期刊
BIOSENSORS & BIOELECTRONICS
卷 126, 期 -, 页码 269-274出版社
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2018.10.054
关键词
SPR; Methyltransferase activity; Inhibitor screening; Methylation-specific ds-DNA consensus sites; P53 protein
类别
资金
- National Natural Science Foundation of China [21575166, 21876208, 21705166, 21545010, 31527803, 21305085]
- Open Research Fund of Henan Key Laboratory of Biomolecular Recognition and Sensing (Shangqiu Normal University) [HKLBRSK1701]
Abnormal DNA methylation is closely related to cancer initiation and progression, and strategies to assay methyltransferase activity and screen its inhibitors are essential for cancer diagnosis and therapy. In this work, surface plasmon resonance (SPR)-based assay for real-time and sensitive monitoring of DNA methyltransferase activity and screening of its inhibitors was conducted via methylation of double-stranded (ds)-DNA consensus sites and the follow-up p53 protein recognition. The consensus ds-DNA possesses a specific sequence of 5'-CCGG-3' in which the second C base can be methylated by M.SssI methyltransferase (M.SssI MTase) and the methylation process impedes the recognition and cleavage of the ds-DNA by HpaII endonuclease, thus, the attachment of p53 protein leads to remarkable SPR signals. In contrast, inhibition of M.SssI MTase activity by a potent inhibitor leaves the consensus ds-DNA unmethylated, and the cleavage of the ds-DNA by HpaII prevents p53 protein from adsorbing onto the chip surface, leading to tiny SPR signals. The binding affinity (K-D) between p53 protein and the methylated consensus ds-DNA was deduced to be 3.04 nM, evidencing the strong binding capability. Two nucleoside inhibitors of 5-Azacytidine (5-Aza) and 5-aza-2'-deoxycytidine (5-Aza-dC), and a non-nucleoside inhibitor of procaine were examined, and their half-maximal inhibiting concentration (IC50) values were highly comparable with those by other methods. The sensing protocol has been successfully utilized for the assay of M.SssI MTase activity in normal and cancer cell lysates. The proof-of-concept experiments demonstrate that SPR serves as a viable means for sensitive detection of methyltransferase activity and screening of its inhibitors using p53 protein bound to methylation-specific ds-DNA consensus sites.
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