Highly sensitive homogeneous electrochemical assay for methyltransferase activity based on methylation-responsive exonuclease Ⅲ-assisted signal amplification

摘要

DNA methylation catalyzed by methyltransferase(MTase) plays a critical role in many biological processes. In this paper, a novel and highly sensitive homogeneous electrochemical assay was developed for the detection of DNA MTase based on methylation-responsive exonuclease Ⅲ-assisted signal amplification. Upon the action of MTase/endonuclease on hairpin probe 1(HP 1) containing the methylation-responsive sequence, single-stranded DNA segments are generated to hybridize with methylene blue(MB)-labeled hairpin probe 2(HP 2). Then the digestion of HP 2 from the blunt 3' terminus by exonuclease Ⅲ is activated, resulting in the release of MB-labeled mononucleotides and the complementary DNA segment which could hybridize with another HP 2 to initiate the signal amplification process. The MB-labeled mononucleotide, due to its less negative charge and smaller size, diffuses easily to the negatively charged indium tin oxide(ITO) electrode, generating an amplified electrochemical signal. The detection limit of the proposed assay was estimated to be 0.04 U/mL, which was better than or comparable to that of the biosensors previously reported. To the best of our knowledge, it is the first time to adopt exonuclease Ⅲ-assisted signal amplification for homogeneous electrochemical assay of MTase activity, and this strategy exhibits the advantages of high sensitivity as well as simplicity. Since this assay is carried out in a homogeneous solution phase instead of on an electrode/solution interface, sophisticated probe immobilization processes could be avoided. The as-proposed strategy exhibits promising potential for MTase functional studies and related researches.