Wang Li-Juan, Han Xiao, Qiu Jian-Ge, Jiang BingHua, Zhang Chun-Yang
College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University Jinan 250014 China
Academy of Medical Sciences, Zhengzhou University Zhengzhou 450000 China
Chem Sci. 2020 Aug 25;11(35):9675-9684. doi: 10.1039/d0sc03240a.
DNA methylation at cytosine/guanine dinucleotide islands (CpGIs) is the most prominent epigenetic modification in prokaryotic and eukaryotic genomes. DNA methyltransferases (MTases) are responsible for genomic methylation, and their aberrant activities are closely associated with various diseases including cancers. However, the specific and sensitive detection of multiple DNA MTases has remained a great challenge due to the specificity of the methylase substrate and the rareness of methylation-sensitive restriction endonuclease species. Here, we demonstrate for the first time the cytosine-5 methylation-directed construction of a Au nanoparticle (AuNP)-based nanosensor for simultaneous detection of multiple DNA MTases at the single-molecule level. We used the methyl-directed endonuclease GlaI to cleave the site-specific 5-methylcytosine (5-mC). In the presence of CpG and GpC MTases (, M.SssI and M.CviPI), their hairpin substrates are methylated at cytosine-5 to form the catalytic substrates for GlaI, respectively, followed by simultaneous cleavage by GlaI to yield two capture probes. These two capture probes can hybridize with the Cy5/Cy3-signal probes which are assembled on the AuNPs, respectively, to form the double-stranded DNAs (dsDNAs). Each dsDNA with a guanine ribonucleotide can act as the catalytic substrate for ribonuclease (RNase HII), inducing recycling cleavage of signal probes to liberate large numbers of Cy5 and Cy3 molecules from the AuNPs. The released Cy5 and Cy3 molecules can be simply quantified by total internal reflection fluorescence (TIRF)-based single-molecule imaging for simultaneous measurement of M.SssI and M.CviPI MTase activities. This method exhibits good specificity and high sensitivity with a detection limit of 2.01 × 10 U mL for M.SssI MTase and 3.39 × 10 U mL for M.CviPI MTase, and it can be further applied for discriminating different kinds of DNA MTases, screening potential inhibitors, and measuring DNA MTase activities in human serum and cell lysate samples, holding great potential in biomedical research, clinical diagnosis, drug discovery and cancer therapeutics.
胞嘧啶/鸟嘌呤二核苷酸岛（CpGIs）处的DNA甲基化是原核生物和真核生物基因组中最显著的表观遗传修饰。DNA甲基转移酶（MTases）负责基因组甲基化，其异常活性与包括癌症在内的各种疾病密切相关。然而，由于甲基化酶底物的特异性和甲基化敏感限制性内切酶种类的稀少，多种DNA MTases的特异性和灵敏检测仍然是一个巨大的挑战。在此，我们首次展示了基于金纳米颗粒（AuNP）的纳米传感器的胞嘧啶-5甲基化导向构建，用于在单分子水平上同时检测多种DNA MTases。我们使用甲基导向的内切酶GlaI切割位点特异性的5-甲基胞嘧啶（5-mC）。在存在CpG和GpC MTases（M.SssI和M.CviPI）的情况下，它们的发夹底物在胞嘧啶-5处被甲基化，分别形成GlaI的催化底物，随后被GlaI同时切割产生两个捕获探针。这两个捕获探针可以分别与组装在AuNPs上的Cy5/Cy3信号探针杂交，形成双链DNA（dsDNAs）。每个带有鸟嘌呤核糖核苷酸的dsDNA可以作为核糖核酸酶（RNase HII）的催化底物，诱导信号探针的循环切割，从AuNPs上释放大量的Cy5和Cy3分子。释放的Cy5和Cy3分子可以通过基于全内反射荧光（TIRF）的单分子成像简单定量，用于同时测量M.SssI和M.CviPI MTase活性。该方法具有良好的特异性和高灵敏度，M.SssI MTase的检测限为2.01×10 U/mL，M.CviPI MTase的检测限为3.39×10 U/mL，并且可以进一步应用于区分不同种类的DNA MTases、筛选潜在抑制剂以及测量人血清和细胞裂解物样品中的DNA MTase活性，在生物医学研究、临床诊断、药物发现和癌症治疗中具有巨大潜力。
