title: Quencher-free fluorescence strategy for detection of DNA methyltransferase activity based on exonuclease III-assisted signal amplification.
abstract: This work demonstrates a novel method for DNA methyltransferase (MTase) activity detection with a quencher-free molecular beacon (MB) probe based on exonuclease (Exo) III-assisted signal amplification. In the presence of Dam MTase and DpnI endonuclease, the elaborately designed hairpin substrate (MB1) was cleaved into two parts (part A and part B). Exo III can then digest part A and release a single-stranded target of the 2-aminopurine-labeled MB (MB2). Subsequently, the MB2 can hybridize with its target to form a double-stranded structure with a protruding 3'-terminus and then trigger the digestion of MB2 by Exo III. During the digestion of MB2, the 2-aminopurine is separated from the DNA strands and released free in solution, inducing an increase of the fluorescent signal. Owing to the presence of a recessed 3'-terminus in the formed double-stranded DNA, Exo III-assisted recyclable cleavage of MB2 was achieved. Therefore, an amplified fluorescence signal was observed. Under the optimized conditions, Dam MTase can be detected in the range of 0.2-40 units/mL with a limit of detection of 0.2 units/mL and good selectivity. Furthermore, the present assay can be used for screening potential DNA MTase inhibitors. Graphical Abstract A quencher-free fluorescence assay for sensitive detection of DNA methyltransferase activity based on exonuclease III-assisted signal amplification is reported.
authors.affiliation: State Key Laboratory of Medical Genetics & School of Life Sciences, Central South University, Changsha, Hunan, 410013, China.
authors.forename: Haisheng
authors.initials: H
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Medical Genetics & School of Life Sciences, Central South University, Changsha, Hunan, 410013, China. macb2012@csu.edu.cn.
authors.forename: Changbei
authors.initials: C
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Medical Genetics & School of Life Sciences, Central South University, Changsha, Hunan, 410013, China.
authors.forename: Meijuan
authors.initials: M
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Medical Genetics & School of Life Sciences, Central South University, Changsha, Hunan, 410013, China.
authors.forename: Hanchun
authors.initials: H
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Medical Genetics & School of Life Sciences, Central South University, Changsha, Hunan, 410013, China.
authors.forename: Hailun
authors.initials: H
authors.lastname: He
authors.affiliation: State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, Hunan, 410081, China.
authors.forename: Kemin
authors.initials: K
authors.lastname: Wang
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry; D019175:DNA Methylation; D015342:DNA Probes / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D004789:Enzyme Activation; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D005240:Feasibility Studies; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D015342:DNA Probes; D015075:2-Aminopurine; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: 2-Aminopurine probe; DNA methyltransferase; Exonuclease III-assisted signal amplification; Quencher-free