Luminescence Sensing for Qualitative and Quantitative Detection of 5

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Luminescence sensing for qualitative and quantitative detection of 5-methylcytosine Yushu Yuan, Tingting Hong, Yi Chen, Yafen Wang, Xueping Qiu, Fang Zheng, Xiaocheng Weng, and Xiang Zhou Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 26 Jul 2018 Downloaded from http://pubs.acs.org on July 26, 2018

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Analytical Chemistry

Luminescence sensing for qualitative and quantitative detection of 5methylcytosine Yushu Yuan,‡, † Tingting Hong,‡, † Yi Chen,† Yafen Wang,† Xueping Qiu,§ Fang Zheng,§ Xiaocheng Weng, *,† and Xiang Zhou *,† College of Chemistry and Molecular Sciences, Key Laboratory of Biomedical Polymers of Ministry of Education, Wuhan University, Wuhan, Hubei, 430072, P. R. China †

§

Zhongnan Hospital, Wuhan University, Wuhan, Hubei, 430071, P. R. China

‡

These authors contributed equally.

ABSTRACT: 5-methylcytosine (5mC) is revealed as a heritable epigenetic modifiation in genomic DNA. It has been reported that cytosine/guanine dinucleotides (CpG) hypermethylation in the promoter regions of tumor suppressor genes are related with inappropriate gene silencing, so the determination of 5mC in the CpG islands of mammals has attracted much attention. In this paper, a luminescence sensing strategy based on bisulfite treatment, asymmetric PCR and ATP-releasing nucleotide was proposed. With little background, this method can provide accurate quantitative information about methylation changes at CpG sites, even at a specific site. The proposed method can be successfully employed to determine the methylation status of three hepatocellular carcinomas (HCC) related genes in clinical tissues.

5-methylcytosine (5mC) is the most important epigenetic modification and plays essential roles in gene regulation, embryogenesis and cellular differentiation.1-3 What’s more, aberrant DNA methylation levels can lead to genomic instability as well as the initiation of cancer.4, 5 Increased methylation of tumour suppressor genes is an early event of the process of carcinogenesis, suggesting that altered DNA methylation patterns could be early detectable neoplastic changes associated with tumorigenesis, also benefit for the treatment evaluation, and prognosis.1, 6, 7 A recent research demonstrated that DNA methylation detection could be applied in precise classification of the central nervous system tumors which is particularly challenging by traditional methods.8 Consequently, among all the epigenetic study conducted so far, 5mC is most relevant to clinical research and it is very significant to develop better techniques for its content or pattern analysis.9-13 Currently, accurate global 5mC analysis were performed by LCMS system and whole genome sequencing technology, which provide integrated information but accompanied with high costs and complicated operation.14, 15 On the other hand, the 5mC detection in specific-loci gene is another research hot spot which is very useful in further biomedical study and clinical application. Among the various assays for the regional methylation analysis, bisulfite induced deamination based methods become the most widely used.16, 17 Assisted with bisulfite-mediated cytosine conversion to uracil, Methylation-specific PCR (MS-PCR) was developed which facilitates the precise mapping of methylation site in CpG islands by gel electrophoresis.18 After that, other techniques were combined with the principle of MS-PCR to improve the sensitivity of 5mC detection.19 Moreover, the cooperation of bisulfite with DNA ligase also successfully used in 5mC sites identification.20 However, all these methods need intricate design of specific primers. The false-positive possibility should also be considered, and all of these methods are difficult to be used in partial methylation detection. Combined bisulfite restriction analysis (COBRA) is another choice employing restriction enzyme digestion to detect sequence variation in PCR products,21 but the requirement of enzyme recognition sites has largely limited its application. It’s urgent to develop a simple and universal method

