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Simultaneous and Sensitive detection of multi-site 5methylcytosine including non-CpG sites at single-5mC-resolution Shuang Rao, Yuqi Chen, Tingting Hong, Zhiyong He, Shan Guo, Han Lai, Ge Guo, Yuhao Du, and Xiang Zhou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02625 • Publication Date (Web): 07 Oct 2016 Downloaded from http://pubs.acs.org on October 10, 2016

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Analytical Chemistry

Simultaneous and Sensitive detection of multimulti-site 55methylcytosine including nonnon-CpG sites at singlesingle-5mC5mC-resolution Shuang Rao, Yuqi Chen, Tingting Hong, Zhiyong He, Guo Shan, Han Lai, Ge Guo, Yuhao Du*, Xiang Zhou* College of Chemistry and Molecular Sciences, Institute of Advanced Studies, Key Laboratory of Biomedical Polymers of Ministry of Education, Wuhan University, Wuhan 430072, Hubei Province, China ABSTRACT: The methylation status of multiplexed methylcytosine sites can be simultaneously monitored by ligationdepended PCR assay. The ability of quantitative detection of multiplexed sites in one PCR reaction makes it a good choice for detecting methylation at both CpG and non-CpG sites for research and diagnosis of disease compared with others. The assay can determine as low as 20 aM methylated DNA and has been successfully applied to the genomic DNA sample derived from cancer cell lines.

Epigenetic modifications of DNA have been recognized as a common hallmark of human disease, including cancer. DNA methylation is one epigenetic mechanism that involves 5-methylcytosine (5mC) in cytosine/guanine dinucleotides (CpG), particularly in CpG islands, which are located in or near the promoter region of many genes that function to modulate cellular processes, including embryonic development, transcription, chromatin structure, X-chromosome inactivation, genomic imprinting and chromosome stability.1-3 Recently, DNA methylation of non-CpG cytosines (CpA, CpT, CpC) has been reported, and accumulating evidence indicates there is a potential functional significance of non-CpG methylation.4-5 For example, studies have reported that in human mature oocytes, there are significant levels of non-CpG methylation within gene bodies in the genome, which is correlated with the level of expression of the corresponding genes.6 More recently, there have been reports identifying a possible mechanism whereby mCH may regulate neuronal gene expression through its recognition by methyl CpG binding protein 2 (MeCP2), which is implicated in the neurological disorder Rett syndrome.7 To date, the mechanism by which non-CpG methylation controls transcription has not been identified but has drawn considerable interest among both researchers and clinicians. The recent development of next-generation DNA sequencing technology has enabled global mapping of methylation patterns at single-base resolution. The analyses of DNA methylation patterns have illustrated that specific cancers are associated with aberrant methylation patterns (hypermethylation and hypomethylation) in promoter regions of multiple genes.8 Furthermore, studies have shown that the simultaneous testing of multiple CpG sites can greatly enhance the accuracy of cancer diagnosis.9 Therefore, in a genomic sample, the multiplexed and sensitive detection of alterations in DNA methylation, including non-CpG sites, is essential for research, diagnosis or prognosis of disease.

teins such as methyl-binding domain (MDB) or antibodies raised against 5mC.10-12 However, most of these methods only allow the study of restricted sites due to the limited availability of the enzyme-recognition sites. Most importantly, to detect DNA methylation, these approaches only allow the analysis of methylation of one site at a time. Sodium bisulfite treatment converts unmethylated cytosines into uracil, whereas 5mC remains unaffected. Therefore, bisulfite treatment can effectively differentiate between the unmethylated and methylated cytosine residues in genomic DNA and is now regarded as the goldstandard method for 5mC detection. By combining bisulfate treatment and high-throughput sequencing, a methylation map can be generated to single-base resolution across the entire genome.6 While this approach is excellent for researchers, simpler and more convenient methods to detect genomic methylation for routine diagnostics are still lacking due to the high cost and high levels of background noise of next generation sequencing. Given the demands of simultaneous detection of multiplexed methylation sites at single-5mC-resolution in a genomic DNA sample, we have developed a novel ligation-depended PCR assay. Through the amplification of bisulfite-treated DNA targets using PCR, methylated cytosines can be simultaneously detected at single-5mCresolution with high sensitivity. The methylated DNA target can be quantitatively determined as low as 20 aM. More importantly, by encoding the ligation probe with a unique length for each methylation site, the different methylated sites can be distinguished based on the length of the PCR products. Therefore, the simultaneous analysis of multiplexed DNA methylation sites, including nonCpG sites, can be determined in a simple PCR reaction. In this study, we show that this novel technique can be applied successfully to DNA samples derived from HepG-2 and MCF-7 cell lines.

