Fluorescence monitoring of STING using a coumarin-based chemigenetic probe

Abstract

Protein condensate, a fundamental biological structure in biomolecular research, plays an important role in cell fate decision and cellular homeostasis. However, the lack of specific, stimulus-responsive tools to track the dynamics of proteins has hindered the in-depth investigation into their regulatory functions in cells. Herein, we report a fluorescent chemigenetic probe Y6_STING, where coumarin derivative Y6 is specifically conjugated onto the stimulator of interferon genes (STING) protein, enabling H₂O₂-mediated fluorescence monitoring of the dynamics of STING in living cells. Through structural modification of the coumarin backbone, substituent effects reveal that electron density at the C7 position of coumarin dictates the initial fluorescence intensity of Y1–Y3. By introducing a boron-cage onto the hydroxyl group of coumarin, Y4–Y6 exhibit a dual fluorescence response upon H₂O₂ stimulation. Upon reaching the proteins of interest (POIs), the combination of Y6 containing a PEG-linked HaloTag ligand and a genetically encoded STING protein allows for H₂O₂-triggered STING-labeling and fluorescence activation in both gels and aqueous solution. This stimulus-mediated response of Y6_STING enables monitoring the dynamics of STING in living cells. We evaluate the intracellular tracing ability of Y6 and demonstrate that Y6 could be retained in cells for long-term tracking of STING. Hence, this novel strategy would greatly facilitate the advancement of monitoring protein dynamics in proteostasis and medicine research.

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Introduction

Fluorescent materials that provide dynamic and quantitative information have become indispensable for biological analysis and clinical diagnosis.^1–3 Small molecular fluorescent probes are extensively developed for biosensing and bioimaging because of their brightness and sensitivity.^4–6 Fluorescent proteins (FPs) also emerge as powerful tools to be fused with proteins of interest (POIs) for visualizing the localization and trafficking of POIs.^7–9 However, a single tool alone has limitations in meeting the requirements for accurate monitoring of POIs in complex biological environments. Therefore, the construction of a hybrid fluorescent system by taking advantage of the merits of both organic fluorescent dyes and FPs is urgent. To date, fluorogen-conjugated POIs have gained much attention for developing protein-based fluorescent macromolecular materials.^10,11 The covalent or non-covalent modification of proteins with fluorophores can render a POI fluorescent without compromising its function.^12–14 Nevertheless, it is still challenging to construct a fluorophore-fused POI with multiple stimuli-mediated fluorescence responses in vitro and in cells.

The dynamic behavior of POIs is often closely associated with protein condensates—membrane-less organelles that play a pivotal role in regulating the dynamic signaling of proteins.^15,16 Abnormal protein aggregation has been associated with several human diseases,^17,18 underscoring the critical necessity of developing a POI-specific fluorescence labeling platform for spatiotemporal tracking of intracellular protein dynamics.^19–21 Multiple strategies such as click reactions,^22,23 protein coupling modifications,²⁴ or genetic codon expansion²⁵ have significantly increased the labeling accuracy of POIs. Among them, HaloTag technology offers a facile approach for rapidly tagging POIs through the nucleophilic displacement of the terminal chloride from the HaloTag ligand with the Asp106 residue of the HaloTag protein.^26–28 The covalent conjugation between the HaloTag protein and the chloroalkane ligand is highly specific and irreversible under physiological conditions.²¹ Therefore, it is desirable that the HaloTag system is used to develop protein-conjugated sensors for multiple applications.

Herein, we report a unique strategy to construct a fluorescent chemigenetic probe Y6_STING for stimulus-mediated fluorescence monitoring and tracking of the STING protein in vitro and in cells. Firstly, we designed a series of coumarin-derived probes (Y1–Y5) by modifying the hydroxyl and aldehyde groups of the coumarin backbone (Fig. 1). Among them, Y4 and Y5, featuring a H₂O₂-sensitive boron-cage at the C7 position, exhibited the desired stimulus responsiveness. This property was retained in Y6, which we further conjugated with a PEG-linked HaloTag ligand for specific labeling of POI. Using HaloTag technology, we then assessed the fluorescence labeling and responding ability of Y6 toward STING and GFP proteins. Y6_POIs exhibited bright fluorescence in both gels and aqueous solution. In particular, Y6_GFP presented a fluorescence resonance energy transfer (FRET) signal in response to H₂O₂ stimulation. Moreover, we further evaluated the intracellular tracing ability of Y6 to STING and demonstrated its capacity to persist within cells, enabling long-term tracking of STING in cells. Hence, it provides a novel strategy for Y6 to monitor the dynamic process of STING protein via fluorescence, paving a way to identify the key effectors that mediate innate immunity in living cells.

