the Biosciences Central Research Facility of HKUST (Clear Water Bay) for supporting the work in the microscopy core. This work is supported by the General Research Fund from the Research Grants Council of Hong Kong (#16300521) and Bridge Gap Fund from HKUST (BGF.2025.023).

Author contributions

Y.K., Y.H., and C.Y.L. designed the research. Y.H. and C.Y.L. executed the experiments and analyzed the results. L.F. assisted with the experiments and analyzed the results. Y.S., M.Z., and T.M.Y. assisted with data processing and validation. C.X. and P.S. assisted with RNA characterization. Y.K., Y.H., C.Y.L., and L.F. wrote the manuscript. Y.K. coordinated and supervised the project. All authors have read and approved the manuscript and its content.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Anti-DYKDDDDK tag antibody	Abcam	Cat#ab205606; RRID:AB_2916341
GAPDH Polyclonal Antibody	BIOSS	Cat#bs-10900R: RRID: AB_3661955
Goat anti-Rabbit IgG Secondary Antibody [HRP]	Novus Biologicals	Cat#HAF008: RRID: AB_357235

Chemicals, peptides, and recombinant proteins

Q5® High-Fidelity 2X Master Mix	NEB	Cat#M0493
MEGAscript™ T7 Transcription Kit	Thermo Fisher	Cat#AM1334
N1-methylpseudouridine-5′-triphosphate	TrikLink BioTechnologies	Cat#N-1081
ARCA (Anti-Reverse Cap Analog)	TrikLink BioTechnologies	Cat#N-7003-10
G(5′)ppp(5′)A RNA Cap Structure Analog	NEB	Cat#S1406L
TURBO^TM DNase	Thermo Fisher	Cat#AM2238
Antarctic Phosphatase	NEB	Cat#M0289L
DMEM, high glucose	Thermo Fisher	Cat#11965092
Fetal Bovine Serum	Sigma-Aldrich	Cat#F2442
MEM Non-essential Amino Acid Solution (100×)	Sigma-Aldrich	Cat#M7145
Gibco^TM Penicillin-Streptomycin	Thermo Fisher	Cat#A5955
Mycolor One-Step Mycoplasma Detector	Vazyme	Cat#D201-02
Dulbecco’s Phosphate Buffered Saline	Sigma-Aldrich	Cat#D8537
Trypsin-EDTA (0.25%), phenol red	Thermo Fisher	Cat#25200072
Lipofectamine MessengerMAX Transfection Reagent	Thermo Fisher	Cat#LMRNA008
Lipofectamine 3000	Thermo Fisher	Cat#L3000008
OptiMEM Reduced Serum Medium	Thermo Fisher	Cat#31985-070
Puromycin dihydrochloride	MERCK	Cat#P9620
Geneticin™ Selective Antibiotic (G418 Sulfate), Powder	Thermo Fisher	Cat#11811023
eBioscience^TM 7-AAD Viability Staining Solution	Thermo Fisher	Cat#00-6993-50
Attune Performance Tracking Beads	Thermo Fisher	Cat#4449754
CelLytic M Cell Lysis Reagent	Sigma-Aldrich	Cat#C2978
Halt Protease Inhibitor Cocktail (100X)	Thermo Fisher	Cat#78430
Pierce BCA Protein Assay Kits	Thermo Fisher	Cat#23225
Immun-Blot PVDF Membrane	Bio-Rad	Cat#1620177
ECL Prime Western Blotting System	Merck	Cat#GERPN2232

Critical commercial assays

QIAquick Gel Extraction Kit	Qiagen	Cat#28706X4
QIAquick PCR Purification Kit	Qiagen	Cat#28104
RNeasy MinElute Cleanup kit	Qiagen	Cat#74204
μ-Dish 35 mm, high	ibidi	Cat#81156

