Site-specific immunochemical methylation assessment from genome DNA utilizing a conformational difference between looped-out target and stacked-in nontarget methylcytosines
Site-specific immunochemical methylation assessment from genome DNA utilizing a conformational difference between looped-out target and stacked-in nontarget methylcytosines
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利用环出靶标和堆积非靶标甲基胞嘧啶之间的构象差异，对基因组 DNA 进行位点特异性免疫化学甲基化评估
DOI：
10.1016/j.bios.2015.03.061
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发表时间：
2015
影响因子：
12.6
通讯作者：
Hiroyuki Yanagisawa Kyoko Yoshioka and Osamu Niwa
中科院分区：
文献类型：
--
作者：
Ryoji Kurita;Hiroyuki Yanagisawa Kyoko Yoshioka and Osamu Niwa
我们报告了基因组DNA中甲基胞嘧啶的序列选择性免疫化学区分，我们利用选择性抗体结合在凸起区域的环出甲基胞嘧啶，而不使用亚硫酸氢盐处理，甲基化敏感限制性内切酶或PCR。首先，我们使用表面等离子体共振测量和微滴板研究了抗甲基胞嘧啶抗体对甲基胞嘧啶在完全匹配、突出、错配和基本位点的亲和力，以探索抗体与目标甲基胞嘧啶结合的差异。在凸起区域观察到最高的亲和力，与单链DNA的亲和力相当。特别是，在完全匹配位点没有观察到亲和力。这是因为在凸起的甲基胞嘧啶中没有氢键或π -π堆叠等相互作用，因此只能使凸起中的目标环出。将甲基化和未甲基化的基因组DNA混合，形成一个模型DNA，用来评估特定位点的甲基化比率。片段DNA与生物素化探针DNA杂交，其序列能够在目标处形成单个碱基凸起。探针的设计很简单，因为它只包括从一个完整的匹配序列中消除与目标胞嘧啶配对的鸟嘌呤。结果，我们成功地获得了免疫分析信号与特定位点在4 h内的甲基化比率之间的线性关系（r2=0.9962）。
We report the sequence-selective immunochemical discrimination of methylcytosine from genomic DNA that we achieved by utilizing selective antibody binding to a looped-out methylcytosine in a bulge region and without using bisulfite treatment, a methylation-sensitive restriction enzyme, or PCR. First, we investigated the affinity of an anti-methylcytosine antibody for methylcytosine in full match, bulge, mismatch and abasic sites using surface plasmon resonance measurement and a microtiter plate to explore the differences as regards antibody binding to the target methylcytosine. The highest affinity, which was comparable to that in a single strand of DNA, was observed in the bulge region. In particular, no affinity was observed in a full match site. This is because there is no interaction such as hydrogen bond or π–π stacking for the bulged methylcytosine, thus enabling only the target in the bulge to be looped out. Methylated and unmethylated genomic DNA were blended to form a model DNA with which to assess the methylation ratio at a specific site. Fragmented DNA was hybridized with a biotinylated probe DNA, which has a sequence capable of forming a single base bulge at the target. The probe design is simple because it consists solely of the elimination of guanine paired with the target cytosine from a full match sequence. As a result, we successfully obtained a linear relationship (r2=0.9962) between the immunoassay signal and the methylation ratio of a specific site within 4 h.