that can provide accurate quantitative information about methylation changes at CpG sites. In our previous study, a fluorescein-dGTP incorporated asymmetric PCR assay strategy was developed for qualitative and quantitative detection of methylation at CpG sites.22 This method can monitor the methylation status at each CpG site without the limits in MS-PCR or COBRA, but autofluorescence of the modified dGTP brings in high background, making it troublesome for fluorescence measurement. So gel electrophoresis was mainly used in that study, largely limiting its applications. Luminescence sensing, which has the advantage of lower background, demonstrates the important role in DNA synthesis or cell biology. Inspired by the innovative work of ATP-releasing nucleotides by Eric Kool’s group,23 and taking advantages of the modified dGTP (called dGppppA for short), we designed a luminescence turn on approach to qualitatively and quantitatively analyze the methylation status of CpG sites. EXPERIMENT SECTION Materials and Equipments. Reagents and starting materials for chemical syntheses were obtained from commercial suppliers (Sigma-Aldrich or Alfa Aesar) unless otherwise indicated. All of the oligonucleotides were synthesized and purified by GeneCreate Co., Ltd. (Wuhan, China) and their sequences were listed in Table S1 and S2. Hot Start Taq polymerase, 2'-Deoxycytidine 5'triphosphate (dCTP), 2'-Deoxythymidine 5'-triphosphate (dTTP), 2'-deoxyadenosine-5'- triphosphate (dATP) and dNTP mix (2.5 mM for each) were purchased from Takara Biotech (China). Bisulfite Conversion Kit was purchased from New England BioLabs (USA). A General AllGen Kit was purchased from CWBIO. Klenow Fragment (exo-) was purchased from thermo Fisher Scientific. DNA Clean & ConcentratorTM-5 kit was purchased from ZYMO RESEARCH. The extended bands analyzed by polyacrylamide gel electrophoresis were scanned with a Pharos FX Molecular imager (Bio-Rad, USA) operated in the fluorescence mode. DNA concentrations were quantified by NanoDrop 2000c (thermo scientific, USA). The pEASY-T5 Zero Cloning Kit was purchased from TransGen Biotech. ATP Determination Kit

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(A22066) from Life Technologies (Invitrogen) was used for the bioluminescence assay. The luminescence was measured in a luminometer (GloMax® 20/20, Promega). Extracted Genomic DNA from Cells and Tissues. HepG2, MDA-MB-231 and MCF-7 were cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 5% penicillinstreptomycin. Genomic DNA were extracted and purified by CWBIO Genomic DNA kit according to the manufacturer’s instruction. The hepatic cancerous or paracancerous tissues in patients with HCC were obtained from Zhongnan Hospital. All tissues were obtained with the approval of patients. All the patients were treated with surgery recently. Genomic DNA was extracted according a standard proteinase K digestion and phenol/chloroform extraction method. Bisulfite treatment. For 76-mer templates, bisulfite conversion was performed according to the previous reported22. About 1 μg DNA in a final volume of 50 μL were mixed with 5.5 μL NaOH (3 M) and incubated at 42 °C for 30 mins. Then 30 μL freshly prepared hydroquinone (10 mM) was added to obtain a yellow solution; followed by the addition of 520 μL freshly prepared sodium bisulfite (3.6 M, pH 5.0, freshly prepared). The reaction tube was turned up and down gently for ixing. 200 μL liquid paraffin was added to cover the mixture and the mixture was incubated at 50 °C for 16 h. After reaction, we used Millipore to remove salts. The modified DNA was dissolved in 50 μL ultrapure water. As to genomic DNA extracted from cells and tissues, we used a Bisulfite Conversion Kit according to the manufacturer’s instructions. PCR amplification. Bisulfite treated 76-mer template was amplified using asymmetry PCR directly. The PCR system contains 5 μL 10 × PCR reaction buffer, 4 μL dNTPs (2.5mM each), 2.5 U Hotstart Taq polymerase, 10 μL forward primer (10 μM), 1 μL reverse primer (1 μM) and 1 ng bisulfite-treated DNA. The amplification was carried out under the following conditions: 95 °C for 5 min, and 40 cycles of PCR at 94 °C for 30s, 48 °C for 30s, 72 °C for 30 s, then extended at 72 °C for 5 min. For bisulfite modified genomic DNA, we used a two-step PCR. The first PCR system contains 1μL 10 × PCR reaction buffer, 0.8 μL dNTPs (2.5 mM each), 0.5 U Hotstart Taq polymerase, 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), and 4 μL bisulfite treated genomic DNA in a total volume of 10 μL. The amplification was carried out under the following conditions: 95 °C for 5 min, and 40 cycles of PCR at 94 °C for 30 s, 54 °C for 30 s (for E-cadherin, the annealing temperatures for ACP1 and TRIM58 are 49 °C and 56 °C respective), 72 °C for 30 s, then extended at 72 °C for 5 min. 2 μL PCR product was used as template in the second PCR. The second PCR system contains 5 μL 10 × PCR reaction buffer, 4 μL dNTPs (2.5 mM each), 2.5 U Hotstart Taq polymerase, 10 μL forward primer (10 μM), 1 μL reverse primer (1 μM). The amplification was carried out at a similar condition in 30 cycles. The PCR products are purified using a DNA Clean & ConcentratorTM-5 kit according to the manufacturer’s instructions. The PCR product was confirmed by 3% agarose gel. DNA concentration was quantified by NanoDrop 2000c (Thermo Scientific, USA). Primer extension reaction and luminescence detection. To make sure that the amounts of the amplified templates used in the extension reactions are almost equal, NanoDrop 2000c was used to quantify their concentration. The amplified products are mixed with 3 μL 100 μM reverse primer, then heated at 95 °C for 10 min and cooled to 4 °C immediately. After that, we add 1 μL 10 × Klenow Buffer, 0.6 μL 500μM dNTP mix (we used dGppppA to replace dGTP, and for the single base extension reaction, only 0.4 μL 500μM dGppppA was added), 1 Unit KF exo- DNA polymer-