Fig. 1 A schematic diagram showing the structural design of Y1, Y2, Y3, Y4, Y5, and Y6.

Results and discussion

Structural design and fluorescence characterization of coumarin-based Y1–Y6 with multiple active sites

By structurally tailoring the electron donating/withdrawing groups of a natural coumarin product, we designed a “step-by-step” strategy to obtain six chemical probes with stimuli-mediated fluorescence responses (Fig. 1 and Scheme S1). Specifically, two different functional groups were installed on the C7 hydroxyl position (R₁) and C8 aldehyde group (R₂) of coumarin, yielding Y1, Y2, Y3, Y4, Y5, and Y6. We then performed the optical experiments to compare the fluorescent properties of probe Y1–Y6. In comparison with the coumarin building block (compound 1), Y1 showed negligible fluorescence peaking at 452 nm (Fig. 2A, B and Fig. S1), possibly due to the incorporation of electron-withdrawing acetyl group onto the C7 position of compound 1 disrupted the electron distribution and quenched the fluorescence. When the acetyl group was replaced with a benzyl group at the C7 hydroxyl moiety, Y2 and Y3 exhibited a blue-shifted fluorescence peak at 373 nm with a 1.5- or 3.5-fold higher intensity than compound 1 (Fig. 2A and B), respectively. Notably, Y3 outperformed Y2 in fluorescence brightness (2.2-fold) at the same concentration, possibly because the hydroxymethyl group at the C8 position of coumarin enhanced the hydrophilicity of Y3 in aqueous solution.

Fig. 2 The fluorescent properties of probe Y1–Y6. (A) The fluorescence intensity of 1, Y1, Y2, and Y3 at 5 µM in DMSO/PBS solution (v/v = 1 : 99), E_x = 320 nm. (B) The fluorescence ratio (I/I₀) was plotted as a function against compound 1, Y1, Y2 and Y3. I₀ was the initial fluorescence intensity of compound 1 at 5 µM in DMSO/PBS solution (v/v = 1 : 99), E_x = 320 nm. (C) and (E) Fluorescence response of Y5 ((C), 5 µM) or Y6 ((E), 5 µM) against 1 mM H₂O₂ in DMSO/PBS solution (v/v = 1 : 99) for different periods of time, E_x = 320 nm. (D) and (F) The fluorescence ratio (I/I₀) was plotted as a function against each time point in DMSO/PBS (v/v = 1 : 99) solution. I₀ was the initial fluorescence intensity of Y5 (D) or Y6 (F) at 5 µM.

Y4 and Y5, featuring a H₂O₂-sensitive borate-cage at the C7 position of coumarin, exhibited stimuli-responsive behaviors in aqueous solution. The initial fluorescence of Y4 and Y5 exhibited a peak at 376 nm in the DMSO/PBS mixture solution (Fig. 2C and Fig. S2, S3). Upon addition of 1 mM H₂O₂, both Y4 and Y5 showed a time-dependent fluorescence decrease in the initial 375 nm and a concomitant fluorescence increase peak at 450 nm (Fig. 2D and Fig. S3). Leveraging the excellent fluorescence properties and functional modification site of Y5, we incorporated a polyethylene glycol (PEG)-linked chloroalkane onto the 1,3-dioxane group of Y5 to develop a biorthogonal probe Y6 (Fig. 1). The modified HaloTag ligand enables the covalent conjugation of Y6 to HaloTag-fused POIs for fluorescence activation. The fluorescence response of Y6 towards H₂O₂ was measured by adding 1 mM H₂O₂ to a 5 µM Y6 solution. Y6 revealed a 375-nm fluorescence decrease and a concomitant 5.4-fold increase at 440 nm within 30 min, indicating the excellent fluorescence sensitivity of Y6 to H₂O₂ in an aqueous environment (Fig. 2E and F). There is no pH change when incubating Y6 with 1 mM H₂O₂ for different periods of time or different amounts of H₂O₂ for 30 min (Fig. S4). Y6 showed excellent selectivity for H₂O₂ over various other reactive oxygen species (ROS) and other interfering substances (Fig. S5). This selectivity primarily stems from the specific oxidation of the phenylboronic pinacol ester moiety on Y6 by H₂O₂, which generates the corresponding phenol product and releases a p-quinone methide intermediate (Fig. S6).

