DNA POLYMORPHISM IN SOLUTION--A THERMODYNAMIC STUDY

我们建议继续我们的热力学特征的 控制稳定性和构象的分子力 核酸分子在溶液中的偏好。 我们的最终 目的是建立一个综合热力学图书馆， 提供了评估序列特异性，结构， 特异性和溶剂特异性构象偏好 天然存在的核酸内的功能重要的结构域。 随着目前正在生成的令人印象深刻的序列数据量， 人类基因组计划，这种能力正变得越来越 重要. 最后，人们想评估局部序列是否 基因组中的结构域有利于特定的结构基序， 生物作用或控制的功能位点。 相同的 热力学数据也需要合理设计的解决方案 条件和第三链寡核苷酸序列 反义/反基因治疗策略和基于核酸的 诊断协议。 这些应用所需的热力学数据将通过以下方法获得： 使用微量热法（等温混合和温度扫描） 为了表征螺旋形成事件、螺旋破坏事件和螺旋- 在特别设计的寡聚核酸中的到螺旋的转化 序列会有系统性变化的分子。 这 这种方法使我们能够将测得的热力学参数 其具有由UV和/或UV光定义的特定结构和/或构象特征， CD光谱以及高场NMR。 事实上，在此前的 在预算期间，我们使用了这种光谱和 量热技术来表征化学上的宽范围 DNA的二级结构形式。 期间 下一个预算期，我们建议通过以下方式建立和扩大这一基础： 将我们的量热研究集中在最近发现或重新发现的 具有生物学和生物医学意义的核酸结构， 还没有或者还没有充分地进行化学表征。 具体而言，在所要求的预算期间，我们提议 根据碱基序列、碱基修饰和溶液确定 条件的相对稳定性（deltaGo），温度依赖 转变（Δ Ho，Δ Cp）和熔融协同性 （deltaHvH/deltaHcal）： 致突变损伤; DNA三链体; DNA和RNA四链体; DNA/RNA 杂交双链体和弯曲DNA。 所产生的数据将大大 扩展现有的热力学图书馆。 最终，我们打算 建立核酸相图，定义相对 稳定性和映射温度和溶剂诱导的相互转化 序列特异性构象状态。 考虑到碱基修饰和/或 选择性局部控制机制中的构象异质性 例如蛋白质-核酸相互作用，药物-DNA结合， 基因表达，DNA包装，预测序列的能力， DNA中的依赖性、局部构象偏好和转化 在DNA/RNA杂交体中是至关重要的。 量热 本提案中描述的实验旨在提供 建立这种预测能力所需的热力学数据， 可以鉴定有利于特定结构形式的序列， 与特定的功能角色相关。 简而言之，热力学 我们计划获得的数据对于解释人类基因组非常重要 结构-功能关系方面的序列数据，以及 制定合理的方法来设计有效的第三 用于治疗和诊断方案链寡聚体。

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. With the impressive volume of sequence data currently being generated by the Human Genome Project, such a capacity is becoming increasingly important. Ultimately, one would like to assess if local sequence domains in the genome favor specific structural motifs which correspond to functional sites of biological action or control. The same thermodynamic data also required for the rational design of solution conditions and third strand oligonucleotide sequences for use in antisense/antigene therapeutic strategies and nucleic-acid based diagnostic protocols. The thermodynamic data needed for these applications will be obtained by using microcalorimetry (both isothermal mixing and temperature scanning) to characterize helix forming events, helix disrupting events, and helix- to-helix transformations in specially designed oligomeric nucleic acid molecules with 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 by high field NMR. In fact, during the previous budget periods, we have used this combination of spectroscopic and calorimetric techniques to characterize thermodynamically a wide range of DNA secondary structural forms of biological interest. During the next budget period, we propose to build and to expand this foundation by focusing our calorimetric studies on recently discovered or rediscovered nucleic acid structures of biological and biomedical significance which have yet to be or are insufficiently 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 (deltaHvH/deltaHcal) of the following nucleic acid systems: DNA duplexes with mutagenic lesions; DNA triplexes; DNA and RNA tetraplexes; DNA/RNA hybrid duplexes; and bent DNA. The resulting data will substantially expand the existing thermodynamic library. Ultimately, we intend to establish nucleic acid phase diagrams which 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 hybrids is of the utmost importance. The calorimetric experiments described in this proposal are 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. In short, the thermodynamic data we propose to obtain will be important for interpreting Human Genome sequence data in terms of structure-function relationships, as well as for developing a rational approach to the design of effective third strand oligomers for use in therapeutic and diagnostic protocols.