Photoswitchable intein for light control of covalent protein binding and cleavage

Baloban, Mikhail; Manoilov, Kyrylo Yu.; Karasev, Maksim M.; Verkhusha, Vladislav V.; Shcherbakova, Daria M.

doi:10.1038/s41467-025-63595-9

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Published: 11 September 2025

Photoswitchable intein for light control of covalent protein binding and cleavage

Nature Communications volume 16, Article number: 8263 (2025) Cite this article

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Abstract

Precise control of covalent protein binding and cleavage in mammalian cells is crucial for manipulating cellular processes but remains challenging due to dark background, poor stability, low efficiency, or requirement of unnatural amino acids in current optogenetic tools. We introduce a photoswitchable intein (PS Intein) engineered by allosterically modulating a small autocatalytic gp41-1 intein with tandem Vivid photoreceptor. PS Intein exhibits superior functionality and low background in cells compared to existing tools. PS Intein-based systems enable light-induced covalent binding, cleavage, and release of proteins for regulating gene expression and cell fate. The high responsiveness and ability to integrate multiple inputs allow for intersectional cell targeting using cancer- and tumor microenvironment-specific promoters. PS Intein tolerates various fusions and insertions, facilitating its application in diverse cellular contexts. This versatile technology offers efficient light-controlled protein manipulation, providing a powerful tool for adding functionalities to proteins and precisely controlling protein networks in living cells.

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Introduction

Precise control of peptide bond formation is valuable for spatiotemporal precise regulation of protein functions and triggering of signaling pathways. Covalent bond formation and cleavage can be used to stabilize and amplify transient events in living cells, such as protein-protein interactions¹ and gene expression. Covalent bond modifications can be employed for signal amplification due to the accumulation of irreversible reaction products. Additionally, covalent protein binding and cleavage are used in synthetic biology^2,3,4.

Several genetically encoded tools can be used to covalently modify proteins post-translationally, such as sortase A⁵, SpyTag and SpyCatcher⁶, and inteins^7,8,9,10. Sortase A is a transpeptidase enzyme that catalyzes a covalent peptide bond formation between two molecules connected to the C-terminal LPXTG motif and the N-terminal poly-Gly motif¹¹. In contrast, inteins and SpyTag/SpyCatcher are polypeptides that form a covalent bond autocatalytically. SpyTag is a short peptide and SpyCatcher is a protein that recognizes SpyTag and forms an isopeptide bond with it¹². Inteins are internal protein domains that catalyze the formation of a polypeptide bond between the flanking N- and C-terminal polypeptides termed exteins¹⁰. The reaction catalyzed by inteins is called protein splicing. The first Cys or Ser at the N-terminus of an intein is required for its function. The C-terminal Asn of an intein and Ser, Cys, or Thr at the beginning of a C-extein were also shown to be critical¹³. Inteins were found in many organisms, including bacteria, fungi and lower plants¹⁴. The most interesting for protein engineering are so-called mini-inteins that are small domains with no endonuclease activity^15,16. In addition to single-chain contiguous inteins, there are naturally and artificially split inteins, which can be used to covalently join two separate polypeptides attached to the N- and the C-terminal intein fragments (Int^N and Int^C)^17,18. Inteins are versatile tools as they allow covalent binding, release, and cyclization of polypeptides^9,10. They leave the final protein product with few or no extra amino acids.

Inteins are used in synthetic biology and beyond for integrating multiple inputs into a single output. Examples include selection of bacterial and mammalian cells containing two expression constructs^19,20, trans-splicing of large proteins, such as Streptococcus pyogenes Cas9 (SpCas9), from split fragments delivered in separate recombinant adeno-associated viruses²¹, intersectional reporters in the mouse brain²², and engineering of orthogonal signaling pathways with desired logic gates. Protein splicing can also be used for targeted modification of cellular proteins, including labeling²³, and insertions of designed chemically modified peptides into natural proteins^24,25,26. Inteins were also used to synthesize biocompatible polymers in living cells²⁷.

Researchers tried to implement control over intein-mediated reaction to allow so-called conditional protein splicing¹⁸. Inteins were engineered to be controlled by small cell permeable drugs^28,29, temperature^30,31, proteases^32,33, and salt^34,35. Several optogenetic tools (OT) for light control were also developed^{33,36,37,38,39}. OTs based on “caging” of the reactive interface between IntN and IntC of NpuDnaE intein^38,39 with light-oxygen voltage (LOV) domain from Avena sativa AsLOV2 suffer from substantial dark background. To obtain functional OTs with AsLOV2 as a photoreceptor, researchers had to introduce mutations in AsLOV2 and truncations in inteins that should reduce their activity. Earlier reported light-induced reconstitution of artificially split Saccharomyces cerevisiae VMA intein³⁷ performs best in yeast and was described as not efficient in mammalian cells due to poor solubility of intein fragments and poor tolerance to various exteins¹⁸. Far-red light-sensing plant photoreceptors used in the above-mentioned tool also require a supply of an exogenous phycocyanobilin (PCB) chromophore. A recent system for control of protein splicing with near-infrared light-sensing DrBphP photoreceptor and its partner Affibody was used in mammalian cells⁴⁰ without exogenous chromophores, because it relies on biliverdin that is already present in cells. However, this system is characterized by noticeable background caused by the interfering DrBphP-Affibody interaction in the darkness. Tools relying on the incorporation of photocaged unnatural amino acids^33,36 are efficient, precise, and have minimal background in the darkness. However, they require chemically synthesized molecules, their delivery or encoding a complex machinery for their incorporation in living cells.

Non-intein-based OTs for covalent bond formation based on SpyTag and SpyCatcher also suffer from the above limitations, making them suboptimal for applications in mammalian cells^41,42. The AsLOV2-based tool has a noticeable background and is relatively inefficient⁴¹, whereas the recently reported tool used in cells requires an unnatural amino acid⁴². An OT, named PhoCl, and its derivatives are effective for light-controlled autocatalytic protein cleavage and have negligible dark background^43,44. PhoCl-based tools are derived from fluorescent proteins and function by light-induced chromophore conversion accompanied by polypeptide chain breakage. PhoCl tools do not allow covalent protein binding. Also, PhoCl depends on violet light that has considerable cytotoxicity^45,46.

To overcome limitations of the existing OTs, we searched for alternative means of light-controlled intein regulation. The use of photoreceptors that act through light-induced heterodimerization in OTs, especially when acting irreversibly, requires careful engineering and adjustment of expression of system components (example in ref. ⁴⁷) because even a minor dark background can accumulate. Also, there are only a handful of examples of OTs acting through light-induced heterodimerization in a single polypeptide chain⁴⁸. Steric block of the reactive protein fragment with AsLOV2 is not always effective for caging of structured protein domains, and, therefore, the background problem is also an issue. The use of AsLOV2 for allosteric control was shown to be effective. However, it works best for light-inactivation^49,50. We select a recently reported approach for allosteric control of a protein based on insertion of a loop containing tandem Vivid (VVD) photoreceptor domains from Neurospora crassa connected by a flexible linker⁵¹. This loop, named LightR, introduces distortion into the protein in the darkness, resulting in its deactivation. Light induces VVD homodimerization that leads to the loop closure and restoration of a protein function.

To install allosteric light control, we selected gp41-1 intein⁵², which is among the smallest and fastest². Its size is just 125 amino acid residues. Gp41-1 is naturally split at position 88 (hereafter numbering of gp41-1 amino acid residues follows that in PDB ID: 6QAZ⁵³), and also can function as a single-chain intein when its two fragments IntN and IntC are covalently linked. An additional advantage is that it uses Ser instead of Cys at position +1 of the C-extein (first C-extein residue after the intein sequence), which makes it more suitable for seamless ligation of polypeptide chains⁵².