Bioorthogonal conjugation of Y6 onto the HaloTag recombinant protein

We evaluated the protein labeling ability of Y6 to HaloTag-fused STING and GFP as model proteins (Fig. 3A). It's well-established that STING139-379 represents the soluble truncate of the STING protein, in which the last 40 amino acids of STING play a crucial role in the STING/IFNs signaling pathway.¹⁵ Hence, we chose STING139-379 and STING139-340 truncates as the model proteins. Two soluble STING truncates and GFP were successfully fused with HaloTag protein and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Fig. S7–S9). After incubating STING139-340_Halo, STING139-379_Halo, or GFP_Halo with Y5 or Y6 for 1 h, the resultant reaction was further treated with 1 mM H₂O₂ for 30 min (Fig. 3A). Fluorescence analysis was then applied to the SDS-PAGE gel, followed by Coomassie staining. As shown in Fig. 3B, the signal location of both methods matched well, with only Y6-treated groups exhibiting strong fluorescence, indicating a successful coupling of the probe Y6 to the HaloTag-fused POIs. We further incubated Y6 with POIs_Halo at various concentrations (0.25–1 µg µL⁻¹). The fluorescence intensity of Y6_POI correlated linearly with protein concentration in the gel (Fig. 3C and Fig. S10), demonstrating a dose-dependent conjugation efficiency.

Fig. 3 Covalent conjugation of Y6 with STING139-340_Halo, STING139-379_Halo or GFP_Halo protein to form Y6_POI. (A) A schematic diagram showing how to set up a conjugation reaction to obtain Y6_POI. (B) The SDS-PAGE results from the reaction between POI (1 µg µL⁻¹) with Y5 (100 µM) or Y6 (100 µM) at room temperature for 1 h. Aliquots were taken, treated with 1 mM H₂O₂ for 30 min, boiled, and loaded on the SDS-PAGE. The fluorescence gel was taken by irradiating the gel at 302 nm. (C) The SDS-PAGE results from the reaction between different amounts of STING139-340_Halo or GFP_Halo protein with Y6 (100 µM) at room temperature for 1 h. Aliquots were taken and analyzed as in panel B. Note: The same amount of protein without Y6 in the labeling reaction was used as control.

Stimulus-mediated fluorescence response of the chemigenetic probe Y6_POI

To analyse the fluorescence activity of Y6_POI as a coumarin-labelled macromolecule in an aqueous solution, we first obtained the purified Y6_STING139-379 protein complex (Fig. 4A). In aqueous solution, the fluorescence of Y6_STING139-379 at 375 nm decreased when incubated with H₂O₂ at 1 mM for 30 min (Fig. 4B and Fig. S11–S12). At the same time, the intensity of Y6_STING139-379 at 440 nm increased 7.3-fold compared with its initial fluorescence (Fig. 4B and Fig. S11–S12). The fluorescence of Y6_STING139-379 maintained 95% (4 °C) and 93% (25 °C) of the initial fluorescence intensity in PBS for 24 h (Fig. 4C), indicating the good photostability of Y6_STING139-379 in aqueous solution.

Fig. 4 The fluorescence response of chemigenetic Y6_POI. (A) A schematic diagram showing the stimulus-mediated fluorescence response of chemigenetic probe Y6_POI. (B) Fluorescence response of Y6_STING139-379 in aqueous solution upon incubation of Y6_STING139-379 with increasing concentrations of H₂O₂ at room temperature for 30 min. (C) The photostability of Y6_STING139-379 at 4 °C or 25 °C for 24 h. I₀ was the initial fluorescence intensity of Y6_STING139-379 at 0 h, E_x = 320 nm. (D) The fluorescence response of Y6_GFP to 1 mM H₂O₂ at room temperature for 10 min, E_x = 320 nm. Note: Y6_GFP was purified using an ultrafiltration tube (10 kD) and washed with PBS 3 times to remove unbound Y6 after the covalent reaction. (E) The fluorescence ratio (I/I₀) was plotted as a function of the concentration of GFP_Halo in aqueous solution. I₀ was the fluorescence intensity of Y6_GFP when the concentration of GFP_Halo at 0.25 µg µL⁻¹.

