Label-free raman observation of TET1 protein-mediated epigenetic alterations in DNA
Label-free raman observation of TET1 protein-mediated epigenetic alterations in DNA
复制标题
无标记拉曼观察 TET1 蛋白介导的 DNA 表观遗传改变
DOI：
10.1021/acs.analchem.9b01004
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发表时间：
2019
影响因子：
7.4
通讯作者：
Yu Qiuming
中科院分区：
文献类型：
--
作者：
Luo Xiaojun;Jiang Lijuan;Kang Tuli;Xing Yingfang;Zheng Erjin;Wu Ping;Cai Chenxin;Yu Qiuming
众所周知，DNA的表观遗传修饰可以调节基因的活性和表达，并被认为会导致遗传性疾病，如癌症。在哺乳动物基因组中发现了四种修饰的胞嘧啶：5-甲基胞嘧啶(5mC)、5-羟甲基胞嘧啶(5hmC)、5-甲酰胞嘧啶(5fC)和5-羧基胞嘧啶(5caC)。它们被认为是DNA表观遗传标记，在调节DNA甲基化和去甲基化之间的动态平衡方面发挥着关键作用。虽然检测5mC的方法无处不在，但很少有检测方法同时检测所有四种修饰的胞嘧啶，以及监测它们的动态变化。在这里，我们发展了一种基于无标记表面增强拉曼光谱(SERS)的方法，利用具有良好控制的热点和开放表面的等离子体金纳米孔阵列(PGNA)作为衬底，直接检测这四种DNA修饰。该方法基于识别DNA碱基修饰引起的SERS光谱特征。我们的研究表明，5mC、5hmC、5fC和5caC分别在785、660、1450和1680 cm-1处有明显的拉曼光谱特征。此外，该方法还可用于跟踪Tet蛋白介导的DNA中这四种修饰胞嘧啶之间的动态变化。进一步证明了从5mC到5hmC、5fC和5caC的动态逐级转化是一个典型的三步连续反应，其速率常数分别为0.6、0.25和0.15min-1，这是以前用SERS方法所没有得到的。
Epigenetic modifications of DNA are known to modulate gene activity and expression and are believed to result in genetic diseases, such as cancer. Four modified cytosines were discovered in mammalian genomes: 5-methycytoine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). They are regarded as DNA epigenetic markers and play key roles in the regulation of the dynamic balance between DNA methylation and demethylation. Although detection approaches toward 5mC are ubiquitous, few assays have reported the simultaneous determination of all four modified cytosines as well as monitoring of their dynamic alterations. Here, we developed a label-free surface enhanced Raman spectroscopy (SERS)-based method for directly sensing the four DNA modifications by using a plasmonic gold nanohole array (PGNA) with well-controlled hot spots and an open surface as the substrate. This method is based on identifying SERS spectral features resulting from DNA base modifications. Our study shows that 5mC, 5hmC, 5fC, and 5caC exhibit distinct Raman spectroscopic signatures at 785, 660, 1450, and 1680 cm–1, respectively. Moreover, the developed method can be used for tracking of the dynamic alterations among these four modified cytosines in DNA mediated by the ten-eleven translocation (TET) protein. The dynamic stepwise conversion from 5mC into 5hmC, 5fC, and 5caC is further demonstrated to be a typical three-step consecutive reaction with rate constants of 0.6, 0.25, and 0.15 min–1, respectively, which has not been achieved before via a SERS-based method.