Yamayoshi Asako, Matsuyama Yohei, Kushida Mikihiko, Kobori Akio, Murakami Akira
Department of Biomolecular Engineering, Kyoto Institute of Technology, Matsugasaki, Sakuo-ku, Kyoto, Japan.
Photochem Photobiol. 2014 May-Jun;90(3):716-22. doi: 10.1111/php.12232. Epub 2014 Jan 21.
DNA methylation and demethylation significantly affect the deactivation and activation processes of gene expression significantly. In particular, C-5-methylation of cytosine in the CpG islands is important for the epigenetic modification in genes, which plays a key role in regulating gene expression. The determination of the location and frequency of DNA methylation is important for the elucidation of the mechanisms of cell differentiation and carcinogenesis. Here we designed a psoralen-conjugated oligonucleotide (PS-oligo) for the discrimination of 5-methylcytosine (5-mC) in DNA. The cross-linking behavior of psoralen derivatives with pyrimidine bases, such as thymine, uracil and cytosine has been well discussed, but there are no reports which have examined whether cross-linking efficiency of psoralen with cytosine would be changed with or without C-5 methylation. We found that the cross-linking efficiency of PS-oligo with target-DNA containing 5-mC was greatly increased compared to the case of target-DNA without 5-mC, approximately seven-fold higher. Here we report a new aspect of the photocross-linking behavior of psoralen with 5-mC that is applicable to a simple, sequence-specific and quantitative analysis for the discrimination of 5-mC in DNA, which can be applicable to study the epigenetic behavior of gene expressions.
DNA甲基化和去甲基化对基因表达的失活和激活过程有显著影响。特别是，CpG岛中胞嘧啶的C-5甲基化对于基因的表观遗传修饰很重要，它在调节基因表达中起关键作用。确定DNA甲基化的位置和频率对于阐明细胞分化和致癌机制很重要。在此，我们设计了一种补骨脂素缀合的寡核苷酸（PS-寡核苷酸），用于区分DNA中的5-甲基胞嘧啶（5-mC）。补骨脂素衍生物与嘧啶碱基（如胸腺嘧啶、尿嘧啶和胞嘧啶）的交联行为已得到充分讨论，但尚无研究考察补骨脂素与胞嘧啶的交联效率在胞嘧啶C-5甲基化与否的情况下是否会发生变化。我们发现，与不含5-mC的靶DNA相比，PS-寡核苷酸与含5-mC的靶DNA的交联效率大大提高，约高7倍。在此，我们报道了补骨脂素与5-mC光交联行为的一个新方面，它适用于对DNA中5-mC进行简单、序列特异性和定量分析，可用于研究基因表达的表观遗传行为。