Insertion of the double VVD loop results in an OT termed PhotoSwitchable Intein (PS Intein). We first demonstrate that PS Intein enables light-controlled reconstitution of the fluorescent protein mCherry from its split dark fragments. This is achieved with all interacting protein domains linked in a single polypeptide or expressed as two polypeptides split at the natural gp41-1 split site. We then compare PS Intein with the previously reported AsLOV2-based tool, LOVInC³⁸, demonstrating PS Intein’s functionality in mammalian cells and its low background. We confirm that the two polypeptides of Split PS Intein do not interact until illuminated. Our tool can also be used for light-controlled protein cleavage. As a functional output, we perform light-induced cleavage of the Bid protein to induce apoptosis. Furthermore, by designing a circular permuted (cp) PS Intein, we achieve light-controlled release of an active protein from the cytoplasm to the nucleus, activated through its cleavage from a cytoplasm-localized construct. We utilize cp PS Intein to release various transcriptional activators, resulting in light-controlled gene expression systems. These systems benefit from signal amplification resulting from the accumulation of a constitutively active transactivator. Lastly, we demonstrate that PS Intein-based gene expression systems can function as logic gates for specific intersectional cell targeting, using selection with specific promoters and light. The developed PS Intein optogenetic module offers versatile options for light-controlled covalent protein manipulations with high efficiency and low background, thus serving as a powerful platform for precise spatiotemporal control of cellular processes.

Results

Engineering of photoswitchable intein

For engineering of light-controlled protein splicing (Fig. 1), we tested several sites to insert the double VVD (LightR) allosteric switch⁵¹ into gp41-1. We selected insertion sites targeting the loops connecting beta sheets in the available crystal structure⁵³. We also tested sites located in the beta-sheets, rather than in the loops connecting them, as this approach previously allowed tight drug-dependent control of a split intein¹ (Fig. 1c). We also included one insertion at position 87 corresponding to the natural gp41-1 split site between the N-terminal fragment called IntN and the C-terminal fragment called IntC here. This variant was used as a light-insensitive control.

**Fig. 1: Engineering of light-controlled protein splicing.**

To facilitate splicing⁵⁴, we added extein amino acid sequences SGY (N-extein) and SSS (C-extein) at the N- and C-termini of gp41-1, respectively (Fig. 1b). For the readout of protein splicing, we used intein-mediated reconstitution of a split fluorescent protein mCherry (Fig. 1a). We selected the split mCherry variant that was reported to become fluorescent only when covalently linked, as opposed to variants that reconstitute mCherry when brought in close proximity³. Resulting cellular mCherry fluorescence was assessed by flow cytometry 24 h post-transfection (Fig. 1d, Supplementary Fig. 1). The samples were illuminated with pulsed 450-nm LED light for 6 h prior to flow cytometry analysis.

As expected, the variant with insertion at position 87 spliced efficiently in both light and dark conditions, consistent with this position being a natural split site of gp41-1. Three other tested positions showed light-to-dark ratios around 3. Position 103 yielded the ratio of 9 (Fig. 1d). We chose the variant with insertion at position 103 for further PS Intein development, as it demonstrated the lowest spontaneous activation in darkness, yet efficient activation upon illumination.

To further optimize the initial construct, we varied the lengths of linkers L1 and L3 connecting VVD domains with the intein (Fig. 1a, b). We found that linkers GTGSGG (L1) and GSGS (L3) allowed achieving the highest 12.1-fold light-to-dark contrast (Fig. 1e). This final version of the light-regulated intein was named PS (PhotoSwitchable) Intein.

Characterization of PS Intein

First, we compared the performance of PS Intein with the positive and negative controls. The single-chain gp41-1 without the double VVD insert attached to the split mCherry was used as a constantly active positive control. The same construct with C1A mutation in gp41-1 abolishing its autocatalytic splicing activity⁵² was used as a negative control. We did not observe any light activation in control samples (light-to-dark ratios were close to 1). PS Intein demonstrated negligible dark background activity, close to the negative control (Fig. 2a) (less than 1.5-fold difference). This was confirmed by microscopy (Fig. 2b). We also confirmed that PS Intein can be delivered to cells via AAV (Supplementary Fig. 2) that should make it possible to use the system in primary cells or in vivo.

**Fig. 2: Characterization of the photoswitchable intein, PS Intein.**

Further, we compared PS Intein with controls (gp41-1 without the double VVD insert was a positive control, and PS Intein with C1A mutation was a negative control) using Western blot (Supplementary Fig. 3). Total protein samples from mammalian cell lysates were analyzed using anti-mCherry and anti-GFP primary antibodies. As expected, we detected formation of mCherry in illuminated PS Intein sample and positive control samples, as opposed to the dark sample and the negative control.

Next, we tested different illumination times applied to reconstitution of mCherry. After illumination, cells were incubated for 6 h to allow mCherry maturation (Fig. 2c, top). We observed that the mCherry fluorescence signal plateaued after 2 h of illumination (Fig. 2c, bottom). We observed ~25% signal increase already after 5 min of illumination (Fig. 2c, bottom).

We further tested if PS Intein can be used for light-induced intracellular targeting of mCherry (or other protein of interest). We tagged split mCherry with nuclear localization signal (NLS) to be released to the nucleus from the molecule localized in the cytoplasm via nuclear export signal (NES). This construct was highlighted by msfGFP inserted in the loop between IntN and IntC, i.e. in the natural gp41-1 split site. We attached a MycA1 NLS⁵⁵ to the N-terminal part of split mCherry. As NES, we used a strong Super-PKI2 NES⁵⁶ fused with msfGFP between IntN and IntC (Fig. 2d). The strengths of NES and NLS were selected in a way, so that the molecule is localized in cytoplasm in darkness⁵⁷, as visualized by msfGFP (Fig. 2d, e). This also indicates that PS Intein tolerates an insertion of a functional protein in the loop between IntN and IntC. As expected, light triggered the reconstitution of mCherry and its relocalization to the nucleus (Fig. 2e).

Split PS Intein and light-induced protein ligation

We tested if PS Intein can be split using the natural split site of the gp41-1. This design should enable covalent binding of two separate polypeptides upon illumination. We inserted T2A peptide between IntN and IntC in the PS Intein (Fig. 3a). As above, we used co-expressed EGFP for selection of expressing cells. Compared to a single molecule PS Intein, Split PS Intein retained more than 60% response with a low dark background (Fig. 3b) and a light-to-dark ratio of ~9. We also tested split PS Intein with split mCherry by Western blot (Supplementary Fig. 3). In accordance with the flow cytometry analysis, we detected formation of mCherry in illuminated Split PS Intein sample, as opposed to the dark sample.

**Fig. 3: Split PS Intein benchmarking and application to protein ligation.**

Next, we directly compared Split PS Intein with the earlier reported genetically encoded tool for blue light-controlled protein splicing based on AsLOV2 domain³⁸. In our construct used for reconstitution of split mCherry, we replaced IntN with the so-named InN of NpuDnaE and light-sensing IntC with the LOVInC containing mutated AsLOV2 domain fused to truncated InC of NpuDnaE as described in ref. ³⁸ (Fig. 3c). We used the same illumination conditions as in our other experiments (450 nm LED (3 mW cm⁻², 2 s On, 10 s Off) for 6 h prior to flow cytometry analysis). We observed low activation and no significant difference between dark and illuminated samples for the LOVInC-containing tool, resulting from its relatively high dark background. (Fig. 3d and Supplementary Fig. 4). We also compared split PS Intein with LOVInC using illumination conditions similar to those described in ref. ³⁸ (overnight for 1 s On, 30 s Off intervals using 450 nm, 25 mW cm⁻²) (Supplementary Fig. 5). We observed similar results as with the low light power.