Early approaches to detect DNA methylation involved methylation sensitive restriction enzymes, pro-

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strate. To improve the specificity of the ligation, a guanine was placed at the 3’ end of ligation probe to reduce the tolerance of the ligase.13 Each ligated probe pair has a unique length that acts as a “size code” for its target. All ligated probes have identical sequences at the 5’ and 3’ ends, which permit simultaneous amplification in one PCR reaction with only one PCR primer pair. What’s more, compared with traditional PCR-depended methods for the detection of DNA methylation which need careful design of PCR primes, the use of uniform end in the probe pair ensure the success of the PCR primes used in the assay. Then, the ligated probe pairs with different lengths can be amplified using PCR and the PCR products are analyzed using gel electrophoresis. Therefore, different methylated sites will produce PCR products with different lengths. When the target site is not methylated, no amplification product is observed.

EXPERIMENTAL SECTION Materials. Ligase-65 enzyme was purchased from MRC-Holland and CpG Methyltransferase from New England Biolabs. All water used in this study was sterilized and deionized. HPLC purified DNA probes and ULTRAPAGE purified DNA oligonucleotides were obtained from Shanghai Sangon Biotech (Shanghai, China). The sequences of all synthetic oligodeoxynucleotides used in the study were listed in Table S1. Plasmid pUC 19 DNA was purchased from Fermentas. PCR mix and General AllGen Kit was purchased from CWBIO. All other reagents were of analytical reagent grade and were used without further purification. The PCR reaction was carried out in a Biometra Thermocycler(Bio-Rad,USA). Polyacrylamide gel electrophoresis products were scanned with Pharos FX Molecular imager(Bio-Rad,USA). Figure 1 The principle of the ligation-depended PCR assay to detect multiplexed methylcytosine sites in a region of interest DNA.(a)The assay for single site (b)The assay for multiple site

The general outline of our strategy for simultaneous detection of multiplexed methylated cytosines in genomic DNA samples is shown in Figure 1. First, the DNA sample was treated with sodium bisulfite after denaturation, which converts the unmethylated cytosines to uracil but has no effect on 5 mC. Then, we added the ligation probe pairs, which consisted of two oligonucleotides that can be ligated to each other when adjacently hybridized to the specific sequence of genomic DNA at the methylated site. One probe contains a guanine at the 3’ end to differentiate between the methylated and unmethylated DNA. Finally, the ligation probe-DNA complex was simultaneously ligated in the presence of a DNA ligase. The DNA ligase used in this study is sensitive to DNA mismatch, and only the methylated target DNA that correctly matches the probe pair can be ligated. The selectivity of our method is achieved not only by the specific hybridization of the ligation probe pairs with methylated DNA but also by the highly discriminative ligation activity of the ligase toward the perfectly matched sub-

