Protein Hybrid Probe with Fluorogenic Switch for

proteins are powerful tools for detecting biological molecules and signals in living cells. To date, most targets of the hybrid probes have been limit...
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Synthetic-Molecule/Protein Hybrid Probe with Fluorogenic Switch for Live-Cell Imaging of DNA Methylation Yuichiro Hori,†,‡ Norimichi Otomura,† Ayuko Nishida,† Miyako Nishiura,† Maho Umeno,† Isao Suetake,§,||,⊥ and Kazuya Kikuchi*,†,‡ †

Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan § Laboratory of Epigenetics, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan || Center for Twin Research, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan ⊥ College of Nutrition, Koshien University, Takaraduka, Hyogo 665-0006, Japan ‡

S Supporting Information *

ABSTRACT: Hybrid probes consisting of synthetic molecules and proteins are powerful tools for detecting biological molecules and signals in living cells. To date, most targets of the hybrid probes have been limited to pH and small analytes. Although biomacromolecules are essential to the physiological function of cells, the hybrid-probe-based approach has been scarcely employed for live-cell detection of biomacromolecules. Here, we developed a hybrid probe with a chemical switch for live-cell imaging of methylated DNA, an important macromolecule in the repression of gene expression. Using a protein labeling technique, we created a hybrid probe containing a DNA-binding fluorogen and a methylated-DNA-binding domain. The hybrid probe enhanced fluorescence intensity upon binding to methylated DNA and successfully monitored methylated DNA during mitosis. The hybrid probe offers notable advantages absent from probes based on small molecules or fluorescent proteins and is useful for live-cell analyses of epigenetic phenomena and diseases related to DNA methylation.

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INTRODUCTION Recent progress in protein labeling technology has enabled the development of synthetic-molecule/protein hybrid probes for detecting various biological molecules and signals in living cells.1,2 These probes are created by specifically labeling proteins with synthetic molecules. Among protein labeling techniques, a protein-tag-based system using SNAP-tag,3,4 HaloTag,5,6 fluorogen-activating protein,7,8TMP-tag,9,10 PYPtag,11,12 and so on, is a useful and versatile method for creating hybrid probes in living cells. In this system, a protein fused to a protein tag is specifically modified with a fluorophore conjugated to a ligand of the protein tag. To date, various hybrid probes have been exploited using the protein-tag-based methods with functional fluorophores that change fluorescence property in response to pH,13,14 metal ions,15−17 or gas molecules.18,19 Compared with small-molecule-based probes, these hybrid probes have the following attractive advantages. First, local biosignals are easily detected in a target organelle using genetic engineering techniques by which a signal sequence is fused to a protein tag in a hybrid probe. Second, high molecular-recognition ability of a protein moiety in a hybrid probe allows for specific detection of small molecules such as glutamate, acetylcholine, GABA, and their related compounds.20−22 However, most targets of hybrid probes are limited to small molecules. Although biomacromolecules are essential for cell © XXXX American Chemical Society

homeostasis and function, fewer studies of hybrid probes for live-cell detection of biomacromolecules have been reported. Previously, protein kinase activity was imaged using hybrid probes.23,24 However, these probes were inserted into cells by microinjection, as protein labeling was conducted in vitro using a classical method based on cysteine-reactive iodoacetamide. Thus, sensing of biomacromolecules using protein-tag-based hybrid probes has been scarcely examined. Herein, to overcome this current limitation, we developed a hybrid probe for imaging of biomacromolecules, focusing on epigenetically modified DNA. Among the several types of DNA modifications, methylation plays crucial roles in epigenetic repression of gene expression.25 This modification typically occurs in cytosines in CpG sequences and is catalyzed by DNA methyltransferase (Dnmt).25 DNA methylation is involved in many biological phenomena such as embryogenesis, development, and differentiation.26 Since aberrant DNA methylation causes various diseases including cancer and neurological disorders, DNA methylation is an important drug target for these diseases.27 Indeed, Dnmt inhibitors are clinically used for the treatment of cancer. 28 Recent studies have also shown that DNA methylation inhibition leads to immune activation for cancer Received: September 18, 2017