Inspired by the spectral overlap between the absorption of GFP protein and the emission of H₂O₂-cleaved Y6 (Fig. S13), we investigated the potential fluorescence resonance energy transfer (FRET) in the Y6_GFP complex. Upon excitation at 320 nm, Y6_GFP, purified from the reaction containing 0.25 µg µL⁻¹ GFP-Halo protein and 100 µM Y6, exhibited a main fluorescence peak at 440 nm and a shoulder peak at 510 nm after incubation with 1 mM H₂O₂ (Fig. 4D). When the protein concentration increased from 0.25 to 1 µg µL⁻¹, the fluorescence of Y6_GFP at 440 nm only increased about 1.3-fold, while the fluorescence peak at 510 nm increased about 2.8-fold (Fig. 4E), indicating an efficient intramolecular FRET in the Y6_GFP complex. These findings underscore the stimulus-mediated response of Y6_POI, not only in its capacity to light up protein of interest, but also in its ability to achieve multiple fluorescence emission in the fluorophore-conjugated macromolecular materials.

Cell imaging and detection of endogenous H₂O₂ in different cells

To systematically characterize the cell imaging efficacy of diverse fluorogenic probes, we first evaluated their cytotoxic profiles in non-tumorigenic 293T and tumorigenic HeLa cell lines. Following 24 h incubations at 20 µM, all probes exhibited the negligible cytotoxicity with above 95% cell viability in 293T and HeLa cells (Fig. 5A). Next, we incubated HeLa cells with each probe at 20 µM for 2 h and found that there was progressive fluorescence increase from cells treated with Y1 to Y3, which is consistent with in vitro assay in aqueous solution (Fig. S14). Notably, Y4 and Y5 emitted bright fluorescence signals in cells, likely attributed to the endogenous H₂O₂-mediated cleavage of their boron-caged moieties, triggering fluorescence “off–on” activation (Fig. S14). Given the elevated H₂O₂ production in tumor cells (Fig. 5B), we hypothesized that Y4 and Y5 could distinguish the different H₂O₂ levels between HeLa and 293T cells. To test this, 20 µM of each probe was incubated with HeLa or 293T cells for 2 and 12 h, respectively. As shown in Fig. 5C, HeLa cells treated with Y4 and Y5 displayed significantly higher fluorescence than 293T cells under the same conditions, indicating that the overexpression of H₂O₂ in HeLa cells leads to the highly efficient cleavage of the boron-cage and activation of Y4 and Y5. These results validate our hypothesis and further support the elevated oxidative stress in tumor cells.

Fig. 5 The cell imaging of Y4 and Y5 in 293T and HeLa cells. (A) 293T and HeLa cells were treated with Y1–Y5 at 20 µM for 24 h and subjected to CCK8 assays. Data from three independent groups receiving the same treatment were plotted as means ± SD. (B) The levels of H₂O₂ produced by 293T and HeLa cells were measured by a commercial H₂O₂ detection kit. (C) 293T and HeLa cells were treated with Y4 or Y5 at 20 µM for 2 h or 12 h, cells were fixed and analyzed by confocal microscopy. The fluorescence was recorded in the range of 500–550 nm. Scale bar = 10 µm.

