Ashida Koudai, Kitabayashi Ayumi, Nishiyama Kazuki, Nakano Shu-Ichi
Department of Nanobiochemistry, Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, 7-1-20, Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.
Molecules. 2025 Apr 18;30(8):1831. doi: 10.3390/molecules30081831.
Cytosine-rich DNA sequences form i-motif structures associated with various cellular functions including gene regulation. DNA sequences containing consecutive C residues are widely deemed essential for i-motif formation; however, some sequences lacking C-tracts have been reported to form minimal i-motif structures. We systematically investigated the variability in the minimal i-motif-forming DNA sequence comprising two TCGTTCCGT sequence units, which forms two C:C pairs and two G:C:G:T base tetrads. A comprehensive analysis of structural stability by DNA thermal melting temperature measurements revealed that oligonucleotides disrupting the formation of the base tetrad or its stacking interactions with a C:C pair prevent stable i-motif formation, and modifications to the sequence context and length of the lateral loops are difficult. This study further demonstrated that spermine effectively restores the stability reduction caused by creating a bulge, long loop, or dangling end within the minimal i-motif structure, which is less pronounced in the C-rich i-motif. The results suggest that the formation of minimal i-motifs with various sequences is facilitated in polyamine-rich environments, such as the nucleus of mammalian cells. These findings are valuable for identifying potential i-motif-forming sites lacking C-tracts in genomes and provide insights into the electrostatic interactions between i-motif structures and biological polyamines.
富含胞嘧啶的DNA序列形成与包括基因调控在内的各种细胞功能相关的i-基序结构。含有连续C残基的DNA序列被广泛认为是i-基序形成所必需的；然而，据报道一些缺乏C链的序列也能形成最小的i-基序结构。我们系统地研究了由两个TCGTTCCGT序列单元组成的最小i-基序形成DNA序列的变异性，该序列形成两个C:C对和两个G:C:G:T碱基四重体。通过DNA热解链温度测量对结构稳定性进行的全面分析表明，破坏碱基四重体形成或其与C:C对的堆积相互作用的寡核苷酸会阻止稳定的i-基序形成，并且对侧向环的序列背景和长度进行修饰也很困难。这项研究进一步表明，精胺能有效恢复因在最小i-基序结构内产生凸起、长环或悬垂末端而导致的稳定性降低，这种现象在富含C的i-基序中不太明显。结果表明，在富含多胺的环境中，如哺乳动物细胞的细胞核中，有助于形成具有各种序列的最小i-基序。这些发现对于识别基因组中缺乏C链的潜在i-基序形成位点具有重要价值，并为i-基序结构与生物多胺之间的静电相互作用提供了见解。
