Assay of DNA methyltransferase 1 activity based on uracil-specific excision reagent digestion induced G-quadruplex formation
Assay of DNA methyltransferase 1 activity based on uracil-specific excision reagent digestion induced G-quadruplex formation
复制标题
基于尿嘧啶特异性切除试剂消化诱导 G-四链体形成的 DNA 甲基转移酶 1 活性测定
DOI：
10.1016/j.aca.2017.07.021
复制
发表时间：
2017
影响因子：
6.2
通讯作者：
Xiang Yang
中科院分区：
文献类型：
--
作者：
Li Jinlong;He Guangwu;Mu Chaoli;Wang Keming;Xiang Yang
DNA甲基转移酶催化的DNA甲基化在基因转录、基因组印迹和细胞分化等多种生物学过程中发挥着重要作用。因此，利用尿嘧啶特异性切割试剂(USER)诱导的G-四链体的形成，成功地建立了一种新的、有效的检测DNA甲基转移酶1(DNMT1)活性的电化学方法。简单地说，将含有DNMT1识别序列的双链DNA固定在电极上。其中，一条链(DNAS1)含有富含G的序列和一个胞嘧啶碱基，而补链(DNAS2)含有富含C的序列和甲基化的胞嘧啶。通过DNMT1的活性，双链DNA的半甲基化CG识别序列被甲基化，DNA S2链被切割，随后用EpiTect快速亚硫酸氢盐转换试剂盒和用户处理后，留下DNA S1形成G-四链-氯化血红素DNAzyme用于信号扩增。在最佳条件下，该方法的线性范围为0.1U/mLDNMT1，检出限为0.06U/mLDNMT1。抑制实验表明，在160U/−M S-腺苷蛋氨酸存在下，−-1027对DNMT1的IC50值为6μM。由于该方法可以有效地检测人DNMT1活性，并已成功应用于复杂的生物样品，因此在DNA甲基化相关的临床实践和生化研究中具有很大的应用潜力。
DNA methylation catalyzed by DNA methyltransferase plays an important role in many biological processes including gene transcription, genomic imprinting and cellular differentiation. Herein, a novel and effective electrochemical method for the assay of DNA methyltransferase 1(DNMT1) activity has been successfully developed by using uracil-specific excision reagent (USER) induced G-quadruplex formation. Briefly, double stranded DNA containing the recognition sequence of DNMT1 is immobilized on the electrode. Among them, one strand (DNA S1) contains G-rich sequence and a cytosine base, while the supplement strand (DNA S2) cotains C-rich sequence and a methylated cytosine. Through the activity of DNMT1, the hemimethylated CG recognition sequence of the double stranded DNA are methylated and DNA S2 strand is cleaved and removed after the subsequently treatment with EpiTect fast bisulfite conversion kits and USER, leaving the DNA S1 to form the G-quadruplex-hemin DNAzyme for signal amplification. Under optimal-conditions, the method shows wide linear range of 0.1–40 U mL−1with a detection limit of 0.06 U mL−1. Furthermore, the inhibition assay study demonstrates that SGI-1027 can inhibit the DNMT 1 activity with the IC50 values of 6 μM in the presence of 160 μM S-adenosylmethionine. Since this method can detect human DNMT1 activity effectively and has successfully been applied in complex biological samples, it may have great potential in the applications in DNA methylation related clinical practices and biochemical researches.