Intracellular labeling ability of Y6 for precise tracking of the STING protein

As mentioned above, STING is known as an endoplasmic reticulum-resident protein critical for cellular innate immunity,²⁹ which requires dimerization or oligomerization to execute its biological function. Given the excellent stimulus-mediated fluorescence activation and biorthogonal conjugation of Y6in vitro, we investigated the intracellular tracking ability of Y6 to STING (Fig. 6A). We first examined the cytotoxicity of Y6 on normal 293T cells and cancerous HeLa cells. After 24 h of treatment at 20 µM, Y6 showed negligible cytotoxicity to both 293T and HeLa cells (Fig. 6B), thereby indicating its excellent biocompatibility in cells. Next, we constructed a STING_Halo plasmid and transfected it into 293T and HeLa cells, achieving the successful expression of the STING_Halo protein within the cells (Fig. 6C and Fig. S15). For intracellular labeling, STING_Halo expressing 293T and HeLa cells were treated with 20 µM Y5 or Y6 for 2 h, followed by medium replacement with fresh DMEM and further incubation for 2 h. After removal of the fluorophore supply from the culture media, fluorescence microscopy showed negligible signal in Y5-treated cells, whereas Y6 maintained persistent fluorescence activation (Fig. 6D and E). This disparity arises from the HaloTag ligand of Y6, which mediates covalent conjugation to STING protein, enabling the stable intracellular retention and precise tracking of STING in cells. All these results highlighted that Y6 could precisely label and track STING protein, enabling effective monitoring of STING dynamics in cells.

Fig. 6 Y6 exhibited fluorescence tracking ability to STING protein in cells. (A) A schematic diagram showing cells overexpressing STING_Halo protein were labeled with Y5 or Y6 for fluorescence tracking in cells. (B) 293T and HeLa cells were treated with Y6 at 20 µM for 24 h and subjected to CCK8 assays. Data from three independent groups receiving the same treatment were plotted as means ± SD. (C) 293T cells were transfected with STING_Halo plasmid for 36 h, lysed, and analyzed for indicated proteins by immunoblots. (D) 293T and HeLa cells transfected with STING_Halo plasmid were treated with Y5 or Y6 at 20 µM for 2 h and then refreshed with DMEM medium and cultured for an additional 2 h, cells were fixed and analyzed by confocal microscopy. The fluorescence was recorded in the range of 500–550 nm. Scale bar = 10 µm. (E) The quantitative data show the fluorescence ratio in panel D. I₀ is the background signal of untreated cells. Data with error bars are expressed as mean ± SD.

Conclusions

In summary, we rationally developed six coumarin-based fluorescent probes Y1 to Y6 using the structure–activity relationship to evaluate the fluorescence labeling and responding ability towards HaloTag-fused POIs in vitro and in cells. The electron density at the C7 position of coumarin contributed to the initial fluorescence intensity, while the hydroxymethyl moiety on 1,3-dioxane changed the hydrophilicity of Y1–Y3. By installing a boron-cage on the hydroxyl group of coumarin, Y4–Y6 exhibited a dual fluorescence response upon H₂O₂ stimulation. We further examined the labeling specificity and stimuli-responsive fluorescence properties of Y6 to proteins of interest (POIs) and demonstrated that Y6 containing a PEG-linked HaloTag ligand could covalently conjugate to STING and GFP proteins. Y6_POIs exhibited bright fluorescence both in gels and aqueous solution, in which Y6_GFP presented a FRET signal in response to H₂O₂ stimulation. Moreover, we evaluated the intracellular tracing ability of Y6 and indicated that Y6 could be retained in cells for long-term tracking of STING in cells. Hence, we present a novel fluorescence platform for the advancement of monitoring protein activation in proteostasis and medicine research.

Author contributions

Xuekun Zhang: project administration; Yu Jiang: project administration; Ying Wang: project administration; Yaxi Li: supervision, writing – reviewing and editing; Haozhou Tang: investigation, validation; Andong Shao: conceptualization, methodology, validation, writing – original draft, supervision; Jianming Ni: supervision, writing – reviewing and editing.

Conflicts of interest

All authors have declared that no conflict of interest exists.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information: additional experimental data including experimental details, synthesis of probes, absorption spectra, protein gel, cell imaging, and characterization of probes. See DOI: https://doi.org/10.1039/d5nj03804a.

Acknowledgements

This work was partially supported by grants from the Postgraduate Research & Practice Innovation Program of Jiangsu Province (SJCX24_1373), the National Natural Science Foundation of China (22107036), the Basic Research Program of Jiangsu (BK20240303 and BK20252081), the Wuxi “Taihu Light” Science and Technology Research Project (Y20232029), and the Youth Projects of Wuxi Health Commission (Q202324).

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Footnote

† X. Zhang and Y. Jiang contributed equally to this work.

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