Genomic DNA extraction and pUC 19 DNA methylation. HepG-2 and MCF-7 were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 5% penicillin-streptomycin. Genomic DNA were extracted and purified by CWBIO Genomic DNA kit according to the manufacturer’s instruction. Plasmid pUC 19 DNA was linearized with AlwNI endonuclease and then methylated with methyltransferase. Bisulfite treatment. About 1 µg DNA in a final volume of 10 µL was denatured by 0.2 M NaOH for 10 minutes at 37 °C for 10 minutes. 6 µL of 10 mM hydroquinone (Sigma) and 104 µL of 3 M sodium bisulfite (Sigma) at pH 5, both freshly prepared, were added and mixed, and samples were incubated under mineral oil at 50 °C for 16 hours. Modified DNA was desalted by Millipore membranes for three times according to the manufacturer (Promega) and eluted into 50 µL of water. Modification was completed by NaOH (final concentration, 0.3 M) treatment for 15 minutes at 37 °C, followed by desalting with Millipore membranes for three times. DNA was resuspended in water and used immediately or stored at 20 °C. Ligation specificity of Ligase-65 enzyme. Short single strand DNA oligonucleotides, ligation probe pair


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(each final concentration of 10 nM) were mixed with 20 aM target DNA in Tris-HCl buffer (200 mM, pH 7.4) in a final volume of 20 µL. The mixture was heated to 95 °C for 5 minutes and then cooled to room temperature in a thermal cycler. After annealing, ligation was performed by diluting samples to 40 µL with dilution buffer, which contain 1 µL ligation-65 enzyme, 3 µL ligase buffer A and 3 µL ligase buffer B, and incubation at 54 °C for 15 minutes. The ligase enzyme was inactivated by heating at 98 °C for 5 minutes. Then, the reaction mixture was analyzed through denatured polyacrylamide electrophoresis gel. Ligation-depended PCR. For short single strand DNA oligonucleotides, ligation probe pair (each final concentration of 10 nM) was mixed with 20 aM target DNA in Tris-HCl buffer (200 mM, pH 7.4) in a final volume of 20 µL. The mixture was heated to 95 °C for 5 minutes and then cooled to room temperature in a thermal cycler. For pUC 19 DNA and genomic DNA ， appropriate amount of DNA samples were diluted to 10 µL and were heated at 98 °C for 5 minutes in 200 µL tubes in a thermalcycler with a heated lid. After addition of 1.5 µL salt solution mixed with 1.5 µL probe mix, samples were heated at 95 °C for 1 minute and then incubated for 16 hours at 60 °C. For both the annealed nucleotide, ligation was performed by diluting samples to 40 µL with dilution buffer, which contain 1 µL ligation-65 enzyme, 3 µL ligase buffer A and 3 µL ligase buffer B, and incubation at 54 °C for 15 minutes. The ligase enzyme was inactivated by heating at 98 °C for 5 minutes. 23 µL of the ligation reaction, 25 µL PCR mix and 1 µL for PCR primer pairs were mixed. PCR was for 20 cycles (30 s at 95 °C, 30 s at 61 °C, 35 s at 72 °C) for short single strand DNA and 30 cycles (30 s at 95°C, 30 s at 61 °C, 35 s at 72 °C) for pUC19 DNA and genomic DNA. PCR reactions were analyzed through polyacrylamide gel electrophoresis (PAGE).

RESULTS AND DISCUSSION Ligation specificity. Before the analysis of the methylation site, we have tested the specificity of ligation. The ligation under optimized conditions is sensitive to single base pair mismatch (Figure S1). Only the ligation of base pair hybridized to bisulfite treated methylated target which was fully matched with probe pair produced the ligation product. For the unmethylated target, one mismatch was located at the 3’ end of ligation probe, no ligation product was formed. Considering G-U mismatch is the most stable mismatches. The target sequence containing a G-U mismatch at the ligation site for the DNA ligase was used. As a result, clearly, no ligation product was observed for the G-U mismatch (Figure S2). DNA sequences with one methylation site. To test the feasibility of our method, we began with synthetic oligonucleotides containing one methylated cytosine. The DNA samples were subjected to bisulfite treatment using standard protocols, followed by ligation and PCR using a ligation probe and PCR primers. As shown in Figure 2, the

