Sensitive surface plasmon resonance detection of methyltransferase activity and screening of its inhibitors amplified by p53 protein bound to methylation-specific ds-DNA consensus sites

• Published in: Biosensors & bioelectronics 2019-02, Vol.126, p.269-274
• Main Authors: Xia, Yonghong, Wu, Ling, Hu, Yuqing, He, Yuhan, Cao, Zhong, Zhu, Xu, Yi, Xinyao, Wang, Jianxiu
• Format: Article
• Language: English
• Subjects: Inhibitor screening; Methylation-specific ds-DNA consensus sites; Methyltransferase activity; P53 protein; SPR
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2018.10.054

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 (KD) 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. •Real-time and sensitive SPR assay of DNA methyltransferase activity and screening of its inhibitors are proposed.•The method relies on the methylation of double-stranded (ds)-DNA consensus sites and the follow-up p53 protein recognition.•The strong binding capability between the methylated consensus ds-DNA and p53 protein is demonstrated.•The method serves as a viable means for screening of potent inhibitors of DNA methyltransferase.