Deposited data

Raw data	This paper	https://doi.org/10.17632/wsj22ttzvd.1

Experimental models: cell lines

Human: HEK293T	ATCC	Cat#CRL-3216
Human: HeLa	ATCC	Cat#CRL-CCL-2

Oligonucleotides

5′ UTR sequence, see Table S1	Guangzhou Ruibo Biotechnology Co., Ltd.	N/A
3′ UTR sequence, see Table S2	Guangzhou Ruibo Biotechnology Co., Ltd.	N/A
PCR primers	Guangzhou Ruibo Biotechnology Co., Ltd.	N/A

Recombinant DNA

Plasmid mCherry-Mito-7	Olenych et al.⁹¹	Addgene#55102
Plasmid pEBFP2-Nuc	Ai et al.⁹²	Addgene#14893
Plasmid miRFP670nano	Oliinyk et al.⁹³	Addgene#127443
Plasmid pCMV-N1-mouse-superfolderGFP (mGRASP)	Yamagata et al.⁷¹	Addgene#42143
Plasmid pcDNA3.1_pMagFast2(3x)-iRFP	Kawano et al.⁵³	Addgene#67297
Plasmid pcDNA3.1_nMagHigh1-EGFP-CAAX	Kawano et al.⁵³	Addgene#67300
Plasmid pSKDuet16	Ellila et al.⁵⁸	Addgene#41684
Plasmid pETZZtevPP7CP(C68A,C73A)	Hogg et al.⁷²	Addgene#27548
Plasmid encoding CRY2	Song et al.⁵⁴	N/A
Plasmid encoding CIBN	Song et al.⁵⁴	N/A
Plasmid encoding EGFP	Shibata et al.⁹⁴	N/A
Plasmid encoding MS2CP	Nakanishi et al.²³^,⁵¹	N/A

Software and algorithms

Microsoft Excel for Microsoft 365	Microsoft	N/A
FIJI	ImageJ	https://fiji.sc/
Pymol	Schrödinger, LLC	https://www.pymol.org/
GraphPad Prism	GraphPad Software	https://www.graphpad.com
TM-score on-line	Zhang Lab, NUS	https://aideepmed.com/TM-score/
AlphaFold 3	Google DeepMind and Isomorphic Labs	https://alphafoldserver.com/
LAS X Life Science Microscope Software	Leica	https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/
Attune NxT Flow Cytometer Software version 4.2	Thermo Fisher	https://www.thermofisher.com/hk/en/home/life-science/cell-analysis/flow-cytometry/flow-cytometers/attune-acoustic-focusing-flow-cytometer/attune-cytometer-software.html

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Experimental model and study participant details

Cell lines

All procedures were carried out under aseptic conditions meeting biological safety requirements. HEK293T cells (ATCC, Cat#CRL-3216) are human embryonic kidney cells derived from a female fetus. HeLa cells (ATCC, Cat# CCL-2) are human cervical carcinoma cells derived from a 31-year-old female patient. HEK293T cell lines used in this study were kindly provided by Prof. Pak Hang Peter Cheung at HKUST. HeLa cell lines used in this study were kindly provided by Prof. Ben Zhong Tang at HKUST. They were originally obtained from ATCC and used after receipt from established laboratory stocks. Independent cell line authentication was not performed in this study. Cell lines were routinely tested for mycoplasma contamination using Mycolor One-Step Mycoplasma Detector (Vazyme, Cat#D201-02). All cell culture procedures and conditions are described in the method details section below.

Ethical statement and study approval

The acquisition and use of the above cell lines for the experiments described herein were approved by the Committee on Research Practices (CRP) Safety Panel and the Health, Safety and Environment Office (HSEO) at The Hong Kong University of Science and Technology (HKUST) (Safety Protocol: SP-2023-0229). As these studies utilized established human cell lines derived from commercial repositories, individual informed consent was not applicable. All cell lines were handled in accordance with institutional biosafety and ethical guidelines.

