Direct labeling of 5-methylcytosine and its applications
Direct labeling of 5-methylcytosine and its applications
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DOI：
10.1021/ja068660c
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发表时间：
2007-05-02
影响因子：
15
通讯作者：
Okamoto, Akimitsu
中科院分区：
文献类型：
--
作者：
Tanaka, Kazuo;Tainaka, Kazuki;Okamoto, Akimitsu
胞嘧啶甲基化是最重要的表观遗传事件之一，并且已经进行了大量努力来开发用于甲基胞嘧啶检测的简单反应。在本文中，我们描述了设计的标签可连接的配体直接甲基胞嘧啶标记和它们的应用荧光和电化学分析。初步研究了联吡啶取代基的位置对锇在甲基胞嘧啶上络合效率的影响。结果，在C4位具有取代基的联吡啶衍生物在含有锇酸钾和六氰合铁（III）酸钾的反应混合物中显示出在单链DNA的甲基胞嘧啶残基处的有效络合。基于该结果，合成了在C4位具有可附接标签的氨基接头的联吡啶衍生物。在锇酸盐和合成配体的存在下，金属络合物形成的效率明显地通过胞嘧啶的C5位置处的甲基的存在/不存在而改变。然后将功能标记单元的琥珀酰亚胺酯连接到固定在甲基胞嘧啶上的联吡啶配体上。这些标签连接到甲基胞嘧啶使我们能够检测目标甲基胞嘧啶在DNA中的荧光和电化学。例如，我们能够通过从杂交的荧光DNA探针到甲基胞嘧啶上的荧光标记的荧光共振能量转移来荧光测定地获得关于特定位点处的甲基化状态的信息。此外，通过电化学标记的甲基胞嘧啶和探针DNA修饰的电极的组合，观察到甲基胞嘧啶选择性的特征电流信号。这种甲基胞嘧啶的直接标记是一种新的甲基化检测方法，具有许多不同于传统方法的优点。
Cytosine methylation is one of the most important epigenetic events, and much effort has been directed to develop a simple reaction for methylcytosine detection. In this paper, we describe the design of tag-attachable ligands for direct methylcytosine labeling and their application to fluorescent and electrochemical assays. The effect of the location of bipyridine substituents on the efficiency of osmium complexation at methylcytosine was initially investigated. As a result, a bipyridine derivative with a substituent at the C4 position showed efficient complexation at the methylcytosine residue of single-stranded DNA in a reaction mixture containing potassium osmate and potassium hexacyanoferrate(III). On the basis of this result, a bipyridine derivative with a tag-attachable amino linker at the C4 position was synthesized. The efficiency of metal complex formation in the presence of the osmate and the synthetic ligand was clearly changed by the presence/absence of a methyl group at the C5 position of cytosine. The succinimidyl esters of functional labeling units were then attached to the bipyridine ligand fixed on the methylcytosine. These labels attached to methylcytosine enabled us to detect the target methylcytosine in DNA both fluorometrically and electrochemically. For example, we were able to fluorometrically obtain information on the methylation status at a specific site by means of fluorescence resonance energy transfer from a hybridized fluorescent DNA probe to a fluorescent label on methylcytosine. In addition, by the combination of electrochemically labeled methylcytosine and an electrode modified by probe DNAs, a methylcytosine-selective characteristic current signal was observed. This direct labeling of methylcytosine is a conceptually new methylation detection assay with many merits different from conventional assays.