title: Detection and discrimination of maintenance and de novo CpG methylation events using MethylBreak.
abstract: Understanding the principles governing the establishment and maintenance activities of DNA methyltransferases (DNMTs) can help in the development of predictive biomarkers associated with genetic disorders and diseases. A detection system was developed that distinguishes and quantifies methylation events using methylation-sensitive endonucleases and molecular beacon technology. MethylBreak (MB) is a 22-mer oligonucleotide with one hemimethylated and two unmethylated CpG sites, which are also recognition sites for Sau96I and SacII, and is attached to a fluorophore and a quencher. Maintenance methylation was quantified by fluorescence emission due to the digestion of SacII when the hemimethylated CpG site is methylated, which inhibits Sau96I cleavage. The signal difference between SacII digestion of both MB substrate and maintenance methylated MB corresponds to de novo methylation event. Our technology successfully discriminated and measured both methylation activities at different concentrations of MB and achieved a high correlation coefficient of R2=0.997. Additionally, MB was effectively applied to normal and cancer cell lines and in the analysis of enzymatic kinetics and RNA inhibition of recombinant human DNMT1.
authors.affiliation: Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li 320, Taiwan.
authors.forename: William
authors.initials: W
authors.lastname: Hsu
authors.affiliation: Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li 320, Taiwan; Department of Chemistry, Chung Yuan Christian University, Chung-Li 320, Taiwan.
authors.forename: Augustus T
authors.initials: AT
authors.lastname: Mercado
authors.affiliation: Graduate Institute of Medical Sciences and Department of Pharmacology, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110-31, Taiwan.
authors.forename: George
authors.initials: G
authors.lastname: Hsiao
authors.affiliation: Department of Chemistry, Chung Yuan Christian University, Chung-Li 320, Taiwan; Center for Nanotechnology and Institute of Biomedical Technology, Chung Yuan Christian University, Chung-Li 320, Taiwan. Electronic address: juiming@cycu.edu.tw.
authors.forename: Jui-Ming
authors.initials: JM
authors.lastname: Yeh
authors.affiliation: Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li 320, Taiwan; Center for Nanotechnology and Institute of Biomedical Technology, Chung Yuan Christian University, Chung-Li 320, Taiwan. Electronic address: cychen@cycu.edu.tw.
authors.forename: Chung-Yung
authors.initials: CY
authors.lastname: Chen
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D045744:Cell Line, Tumor; D018899:CpG Islands; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation; D006801:Humans; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human
keywords: DNA methyltransferases; Maintenance methylation; MethylBreak; Methylation-sensitive endonucleases; Molecular beacon; de novo methylation