Kawamoto Yusuke, Sasaki Asuka, Hashiya Kaori, Ide Satoru, Bando Toshikazu, Maeshima Kazuhiro, Sugiyama Hiroshi
Department of Chemistry , Graduate School of Science , Kyoto University , Kyoto 606-8502 , Sakyo , Japan . Email:
Biological Macromolecules Laboratory , 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 . Email:
Chem Sci. 2015 Apr 1;6(4):2307-2312. doi: 10.1039/c4sc03755c. Epub 2015 Jan 20.
The binding of molecules to specific DNA sequences is important for imaging genome DNA and for studying gene expression. Increasing the number of base pairs targeted by these molecules would provide greater specificity. -Methylpyrrole--methylimidazole (Py-Im) polyamides are one type of such molecules and can bind to the minor groove of DNA in a sequence-specific manner without causing denaturation of DNA. Our recent work has demonstrated that tandem hairpin Py-Im polyamides conjugated with a fluorescent dye can be synthesized easily and can serve as new probes for studying human telomeres under mild conditions. Herein, to improve their selectivities to telomeres by targeting longer sequences, we designed and synthesized a fluorescent tandem trimer Py-Im polyamide probe, comprising three hairpins and two connecting regions (hinges). The new motif bound to 18 bp dsDNA in human telomeric repeats (TTAGGG) , the longest sequence for specific binding reported for Py-Im polyamides. We compared the binding affinities and the abilities to discriminate mismatch, the UV-visible absorption and fluorescence spectra, and telomere staining in human cells between the tandem trimer and a previously developed tandem hairpin. We found that the tandem trimer Py-Im polyamide probe has higher ability to recognize telomeric repeats and stains telomeres in chemically fixed cells with lower background signal.
分子与特定DNA序列的结合对于基因组DNA成像和基因表达研究至关重要。增加这些分子靶向的碱基对数量将提供更高的特异性。-甲基吡咯-甲基咪唑（Py-Im）聚酰胺是这类分子中的一种，能够以序列特异性方式结合到DNA小沟，而不会导致DNA变性。我们最近的工作表明，与荧光染料共轭的串联发夹Py-Im聚酰胺能够轻松合成，并可作为在温和条件下研究人类端粒的新型探针。在此，为了通过靶向更长序列提高其对端粒的选择性，我们设计并合成了一种荧光串联三聚体Py-Im聚酰胺探针，其包含三个发夹和两个连接区域（铰链）。这种新基序与人类端粒重复序列（TTAGGG）中的18 bp双链DNA结合，这是Py-Im聚酰胺报道的特异性结合的最长序列。我们比较了串联三聚体与先前开发的串联发夹在结合亲和力、区分错配的能力、紫外可见吸收和荧光光谱以及人类细胞中端粒染色方面的差异。我们发现，串联三聚体Py-Im聚酰胺探针具有更高的识别端粒重复序列的能力，并且能够在化学固定细胞中以较低背景信号对端粒进行染色。