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DOI: 10.1021/jacs.7b09713 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Figure 1. Principle of fluorogenic detection of methylated DNA using a hybrid probe created by a PYP-tag labeling system.

therapy.29 Due to the importance of DNA methylation, techniques for its detection are in high demand in the fields of biological, pharmaceutical, and medical sciences. To date, a number of methods have been utilized for in vitro detection of DNA methylation;30 however, live-cell detection is limited to a method utilizing a fluorescent protein (FP).31−33 In this method, the localization of a FP fused to a methyl-CpGbinding domain (MBD) that specifically recognizes the 5methylcytosine of CpG-containing DNA34 is observed using a fluorescence microscope. We previously used a similar approach involving a protein tag.35 However, their problem is that fluorescence is detected from the fusion proteins unbound to DNA. Consequently, the fluorescence of the free proteins cannot be distinguished from that of the DNA-bound proteins.33,35 Recently, there has been much interest in the mechanism of how DNA is methylated in concert with histone modification after DNA replication during the cell cycles.36 In such a mechanistic study, live-cell analyses are valuable to verify spatiotemporal information on DNA methylation, and it is thus necessary to design a probe that detects methylated DNA in living cells without the interference of undesirable signals derived from a free probe. To solve this problem, by applying a protein-tag system, we developed a synthetic-molecule/protein hybrid probe that enhances fluorescence intensity when the hybrid probe binds to methylated DNA in living cells (Figure 1). By taking advantage of the switching function of a synthetic molecule and molecular-recognition ability of a protein, specific and fluorogenic detection of methylated DNA was achieved. Importantly, this chemistry-driven fluorogenic switch solved the problem of the FP probes, which are fluorescent in a DNAunbound form. Using this hybrid probe, we successfully monitored fluorescence signals of methylated DNA during mitosis in living cells.

detect methylated DNA, we employed oxazole yellow (YO), which enhances fluorescence upon DNA binding.38 The PYPtag system was used to specifically label PYP3R-MBD1−75 with PYP-ligand-conjugated YO (YOCNB) and generate the hybrid probe in living cells. PYP3R is a PYP-tag mutant with neutral pI, in which three acidic amino acids were converted to arginine residues.11 In a previous study, a synthetic YO conjugate with a DNA-binding peptide selectively bound to its target DNA.39 However, this was limited to in vitro study, because the conjugate does not cross the cell membrane. Thus, a proteintag-based labeling system is required for generation of the hybrid probe that allows live-cell imaging. We envisioned that the hybrid probe specifically binds to methylated DNA by virtue of the MBD module in living cells, and thereby the YO module becomes proximal to methylated DNA, resulting in DNA binding of the YO module, which enhances fluorescence. Since YO itself does not bind to DNA with high affinity,38,39 we expected that the YO module binds to DNA due to proximity effects only when the YO is recruited near methylated DNA by the hybrid probe, while nonspecific DNA binding of the YO is restrained. Fluorogenic Response for Detecting DNA Methylation in Vitro. The PYP-tag labeling molecule, YOCNB, was synthesized by connecting YO with a PYP-tag ligand (Figure 1, Scheme S1). We measured the absorption spectra to confirm the labeling of PYP3R-MBD1−75 with YOCNB (Figure S1). The absorption bands of free YOCNB (red line) at ∼350 and ∼480 nm were derived from the ligand (4-hydroxycinnamic acid thioester) and the YO moieties, respectively, whereas no protein absorption band was detected in these regions. The reaction of YOCNB with PYP3R-MBD1−75 increased the absorbance of the ligand moiety at ∼450 nm. This spectroscopic change is characteristic of the formation of the PYP-tag−ligand complex, as reported previously.37 These results indicate that a hybrid probe consisting of a YOCNB/ PYP3R-MBD1−75 complex was generated. YOCNB showed similar labeling kinetics to TP-CA, which was previously synthesized as a PYP-tag ligand without a fluorophore (Figure S1). This indicates that the conjugation of YO to the PYP-tag ligand did not influence the labeling reactions. After the labeling reaction, a gel shift assay was conducted to examine whether the hybrid probe selectively binds to methylated DNA. In this assay, the gel was stained using SYBR Green. PYP3R labeled with YOCNB did not bind to methylated or unmethylated DNA (Figure 2a). In contrast, the hybrid probe was selectively bound to methylated DNA (Figure

