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

构象稳定性

• 批准号: 7579900
• 负责人: Lesley Davenport
• 金额: $ 16.19万
• 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/7579900
• 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; Series; Site; Structure; Tandem Repeat Sequences; Telomerase; Thermodynamics; Titrations; 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串联重复端粒序列可以假设 通过鸟嘌呤残基的Hoogsteen碱基配对的高度稳定的G-四链体结构。这些 二级DNA结构可以抑制端粒酶的活性，端粒酶是一种对 肿瘤发生人们对开发所谓的G-四链体交互剂（QIA）有浓厚的兴趣， 其能够稳定G-四链体结构作为潜在的化学治疗剂。然而，在这方面， 这一有前途的研究领域的进展受到普遍缺乏知识的阻碍， 控制G-四链体形成、构象和动力学的基本规则， 影响生产性和选择性QIA配体相互作用。因此，迫切需要了解 G-四链体的基本化学和物理特性。本研究申请的目的是： (1)检查G-四链体内特定位点的构象稳定性，并评估 QIA结合这些参数; 2）检查分子内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 用荧光核苷取代单个鸟嘌呤残基的人端粒序列 G-四重体内不同位置的模拟。G-四链体的构象和动力学将 使用最先进的荧光方法进行研究。等温滴定量热法（ITC）将 用于检查QIA配体结合中涉及的热力学量;以及电泳和UV 分析以确认四链体形成。这些组合提供的信息 表征方法将使我们能够映射四链体的动态结构，并确定 QIA约束力的影响。拟议研究的广泛、长期目标是了解 基本的“规则”，管理的构象和动力学的G-四链体DNA，并开始开始， 了解特定配体结合对它们的影响，以最终应用于合理设计， QIA。