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ase in a final volume of 10 μL. The mixtures were incubated at 37 °C for 1 h, then denatured at 65 °C for 10 min. The luminescence detection was carried out by adding 5 μL extension reaction solution to 50 μL luciferase reaction solution (prepared as instructed by the ATP determination kit) in 1.5 mL tubes. RESULTS AND DISCUSSION Principle of detection of 5-methylcytosine. As illustrated in figure 1, bisulfite treatment converts the unmethylated cytosine to uracil, which will read as thymine during PCR amplification, while methylcytosine is maintained as cytosine. The bisulfite treated target DNA was amplified by asymmetric PCR and followed by primer extension. In the presence of dGppppA, one methylcytosine in the original DNA will generate one ATP, which can be easily measured by commercial luciferase + luciferin reaction assay, but the cytosine one cannot.24 In this way, we can accurately quantify all 5mC quantity sites in specific gene region without cumbersome design of multiple primers. What’s more, the methylation status at specific gene site can also be identified by single base extension. With the features of simplicity and high sensitivity, this method was used to examine the methylation status in the pro-

moter regions of the three target genes related to HCCs. Figure 1. Schematic illustration of ATP-releasing dGppppA facilitating assay for quantitative detection of methylation in a region of interest. (a) Target DNA containing 5mC, which will generate luminescence signal. (b) Target DNA without 5mC sites, no luminescence was generated. The ATP-releasing nucleotide (dGppppA) indeed participates in the primer extension reaction. To confirm that this strategy is able to identify diverse methylation statuses in a region of interest, we chose five DNA oligonucleotides containing diverse methylated CpG sites as target gene model (sequences listed in Table S1). After bisulfite conversion, asymmetric PCR and DNA purification, the purified PCR products were directly used to perform primer extension reaction by the addition of primers (reverse primer was used as the extension primer), modified dNTP mixtures (we used dGppppA to replace nature dGTP), KF exo-. Because the residual dNTPs from the PCR process may interfere the primer extension reaction, we should confirm that dNTPs was totally removed and only dGppppA could participate in the extension reaction to pair with cytosine. For this purpose, denature PAGE gel electrophoresis was used to analysis the extension products with the FAMlabeled extension primer. The results showed that no new extended bands were observed without extra addition of dNTPs (Figure S1), which indicated the primer extension couldn’t proceed because of dNTPs from the asymmetric PCR step was removed totally. Upon the addition of dNTPs mixture, containing nature dGTP or dGppppA, all of the primers extended to get the fulllength products. In the groups with dATP, dTTP and dCTP but without dGTP or dGppppA, only control sequence bearing no 5mC sites can reveal a full-length extension product. As to the DNA sequences bearing different numbers of 5mC sites, the extensions were stopped at their first methylation site. All of these


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Analytical Chemistry results indicate that dNTPs residuals from the PCR reaction were completely eliminated by PCR product purification and could not interfere the following steps. Finally, the remained cytosines that survived from the bisulfite treatment will only pair with dGppppA, then release ATP after the primer extension process. It means that the amount of generated ATP in this strategy is positive correlated with the ratio of 5mC in target gene, so it is possible that the quantitative detection of 5mC in specific gene-loci can be achieved by precise determination of ATP through luminescence signal. Linear relationship between numbers of 5mC sites and luminescence intensity. After confirming that dGppppA has participated in this strategy, we should further verify that it’s feasible to determine the methylation level by measuring the luminescence. We first investigate the relationship between the diverse number of methylation sites and the luminescence intensity. The same five DNA oligonucleotides containing 0, 1, 2, 4, 6 methylation sites (we called them con, 1mC, 2mC, 4mC, 6mC in figure 2a) were also investigated in these experiments. As mentioned above, the amounts of the generated ATP are related to the extent of methylation. To transform the generated ATP into luminescence signal, luciferase + luciferin reaction mixture was used to determine ATP produced in this process. Figure 2a indicated that the increased luminescence signal was obvious when there is only one methylated cytosine site in target DNA template. In addition, with the increasing number of methylation sites, the bioluminescence signals was enhanced sharply but with a perfect linear relationship between the bioluminescence signals and the number of methylation site (figure 2b, R2=0.9845). These results indicate that this luminescence measuring method will be competent in the 5mC detection within specific gene-loci with high sensitivity and excellent linear relationship.