Split PS Intein should be useful in OTs working by light-induced ligation of two polypeptides. However, IntN and IntC of split gp41-1 spontaneously interact⁵². We decided to test if IntN and VVD-containing IntC in Split PS Intein could interact before illumination. If it occurs before, then two polypeptides are already bound to each other and illumination results in covalent joining of respective exteins (Fig. 3e, mechanism 2). If it occurs after, then two polypeptides are not bound to each other, until both interaction and splicing are triggered by light (Fig. 3e, mechanism 1). To test this, we replaced mCherryN and mCherryC in Split PS Intein with the DNA-binding protein TetR and transcription activator vp16, respectively (Fig. 3e). Together, TetR and vp16 constitute tetracycline-controlled transactivation protein (tTA)⁵⁸, which activates gene expression from tetracycline-responsive promoter elements (TRE). As a gene expression reporter, we used pTRE-mCherry. We observed that mCherry fluorescence of HeLa cells co-transfected with Split PS Intein and the reporter in darkness corresponded to the minimal fluorescence of the reporter-only expressing cells (Fig. 3f, Supplementary Fig. 6). Thus, spontaneous interaction of IntN and VVD-containing IntC in darkness can be excluded.

Light-induced protein cleavage: apoptosis via Bid activation

To demonstrate the use of PS Intein in OTs working by protein cleavage, we developed a light-induced Bid cleavage tool initiating apoptosis. Bid is a member of the Bcl-2 family of pro-apoptotic factors; it is composed of two functional domains: the C-terminal BH3 domain that induces cytochrome c release from mitochondria and the N-terminal domain that interacts with the BH3 domain and inhibits its action. Upon Bid cleavage by caspase 8, its N-terminal domain is removed and degraded. Then, unblocked BH3 performs its action resulting in subsequent activation of caspase 3 and cell death⁵⁹.

We tested different designs of apoptosis regulating OTs. In the first design called PS Intein Bid, NBid and CBid are placed in the PS Intein polypeptide close to each other, separated only by IntN (Fig. 4a). In another design, Split PS Intein Bid, NBid was placed at the N-terminus, while CBid was placed at the C-terminus of the IntC (Supplementary Fig. 7). We also created a split version with a long 20 amino acid linker between IntC and CBid (named Split Intein Bid L). To test our constructs, we monitored cell death by cell viability assay with SYTOX Green dye (Fig. 4b, Supplementary Fig. 8). We co-transfected cells with mCherry. To confirm that cells died from apoptosis, we tested if permeable pan-caspase inhibitor Z-VAD-FMK inhibits apoptosis. As a negative control of light activation, we used C1A mutant. We observed a high mortality rate for both Split PS Intein Bid design samples kept in darkness (Fig. 4b), ~20% for both. Therefore, they were excluded from further analysis. PS Intein Bid expressing cells showed ~8.8% cell death in darkness that is close to the negative controls (PS Intein Bid + Z-VAD-FMK or PS Intein/C1A Bid + Z-VAD-FMK) and mCherry only expressing cells, ~7%. 20% of cells died after being illuminated for 30 min, while more than a half (~53%) died after being illuminated for 6 h. We also visualized activation of apoptosis in cells expressing either PS Intein Bid or PS Intein/C1A Bid mutant (Fig. 4c, d, Supplementary Fig. 9). For this, we used NES-DEVD-mCardinal-NLSx3⁴³. In darkness, mCardinal fluorescence was detected both in the cytoplasm and the nucleus. After illumination, activated caspase 3 should cleave the reporter protein at DEVD site, leading to translocation of mCardinal-NLSx3 into the nucleus (Fig. 4c, d, top). We observed such translocation for PS Intein Bid-expressing cells (Fig. 4c, Supplementary Fig. 9), as opposed to PS Intein/C1A Bid mutant-expressing cells (Fig. 4d, Supplementary Fig. 9).

**Fig. 4: PS Intein Bid for light-induced apoptosis.**

Light-induced protein release: activation of gene expression

Light-induced protein cleavage can be used for activation of protein of interest via its controlled release from sequestered localization within a cell. Here, we decided to release tTA from the cytoplasm to the nucleus. To sequester our constructs in the cytoplasm, we used mutated estrogen receptor ligand-binding domain (ER^T2)⁶⁰.

Using Split PS Intein, we placed ER^T2 at the N-terminus of IntC and tTA at its C-terminus (Fig. 5a, c). We also designed a construct that allows the use of two ER^T2 domains attached at both the N- and C-termini of the caged protein, since earlier studies indicated to more efficient caging with double ER^T2 ^44,61. For this design, we used a circular permuted version of PS Intein (cp PS Intein) (Fig. 5b, d). tTA should be released without any attached domains, in a circularized form (Fig. 5b). The constructs were tested in HeLa cells, which were illuminated for 6 h with pulsed light and analyzed 24 h after illumination (Fig. 5e, left). As expected, caging with one ER^T2 resulted in higher background than caging with two ER^T2 domains. Interestingly, the magnitudes of response were comparable, with even higher response for the double ER^T2 construct containing cp PS Intein. Primarily due to lower background, the light activation contrast for cp PS Intein tTA was ~16 fold higher than for Split PS tTA (Fig. 5e, Supplementary Fig. 10). Overall, for the best performing cp PS Intein tTA system, we obtained light activation contrast of more than 590-fold with 6 h of illumination. We then confirmed that cp PS Intein tTA system produced high light-to-dark contrasts in various cell lines and expression conditions (Fig. 5f, Supplementary Fig. 11).

**Fig. 5: PS Intein and Split PS Intein for light-induced gene expression.**

We hypothesized that the high dynamic range of light-induced gene expression can be used to obtain spatial patterns of gene expression at a tissue scale. To demonstrate this, we performed spatial illumination of the dish with a monolayer of transiently transfected HEK293T cells using a simple photomask (Supplementary Fig. 12). We showed that a complex pattern on monolayer of cells expressing cp PS Intein tTA can be obtained at cellular resolution.

To test the versatility of our approach for caging, we created constructs that should release Gal4-vp16. We replaced TetR with Gal4 transcriptional activator, recognizing yeast-derived upstream activation sequence (UAS)⁶². We used pG5-EGFP reporter plasmid with five UAS repeats as a reporter. As with tTA above, we obtained lower background for the cp PS Intein Gal4-vp16 with double ER^T2 than for Split PS Intein Gal4-vp16 with a single ER^T2 (Fig. 5e, Supplementary Fig. 13). The contrast for cp construct was ~10-fold higher than for the Split version. Compared to tTA, Gal4-vp16 systems produced lower backgrounds, although also lower responses and contrasts. The cp PS Intein Gal4-vp16 system produced light activation contrast of more than 140-fold with 6 h of illumination.

To extend our approach for light-induced protein release, we decided to activate gene expression using dCas9. As a transactivator, we selected VP64-p65-Rta (VPR) transactivator because a combination of dCas9 with VPR demonstrated relatively strong gene expression⁶³. For dCas9-VPR, we tested both cp PS Intein and Split PS Intein based designs. The large size of dCas9-VPR fusion could create a challenge for its proper delivery to the nucleus. Therefore, we tested three design variants with differences in attached NLS. In cp PS Intein based design (Supplementary Fig. 14a), we replaced two SV40 NLS within VPR transactivator with flexible linkers to avoid dCas9 relocalization into the nucleus before intein activation takes place. We also tested two designs based on Split PS Intein (Supplementary Fig. 14b). In variant 1, two NLSs should be covalently attached to dCas9-VPR under illumination. In variant 2, we tried a release of original dCas9-VPR that contained all NLSs. To test our PS Intein dCas9-VPR constructs, we used them together with gRNA-expressing plasmid and an EGFP reporter⁶⁴. As for tTA and Gal4-vp16, cp PS Intein design resulted in the highest light-activation contrast and the lowest background of all variants (Supplementary Fig. 14c, Supplementary Fig. 15). However, this contrast and the response were relatively low (24-fold), compared to other transactivators.

