College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, PR China.
Hunan Provincial Key Laboratory of Materials Protection for Electric Power and Transportation, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410004, Hunan, PR China.
Biosens Bioelectron. 2019 Feb 1;126:269-274. doi: 10.1016/j.bios.2018.10.054. Epub 2018 Oct 26.
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) 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 (IC) 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.
异常的 DNA 甲基化与癌症的发生和发展密切相关，因此，测定甲基转移酶活性并筛选其抑制剂的策略对于癌症的诊断和治疗至关重要。在这项工作中，通过双链 (ds)-DNA 共有序列的甲基化以及随后的 p53 蛋白识别，基于表面等离子体共振 (SPR) 的测定法被用于实时、灵敏地监测 DNA 甲基转移酶的活性并筛选其抑制剂。该共有 ds-DNA 具有特定的 5'-CCGG-3'序列，其中第二个 C 碱基可被 M.SssI 甲基转移酶 (M.SssI MTase) 甲基化，而甲基化过程会阻碍 HpaII 内切酶识别和切割 ds-DNA，因此，p53 蛋白的结合会导致显著的 SPR 信号。相反，通过一种有效的抑制剂抑制 M.SssI MTase 活性会使共有 ds-DNA 保持非甲基化状态，而 HpaII 对 ds-DNA 的切割会阻止 p53 蛋白吸附到芯片表面，导致微小的 SPR 信号。p53 蛋白与甲基化共有 ds-DNA 之间的结合亲和力 (K) 推断为 3.04 nM，证明了其强大的结合能力。两种核苷抑制剂 5-氮杂胞苷 (5-Aza) 和 5-氮杂-2'-脱氧胞苷 (5-Aza-dC)，以及一种非核苷抑制剂普鲁卡因被进行了检测，其半最大抑制浓度 (IC) 值与其他方法非常相似。该传感方案已成功用于测定正常和癌细胞裂解物中的 M.SssI MTase 活性。概念验证实验证明，SPR 可作为一种可行的方法，用于通过与甲基化特异性 ds-DNA 共有序列结合的 p53 蛋白来灵敏地检测甲基转移酶的活性并筛选其抑制剂。
