DNA POLYMORPHISM IN SOLUTION--A THERMODYNAMIC STUDY

我们建议继续我们的分子热力学特征 控制稳定性和构象偏好的力 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， 评估序列特异性、结构特异性和溶剂特异性 重要功能域的构象偏好 天然存在的核酸。 实现这些目标所需的热力学数据将通过以下方法获得： 应用微量热技术（等温混合和 温度扫描）以表征螺旋形成和螺旋破坏 事件以及螺旋到螺旋的构象转变，特别是 设计和合成的寡聚和多聚核酸分子 其具有将被系统地改变的序列。 这种方法 使我们能够将测量的热力学参数与特定的 通过UV和CD光谱确定的结构和/或构象特征 以及通过高场NMR。 事实上，在上一个预算期间， 我们结合了光谱和量热技术， 描述所有十个最近邻的Waston-Crick 相互作用以及各种DNA二级结构形式， 生物学意义（例如发夹、具有悬挂末端的双链体、双链体 具有脱碱基位点、固定连接、“哑铃”等）。 在下一个 在预算期间，如下所述，我们将把我们的热量研究 生物学的其它核酸核酸结构 这一点，尚待考证。 具体而言，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），温度依赖性转变 （Δ Ho，Δ Cp）和熔融协同性（Δ Hv.H/Δ Hcal） 具有碱基修饰的DNA双链体 诱变损伤（例如环外和烷基化加合物）; DNA三链体; 当适当定相时产生畸变的DNA双链体 “弯曲”; DNA发夹; DNA哑铃状结构; DNA双链体， 悬挂末端;和DNA/RNA杂合双链体。 热力学数据我们 从这些拟议的研究中获得的成果将大大扩大我们现有的 图书馆，从而为我们提供了一个扩大和改善的经验基础 用于评估相对稳定性和结构，以及解决方案 条件 热力学数据也将帮助我们评估 序列、结构和溶剂诱导的构象变化程度 扭曲和转型有助于整体的驱动力 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杂合双链体）的相图， 定义相对稳定性并绘制温度和溶剂 诱导序列特异性构象状态的相互转化。 考虑到碱基修饰和/或构象修饰的潜在作用， 选择性局部控制事件的机制的异质性， 蛋白质-核酸相互作用、药物-DNA结合、基因表达和 DNA包装，一种预测序列依赖性，局部 DNA和DNA/RNA中的构象偏好和转化 聚合物是最重要的。 量热实验 在这个建议中描述的ar旨在提供热力学数据 需要建立这种预测能力，以便有利于序列 特定的结构形式可以被识别并与特定的 功能角色。

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.