Then we benchmarked single-component PS Intein-based light-controlled gene expression systems against a single-component LightOn system, which also uses VVD as a light-sensing module⁶⁵. For correct comparison, we tested systems in parallel experiments using the same reporter amounts and the same molar ratios of plasmids encoding a system to a reporter (1:1) for different systems. We benchmarked our two systems: cp PS Intein tTA with pTRE-EGFP reporter and cp PS Intein Gal4-vp16 system that uses the same UAS-based Gal4-vp16 responsive reporter pG5-EGFP, since LightOn is also based on Gal4 DNA-binding domain. We observed light-to-dark contrasts of ~300 for both PS Intein based systems and contrasts below 40 for LightOn (Fig. 5g, Supplementary Fig. 16).

Irreversible release of a transactivator should result in accumulation of the signal in the darkness with no additional illumination, thus resulting in signal amplification. We decided to estimate gene expression responses to shorter illumination times of the cp PS Intein tTA system, which demonstrated the highest activation contrast in the experiments above, with pTRE-SEAP reporter (Fig. 5h). First, we tested time intervals from 5 min up to 12 h (Fig. 5i). We found that 5 min of illumination was already sufficient to obtain more than 10-fold signal increase, after background subtraction. Thus, we went forward with testing shorter time intervals, down to seconds. We observed that few seconds illumination resulted in the contrast of ~3.7 fold between the light-to-dark samples. 30 s illumination produced ~7-fold increase (Fig. 5j).

The low dark background of the cp PS Intein tTA gene expression system prompted us to test if we could integrate it into the genome of a stably expressing cell line. For this, we obtained stably expressing HeLa cells using puromycin selection. After a month of maintaining in the dark, the cells were transfected with the reporter plasmid and tested for light-induced gene expression as above (6 h with pulsed light and with flow cytometry analysis 24 h after illumination) (Supplementary Fig. 17). The light-to-dark contrast for the stably expressing cells was lower than for the transiently expressing cells, most possibly because of the lower cp PS Intein tTA gene copy number in stables. However, the fact that our PS Intein-based gene expression system can operate in a genetically stable cell line with a few genomic copies is encouraging for future potential applications in tissue engineering⁶⁶.

Specific cell targeting with light-induced gene expression

Strong response of PS Intein-based systems for light-induced activation of gene expression prompted us to test if they can be used for cell selection using cell-type specific promoters and light (Fig. 6). First, we tested performance of both cp PS Intein tTA and LightOn systems expressed from hTERT promoter (phTERT) (Fig. 6b), which is active in cancer and germline cells^67,68. Microscopy analysis demonstrated much stronger EGFP reporter expression in case of cp PS Intein tTA compared to LightOn (Fig. 6c). Quantification by flow cytometry confirmed low response in case of LightOn similar to that of reporter-only background for pTRE-EGFP reporter (Fig. 6d and Supplementary Fig. 18). Both systems were comparably expressed as visualized by co-produced mCherry (Fig. 6e).

**Fig. 6: PS Intein-based gene expression systems for specific cell targeting.**

Split PS Intein-based gene expression systems can be used for specific targeting of cells selected by two inputs and light. We tested two cancer- and fetal-specific promoters (phTERT and BIRC5 promoter (pBIRC5))⁶⁹), and an inducible NFκB promoter associated with tumor microenvironment⁷⁰, which can be activated with TNF-α. Activation of gene expression should occur if three conditions are met: two promoters are active and cells are illuminated (Fig. 6f). Indeed, we observed reporter expression in samples that were illuminated and treated with TNF-α (Fig. 6h, Supplementary Fig. 19) activating NFκB promoter (Fig. 6i, Supplementary Fig. 20). Light-induced gene expression response was higher for the construct expressed from pBIRC5 than the one expressed from phTERT, in accordance with the strengths of these promoters correlating with the fluorescence of co-expressed mTagBFP2 (Fig. 6j, Supplementary Fig. 21).

Discussion

Here we designed an OT for protein splicing robustly functional in mammalian cells. PS Intein is characterized by a low dark background (Fig. 2a), a feature that was difficult to achieve for light-controlled tools catalyzing an irreversible reaction. We achieved this by using allosteric regulation of the gp41-1 intein via the inserted double VVD loop, which should restore activity of a distorted protein. However, low background from the distorted intein came at a cost of a slower response than that of unmodified gp41-1 acting in seconds²³. Still, illumination for 1-2 h was sufficient to obtain the highest light-to-dark contrast for reconstitution of mCherry (Fig. 2c).

Similar to gp41-1, PS Intein can function in contiguous or split form (Fig. 3b). Split PS Intein can be used to covalently stitch two polypeptides of interest using light control. Using reconstitution of tTA transcriptional activator from DNA binding TetR and transactivator vp16 domains, we proved that two Split PS Intein containing polypeptides do not interact in the dark (Fig. 3f).

In benchmarking of Split PS Intein against the reported AsLOV2-based tool for blue light control of protein splicing LOVInC³⁸, we demonstrated that LOVInC is prone to high dark background resulting in almost no light-to-dark contrast for LOVInC, as opposed to Split PS Intein (Fig. 3d and Supplementary Fig. 5).

By designing PS Intein Bid for activation of apoptosis, we showed that PS Intein can be applied to light-induced cleavage of a protein of interest. The protein to be cleaved should consist of separately folding domains or tolerate an insertion. PS Intein module allows different arrangements of protein domains in a polypeptide, with a possibility to place functional protein domains at the N-terminus, in the loop between IntN and IntC, or at the C-terminus. We showed the importance of screening different domain arrangements to identify a construct with optimal protein caging in the darkness. While placing NBid and CBid at termini of IntC yielded high dark background, insertion of CBid in the loop between IntN and IntC allowed effective caging of CBid in the darkness (Fig. 4a, b).

We demonstrated that PS Intein can be used for light-controlled release of intracellularly caged proteins. While we tested the cytoplasmic caging here, we suggest that the approach can be extended to protein caging at other subcellular locations, such as the mitochondria or the nucleus. We tested two ways for light-controlled release of transcriptional activators from the cytoplasm to the nucleus. Cytoplasmic caging with two ER^T2 domains attached at both N- and C-termini of cp PS Intein-containing construct was the most effective design showing low dark background and highest light activation contrasts (Fig. 5e). Cytoplasmic caging of dCas9-VPR was not as effective as caging of tTA or Gal4-vp16. The primary reason for this is the ability of the large Cas9 to enter the nucleus without exogenous NLS that was just recently explicitly shown and studied^71,72. Our data also indicate that Gal4-based gene expression systems are characterized by both lower response and lower background than TetR-based systems, while the light-to-dark contrasts are comparable.

We obtained high light-to-dark contrasts (mostly > 100-fold, with the highest > 500-fold for cp PS Intein tTA) for PS Intein-based gene expression systems (Fig. 5). Although contrasts depend on experimental conditions, the light-to-dark contrast obtained here are among the highest reported for gene expression systems using TetR or Gal4^65,73,74. We directly compared different ways of flow cytometry data processing for the same dark and illuminated samples co-transfected with cp PS Intein tTA, mCherry encoding plasmid, and pTRE-EGFP reporter, and confirmed that different methods of data analysis produced comparable results (Supplementary Fig. 22).

In direct side-by-side benchmarking of single-component cp PS Intein gene expression systems against the single-component LightOn system, we observed several-fold higher light-to-dark responses for PS Intein-based systems than for LightOn (Fig. 5g). Since both LightOn and PS Intein use the VVD light-sensing module, slow dark deactivation of VVD⁷⁵ is not the main reason for the strong response observed in PS Intein-based systems. We hypothesize that higher light-to-dark contrasts for PS Intein-based systems result from irreversible formation and accumulation of a constitutively active transcriptional activator (schematics in Fig. 5a, b, h) that allows signal amplification. In accordance with this, short illumination times down to seconds were sufficient for several-fold activation of reporter expression detected for cp PS Intein tTA system (Fig. 5j). Short illumination time should be useful for optical control of cells involved in dynamic movements in the body or in a device, such as metastasizing cancer cells or engineered immune cells at a particular location. The ability to incorporate cp PS Intein tTA system into a stable cell line should also be useful in cell and tissue engineering applications.

