title: Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay.
abstract: We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5'-CmCGG-3', to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05±0.01) ng mL(-1) (at a signal/noise of 3) of MBD2 with a linear range of 0.2-300 ng mL(-1) and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no requirement of bisulfite conversion, PCR amplification, radioisotope labeling, or separation.
authors.affiliation: Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, National and Local Joint Engineering Research Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210097, PR China.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Ji
authors.affiliation: Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, National and Local Joint Engineering Research Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210097, PR China.
authors.forename: Yingdan
authors.initials: Y
authors.lastname: Qian
authors.affiliation: Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, National and Local Joint Engineering Research Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210097, PR China.
authors.forename: Ping
authors.initials: P
authors.lastname: Wu
authors.affiliation: Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, National and Local Joint Engineering Research Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210097, PR China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, National and Local Joint Engineering Research Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210097, PR China. Electronic address: cxcai@njnu.edu.cn.
authors.forename: Chenxin
authors.initials: C
authors.lastname: Cai
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D006108:Graphite / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010087:Oxides / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; C115368:MBD2 protein; D010087:Oxides; D006108:Graphite; D004247:DNA; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases; D004262:DNA Restriction Enzymes
keywords: Deoxyribonucleic acid demethylase; Deoxyribonucleic acid methylation; Fluorescence quenching; Graphene oxide; Restriction endonuclease