Kawamoto Yusuke, Sasaki Asuka, Chandran Anandhakumar, Hashiya Kaori, Ide Satoru, Bando Toshikazu, Maeshima Kazuhiro, Sugiyama Hiroshi
Department of Chemistry, Graduate School of Science, Kyoto University , Sakyo, Kyoto 606-8502, Japan.
Structural Biology Center, National Institute of Genetics, and Department of Genetics, School of Life Science, Graduate University for Advanced Studies (Sokendai) , Mishima, Shizuoka 411-8540, Japan.
J Am Chem Soc. 2016 Oct 26;138(42):14100-14107. doi: 10.1021/jacs.6b09023. Epub 2016 Oct 13.
Synthetic molecules that bind sequence-specifically to DNA have been developed for varied biological applications, including anticancer activity, regulation of gene expression, and visualization of specific genomic regions. Increasing the number of base pairs targeted by synthetic molecules strengthens their sequence specificity. Our group has been working on the development of pyrrole-imidazole polyamides that bind to the minor groove of DNA in a sequence-specific manner without causing denaturation. Recently, we reported a simple synthetic method of fluorescent tandem dimer polyamide probes composed of two hairpin moieties with a linking hinge, which bound to 12 bp in human telomeric repeats (5'-(TTAGGG)-3') and could be used to specifically visualize telomeres in chemically fixed cells under mild conditions. We also performed structural optimization and extension of the target base pairs to allow more specific staining of telomeres. In the present study, we synthesized tandem tetramer polyamides composed of four hairpin moieties, targeting 24 bp in telomeric repeats, the longest reported binding site for synthetic, non-nucleic-acid-based, sequence-specific DNA-binding molecules. The novel tandem tetramers bound with a nanomolar dissociation constant to 24 bp sequences made up of four telomeric repeats. Fluorescently labeled tandem tetramer polyamide probes could visualize human telomeres in chemically fixed cells with lower background signals than polyamide probes reported previously, suggesting that they had higher specificity for telomeres. Furthermore, high-throughput sequencing of human genomic DNA pulled down by the biotin-labeled tandem tetramer polyamide probe confirmed its effective binding to telomeric repeats in the complex chromatinized genome.
已开发出能与DNA序列特异性结合的合成分子，用于多种生物学应用，包括抗癌活性、基因表达调控以及特定基因组区域的可视化。增加合成分子靶向的碱基对数量可增强其序列特异性。我们团队一直致力于开发以序列特异性方式结合到DNA小沟且不会导致变性的吡咯-咪唑聚酰胺。最近，我们报道了一种简单的合成方法，可合成由两个带有连接铰链的发夹部分组成的荧光串联二聚体聚酰胺探针，该探针可与人端粒重复序列（5'-(TTAGGG)-3'）中的12个碱基对结合，并可用于在温和条件下对化学固定细胞中的端粒进行特异性可视化。我们还进行了结构优化和目标碱基对的扩展，以实现对端粒更特异性的染色。在本研究中，我们合成了由四个发夹部分组成的串联四聚体聚酰胺，靶向端粒重复序列中的24个碱基对，这是报道的合成的、非核酸类、序列特异性DNA结合分子最长的结合位点。新型串联四聚体以纳摩尔解离常数与由四个端粒重复序列组成的24个碱基对序列结合。荧光标记的串联四聚体聚酰胺探针能够在化学固定细胞中可视化人类端粒，其背景信号比先前报道的聚酰胺探针更低，这表明它们对端粒具有更高的特异性。此外，生物素标记的串联四聚体聚酰胺探针下拉的人类基因组DNA的高通量测序证实了其在复杂染色质化基因组中与端粒重复序列的有效结合。