We demonstrated that the potent cp PS Intein tTA system can be expressed from cell-specific promoters for cell targeting (Fig. 6). We also demonstrated that Split PS Intein tTA system can be applied to targeting cells using two specific promoters and light (Fig. 6f, g). This can be used for intersectional targeting of cell populations. Here we utilized cancer- and germline-specific (phTERT, pBIRC5), and tumor microenvironment-specific (pNFκB) promoters. This approach complements intersectional labeling strategies using recombinase expression from weak cell-specific promoters⁷⁶ and adds light control to logic gates for specific cell selection²². Light-gated specific cell targeting can potentially be useful in cell therapies, such as for specific selection of cancer or immune cells.

To summarize, PS Intein emerges as a platform technology for light-controlled protein splicing in mammalian cells. PS Intein tolerates various fusions and insertions on both sides of IntN and IntC fragments, including their rearrangement in a circular permuted molecule. PS Intein enables efficient light-controlled covalent protein ligation, cleavage, and release from subcellular caging. PS Intein-based systems using caged transactivators are among the most potent light-controlled gene expression systems, due to built-in signal amplification resulting from irreversible formation of constitutively active transactivators. These systems can be used with cell-specific promoters for precise intersectional cell targeting aided by light control. PS Intein has the potential to replace several previously developed tools with narrow scopes of use and poor performance in mammalian cells. With its versatility and low dark background, PS Intein technology promises to become a valuable instrument for adding functionalities to proteins and protein networks.

Methods

Design of mammalian plasmids

Information on plasmids, their sources, and cloning procedures can be found in Supplementary Note 1. The list of primers with accompanying short descriptions can be found in Supplementary Table 1. Amino acid sequences of the used constructs can be found in Supplementary Figs. 23–33.

Mammalian cell culture

HeLa (ATCC, #CCL-2), HEK293T (ATCC, #CRL-3216), and MDA-MB-231 (ATCC, #HTB-26) cells were cultured in Dulbecco’s modified Eagle’s medium (Corning, #MT10013CV) supplemented with 10% fetal bovine serum (GeminiBio, #100-106-500) and penicillin–streptomycin (HyClone, #SV30010) at 37 °C in 5% CO₂. Transient transfections in HeLa cells were primarily performed using Effectene reagent (Qiagen, #301425) following the manufacturer’s protocol, or, when specified, using PEI according to the method described by Challis et al. ⁷⁷, with modifications for smaller volumes. HEK293T and MDA-MB-231 cells were transfected using Lipofectamine 2000 (ThermoFisher Scientific, #11668027). FuGENE (Promega, #E2311) was used for HeLa transfection specifically for Western blotting (see below). When multiple vectors were used for transfection, equimolar concentrations were applied unless otherwise specified.

To induce homodimerization of VVD domains, cells were illuminated with 450 nm light (3 mW cm⁻², 2 s On, 10 s Off) for 6 h if not stated otherwise.

Cp PS Intein tTA Stable Cell Line Preparation

HeLa cells were seeded into two wells of a 6-well plate. The following day, the cells were transfected with the pCSII-cp PS Intein tTA Puro plasmid using Lipofectamine 3000, according to the manufacturer’s instructions. Three days post-transfection, puromycin dihydrochloride (MedChemExpress, #HY-B1743A) was added at a final concentration of 1 μg/mL. The cells were then maintained in the dark, and the medium containing puromycin was replaced every three days. Approximately one month after transfection, experiments with the reporter were performed. For these experiments, cells were first seeded for transfection; the next day, pTRE-EGFP, pmCherry-N1, and pcDNA3.1+ were co-transfected either into the stable HeLa cell line or into unmodified (parental) HeLa cells using PEI, and illuminated as described above.

AAV preparation and analysis

To produce PS Intein AAV particles, HEK293T cells were co-transfected with AAV-DJ, pHelper, and AAV-PS Intein plasmids, following the protocol previously described⁷⁷. Briefly, 40 µg of total DNA (5.7 µg AAV-PS Intein, 22.8 µg AAV-DJ, 11.4 µg pHelper) was mixed with PEI in DPBS per 150-mm dish. The PEI-DPBS mix was added dropwise to the DNA-DPBS solution while gently vortexing, then vortexed for 10 s and incubated at room temperature for 2 min. Finally, 2 ml of the transfection mix was added dropwise to each dish, swirled to mix, and returned to the incubator. The culture medium was replaced 24 h after transfection, and the viral-containing medium was collected one week post-transfection. The medium was then centrifuged, and the supernatant was filtered through a 0.45 μm filter. The filtered supernatant was subsequently concentrated tenfold using Amicon Ultra Centrifugal Filters with a 100 kDa molecular weight cutoff (Sigma-Aldrich, #UFC9100). HeLa cells were treated with the 0.2 μm-filtered, 10x concentrated viral medium. After 24 hours, the medium was replaced with fresh culture medium. ~48 h post-infection, cells were exposed to 450 nm light (3 mW cm⁻², 2 s ON, 10 s OFF) for 6 h before FACS analysis.

DNA isolation from mammalian cells

HeLa CCL-2 cells were cultured to confluency in a 10 cm dish, then scraped from the dish and collected. The cell pellet was centrifuged, and the supernatant was discarded. The cell pellet was resuspended in Buffer P1 from the QIAprep Spin Miniprep Kit (Qiagen, #27106). The manufacturer’s protocol was followed with one modification: the tube was vortexed for 10 s prior to the addition of neutralization Buffer N3 to ensure efficient lysis and release of total DNA into solution. The DNA was then eluted in 30 µL of water, and 1 µL of the eluted DNA was used for the PCR reaction.

Flow cytometry

Flow cytometry analysis was performed using a BD LSRII flow analyzer. Minimally 5 × 10⁴ live cells were analyzed in each cell sample. Data was analyzed using FACSDiva v.8.0.1 or FlowJo v.7.6.2 software. HeLa cell gating was performed using three gates, for intact cells (FSC-A versus SSC-A), single cells (FSC-A vs FSC-W), and live cells (SSC-A vs SSC-W). If mEGFP was co-expressed, then the cells were gated against untransformed cells in green (Ex. 488 nm, Em. 525/50 nm) channel for selecting mEGFP positive cells. The latter cells were used to assess the brightness in green and/or red (mCherry: Ex. 561 nm, Em. 610/20 nm) channels. The gating strategy used for flow cytometry analysis is exemplified in Supplementary Fig. 34.

If experiments involved a reporter plasmid, cells were typically analyzed 48 h post-transfection and 24 h post-illumination, unless otherwise specified. In the case of split mCherry reconstitution experiments, the cells were analyzed 24 h post-transfection, with cells being illuminated for 6 h prior to FACS analysis. For analysis of the influence of illumination times on PS Intein-mediated reconstitution of mCherry, the timing was different, as indicated in the schematics to Fig. 2c.

Fluorescence microscopy

Live HeLa cells were imaged with an Olympus IX81 inverted epifluorescence microscope. The microscope was equipped with a 200 W metal halide arc lamp (Lumen220PRO, Prior), a 60 × 1.35 numerical aperture (NA) oil objective lens (UPlanSApo, Olympus) and an opiMOS sCMOS camera (QImaging). During imaging, cells were incubated in a cell imaging medium (Life Technologies-Invitrogen, #A59688DJ). During imaging, the temperature inside the microscope plexiglass environmental chamber was maintained at 37 °C. The microscope was operated with a SlideBook v 6.0.8 software (Intelligent Imaging Innovations).

