DNA POLYMORPHISM IN SOLUTION--A THERMODYNAMIC STUDY

我们建议继续我们对分子的热力学描述 控制分子稳定性和构象偏好的作用力 溶液中的核酸分子。我们的最终目标是建立 一个全面的热力学库，提供所需的数据库 评估特定序列、特定结构和特定溶剂 内重要功能结构域的构象偏好 自然产生的核酸。 实现这些目标所需的热力学数据将通过以下方式获得 应用微量热技术(等温混合和 温度扫描)来表征螺旋的形成和破坏 事件以及螺旋到螺旋的构象转变 设计和合成寡聚和聚合核酸分子 它们拥有的序列将被系统地改变。这种方法 使我们能够将测量的热力学参数与特定的 由UV和CD光谱确定的结构和/或构象特征 以及高场核磁共振。事实上，在上一个预算期间， 我们使用光谱和量热技术的组合来 从热力学角度描述所有十个最近邻Waston-Crick 相互作用以及多种DNA二级结构形式 生物兴趣(例如，发夹、两端悬挂的复式发夹、复式发夹 具有基本位置、固定连接、“哑铃”等)。在下一次 预算期间，如下所述，我们将重点进行量热研究 论生物的附加核酸核酸结构 它们的意义还有待于热力学表征。 具体地说，在所要求的预算期间，我们提议确定 作为碱基序列、碱基修饰和溶液条件的函数 相对稳定性(DeltaGo)，即温度相关的跃迁 (DeltaHo，DeltaCp)和熔融协作性(DeltaHv.H/DeltaHcal) 下列核酸系统中的一种：碱基修饰的DNA双链 致突变损伤(如外环和烷基加合物)；DNA三联体； 具有扭曲的DNA双链，当适当地分阶段时会引起 “弯曲”；DNA发夹；DNA哑铃状结构；DNA双链 悬垂的末端；以及DNA/RNA杂交双链。我们的热力学数据 从这些拟议的研究中获得的信息将大大扩展我们现有的 图书馆，从而为我们提供了更广泛和更完善的经验基础 用于评估相对稳定性和结构，以及解决方案 条件。热力学数据还将帮助我们评估 序列、结构和溶剂诱导构象的程度 扭曲和NAD转换有助于推动 具有生物学意义的过程。最终，我们希望建立一个 DNA(包括DNA/RNA杂交双链)的相图 定义相对稳定性并绘制温度-和溶剂- 序列特定构象状态的诱导相互转换。 考虑碱基修饰和/或构象的潜在作用 选择性、局部控制事件的机制的异质性，例如 蛋白质-核酸相互作用、药物-DNA结合、基因表达和 DNA包装，一种预测序列依赖、局部的能力 DNA和DNA/RNA中的构象偏好和转换 聚合物是最重要的。量热实验 本建议书中描述的内容旨在提供热力学数据 建立这种预测能力所必需的，以便有利于 特定的结构形式可以被识别并与特定的 职能角色。

We propose to continue our thermodynamic characterizations of the molecular forces that control the stability and the conformational preferences of nucleic acid molecules in solution. Our ultimate objective is to establish a comprehensive thermodynamic library that provides the data base needed to evaluate sequence-specific, structure-specific, and solvent-specific conformational preferences of functionally-important domains within naturally-occurring nucleic acids. The thermodynamic data required to achieve these goals will be obtained by applying the techniques of microcalorimetry (both isothermal mixing and temperature scanning) to characterize helix forming and helix disrupting events as well as helix-to-helix conformational transitions in specially designed and synthesized oligomeric and polymeric nucleic acid molecules which possess sequences that will be systematically varied. This approach has allowed us to correlate measured thermodynamic parameters with specific structural and/or conformational features defined by uv and CD spectroscopy as well as by high field NMR. In fact, during the previous budget period, we used this combination of spectroscopic and calorimetric techniques to characterize thermodynamically all ten nearest-neighbor Waston-Crick interactions as well as a variety of DNA secondary structural forms of biological interest (e.g. hairpins, duplexes with dangling ends, duplexes with abasic sites, immobile junctions, "dumbbells," etc.). During the next budget period, as described below, we will focus our calorimetric studies on additional nucleic acid nucleic acid structures of biological significate which have yet to be thermodynamically characterized. To be specific, during the requested budget period we propose to determine as a function of base sequence, base modification, and solution conditions the relative stabilities (DeltaGo), the temperature-dependent transitions (DeltaHo, DeltaCp), and the melting cooperativities (DeltaHv.H/DeltaHcal) of the following nucleic acid systems: DNA duplexes with base modified mutagenic lesions (e.g. exocyclic and alkylated adducts); DNA triplexes; DNA duplexes with distortions which when properly phased give rise to "bending"; DNA hairpins; DNA dumbbell-shaped structures; DNA duplexes with dangling ends; and DNA/RNA hybrid duplexes. The thermodynamic data we obtain from these proposed studies will substantially expand our existing library, thereby providing us with a broadened and improved empirical basis for evaluating the relative stabilities and structure, and solution conditions. The thermodynamic data also will assist us in evaluating the degree to which sequence-, structure-, and solvent-induced conformational distortions nad transformations contribute to the overall driving forces of biologically significant processes. Ultimately, we hope to establish a phase diagram for DNA (including DNA/RNA hybrid duplexes) in which we define the relative stabilities and map the temperature- and solvent- induced interconversions of sequence-specific conformational states. Considering the potential roles of base modification and/or conformational heterogeneity in mechanisms for selective, local control of events such as protein-nucleic acid interactions, drug-DNA binding, gene expression, and DNA packaging, an ability to predict sequence-dependent, local conformational preferences and transformations in DNA and in DNA/RNA polymers is of the utmost importance. The calorimetric experiments described in this proposal ar designed to provide the thermodynamic data required to establish this predictive ability so that sequences favoring specific structural forms can be identified and correlated with particular functional roles.