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RESULTS AND DISCUSSION Molecular Design of a Hybrid Probe. To develop a synthetic-molecule/protein hybrid probe in living cells, we employed a PYP-tag-based system that we previously developed for specific labeling of proteins using synthetic molecules.11,12,35 The PYP-tag is a small 14 kDa protein and binds to 4hydroxycinnamic acid derivatives as a ligand.37 For fluorogenic detection of methylated DNA, we designed a hybrid probe consisting of a DNA-binding fluorogen and PYP3R11-tagged MBD1−75 (PYP3R-MBD1−75) (Figure 1). In this hybrid probe, MBD1−75 derived from MBD1 was utilized as a module for specific recognition of methylated DNA. As a fluorogen to B

DOI: 10.1021/jacs.7b09713 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Live-Cell Imaging of Methylated DNA. For live-cell imaging of methylated DNA, PYP3R fused to MBD1−112 (PYP3R-MBD1−112) including its native nuclear localization signal sequence was expressed in NIH3T3 cells, which were then incubated with YOCNB together with Hoechst 33342 and MitoTracker (Figure 4a). The cells showed some small

Figure 2. Selective binding of a hybrid probe to methylated DNA. The hybrid probe represents the complex of PYP3R-MBD1−75 and YOCNB. After reaction of (a) PYP3R (5 μM) or (b) PYP3RMBD1−75 (5 μM) with YOCNB (5 μM), the complexes (0, 50, 100, 200, 300, and 400 nM) were incubated with methylated or unmethylated DNA (50 nM) and then analyzed by a gel shift assay.

2b). When SYBR-Green-unstained gel was imaged, fluorescence of the hybrid probe was detected from bands that corresponded to the complex of methylated DNA and the hybrid probe (Figure S2). Fluorescence measurements demonstrated that YOCNB was almost nonfluorescent in the absence or presence of DNA (Figure 3). This indicates that

Figure 4. Live-cell imaging of methylated DNA. (a) Fluorescence images of cells transfected with empty plasmid and plasmid encoding PYP3R-MBD1−112 or PYP3R. YOCNB (2 μM), Hoechst 33342 (300 ng/mL), and MitoTracker Deep Red (2 μM) were added to the cells. Fluorescent colors of the hybrid probe, Hoechst 33342, and their colocalization are shown as green, magenta, and white in the images, respectively. Scale bar, 20 μm. (b) Effect of 5-AzadC on fluorescence in nuclei. PYP3R-MBD1−112-expressing cells were pretreated in the absence or presence of 5-AzadC (25 μM) for 24 h and then incubated with YOCNB (2 μM) for 75 min. Scale bar, 50 μm. (c) Imaging-based quantification of methylated DNA. Cells expressing PYP3R-MBD1−112 were incubated with different concentrations of 5-AzadC for 24 h and were then labeled with YOCNB (2 μM). The normalized fluorescence intensity of YOCNB in nuclei was plotted against the 5-AzadC concentrations.

Figure 3. Fluorogenic detection of methylated DNA using a hybrid probe. Fluorescence spectra of the hybrid probe (400 nM) and YOCNB (400 nM) in the absence or presence of DNA (50 nM), which was methylated or unmethylated.