Method details

Cell culture

HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium, high glucose (Thermo Fisher, Cat#11965092) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich, Cat#F2442) and 1% Non-essential Amino Acid Solution (Sigma-Aldrich, Cat#M7145). All cells were cultured at 37 °C with 5% CO₂ and passaged using standard methods.

dsDNA template generation

DNA sequences encoding mCherry,⁹¹ EBFP,⁹² miRFP,⁹³ sfGFP,⁷¹ pMagFast2,⁵³ nMagHigh1,⁵³ DnaE Npu intein⁵⁸ and PCP⁷² were obtained from plasmids (Addgene plasmid Cat#55102, 14893, 79987, 42143, 67297, 67300, 41684 and 27548). The sequences for CRY2 and CIBN were a gift from Prof. Liting Duan from the Chinese University of Hong Kong.⁵⁴ The sequences for EGFP⁹⁴ and MCP⁵¹ were a gift from Prof. Hirohide Saito from the University of Tokyo. The sequence for DnaE Npu intein mutant was generated as previously described.⁵⁹ For the sequences used in the intein splicing system, a cysteine residue was added to the splicing site to facilitate splicing.⁷⁵^,⁷⁶ The sequence for the α-helix bulky structure consisting of 2 repeats of 13 amino acid residues (DYMERWYRYYNEF) was obtained from a previous work.⁶⁰ The sequence of split mCherry was generated based on a previous work.⁷⁰ The sequences for plasma membrane (PM) localization tag LCK and CAAX were obtained and added to the DNA construct as previously described.⁹⁵ All dsDNA templates were prepared by fusion PCR using Q5 High-Fidelity 2X Master Mix (NEB, Cat#M0493) as previously described.⁹⁶^,⁹⁷ All the 5′- and 3′-UTR sequences used in this project were reported in Tables S1 and S2, respectively. All primers were purchased from RUIBO Biotech. All DNA templates were purified using the QIAquick PCR Purification Kit (QIAGEN, Cat#28104) or QIAquick Gel Extraction Kit (QIAGEN, Cat#28706X4) according to the manufacturer’s protocol. The quality and concentration of the purified templates were determined by agarose gel electrophoresis and NanoVue Plus spectrophotometer (GE Healthcare).

In vitro transcription and purification

All mRNAs were synthesized by in vitro transcription (IVT) using MEGAScript T7 Transcription Kit (Thermo Fisher, Cat#AM1334) with kit supplied ATP, CTP, GTP, N1-methylpseudouridine-5′-triphosphate (TrikLink BioTechnologies, Cat#N-1081) and Anti Reverse Cap Analog (TriLink BioTechnologies, Cat#N-7003-10) or G(5′)ppp(5′)A RNA Cap Structure Analog (NEB, Cat#S1406L) at a molar ratio of 5:5:1:5:4. The reaction mixture was incubated at 37 °C for 5 hours and further digested at 37 °C for 30 minutes with TURBO DNase (Thermo Fisher, Cat#AM2238). The digested products were purified using RNeasy MinElute Cleanup kit (QIAGEN, Cat#74204) following the manufacturer’s protocol. The purified products were then treated with Antarctic Phosphatase (NEB, Cat#M0289L) at 37 °C for 30 minutes and purified again with RNeasy MinElute Cleanup kit (QIAGEN, Cat#74204). The quality and concentration of the purified mRNAs were determined by UREA-PAGE and NanoVue Plus spectrophotometer (GE Healthcare). The mRNAs were stored at -20 °C until use.

Transfection

For confocal imaging, cells were seeded at a density of 5×10⁴ cells/mL in 2 mL DMEM complete medium, using μ-Dish 35 mm, high (ibidi, Cat#81156). For flow cytometry analysis of expression and cell viability, cells were seeded into 48-well plates at a density of 1×10⁵ cells/mL in 0.25 mL. mRNA and plasmid transfection were conducted using Lipofectamine MessengerMAX (Thermo Fisher, Cat#LMRNA008) and Lipofectamine 3000 (Thermo Fisher, Cat#L3000008), respectively, following manufacturer’s instructions. 3 days after plasmid transfection, cells were cultured in medium supplemented with selection antibiotics, 5 μg/mL of puromycin (MERCK, Cat#P9620) or 400 μg/mL of geneticin (Thermo Fisher, Cat#11811023), for 5 days. The cells were then maintained in medium supplemented with 1 μg/mL of puromycin (MERCK, Cat#P9620) or 200 μg/mL of geneticin (Thermo Fisher, Cat#11811023). The mRNA and plasmid amounts used in every experiment were provided in Table S3.

