Conformational Stability & Dynamics of G-Quadruplexed DNA & Ligand Interactions

构象稳定性

• 批准号: 7778905
• 负责人: Lesley Davenport
• 金额: $ 15.31万
• 依托单位: BROOKLYN COLLEGE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7778905
• 关键词: Area; Arts; Base Pairing; Binding; Calorimetry; Characteristics; Chemicals; Chromosomes; DNA; DNA Structure; Electrophoresis; Enzymes; Fluorescence; G-Quartets; Guanine; Human; Knowledge; Label; Ligand Binding; Ligands; Maps; Methodology; Methods; Molecular Conformation; Property; Research; Research Support; Series; Site; Structure; Tandem Repeat Sequences; Telomerase; Thermodynamics; Titrations; college; design; interest; nucleoside analog; tumorigenesis

Guanine-rich DNA tandem repeat telomeric sequences located at the end of the chromosomes can assume highly stable G-quadruplex structures through Hoogsteen base pairing of the guanine residues. These secondary DNA structures can inhibit the activity of telomerase, an enzyme that is important for tumorigenesis. There is intense interest in developing so-called G-quadruplex interactive agents (QIAs), which are able to stabilize the G-quadruplex structure as potential chemotherapeutics. However, advancement of this promising area of research has been hindered by a general lack of knowledge of the fundamental rules that govern G-quadruplex formation, conformation and dynamics, properties which can influence productive and selective QIA ligand interactions. Hence there a critical need for understanding the basic chemical and physical characteristics of the G-quadruplex. The aims of this research application are: (1) to examine the conformational stability at specific sites within the G-quadruplex and to assess the effects of QIA binding on these parameters; and 2) to examine the underlying dynamics of intramolecular G-quadruplex folding and unfolding. To achieve these aims we have prepared a series of fluorescently labeled human telomeric sequences, with replacement of a single guanine residue by a fluorescent nucleoside analog at varying postions within the G-quadruplex. The conformation and dynamics of the G-quadruplex will be studied using state-of-the-art fluorescence methodologies. Isothermal titration calorimetry (ITC) will be used to examine thermodynamic quantities involved in QIA ligand binding; and electrophoresis and UV analyses to confirm quadruplex formation. The information provided by the combination of these characterization methods will allow us to map the dynamic structure of the quadruplex and determine the impact of QIA binding. The broad, long-term objective of the proposed research is to understand the fundamental "rules" that govern the conformation and dynamics of G-quadruplexed DNA, and to begin to understand the effects of specific ligand binding on them for ultimate applications in the rational design of QIAs.

位于染色体末端的富含鸟嘌呤的DNA串联重复序列可以假定 通过鸟嘌呤残基的Hoogsteen碱基配对获得高度稳定的G-四链结构。这些 二级DNA结构可以抑制端粒酶的活性，端粒酶是一种对 肿瘤发生学。人们对开发所谓的G-四链交互代理(QIA)有着浓厚的兴趣， 它们能够稳定G-四链结构作为潜在的化疗药物。然而， 这一前景看好的研究领域的进展受到了普遍缺乏对 控制G-四链体形成、构象和动力学的基本规则，这些性质可以 影响生产性和选择性的QIA配基相互作用。因此，迫切需要了解 G-四链的基本化学和物理特性。本研究申请的目的是： (1)检查G-四链体内特定位置的构象稳定性并评估其影响 2)研究分子内G-四链体的潜在动力学。 折叠和展开。为了实现这些目标，我们制备了一系列荧光标记的 人端粒序列，用荧光核苷取代单个鸟嘌呤残基 在G-四联体中的不同位置的模拟。G-四链遗嘱的构象和动力学 使用最先进的荧光方法进行研究。等温滴定量热法(ITC)将 用于检测QIA配体结合所涉及的热力学参数；以及电泳法和紫外法 分析以确认形成四联体。由这些组合提供的信息 表征方法将使我们能够绘制四链结构的动态结构图，并确定 QIA绑定的影响。拟议研究的广泛、长期目标是了解 控制G-四链DNA构象和动力学的基本规则，并开始 了解特定配基结合对它们的影响，以便在合理设计中最终应用 QIAs。