YOCNB does not bind to DNA by itself under this experimental condition. This is presumably because YO possesses low DNA-binding affinity. Alternatively, steric hindrance around the YO module due to the intramolecular interactions of the YO module with the nitrobenzene moiety of YOCNB might hinder DNA binding. We previously showed that a fluorophore moiety interacts intramolecularly with a nitrobenzene moiety in a PYP-tag probe.40 Thus, this interaction possibly also occurs in YOCNB and suppresses the nonspecific DNA binding activity of YOCNB. The YO module of the hybrid probe emitted weak fluorescence in the absence of DNA (Figure 3), indicating that protein labeling with YOCNB causes the emission. The addition of methylated DNA to the hybrid probe significantly increased the fluorescence intensity of the probe, whereas the fluorescence enhancement was slight in the presence of unmethylated DNA (Figure 3). PYP3R labeled with YOCNB did not increase the fluorescence intensity in the presence of DNA (Figure S3). These results indicate that the hybrid probe selectively binds to methylated DNA with a fluorogenic response.

fluorescent puncta in nuclei. The puncta were almost overlapped with the fluorescence of Hoechst33342, which mainly illuminates heterochromatin rich in methylated DNA.41 Cells transfected with a plasmid encoding PYP3R or an empty vector were incubated with YOCNB to verify if the observed fluorescent puncta resulted from the presence of the MBD module. No significant fluorescence was detected in the nuclei of the cells. These results suggest that the hybrid probe, YOCNB/PYP3R-MBD1−112, is generated in living cells, and specifically illuminates methylated DNA. During the experiments, we noticed that addition of MitoTracker was necessary C

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Journal of the American Chemical Society for efficient labeling in living cells. This could be because MitoTracker promotes the localization of YOCNB to the nuclei. In general, cationic hydrophobic molecules tend to accumulate in the mitochondria.42 Since YOCNB is also cationic, the MitoTracker may expel YOCNB from the mitochondria and prevent the mislocalization of YOCNB, promoting protein-labeling reactions. However, detailed investigation is required to understand the function of the MitoTracker. Further analyses using a DNA methylation inhibitor, 5-azadeoxycitidine (5-AzadC),28 were carried out to confirm whether the puncta arose from the binding reaction of the hybrid probe to methylated DNA (Figure 4b). As a result, cells treated with 5-AzadC lost fluorescence in nuclei. We also conducted live-cell imaging using the YOCNB/PYP3RMBD1−75-based hybrid probe that was investigated in in vitro experiments. We obtained similar results to those of the PYP3R-MBD1−112-based hybrid probe. Fluorescent puncta overlapped with Hoechst 33341 fluorescence were observed in YOCNB-treated cells expressing PYP3R-MBD1−75 and were lost after 5-AzadC treatment (Figure S4). Quantitative analyses using the YOCNB/PYP3R-MBD1−112-based probe were performed to precisely verify the effect of 5-AzadC. The results showed that the loss of fluorescence was dependent on the concentration of 5-AzadC and was consistent with results obtained using a commercially available immunodetection kit (Figure 4c, Figure S5). Thus, it is clearly indicated that the hybrid probe emits fluorescence by specific binding to methylated DNA in living cells and, in addition, allows livecell quantification of DNA methylation. We compared the performance of the hybrid probe with that of a FP-fused MBD (EGFP-MBD1−112) probe reported previously.33 NIH3T3 cells were transfected with a EGFPMBD1−112-encoding plasmid and were imaged (Figure S6). The nuclei of the cells showed fluorescent puncta similar to those detected using the hybrid probe. However, unlike the hybrid probe, significant fluorescence signals of EGFP-MBD1−112 were observed even when cells were treated with 5-AzadC. In certain cells expressing EGFP-MBD11−112, the localization of the fusion protein changed from the Hoechst-stained regions to those not stained with Hoechst. These results indicate that the decrease in DNA methylation level partially changed the localization of the EGFP-based probe but did not suppress the fluorescence signal. This is reasonable because the EGFP-based probe remains fluorescent and would not undergo degradation or fluorescence decay even when the DNA methylation level is low. In this study, this problem has been successfully solved by using the hybrid probe. The advantage of the hybrid probe is that cellular methylated DNA can be clearly identified and quantified owing to its fluorogenic switching mechanism. Imaging of Methylated DNA during Mitosis. Finally, we visualized the dynamics of methylated DNA during mitosis (Figure 5). To estimate the mitotic phase in the cell cycle, we utilized a fluorescent ubiquitination-based cell cycle indicator (FUCCI) system.43 We fused mCherry to hGeminin (hGem) to monitor the S/G2/M phases. To avoid the spectral overlap between mCherry and MitoTracker, we removed the MitoTracker from cells by thorough washing. Cells stably expressing mCherry-hGem1−110 were transfected with the PYP3R-MBD1−112-encoding plasmid and were imaged periodically. Since cell-cycle monitoring requires long-time imaging, we checked the cytotoxicity of the hybrid probe by WST assay. We found that no noticeable cytotoxic effect of addition of YOCNB and expression of PYP3R-MBD1−112 was observed

