School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura-machi, Hachioji, Tokyo, 192-0982, Japan.
Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, 2-10-1 Ookura, Setagaya, Tokyo, 157-0074, Japan.
Sci Rep. 2018 Feb 15;8(1):3116. doi: 10.1038/s41598-018-21514-7.
G-quadruplex (G4) is a DNA secondary structure that has been found to play regulatory roles in the genome. The identification of G4-forming sequences is important to study the specific structure-function relationships of such regions. In the present study, we developed a method for identification of G4 clusters on genomic DNA by high-throughput sequencing of genomic DNA amplified via whole-genome amplification (WGA) in the presence of a G4 ligand. The G4 ligand specifically bound to G4 structures on genomic DNA; thus, DNA polymerase was arrested on the G4 structures stabilised by G4 ligand. We utilised the telomestatin derivative L1H1-7OTD as a G4 ligand and demonstrated that the efficiency of amplification of the G4 cluster regions was lower than that of the non-G4-forming regions. By high-throughput sequencing of the WGA products, 9,651 G4 clusters were identified on human genomic DNA. Among these clusters, 3,766 G4 clusters contained at least one transcriptional start site, suggesting that genes are regulated by G4 clusters rather than by one G4 structure.
四链体（G4）是一种 DNA 二级结构，已被发现可在基因组中发挥调控作用。鉴定形成 G4 的序列对于研究这些区域的特定结构-功能关系非常重要。在本研究中，我们开发了一种通过在存在 G4 配体的情况下对全基因组扩增（WGA）扩增的基因组 DNA 进行高通量测序来鉴定基因组 DNA 上 G4 簇的方法。G4 配体特异性结合到基因组 DNA 上的 G4 结构上；因此，DNA 聚合酶在 G4 配体稳定的 G4 结构上被阻止。我们利用端粒酶抑制剂衍生的 L1H1-7OTD 作为 G4 配体，并证明 G4 簇区域的扩增效率低于非形成 G4 结构的区域。通过对 WGA 产物进行高通量测序，在人类基因组 DNA 上鉴定出 9651 个 G4 簇。在这些簇中，3766 个 G4 簇至少包含一个转录起始位点，这表明基因受到 G4 簇的调控，而不是一个 G4 结构的调控。
