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A High-Affinity Methyl-CpG-Binding Protein for Endonuclease-Free and Label-Free DNA Methyltransferase Activity Detection

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Abstract

DNA methyltransferase (MTase) activity detection has received increasing attention as a promising biomarker and therapeutic target. However, most of these detection methods rely on endonuclease digestion and signal groups labeling. Herein, we present a novel platform for sensing DNA MTase activity that overcomes these limitations. Our approach is both endonuclease-free and label-free, utilizing a combination of a high-affinity streptavidin-methyl-CpG-binding domain (SA-MBD) protein and surface plasmon resonance (SPR) technology. The SA-MBD protein specifically recognizes a hairpin probe containing methylated CpG sites, which is treated with M.SssI MTase. This recognition event generates a corresponding SPR response signal. The limit of detection is as low as 0.016 U/mL, owing to the high-affinity of the SA-MBD protein. Notably, we have demonstrated the feasibility of our method for M.SssI MTase activity analysis in serum and inhibitor screening, which implies the potential prospects for biomedical research.

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Data availability

The data supporting the finding reported herein are available on reasonable request from the corresponding author.

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Acknowledgements

This work was funded by the National Natural Science Foundation of China (61901527, 82300051), National Key Research and Development Program of China (2022YFF0710803, 2022YFF0710800) and Fundamental Research Funds for the Central Universities (2632021ZD02).

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Author notes

  1. Yang Bai and Shulin Tan contributed equally to this work.

Authors and Affiliations

  1. Department of Biomedical Engineering, School of Engineering, China Pharmaceutical University, Nanjing, 210009, China

    Yang Bai, Shulin Tan, Yingsong Sheng, Yueqing Gu & Yunlong Liu

  2. National Center for Respiratory Medicine, Center of Respiratory Medicine, China-Japan Friendship Hospital, Beijing, 100029, China

    Baicun Li

  3. Department of Clinical Pharmacy, Jinling Hospital, State Key Laboratory of Analytical Chemistry for Life Science & Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing, 210002, China

    Haiping Wu

  4. School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China

    Haiping Wu

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  1. Yang Bai
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Correspondence to Haiping Wu, Baicun Li or Yunlong Liu.

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Bai, Y., Tan, S., Sheng, Y. et al. A High-Affinity Methyl-CpG-Binding Protein for Endonuclease-Free and Label-Free DNA Methyltransferase Activity Detection. J. Anal. Test. (2024). https://doi.org/10.1007/s41664-024-00306-1

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