Figure 2. (a) Luminescence measurement of DNA sequence with diverse methylation sites. (b) Linear relationship between luminescence intensity and number of methylation sites, R2 = 0.9845.

Figure 3. (a) Luminescence measurement of mixed samples with diverse proportions of methylated DNA. (b) Luminescence intensity plotted with respect to methylation level, and there is a linear relationship between them, R2 = 0.9767. Error bar, mean ± SEM, n = 3. specific gene represents important function of the DNA epigenetics modification. Next we verify whether this strategy can det-ect mixed samples with diverse methylation levels with guaranteed sensitivity and accuracy. We mixed the non-methylated DNA template with methylated DNA template in a series of ratios range from 0%, 20%, 40%, 60%, 80% to 100%. These model samples were subjected to same experimental procedures as described above. The luminescence signals were measured and the results were shown in increasing proportions of methylated DNA with perfect linear relationship between the ratio of 5mC and the luminescence intensity (figure 3a and 3b). These sensitive and stable features of the output luminescence signal demonstrate that our method is also qualified to quantitatively detect the methylation level changes in the DNA samples. Detection of single site methylation status in the target sequence. Besides the total 5mC ratio within a specific gene region, the research of the methylation status in the single CpG site is also very significant to reveal the regulation function of DNA methylation. Especially in the clinical research, the DNA methylation change in the specific single CpG might provide valuable information for early diagnosis and treatment evaluation.25, 26 Thus, in the biological samples, the single CpG site always presents partial methylation status. Whether our method could be used to determine the methylation statuses at specific site means the application prospect of this strategy. The procedure was similar as mentioned above but some difference in primer extension part. In the primer extension step, a primer complementary to the target at the nucleotide in front of the 5mC sites was designed, and only dGppppA alone was added to perform the single nucleotide extension (Fig 4a). Here the relative amount of released ATP can reflect the methylation status of the single target site. Methylated DNA was mixed with unmethylated control with proportions of 0%, 20%, 40%, 60%, 80% and 100%. Figure 4b and 4c shows that the luminescence intensity increases with the increase of methylation ratio with an excellent linear correlation between the luminescence intensity and the 5mC ratio. These results indicate that this design can be used to qualitative and quantitative detection of 5mC ratio at single site.

Linear relationship between 5mC ratio and luminescence intensity. As we know, the methylation in cytosine is dynamic and ratio of 5mC within specific gene region will be changed frequently, which leads to the coexistence of methylation sequence and demethylation one.24 The diverse methylation level of

Figure 4. (a) Schematic illustration of detection of specific site bearing different methylation status. (b) Luminescence intensity generated by diverse proportions of methylated DNA. (c) Linear



relationship between luminescence intensity and methylation status, R2 = 0.9920. Error bar, mean ± SEM, n = 3. Detection of gene-specific methylation level in genomic DNA from different cell lines. Next, we applied our method to detect some known methylation sites in genome DNA. E-cadherin gene is an important tumor suppressor gene. It was reported that loss of E-cadherin expression caused by epigenetic alteration leads to tumorigenesis.26 In order to investigate its methylation status in human genome, the proposed method was used to analyze the promoter methylation levels of E-cadherin in three human cancer lines (HepG2, MDA-MB-231, and MCF-7). Approximately 100 ng of genomic DNA was subjected to bisulfite treatment after genomic DNA isolation from cultured cells. After that, two PCR steps were performed to generate single-strand DNA as targets for the following primer extension reaction. The first PCR procedure was used to obtain double strand DNA of specific PCR. Then we test the luminescence signal. The results are shown in figure 5a. Just as previously reported, 22, 28 the luminescence produced by HepG2 and MDA-MB-231 was much stronger than MCF-7 (more than 3-fold enhancement). To further confirm the results, we used bisulfite genomic sequencing (BGS) method to analyze endogenous methylation level of the interested region. The results are shown in Figure S2, S3 and S4, among the 16 potential methylation sites, genomic DNA from MDA-MB-231 reads out to be 13.2 cytosines (means they are 5mC in genome) in average, and for HepG2 and MCF-7, there are 11.8 cytosines and 5.0 cytosines respectively. These results indicate that the E-cadherin methylation level of HepG2 and MDA-MB-231 are higher than MCF-7, consistent with luminescence results, indicating that our strategy can be used to detect the methylation status in human genome.