corresponding PCR band with desired length was produced using 20 aM methylated DNA (lane 2), and no PCR product was observed for the unmethylated DNA (lane 3), which suggests that this method can be successfully used to distinguish methylated DNA from unmethylated DNA. When the guanine at the 3’ end of the ligation probe was changed to adenine, which is complementary to the unmethylated DNA site, a PCR product was only observed for unmethylated DNA (lane 4). Sometimes, PCR yields non-specific products which may cause some trouble for the experiment. Because each probe pair has a unique length that acts as a “size code” for its target 5mC site in this assay, we can distinguish the desired PCR product for the target from other non-specific bands, compared to the DNA marker.

Figure 2 Non-denaturing polyacrylamide gel electrophoresis of DNA sequences with one methylation site staining with Gel-Red. Lane 1, DNA ladder marker; Lane 2-3, ligation probe 1 with a guanine at the 3’ end was used as the probe pair; lanes 4–5, ligation probe 2 with a adenine at the 3’ end was used as the probe pair. Lane 6, H2O.

Generally, the conversion efficiency of unmethylated cytosines into uracil is not 100%. In order to further investigate the effect of C-A mismatch caused by the unconverted cytosine after bisulfite treatment, cytosine was introduced to different site in the target oligodeoxynucleotide considering the unconverted cytosine should be evenly spaced in the DNA. As shown in Figure S3, when the unconverted cytosines are not close to the 3’ end of ligation probe, only a small decreasement of the PCR product (about 5%) was observed. Considering the high conversion efficiency of unmethylated cytosines into uracil and slight effect of the C-A mismatch, the conversion efficiency will not cause considerable deviation in our assay even it is not 100%. It is important to evaluate this capability of this assay to detect aberrant methylation in real samples. Therefore, we prepared a series of artificial mixtures composed of methylated and unmethylated DNA at different ratios to determine the methylation level. Samples comprised of 0%, 25%, 50%, 75% and 100% methylated DNA were diluted with unmethylated DNA to a total concentration of 100 aM. All samples were treated with bisulfate and analyzed under the same conditions described above. As shown in Figure, as the proportion of methylated DNA in the mixture increased, there was a parallel increase in the integrated density volume of the corresponding bands.


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Figure 3(b) demonstrates there was a linear relationship between the measured methylation level and the actual input methylation level. This demonstrates the successful application of this ligation-depended PCR assay for the simple and sensitive detection of methylation changes.

Figure 3 (a) Non-denaturing PAGE analysis of mixed samples with diverse proportions of methylated DNA staining with Gel-Red. (b) Integrated density volumes of DNA bands plotted with respect to methylation level, and a linear model fits the experimental data well, R2 =0.97769 .

Plasmid DNA pUC19 modified with multiple 5mC sites. To further confirm the capability of our method for multiplexed methylation sites, we applied the assay to detect methylated and unmethylated sites in plasmid DNA (pUC19). Cytosine methylation was achieved using HhaI methyltransferase (M.HhaI) and MspI methyltransferase (M.MspI), which methylated the first cytosine residue in CG and CC sequences, respectively. After the reaction, methylation of specific cytosines was confirmed using restriction enzyme digestion with R.HhaI and R.MspI (Figure S4). Three methylated CpG sites, one methylated non-CpG site (CpC) and two additional unmethylated cytosine sites in the plasmid DNA were chosen as the targets for detection. Six pairs of ligation probe were designed with different lengths for the six detection targets (87, 98, 112, 128, 146 and 166 nucleotides). As depicted in Figure 4, after ligation and one PCR reaction, the ladder-type bands produced by the different methylated DNA sites were clearly separated and matched the mobility of the corresponding fragments in the DNA ladder, but no bands were observed for the unmethylated sites. These results suggest that this method can be used to detect multiplexed DNA methylation sites with excellent specificity at single-5mC-resolution.

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probes may be amplified with slightly different efficiencies, the PCR product from each detection site will reflect its methylation status.