Blue light irradiation

Cells were incubated under light or dark after transfection. For cells transfected with mRNA constructs encoding light-sensitive protein pairs and their fluorescent reporters only (Figures 1A, 1B, and S1A), blue light irradiation was achieved using the confocal microscope laser (488 nm, 3 mW cm^-2)⁵³ shining from the top for 30 seconds right before imaging. For the rest of the experiment, blue light irradiation was achieved using an LED light source (470 ± 20 nm, 3 mW/cm²)⁵³ shining from the top for 24 hours and removed as cells being put onto the confocal microscope station, or as the cells being detached for flow cytometry.⁹⁸ For cells incubated in the dark, the culture plate was wrapped in aluminum foil.

Live-cell imaging

After 24 hours of transfection, live cell imaging was performed using Nikon C2 confocal microscope (Nikon) or Leica SP8 Confocal Microscope (Leica). The images were taken with a 63× oil immersion objective. Lasers of wavelengths 405 nm, 488 nm, 552 nm, and 638 nm were used together with appropriate emission detection ranges by default settings from the manufacturer.

Cell viability assay

For examining cell viability with respect to different intensities of blue light irradiation, all cell samples were transfected with the same amount of EGFP mRNA and incubated under different intensities of blue light for 24 hours. Viability analysis was performed using 7-AAD Viability Staining Solution (Thermo Fisher, Cat#00-6993-50) according to manufacturer’s instructions. Briefly, 7-AAD staining solution was added to the cell suspension and incubated for 10 minutes. The cells were then analyzed by the Attune NxT Flow Cytometry (Thermo Fisher), where the 7-AAD signals were detected by excitation at 561 nm and emission at 670 ± 14 nm. Living cells (7-AAD negative) were counted for viability calculation, with the cell count of the negative control sample (dark environment) set as 100% viability.

Western blot

Cells were incubated under light or dark conditions overnight after transfection. They were lysed the next day with CelLytic M Cell Lysis Reagent (Sigma-Aldrich, Cat#C2978) supplemented with Protease Inhibitor (Thermo Fisher, Cat#78430) on ice for 15 minutes, and cell debris was removed by centrifugation. The protein concentration in the supernatant was measured by BCA assay (Thermo Fisher, Cat#23225). An equal amount of total protein extract was resolved 12% SDS-PAGE. The samples were then transferred to the PVDF membrane (Bio-Rad, Cat#1620177) by Trans-Blot Turbo Transfer System (Bio-Rad, Cat#1704150). The membrane was blocked with 1× PBST containing 5% skim milk for 1 hour at room temperature, then incubated with Rabbit anti-FLAG primary antibody (1:1,000 dilution) (Abcam, Cat#ab205606) at 4°C overnight. On the second day, the membrane was washed with 1× PBST five times and incubated with HRP conjugated goat anti-rabbit antibody (1:12,000 dilution) (Novus Biologicals, Cat#HAF008) for 1 hour at room temperature. The protein bands were visualized using ECL Prime Western Blotting System (Merck, Cat#GERPN2232) and imaged by ChemiDoc Touch Imaging System (Bio-Rad). After imaging, the membrane was washed twice by stripping buffer (1% Tween 20, 0.1% SDS, pH 2.2) and then washed three times by 1× PBST containing 5% skim milk. The membranes were then stained again by anti-GAPDH primary antibody (1:1,000 dilution) and HRP conjugated goat anti-rabbit antibody (1:12,000 dilution) following the above staining and imaging procedures. The protein band intensities were analyzed using FIJI (Version 2.14.0/1.54f, ImageJ), quantified from three independent experiments (n = 3), and reported as mean ± SD.