To detect EGFP, mCherry or mCardinal fluorescence, 480/40 nm, 570/30 nm or 605/40 nm excitation and 535/40 nm, 615/30 or 640LP nm emission filters were used, respectively.

Typically, cells were analyzed 48 h post-transfection. 24 h before microscopic analysis, cells were transferred on round cover glasses (thickness, 0.13–0.17 mm; diameter, 25 mm; ThermoFisher Scientific, #12-545-102).

Spatial illumination of a monolayer of cells expressing cp PS Intein tTA optogenetic tool

HEK293T cells were seeded onto glass-bottom 35 mm dishes (Greiner, #627860) pre-treated with 50 μg mL⁻¹ Poly-D-lysine hydrobromide (MP Biomedicals, #0215017525). The following day, cells were transfected using Lipofectamine 3000 with a total of 2500 ng DNA per dish: cp PS Intein tTA, pTRE-EGFP reporter (250 ng, 1:1 ratio with the OT plasmid), pmCherry-N1 (250 ng), and pcDNA3.1 + . Six hours post-transfection, the medium was replaced. Cells were illuminated from the bottom with 450 nm LED at 3 mW cm⁻² through a printed photomask, using a cycle of 2 s On and 10 s Off for 6 h. Twenty-four hours after illumination, images were acquired with a ThermoFisher EVOS M7000 Imaging System using a 10x objective lens in green, red, dual green/red, and transmitted light channels. Individual images were automatically stitched by the microscope software.

Protein extraction and Western blotting

The day before transfection, HeLa cells were seeded in a 6-well plate. Transfection was done using FuGENE (Promega, #E2311) according to the manufacturer’s recommendations. Then cells were cultivated for 48 h and medium was changed twice prior to experiment. Illumination was performed for 6 h with pulsed blue light (450 nm LED panel, 3 mW cm⁻², 1 s On, 10 s Off). After illumination, cells were incubated for additional 6 h in dark. Control plates with cells expressing the same constructs were constantly kept in darkness. Then, cells were rinsed with 3 ml of PBS and 500 µl of MPER mammalian protein extraction reagent (ThermoFisher Scientific, #78501) with added Pierce protease inhibitor mini tablets, EDTA-free (ThermoFisher Scientific, #A32955). After a complete cell lysis, resulted solution was centrifuged and the supernatant combined with 4x Laemmli buffer. Then, samples were heated in the boiled water for 3 min and loaded on manually prepared tricine–SDS-PAGE gels for electrophoresis. After the run was over, 0.45 µm nitrocellulose membrane (Bio-Rad, #1620115) was used for gel-to-membrane protein transfer in a transfer buffer with 20% methanol and 0.5% SDS (20 mA for 3.5 h). The membrane with transferred proteins was then incubated for 1 h at 37 °C in the 3% solution of Blotting-Grade Blocker (Bio-Rad, #1706404) in PBS followed by an overnight incubation in a 3% solution of Blotting-Grade Blocker in PBS with 0,04% Tween-20 and 1:1000 dilution of unconjugated polyclonal Rabbit anti-mCherry antibody (Novus Biologicals, #NBP2-25157) at 4 °C with continuous shaking. The next day, the membrane was washed 3 times in PBS at 37 °C with shaking and transferred to a solution of 0.04% Tween-20 in PBS with 1:1000 dilution of polyclonal goat anti-rabbit IgG Alexa Fluor 680-conjugated antibody (ThermoFisher Scientific, #A-21076). Incubation was performed at 4 °C overnight with continuous shaking. Then, the membrane was washed for 30 min with 0.04% Tween-20 in PBS and 3 times with PBS at 37 °C with shaking.

Visualization of protein bands was performed with ODYSSEY CLx infrared imaging system (LI-COR). After visualization of mCherry epitopes, the membrane was further incubated in a 3% solution of the Bio-Rad Blotting-Grade Blocker in PBS with 1:1000-diluted murine monoclonal anti-GFP DyLight 800 4X PEG-conjugated antibody (Thermo Fisher, #MA5-15256-D800). Incubation was performed overnight at 4 °C with shaking. Next day, the membrane was washed with 0.04% Tween-20 in PBS and then 3 times in PBS at 37 °C with shaking before the final visualization using ODYSSEY CLx system.

SEAP kinetics assay

Prior to illumination, medium was replaced with fresh one. Illumination was performed with pulsed blue light (450 nm blue LED panel, 3 mW cm⁻², 1 s On, 10 s Off) for 12 h, 6 h, 2 h, 1 h, 30 min, and 15 min time points and stopped for all the probes 24 h before medium was collected for analysis (48 h post-transfection). Control dishes with cells expressing the same constructs were kept in the dark. SEAP signal was measured in black polystyrene flat-bottom 96-well plates (COSTAR, #3915) using a Great Escape SEAP Fluorescence Detection Kit (Takara Bio, #631704). Registration of fluorescence intensity in wells was performed with a SpectraMax M2 microplate reader (Molecular Devices), Ex. 355 nm and Em. 460 nm.

Cell viability assay

For dead cells staining experiment, transfection was done in 12-well plates. All the cells were co-transfected with pmCherry-N1 (1:9 molar ratio to PS Intein Bid plasmid) and analyzed 48 h post-transfection. Illumination performed either for 30 min or 6 h with pulsed blue light (450 nm blue LED panel, 3 W cm⁻², 2 s On, 10 s Off). Illumination ended 24 h before analysis. Fresh medium with either 20 mM Z-VAD-FMK or DMSO was added 30 min before illumination started. Prior to DEAD staining, to avoid losing floating dead cells, first, cell medium was collected in 1.5 mL Eppendorf tubes, then the cells were trypsinized, resuspended in the same medium, and transferred back to Eppendorf tubes for centrifugation. After centrifugation, the supernatant was removed, and the cells were gently resuspended in 1 mL of FACS buffer. Then 1 µL of SYTOX Green Dead Cell Stain for flow cytometry (ThermoFisher Scientific, #S34860) was added, cells incubated at room temperature for 15 min, and eventually filtered and transferred on ice for FACS analysis.

Statistics and reproducibility

No statistical method was used to predetermine sample size. No data were excluded from the analysis. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

We used transiently transfected cells in fluorescence quantification experiments performed by flow cytometry analysis. For flow cytometry, at least 50,000 cells per sample were recorded. For samples containing co-expressed fluorescent protein, we gated cells by this fluorescent protein signal and analyzed the mean reporter fluorescence in transfected cells containing co-expressed fluorescent protein. For samples not containing a co-expressed fluorescent marker, we calculated the total fluorescence of the reporter in the same number of analyzed cells in different samples. To account for differences in transfection efficiency, we performed three independent experiments. Statistical analysis and data processing were carried out using OriginPro v.9.7.188 software (OriginLab).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data supporting the findings of this study are available within the paper and Supplementary Information. The plasmids constructed in this study, along with their maps and nucleotide sequences, are available at the Addgene repository (#242019 - #242035). Source data are provided with this paper.

