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

•A novel and facile homogeneous electrochemical assay for highly sensitive DNA methyltransferase (MTase) activity detection has been developed.•This signal-on electrochemical biosensing platform for MTase activity assay is the first attempt to combine the homogeneous electrochemical strategy and the methylation-responsive exonuclease III-assisted signal amplification.•This immobilization-free MTase activity assay is carried out in homogeneous solution and easy to implement.

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.

Graphical abstractA novel and highly sensitive homogeneous electrochemical biosensor was proposed for the detection of DNA MTase activity based on methylation-responsive exonuclease III-assisted signal amplification. Methylene blue-labeled mononucleotides can diffuse easily to the negatively charged ITO electrode due to its less negative charge and smaller size, which is responsible for a measurable increase of the redox signal.Figure optionsDownload full-size imageDownload as PowerPoint slide