Department of Chemistry, University of Utah, Salt Lake City, Utah.
Department of Chemistry, University of Utah, Salt Lake City, Utah.
Biophys J. 2018 Apr 24;114(8):1804-1815. doi: 10.1016/j.bpj.2018.03.012.
We have interrogated the isothermal folding behavior of the DNA i-motif of the human telomere, dC, and a high-stability i-motif-forming sequence in the promoter of the human DNA repair gene RAD17 using human physiological solution and temperature conditions. We developed a circular-dichroism-spectroscopy-based pH titration method that is followed by analysis of titration curves in the derivative domain and found that the observed pH-dependent folding behavior can be significantly different and, in some cases, multiphasic, with a dependence on how rapidly i-motif folding is induced. Interestingly, the human telomere sequence exhibits unusual isothermal hysteresis in which the unfolding process always occurs at a higher pH than the folding process. For the RAD17 i-motif, rapid folding by injection into a low-pH solution results in triphasic unfolding behavior that is completely diminished when samples are slowly folded in a stepwise manner via pH titration. Chemical footprinting of the RAD17 sequence and pH titrations of dT-substituted mutants of the RAD17 sequence were used to develop a model of RAD17 folding and unfolding. These results may provide valuable information pertinent to i-motif use in sensors and materials, as well as insight into the potential biological activity of i-motif-forming sequences under stepwise or instantaneous changes in pH.
我们使用人类生理溶液和温度条件，研究了人类端粒 dC 的 DNA i -motif 和人类 DNA 修复基因 RAD17 启动子中高稳定性 i-motif 形成序列的等温折叠行为。我们开发了一种基于圆二色性光谱的 pH 滴定法，随后对导数域中的滴定曲线进行分析，发现观察到的 pH 依赖性折叠行为可能存在显著差异，并且在某些情况下呈多相性，这取决于 i-motif 折叠的诱导速度。有趣的是，人类端粒序列表现出异常的等温滞后现象，其中解折叠过程的 pH 值总是高于折叠过程。对于 RAD17 i-motif，通过快速注入低 pH 溶液进行折叠会导致三相解折叠行为，而当样品通过 pH 滴定以逐步方式缓慢折叠时，该行为会完全消失。RAD17 序列的化学足迹分析和 RAD17 序列的 dT 取代突变体的 pH 滴定用于开发 RAD17 折叠和展开的模型。这些结果可能为 i-motif 在传感器和材料中的应用提供有价值的信息，并深入了解 i-motif 形成序列在 pH 逐步或瞬时变化下的潜在生物学活性。