References

Yao, Z. et al. Split Intein-mediated protein ligation for detecting protein-protein interactions and their inhibition. Nat. Commun. 11, 2440 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Pinto, F., Thornton, E. L. & Wang, B. An expanded library of orthogonal split inteins enables modular multi-peptide assemblies. Nat. Commun. 11, 1529 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Ho, T. Y. H. et al. A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction. Nat. Commun. 12, 2200 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar
Chen, Z. & Elowitz, M. B. Programmable protein circuit design. Cell 184, 2284–2301 (2021).
Article CAS PubMed PubMed Central Google Scholar
Parthasarathy, R., Subramanian, S. & Boder, E. T. Sortase A as a novel molecular “stapler” for sequence-specific protein conjugation. Bioconjug Chem. 18, 469–476 (2007).
Article CAS PubMed Google Scholar
Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl. Acad. Sci. USA 109, E690–E697 (2012).
Article CAS PubMed PubMed Central Google Scholar
Wang, H., Wang, L., Zhong, B. & Dai, Z. Protein splicing of inteins: a powerful tool in synthetic biology. Front Bioeng. Biotechnol. 10, 810180 (2022).
Article PubMed PubMed Central Google Scholar
Mootz, H. D. & Muir, T. W. Protein splicing triggered by a small molecule. J. Am. Chem. Soc. 124, 9044–9045 (2002).
Article CAS PubMed Google Scholar
Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014).
Article PubMed PubMed Central Google Scholar
Shah, N. H. & Muir, T. W. Inteins: nature’s gift to protein chemists. Chem. Sci. 5, 446–461 (2014).
Article CAS PubMed Google Scholar
Pishesha, N., Ingram, J. R. & Ploegh, H. L. Sortase A: a model for transpeptidation and its biological applications. Annu Rev. Cell Dev. Biol. 34, 163–188 (2018).
Article CAS PubMed Google Scholar
Reddington, S. C. & Howarth, M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr. Opin. Chem. Biol. 29, 94–99 (2015).
Article CAS PubMed Google Scholar
Volkmann, G. & Mootz, H. D. Recent progress in intein research: from mechanism to directed evolution and applications. Cell Mol. Life Sci. 70, 1185–1206 (2013).
Article CAS PubMed Google Scholar
Nanda, A., Nasker, S. S., Mehra, A., Panda, S. & Nayak, S. Inteins in science: evolution to application. Microorganisms 8, 2004 (2020).
Chong, S. & Xu, M. Q. Protein splicing of the Saccharomyces cerevisiae VMA intein without the endonuclease motifs. J. Biol. Chem. 272, 15587–15590 (1997).
Article CAS PubMed Google Scholar
Telenti, A. et al. The Mycobacterium xenopi GyrA protein splicing element: characterization of a minimal intein. J. Bacteriol. 179, 6378–6382 (1997).
Article CAS PubMed PubMed Central Google Scholar
Volkmann, G. & Iwai, H. Protein trans-splicing and its use in structural biology: opportunities and limitations. Mol. Biosyst. 6, 2110–2121 (2010).
Article CAS PubMed Google Scholar
Di Ventura, B. & Mootz, H. D. Switchable inteins for conditional protein splicing. Biol. Chem. 400, 467–475 (2019).
Article PubMed Google Scholar
Jillette, N., Du, M., Zhu, J. J., Cardoz, P. & Cheng, A. W. Split selectable markers. Nat. Commun. 10, 4968 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
Palanisamy, N. et al. Split intein-mediated selection of cells containing two plasmids using a single antibiotic. Nat. Commun. 10, 4967 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
Truong, D. J. et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res 43, 6450–6458 (2015).
Article CAS PubMed PubMed Central Google Scholar
Chen, H. S. et al. An intein-split transactivator for intersectional neural imaging and optogenetic manipulation. Nat. Commun. 13, 3605 (2022).
Article ADS CAS PubMed PubMed Central Google Scholar
Bhagawati, M. et al. In cellulo protein semi-synthesis from endogenous and exogenous fragments using the ultra-fast split Gp41-1 intein. Angew. Chem. Int Ed. Engl. 59, 21007–21015 (2020).
Article CAS PubMed PubMed Central Google Scholar
Khoo, K. K. et al. Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing. Nat. Commun. 11, 2284 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Hua, Y. et al. Protein editing using a coordinated transposition reaction. Science 388, 68–74 (2025).
Article CAS PubMed Google Scholar
Beyer, J. N. et al. Intracellular protein editing enables incorporation of noncanonical residues in endogenous proteins. Science 388, eadr5499 (2025).
Article CAS PubMed Google Scholar
Bowen, C. H. et al. Microbial production of megadalton titin yields fibers with advantageous mechanical properties. Nat. Commun. 12, 5182 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar
Mootz, H. D., Blum, E. S., Tyszkiewicz, A. B. & Muir, T. W. Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo. J. Am. Chem. Soc. 125, 10561–10569 (2003).
Article CAS PubMed Google Scholar
Skretas, G. & Wood, D. W. Regulation of protein activity with small-molecule-controlled inteins. Protein Sci. 14, 523–532 (2005).
Article CAS PubMed PubMed Central Google Scholar
Liang, R. et al. A T7-expression system under temperature control could create temperature-sensitive phenotype of target gene in Escherichia coli. J. Microbiol Methods 68, 497–506 (2007).
Article ADS CAS PubMed Google Scholar
Zeidler, M. P. et al. Temperature-sensitive control of protein activity by conditionally splicing inteins. Nat. Biotechnol. 22, 871–876 (2004).
Article CAS PubMed Google Scholar
Gramespacher, J. A., Stevens, A. J., Nguyen, D. P., Chin, J. W. & Muir, T. W. Intein zymogens: conditional assembly and splicing of split inteins via targeted proteolysis. J. Am. Chem. Soc. 139, 8074–8077 (2017).
Article CAS PubMed PubMed Central Google Scholar
Vila-Perelló, M., Hori, Y., Ribó, M. & Muir, T. W. Activation of protein splicing by protease- or light-triggered O to N acyl migration. Angew. Chem. Int Ed. Engl. 47, 7764–7767 (2008).
Article PubMed PubMed Central Google Scholar
Ciragan, A., Aranko, A. S., Tascon, I. & Iwai, H. Salt-inducible protein splicing in cis and trans by inteins from extremely halophilic archaea as a novel protein-engineering tool. J. Mol. Biol. 428, 4573–4588 (2016).
Article CAS PubMed Google Scholar
Heikkinen, H. A., Aranko, A. S. & Iwai, H. The NMR structure of the engineered halophilic DnaE intein for segmental isotopic labeling using conditional protein splicing. J. Magn. Reson 338, 107195 (2022).
Article CAS PubMed Google Scholar
Jung, D. et al. Photo-triggered fluorescent labelling of recombinant proteins in live cells. Chem. Commun. (Camb.) 51, 9670–9673 (2015).
Article CAS PubMed Google Scholar
Tyszkiewicz, A. B. & Muir, T. W. Activation of protein splicing with light in yeast. Nat. Methods 5, 303–305 (2008).
Article CAS PubMed Google Scholar
Wong, S., Mosabbir, A. A. & Truong, K. An engineered split intein for photoactivated protein trans-splicing. PLoS One 10, e0135965 (2015).
Article PubMed PubMed Central Google Scholar
Jones, D. C., Mistry, I. N. & Tavassoli, A. Post-translational control of protein function with light using a LOV-intein fusion protein. Mol. Biosyst. 12, 1388–1393 (2016).
Article CAS PubMed Google Scholar
Karasev, M. M., Verkhusha, V. V. & Shcherbakova, D. M. Near-infrared optogenetic module for conditional protein splicing. J. Mol. Biol. 435, 168360 (2023).
Article CAS PubMed PubMed Central Google Scholar
Hartzell, E. J., Terr, J. & Chen, W. Engineering a blue light inducible spytag system (BLISS). J. Am. Chem. Soc. 143, 8572–8577 (2021).
Article CAS PubMed Google Scholar
Rahikainen, R. et al. Visible light-induced specific protein reaction delineates early stages of cell adhesion. J. Am. Chem. Soc. 145, 24459–24465 (2023).
CAS PubMed PubMed Central Google Scholar
Lu, X. et al. Photocleavable proteins that undergo fast and efficient dissociation. Chem. Sci. 12, 9658–9672 (2021).
Article CAS PubMed PubMed Central Google Scholar
Zhang, W. et al. Optogenetic control with a photocleavable protein, PhoCl. Nat. Methods 14, 391–394 (2017).
Article PubMed Google Scholar
Hockberger, P. E. et al. Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells. Proc. Natl. Acad. Sci. 96, 6255–6260 (1999).
Article ADS CAS PubMed PubMed Central Google Scholar
Ramakrishnan, P., Maclean, M., MacGregor, S. J., Anderson, J. G. & Grant, M. H. Cytotoxic responses to 405nm light exposure in mammalian and bacterial cells: Involvement of reactive oxygen species. Toxicol. Vitr. 33, 54–62 (2016).
Article CAS Google Scholar
Morikawa, K. et al. Photoactivatable Cre recombinase 3.0 for in vivo mouse applications. Nat. Commun. 11, 2141 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Cui, M. et al. A single-component, light-assisted uncaging switch for endoproteolytic release. Nat. Chem. Biol. 20, 353–364 (2024).
Article CAS PubMed Google Scholar
Dagliyan, O. et al. Engineering extrinsic disorder to control protein activity in living cells. Science 354, 1441–1444 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Gil, A. A. et al. Optogenetic control of protein binding using light-switchable nanobodies. Nat. Commun. 11, 4044 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Shaaya, M. et al. Light-regulated allosteric switch enables temporal and subcellular control of enzyme activity. Elife 9, e60647 (2020).
Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–28696 (2012).
Article CAS PubMed PubMed Central Google Scholar
Beyer, H. M., Mikula, K. M., Li, M., Wlodawer, A. & Iwaï, H. The crystal structure of the naturally split gp41-1 intein guides the engineering of orthogonal split inteins from cis-splicing inteins. Febs j. 287, 1886–1898 (2020).
Article CAS PubMed Google Scholar
Oeemig, J. S., Beyer, H. M., Aranko, A. S., Mutanen, J. & Iwaï, H. Substrate specificities of inteins investigated by QuickDrop-cassette mutagenesis. FEBS Lett. 594, 3338–3355 (2020).
Article CAS PubMed Google Scholar
Hodel, M. R., Corbett, A. H. & Hodel, A. E. Dissection of a nuclear localization signal. J. Biol. Chem. 276, 1317–1325 (2001).
Article CAS PubMed Google Scholar
Guttler, T. et al. NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1. Nat. Struct. Mol. Biol. 17, 1367–1376 (2010).
Article PubMed Google Scholar
Karasev, M. M., Baloban, M., Verkhusha, V. V. & Shcherbakova, D. M. Nuclear localization signals for optimization of genetically encoded tools in neurons. Front Cell Dev. Biol. 10, 931237 (2022).
Article PubMed PubMed Central Google Scholar
Baron, U. & Bujard, H. Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances. Methods Enzymol. 327, 401–421 (2000).
Article CAS PubMed Google Scholar
Esposti, M. D. The roles of bid. Apoptosis 7, 433–440 (2002).
Article CAS PubMed Google Scholar
Feil, R., Wagner, J., Metzger, D. & Chambon, P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys. Res Commun. 237, 752–757 (1997).
Article CAS PubMed Google Scholar
Casanova, E. et al. ER-based double iCre fusion protein allows partial recombination in forebrain. Genesis 34, 208–214 (2002).
Article CAS PubMed Google Scholar
Sadowski, I., Ma, J., Triezenberg, S. & Ptashne, M. GAL4-VP16 is an unusually potent transcriptional activator. Nature 335, 563–564 (1988).
Article ADS CAS PubMed Google Scholar
Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).
Article CAS PubMed PubMed Central Google Scholar
Polstein, L. R. & Gersbach, C. A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198–200 (2015).
Article CAS PubMed PubMed Central Google Scholar
Wang, X., Chen, X. & Yang, Y. Spatiotemporal control of gene expression by a light-switchable transgene system. Nat. Methods 9, 266–269 (2012).
Article CAS PubMed Google Scholar
Beyer, H. M. et al. Genetically-stable engineered optogenetic gene switches modulate spatial cell morphogenesis in two- and three-dimensional tissue cultures. Nat. Commun. 15, 10470 (2024).
Article CAS PubMed PubMed Central Google Scholar
Zinn, R. L., Pruitt, K., Eguchi, S., Baylin, S. B. & Herman, J. G. hTERT is expressed in cancer cell lines despite promoter DNA methylation by preservation of unmethylated DNA and active chromatin around the transcription start site. Cancer Res. 67, 194–201 (2007).
Article CAS PubMed Google Scholar
Janknecht, R. On the road to immortality: hTERT upregulation in cancer cells. FEBS Lett. 564, 9–13 (2004).
Article CAS PubMed Google Scholar
Shepelev, M. V., Kopantzev, E. P., Vinogradova, T. V., Sverdlov, E. D. & Korobko, I. V. hTERT and BIRC5 gene promoters for cancer gene therapy: a comparative study. Oncol. Lett. 12, 1204–1210 (2016).
Article CAS PubMed PubMed Central Google Scholar
Cao, Y., Yi, Y., Han, C. & Shi, B. NF-κB signaling pathway in tumor microenvironment. Front. Immunol. 15, 1476030 (2024).
Roggenkamp, E. et al. Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae. G3 (Bethesda) 8, 999–1018 (2018).
Article CAS PubMed Google Scholar
Zhang, W. et al. Hitchhiking of Cas9 with nucleus-localized proteins impairs its controllability and leads to efficient genome editing of NLS-free Cas9. Mol. Ther. 32, 920–934 (2024).
Article CAS PubMed PubMed Central Google Scholar
Kuwasaki, Y. et al. A red light–responsive photoswitch for deep tissue optogenetics. Nat. Biotechnol. 40, 1672–1679 (2022).
Article CAS PubMed Google Scholar
Zhu, L., McNamara, H. M. & Toettcher, J. E. Light-switchable transcription factors obtained by direct screening in mammalian cells. Nat. Commun. 14, 3185 (2023).
Article ADS CAS PubMed PubMed Central Google Scholar
Lee, C. T., Malzahn, E., Brunner, M. & Mayer, M. P. Light-induced differences in conformational dynamics of the circadian clock regulator VIVID. J. Mol. Biol. 426, 601–610 (2014).
Article CAS PubMed Google Scholar
Madisen, L. et al. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85, 942–958 (2015).
Article CAS PubMed PubMed Central Google Scholar
Challis, R. C. et al. Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nat. Protoc. 14, 379–414 (2019).
Article CAS PubMed Google Scholar