methylation of multiple tumor suppressor genes in HCCs may contribute to the pathogenesis of this disease.30,31,32 And numerous studies have shown the potential clinical application value of aberrant methylation in HCC diagnosis and prognosis. 28, 33, 34 Promoter regions of tumor suppressor genes (such as E-cadherin, ACP1 and TRIM58) are reported to be aberrant hypermethylated in HCC tumor tissues.28, 33, 34 In this study, frozen hepatic cancerous or paracancerous tissues from a same patient with HCC were collected, and genomic DNA were extracted using standard methods. Three pairs of tissues were examined. The results presented from figure 5b to 5d clearly demonstrate that the methylation levels of these three genes were higher in tumor tissues compared with adjacent non-tumor tissues, consistent with previous studies. Compared with previously reported 5mC detection, this new strategy is simple and convenient, and it can detect methylation status of several CpG sites simultaneously. Without restrictions to specific primers or enzyme recognition sites, we can detect the CpG sites methylation level in any interested gene. CONCLUSIONS In summary, the current study developed a sensitive luminescence turn on approach for the vital epigenetic modification target namely 5mC detection using modified dGTP. This method can provide accurate quantitative information about methylation level at CpG sites. Owing to the high sensitivity of luciferase signaling, this method can be used in single specific site 5mC detection successfully. Meanwhile, the proposed assay can be used to determine the methylation levels of several CpG sites in genome, including cells and clinical tissues. Moreover，with a corresponding instrument， this strategy can be conducted in a 96 well plate, making it possible for analysis a large number of clinical samples simultaneous. Compared with the reported one,22 we believe this approach has the advantage to provide a quick and potentially highthroughput analysis of 5mC modification in tissues to gain more clinical information.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Eectrophoresis analysis of the primer extension reaction; synthesis of dGppppA; BGS analysis of genomic E-cadherin methylation levels from cancer cell lines MDA-MB-231, HepG2, and MCF-7.

AUTHOR INFORMATION Corresponding Author

Figure 5. (a) Methylation status of the E-cadherin suppressor gene from human cancer cell lines MDA-MB-231, HepG2, and MCF-7. (b-d) Methylation status of E-cadherin (b) TRIM58 (c) and ACP1 (d) from three pairs of tumor tissues or adjacent nontumor tissues, 1, 2 and 3 represent three patients. Error bar, mean ± SEM, n = 3. Detection of gene-specific methylation level in genomic DNA from hepatic cancerous or paracancerous tissue. Encouraged by the above results, we aimed to make practical application of this method in real disease samples. It has been reported that simultaneous detection of multiple CpG sites can enhance the accuracy of cancer diagnosis greatly.29 Several studies have suggested that

*[email protected] *[email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank the National Natural Science Foundation of China (21432008, 91753201 and 21721005 to X. Z.; 21778040, 21572172 to X. W.). Major new drug innovation in the Ministry of science and technology (2017ZX09303013).


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Analytical Chemistry

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(17) Zhao, Y.; Chen, F.; Li, Q.; Wang, L., Fan, C. Isothermal Amplification of Nucleic Acids Chem. Rev. 2015, 115, 12491−12545. (18) Herman, J. G.; Graff, J. R.; Myöhänen, S.; Nelkin, B. D.; Baylin, S. B. Methylation-Specific PCR: a Novel PCR Assay for Methylation Status of CpG Islands P. Natl. Acad. Sci. USA. 1996, 93, 9821-9826. (19) Wang, X.; Chen, F.; Zhang, D.; Zhao, Y.; Wei, J.; Wang, L.; Song, S.; Fan, C.; Zhao, Y. Single Copy-Sensitive Electrochemical Assay for Circulating Methylated DNA in Clinical Samples with Ultrahigh Specificity Based on a Sequential Discrimination-Amplification Strategy Chem. Sci. 2017, 8, 4764-4770.

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