title: Ultrasensitive homogeneous electrochemical strategy for DNA methyltransferase activity assay based on autonomous exonuclease III-assisted isothermal cycling signal amplification.
abstract: DNA methylation catalyzed by methyltransferase (MTase) plays an important role in many biological processes, including gene transcription, genomic imprinting and cellular differentiation. Herein, a simple and novel homogeneous electrochemical strategy for ultrasensitive DNA MTase activity assay has been successfully developed, which is based on methylation-triggered exonuclease (Exo) III-assisted autonomous isothermal cycling signal amplification. A duplex DNA (P1-P2 hybrid) containing the methylation-responsive sequence is ingeniously designed. In the presence of DNA adenine methylation (Dam) methyltransferase (MTase), P1-P2 hybrid is methylated and subsequently recognized and cleaved by Dpn I endonuclease, which triggers the Exo III-catalyzed autonomous cycling cleavage processes. Therefore, a large amount of methylene blue-labeled mononucleotides are released, generating a significantly amplified electrochemical signal toward the Dam MTase activity assay. The directly measured detection limit down to 0.004 U/mL is obtained, which is one or two orders magnitude lower than that of the approaches reported in literature. Since this assay is carried out in homogeneous solution phase under isothermal condition and sophisticated probe immobilization processes are avoided, it is very simple and easy to implement. Due to its advantages of ultrahigh sensitivity, excellent selectivity and simple operation, the as-proposed strategy has great potential in the applications in DNA methylation related clinical practices and biochemical researches.
authors.affiliation: College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, PR China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Liu
authors.affiliation: College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, PR China.
authors.forename: Ting
authors.initials: T
authors.lastname: Hou
authors.affiliation: College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, PR China.
authors.forename: Haiyin
authors.initials: H
authors.lastname: Li
authors.affiliation: College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, PR China. Electronic address: lifeng@qust.edu.cn.
authors.forename: Feng
authors.initials: F
authors.lastname: Li
mesh_terms: D003217:Conductometry / Q000295:instrumentation*; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D015254:DNA Modification Methylases / Q000032:analysis* / Q000737:chemistry*; D004789:Enzyme Activation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D008832:Microchemistry / Q000295:instrumentation; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004247:DNA; D015254:DNA Modification Methylases; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III
keywords: DNA adenine methyltransferase activity; Exonuclease III; Homogeneous electrochemical assay; Signal amplification