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Acknowledgements

This work was supported by grants GM122567 from the US National Institutes of Health, 220011 from the Jane and Aatos Erkko Foundation, and 360277 from the Research Council of Finland (all to V.V.V.).

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Authors and Affiliations

Department of Genetics, and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA
Mikhail Baloban, Kyrylo Yu. Manoilov, Vladislav V. Verkhusha & Daria M. Shcherbakova
Department of Anatomy, Medicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland
Maksim M. Karasev & Vladislav V. Verkhusha

Authors

Mikhail Baloban
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Kyrylo Yu. Manoilov
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Maksim M. Karasev
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Vladislav V. Verkhusha
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Daria M. Shcherbakova
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Contributions

M.B. performed most of the experiments described, with the participation of K.Yu.M. D.M.S. and V.V.V. planned and directed the project. V.V.V. acquired funds. D.M.S. and M.B., with the participation of K.Yu.M. and M.M.K., designed the experiments and analyzed the data. D.M.S., with the participation of M.B. and K.Yu.M., wrote the manuscript. All authors revised the manuscript.

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Correspondence to Vladislav V. Verkhusha or Daria M. Shcherbakova.

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Baloban, M., Manoilov, K.Y., Karasev, M.M. et al. Photoswitchable intein for light control of covalent protein binding and cleavage. Nat Commun 16, 8263 (2025). https://doi.org/10.1038/s41467-025-63595-9

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Received: 14 January 2025
Accepted: 22 August 2025
Published: 11 September 2025
DOI: https://doi.org/10.1038/s41467-025-63595-9