Figure 4 Non-denaturing polyacrylamide gel electrophoresis of plasmid DNA pUC19 used as the detection target. The DNA pUC19 was modified with HhaI methyltransferase (M.HhaI) and MspI methyltransferase (M.MspI) previously to obtain the methylcytosines sites. The red dots indicate the position of the 5mC in the plasmid; the blue dots indicate the position of the normal cytosines in the plasmid. Differ-

ent pairs of ligation probe were designed with different lengths for the six detection sites (87, 98, 112, 128, 146 and 166 bp) Genomic DNA extracted from cell lines. Finally, to demonstrate the application on more complex biological samples, we extended our method to investigate the methylation status of three CpG sites in the E-cadherin promoter region (a tumor suppressor gene) in the human cancer cell lines, HepG-2 and MCF-7. Previous studies have indicated that in HepG-2 cells, E-cadherin is silenced by hypermethylation of CpG islands of the Ecadherin promoter. Moreover, CpG sites in the Ecadherin promoter are unmethylated in MCF-7 cells.15-17 Genomic DNA extracted from these cell lines was subjected to bisulfite treatment and ligation-depended PCR amplification under the optimum conditions. Accordingly, ligation specific probes were designed with different lengths (87, 98 and 112 nucleotides). As shown in Figure 5, the corresponding PCR products with the expected lengths were generated after amplification, and primers were clearly observed in the HepG-2 cells, which indicated that the E-cadherin tumor suppressor gene was hypermethylated in the cells. However, DNA from the MCF-7 cells showed no evidence of methylation in the assay, and no DNA bands were observed during PAGE analysis, which is consistent with previous reports.

PCR efficiency depends on the amplified regions. Unlike the widely used methylation-specific PCR methods for the detection of methylation, it is the ligated probe pair that is amplified, not the DNA sequence in the sample. The DNA sequence derived from target comprises only a small part of PCR amplified region, about 45 bp. The larger part of PCR amplified region is random sequence in the probe, acting as a “size code” for its target. This random sequence is determined by ourselves and normally has an average G-C density. Although different


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Figure 5 Non-denaturing polyacrylamide gel electrophoresis of genomic DNA extracted from human cancer cell lines HepG-2 cell and MCF-7 cell. Lane 1, DNA ladder marker; Lane 2, H2O; Lane 3, genomic DNA extracted from HepG2 cell; Lane 4, genomic DNA extracted from MCF-7 cell.

Methylation status of three CpG sites was detected with this assay. Three pairs of ligation probe were designed with different lengths for the three detection sites (87, 98 and 112 bp)

CONCLUSIONS In this study, we have developed a quantitative method for simultaneous detection of multiplexed methylcytosine sites. As a result, this ligation-depended PCR assay exhibits excellent specificity and high sensitivity with a detection limit of 20 aM, and it can detect multiplexed methylation sites including non-CpG sites at single-5mC-resolution in a single PCR reaction. No specific regions with sufficient methylation density are needed for primer design, reducing false-positive and falsenegative results compared with MSP-associated PCR methods. The method was also extended to cell line derived samples to demonstrate its feasibility as a diagnostic tool. Furthermore, avoiding the use of restriction enzymes, radioactive materials and labeled primers facilitates the determination of methylation status. The use of ligation-depended PCR assay to evaluate methylation status may be beneficial for further applications in the study of epigenetic modification and early diagnosis of methylation related diseases.