Figure 5. Time-lapse imaging of methylated DNA during mitosis. Cells stably expressing mCherry-hGem1−110 were transfected with a PYP3R-MBD1−112-encoding plasmid and were incubated with YOCNB (2 μM) for 40 min. The time-lapse images were obtained using fluorescence microscopy. Scale bar, 5 μm.

(Figure S7). The time-lapse imaging experiments demonstrated that the fluorescent puncta in nuclei gradually aggregated in the center of nuclei in the M phase. Then, the fluorescence signals were segregated as a result of mitosis. These results indicate that the hybrid probe visualized methylated DNA in chromosomes that dynamically change the conformation during mitosis. Importantly, real-time detection of methylated DNA was successfully achieved by combining fluorescence switching of a synthetic molecule and specific recognition of a protein.

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CONCLUSIONS In conclusion, we developed a fluorogenic synthetic-molecule/ protein hybrid probe that images endogenous methylated DNA. The PYP-tag labeling system successfully generated the hybrid probe in living cells. Importantly, the hybrid probe allowed the quantitative analyses of DNA methylation and the real-time imaging of dynamics of methylated DNA during mitosis. Specific fluorogenic detection of methylated DNA was achieved because of the high molecular-recognition ability of the MBD module and switching function of the YO module. The fluorogenic switch of the YO module solved the problem of FP probes that always emit fluorescence regardless of binding reactions with methylated DNA. Since DNA methylation inhibitors are promising drugs for various diseases, this system could be useful for live-cell evaluation of drug potency. Thus, the hybrid probe will contribute to drug discovery as well as basic sciences on epigenetics. Furthermore, this technique could be applied in fluorogenic imaging of D

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genomic loci by replacing the MBD moiety with genome targeting molecules such as TALE.44 It could also be possible to image histone modifications by using a hybrid probe including histone reader domains such as chromodomains,45 since DNA is wound onto histones in nucleosomes. Hybrid probes offer an attractive advantage to overcome problems that are not addressed by small-molecule and FP sensors. We believe that the functional versatility of hybrid probe derivatization will not only lead to the development of sophisticated next-generation tools for live-cell analyses but also pave the way to a new field of chemistry.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b09713. Experimental procedures and supplementary results (PDF)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Kazuya Kikuchi: 0000-0001-7103-1275 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by JSPS KAKENHI (Grant Nos. JP25220207, JP16H00768, JP15K12754, and JP16K13099 to K.K. and JP26282215, JP17H02210, JP16K13088, JP17H06005, and JP16H01428 “Resonance Bio” to Y.H.), by AMED-CREST, and by SICORP from JST.

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DOI: 10.1021/jacs.7b09713 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX