Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, Richmond, VA 23284, USA.
Department of Physics, Virginia Commonwealth University, 701 West Grace Street, Richmond, VA 23284, USA.
Nucleic Acids Res. 2019 Aug 22;47(14):7199-7212. doi: 10.1093/nar/gkz565.
The cytosine (C)-rich sequences that can fold into tetraplex structures known as i-motif are prevalent in genomic DNA. Recent studies of i-motif-forming sequences have shown increasing evidence of their roles in gene regulation. However, most of these studies have been performed in short single-stranded oligonucleotides, far from the intracellular environment. In cells, i-motif-forming sequences are flanked by DNA duplexes and packed in the genome. Therefore, exploring the conformational dynamics and kinetics of i-motif under such topologically constrained environments is highly relevant in predicting their biological roles. Using single-molecule fluorescence analysis of self-assembled DNA duplexes and nanocircles, we show that the topological environments play a key role on i-motif stability and dynamics. While the human telomere sequence (C3TAA)3C3 assumes i-motif structure at pH 5.5 regardless of topological constraint, it undergoes conformational dynamics among unfolded, partially folded and fully folded states at pH 6.5. The lifetimes of i-motif and the partially folded state at pH 6.5 were determined to be 6 ± 2 and 31 ± 11 s, respectively. Consistent with the partially folded state observed in fluorescence analysis, interrogation of current versus time traces obtained from nanopore analysis at pH 6.5 shows long-lived shallow blockades with a mean lifetime of 25 ± 6 s. Such lifetimes are sufficient for the i-motif and partially folded states to interact with proteins to modulate cellular processes.
富含胞嘧啶 (C) 的序列可以折叠成四链体结构，称为 i 型发夹结构，这些序列在基因组 DNA 中很常见。最近对形成 i 型发夹结构的序列的研究表明，它们在基因调控中的作用越来越明显。然而，这些研究大多是在短的单链寡核苷酸上进行的，与细胞内环境相去甚远。在细胞中，形成 i 型发夹结构的序列被 DNA 双链体包围，并包装在基因组中。因此，探索 i 型发夹在这种拓扑约束环境下的构象动力学和动力学对于预测其生物学功能非常重要。我们使用自组装 DNA 双链体和纳米环的单分子荧光分析表明，拓扑环境对 i 型发夹的稳定性和动力学起着关键作用。虽然人类端粒序列（C3TAA）3C3 在 pH5.5 下无论拓扑约束如何都能形成 i 型发夹结构，但在 pH6.5 下，它会在未折叠、部分折叠和完全折叠状态之间发生构象动力学变化。在 pH6.5 下，i 型发夹和部分折叠状态的寿命分别确定为 6±2 和 31±11s。与荧光分析中观察到的部分折叠状态一致，在 pH6.5 下通过纳米孔分析获得的电流与时间关系的检测表明，存在长寿命的浅阻塞，平均寿命为 25±6s。这样的寿命足以使 i 型发夹和部分折叠状态与蛋白质相互作用，从而调节细胞过程。