Flow cytometry analysis

After 24 hours of transfection, cells were harvested in suspension for flow cytometry analysis using Attune NxT Flow Cytometry (Thermo Fisher). The flow cytometry was calibrated with Attune Performance Tracking Beads (Thermo Fisher, Cat#4449754) according to manufacturer’s instructions every time before analysis. The EGFP signal was detected by excitation at 488 nm and emission at 530 ± 30 nm, and the positively transfected cells were gated by the iRFP signal. The 7-AAD signal was detected as mentioned in the previous section.

Protein structure prediction and visualization

The three-dimensional structures of split MCP and PCP were predicted using AlphaFold 3.⁷⁷ The crystal structure of model MCP and PCP were retrieved from the Protein DataBank (PDB ID 1MSC ⁸¹ and 2QUD,⁸² respectively). A cysteine residue was inserted into the split site of the model amino acid sequences and submitted to the AlphaFold Server. The prediction was run using the default settings.⁷⁷ Five models were generated with pLDDT > 70, indicating high accuracy of the predicted structures. The predicted structures were visualized and the RMSD score⁸⁰ with respect to the model protein was calculated using PyMOL⁹⁹ (Version 1.8, Schrödinger, LLC). The TM-score of the predicted structures with respect to the model protein was calculated using TM-score on-line⁷⁸^,⁷⁹ (https://aideepmed.com/TM-score/).

Quantification and statistical analysis

For imaging analysis, 30 cells were analyzed for each sample in each experimental repeat (Experimental repeats: n = 3). PM translocating quantification was employed using the steps described in previous work.¹⁰⁰ The fluorescence signals within the PM and cytosol areas were segmented and measured using FIJI¹⁰¹ (Version 2.14.0/1.54f, ImageJ) and generated as csv files, then plotted as bar graphs in Excel (Version 16.105.1, Microsoft). The fluorescence intensity profiling of single cell was performed following previous work⁵⁶^,⁵⁷ and visualized in GraphPad Prism (Version 10.1.1, GraphPad Software). Significance was analyzed by one-way ANOVA in Excel. The levels of significance are denoted in asterisks as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Detailed statistical information for all experiments can be found in the figure legends and Results.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2026.117671.

Supplemental information

Document S1. Figures S1–S4 and Tables S1–S3

mmc1.pdf^{(31.8MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S4 and Tables S1–S3

mmc1.pdf^{(31.8MB, pdf)}

Data Availability Statement

•
Representative raw image data have been deposited at Mendeley Data at https://doi.org/10.17632/wsj22ttzvd.1 and are publicly available as of the date of publication. Other data reported in this paper will be shared by the lead contact upon request.
•
This paper does not report original code.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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PERMALINK

Bidirectional photoriboswitch for translational regulation in mammalian cells

Yaxin Hu

Cheuk Yin Li

Lala Fu

Yiang Sun

Mingji Zhang

Tak Marx Yau

Chuxiao Xiong

Peng Shi

Yi Kuang

Summary

Graphical abstract

Highlights

Introduction

Results

Evaluation of light-induced protein dimerization and split intein pairs

Figure 1.

Design and evaluation of the Light-OFF split intein system

Figure 2.

Construction of the bidirectional light-sensing protein reconstitution system

Figure 3.

Evaluation of the bidirectional photoriboswitch for orthogonal translational regulation

Figure 4.

Discussion

Limitations of the study

Resource availability

Lead contact

Materials availability

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

STAR★Methods

Key resources table

Experimental model and study participant details

Cell lines

Ethical statement and study approval

Method details

Cell culture

dsDNA template generation

In vitro transcription and purification

Transfection

Blue light irradiation

Live-cell imaging

Cell viability assay

Western blot

Flow cytometry analysis

Protein structure prediction and visualization

Quantification and statistical analysis

Footnotes

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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