Biophysics Department, Research and Clinical Center for Physical Chemical Medicine, Malaya Pirogovskaya str. 1a, Moscow 119435, Russia.
Department of Molecular Virology, FSBI Research Institute of Influenza, Ministry of Health of Russian Federation, prof. Popov str. 15/17, Saint-Petersburg, 197376, Russia.
Nucleic Acids Res. 2018 Apr 6;46(6):2751-2764. doi: 10.1093/nar/gky121.
Non-canonical DNA structures are widely used for regulation of gene expression, in DNA nanotechnology and for the development of new DNA-based sensors. I-motifs (iMs) are two intercalated parallel duplexes that are held together by hemiprotonated C-C base pairs. Previously, iMs were used as an accurate sensor for intracellular pH measurements. However, iM stability is moderate, which in turn limits its in vivo applications. Here, we report the rational design of a new substituted phenoxazine 2'-deoxynucleotide (i-clamp) for iM stabilization. This residue contains a C8-aminopropyl tether that interacts with the phosphate group within the neighboring chain without compromising base pairing. We studied the influence of i-clamp on pH-dependent stability for intra- and intermolecular iM structures and found the optimal positions for modification. Two i-clamps on opposite strands provide thermal stabilization up to 10-11°C at a pH of 5.8. Thus, we developed a new modification that shows significant iM-stabilizing effect both at strongly and mildly acidic pH and increases iM transition pH values. i-Clamp can be used for tuning iM-based pH probes or assembling extra stable iM structures for various applications.
非经典 DNA 结构广泛用于基因表达调控、DNA 纳米技术和新型基于 DNA 的传感器的开发。发夹结构（iMs）是由两个交错的平行双链体组成的，通过半质子化的 C-C 碱基对结合在一起。先前，iMs 被用作细胞内 pH 值测量的精确传感器。然而，iM 的稳定性适中，这反过来又限制了其在体内的应用。在这里，我们报告了一种新的取代苯并恶嗪 2'-脱氧核苷酸（i-clamp）的合理设计，用于 iM 的稳定化。该残基含有一个 C8-氨丙基连接物，与相邻链中的磷酸基团相互作用，而不影响碱基配对。我们研究了 i-clamp 对分子内和分子间 iM 结构的 pH 依赖性稳定性的影响，并找到了修饰的最佳位置。两条位于相反链上的 i-clamp 在 pH 值为 5.8 时提供高达 10-11°C 的热稳定性。因此，我们开发了一种新的修饰方法，在强酸性和弱酸性 pH 条件下均显示出显著的 iM 稳定作用，并增加了 iM 转变 pH 值。i-Clamp 可用于调整基于 iM 的 pH 探针，或组装具有各种应用的额外稳定的 iM 结构。
