4.8 Article

Ultrasensitive and Label-Free Detection of Multiple DNA Methyltransferases by Asymmetric Nanopore Biosensor

Journal

ANALYTICAL CHEMISTRY
Volume 94, Issue 10, Pages 4407-4416

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.1c05332

Keywords

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Funding

  1. National Natural Science Foundation of China [21904093, 21976129]
  2. Zhejiang Province Public Welfare Technology Application Research Project [LGF22B050001]
  3. Zhejiang Provincial Natural Science Foundation of China [LY22B050002, LY22B060001]

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In this study, an ultrasensitive and label-free method for detecting DNA adenine methyltransferase (Dam) and CpG methyltransferase (M.SssI) was developed using the nanopore technique coupled with DNA cascade signal amplification reactions. The method showed high sensitivity and excellent specificity for the detection of methyltransferases.
DNA methylation is catalyzed by a family of DNA methyltransferases that play crucial roles in various biological processes. Therefore, an ultrasensitive methyltransferase assay is highly desirable in biomedical research and clinical diagnosis. However, conventional assays for the detection of DNA methyltransferase activity often involve radioactive labeling, costly equipment, and laborious operation. In this study, an ultrasensitive and label-free method for detecting DNA adenine methyltransferase (Dam) and CpG methyltransferase (M.SssI) was developed using the nanopore technique coupled with DNA cascade signal amplification reactions. A hairpin DNA (HD) comprising of the methylation-responsive sequences was skillfully designed. In the presence of Dam methyltransferase, the corresponding recognition site of hairpin HD was methylated and specifically cleaved by DpnI endonuclease, thus forming a DNA fragment that induces the catalytic hairpin assembly and hybridization chain reaction (CHA-HCR). The generated products could be absorbed onto the Zr4+-coated nanopore, resulting in an ion current rectification signal change. Considering the high sensitivity of the nanopore and excellent specificity toward the recognition of methyltransferase/endonuclease, our developed method could detect both Dam and M.SssI methyltransferases in the same sensing platform. Furthermore, the designed nanopore sensor could realize the multiplex detection of Dam and M.SssI methyltransferases after integration with the cascaded INHIBIT-AND logic gate. This ultrasensitive methyltransferase assay holds great promise in the field of cancer diagnosis.

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