ASSOCIATED CONTENT

AUTHOR INFORMATION

Supporting Information

Corresponding Author

Table S1, sequences of DNA used in this study; Figures S1− S4, polyacrylamide gel electrophoresis for the ligation assay and ligation-depended PCR assay

E-mail: [email protected] E-mail: [email protected]


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Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT The authors thank the National Science Foundation of China (No. 21432008, 21672167, 21402147, 81373256), the National Basic Research Program of China (973 Program) (2012CB720600, 2012CB720603)

REFERENCES (1) Bird, A., Genes & development 2002, 16 (1), 6-21. (2) Li, E.; Beard, C.; Jaenisch, R., Nature 1993, 366 (6453), 3625. (3) Lister, R.; Pelizzola, M.; Dowen, R. H.; Hawkins, R. D.; Hon, G.; Tonti-Filippini, J.; Nery, J. R.; Lee, L.; Ye, Z.; Ngo, Q. M.; Edsall, L.; Antosiewicz-Bourget, J.; Stewart, R.; Ruotti, V.; Millar, A. H.; Thomson, J. A.; Ren, B.; Ecker, J. R., Nature 2009, 462 (7271), 315-22. (4) Lee, T. F.; Zhai, J.; Meyers, B. C., Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (20), 9027-8. (5) Patil, V.; Ward, R. L.; Hesson, L. B., Epigenetics 2014, 9 (6), 823-8. (6) Guo, H.; Zhu, P.; Yan, L.; Li, R.; Hu, B.; Lian, Y.; Yan, J.; Ren, X.; Lin, S.; Li, J.; Jin, X.; Shi, X.; Liu, P.; Wang, X.; Wang, W.; Wei, Y.; Li, X.; Guo, F.; Wu, X.; Fan, X.; Yong, J.; Wen, L.; Xie, S. X.; Tang, F.; Qiao, J., Nature 2014, 511 (7511), 606-10. (7) a)Gabel, H. W.; Kinde, B.; Stroud, H.; Gilbert, C. S.; Harmin, D. A.; Kastan, N. R.; Hemberg, M.; Ebert, D. H.; Greenberg, M. E., Nature 2015, 522 (7554), 89-93; b) Chen, L.; Chen, K.; Lavery, L. A.; Baker, S. A.; Shaw, C. A.; Li, W.; Zoghbi, H. Y., Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (17), 5509-14. (8) Esteller, M., Nature reviews. Genetics 2007, 8 (4), 286-98. (9) Yang, Q.; Dong, Y.; Wu, W.; Zhu, C.; Chong, H.; Lu, J.; Yu, D.; Liu, L.; Lv, F.; Wang, S., Nat. Commun. 2012, 3, 1206. (10) Nair, S. S.; Coolen, M. W.; Stirzaker, C.; Song, J. Z.; Statham, A. L.; Strbenac, D.; Robinson, M. D.; Clark, S. J., Epigenetics 2011, 6 (1), 34-44. (11) Warton, K.; Lin, V.; Navin, T.; Armstrong, N. J.; Kaplan, W.; Ying, K.; Gloss, B.; Mangs, H.; Nair, S. S.; Hacker, N. F.; Sutherland, R. L.; Clark, S. J.; Samimi, G., BMC genomics 2014, 15, 476. (12) Wee, E. J.; Ha Ngo, T.; Trau, M., Sci. Rep. 2015, 5, 15028. (13) Herman, J. G.; Graff, J. R.; Myohanen, S.; Nelkin, B. D.; Baylin, S. B., Proc. Natl. Acad. Sci. U. S. A. 1996, 93 (18), 9821-6. (14) Schouten, J. P.; McElgunn, C. J.; Waaijer, R.; Zwijnenburg, D.; Diepvens, F.; Pals, G., Nucleic Acids Res. 2002, 30 (12), e57. (15) Hiraguri, S.; Godfrey, T.; Nakamura, H.; Graff, J.; Collins, C.; Shayesteh, L.; Doggett, N.; Johnson, K.; Wheelock, M.; Herman, J.; Baylin, S.; Pinkel, D.; Gray, J., Cancer Res. 1998, 58 (9), 1972-7. (16) Kawasaki, H.; Taira, K., Nature 2004, 431 (7005), 211-7. (17) Hong, T.; Wang, T.; He, Z.; Ma, J.; Xiao, H.; Huang, J.; Mao, W.; Zhou, X., Chem. Commun. 2014, 50 (50), 6653-5.


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