STRUCTURAL INVESTIGATION OF DNA QUADRUPLEXES BY NMR

我的博士论文项目的目标是获得关于 一种潜在生物的生物物理特征 重要的高阶DNA结构基序。我们一直在 对其核心结构的DNA四链结构感兴趣 是基于鸟嘌呤四重奏(G-四重奏)。有人建议， 在天然真核细胞染色体上可能形成G-四元组结构 末端(端粒)，并有助于蛋白质结合、调节或 抗降解性。端粒通常由简单的 与端粒相关的富鸟嘌呤序列的串联重复序列 结合蛋白。端粒序列通常被发现是 在系统发育不同的生物中高度保守。更多 然而，最近，许多萌芽中的酵母端粒被显示为 在长度和碱基组成上都有更多的发散序列。 含有一到两个酵母端粒重复序列的寡核苷酸 与这些序列相比，序列的热力学稳定性通常较差 具有更保守的序列，例如来自纤毛虫原生动物的序列。我的 论文项目涉及几个方面的表征 芽殖酵母产生四链富鸟嘌呤寡核苷酸的研究 端粒和相关序列。溶液核磁共振 (核磁共振)和其他光谱方法，以了解其 物理化学特征，影响稳定性的因素 并确定高分辨溶液结构 这些寡核苷酸中。我们核磁共振谱的最新改进 分析工具Sparky对我们的结构分析非常有用 确定过程。程序的可视化能力 内含MidasPlus的结构模型和质子间距离 斯巴基极大地加速了这一进程。成立为法团 用于结构确定过程的图形化工具 分析(Sparky)、距离计算(Mardigras)、结构 解释(琥珀，X-plor)令人兴奋，它已经帮助 这一过程的迭代性质。

The goal of my PhD thesis project was to obtain information about the biophysical characteristics of a potentially biologically significant higher-order DNA structural motif. We have been interested in structures of DNA quadruplexes whose core architecture is based on guanine quartets (G-quartets). It has been suggested that G-quartet structures may form at the natural eukaryotic chromosome ends (telomeres) and contribute to protein binding, regulation, or resistance to degradation. Telomeres are usually composed of simple tandem repeats of guanine-rich sequences associated with telomeric binding proteins. Telomeric sequences are typically found to be highly conserved across phylogenetically diverse organisms. More recently, however, many budding yeast telomeres have been shown to have more divergent sequences both in length and in base composition. Oligonucleotides having one to two repeats of the yeast telomeric sequences are usually less stable thermodynamically compared to those with more conserved sequences, such as ones from ciliate protozoa. My thesis project involved the characterization of several quadruplex-forming guanine-rich oligonucleotides from budding yeast telomeric and related sequences. Solution nuclear magnetic resonance (NMR) and other spectroscopic methods were used to understand their physical chemical features, the factors contributing to the stability of the complexes, and to determine high resolution solution structures of those oligonucleotides. The recent improvement of our NMR spectrum analysis tool, Sparky, has been very useful for our structural determination process. The capability of the program to visualize structural models and inter-proton distances using MidasPlus within Sparky accelerated tis process enormously. The incorporation of graphical tools to the structural determination process, spectrum analysis (Sparky), distance calculation (MARDIGRAS), structure elucidation (Amber, X-PLOR) was exciting and it has already helped the iterative nature of this process.