DNA POLYMORPHISM IN SOLUTION--A THERMODYNAMIC STUDY

我们建议继续我们的热力学描述， 控制稳定性和构象的分子作用力 核酸分子在溶液中的偏好。我们的终极 目标是建立一个全面的热力学图书馆， 提供了评估特定序列、结构-- 特定的和特定于溶剂的构象首选项 自然产生的核酸中具有重要功能的区域。 目前大量的序列数据由 人类基因组计划，这样的能力正在变得越来越强 很重要。最终，人们想要评估局部序列是否 基因组中的结构域倾向于特定的结构基序， 涉及到生物作用或控制的功能部位。相同 溶液的合理设计还需要热力学数据 用于检测的条件和第三链寡核苷酸序列 反义/反基因治疗策略与核酸为基础 诊断方案。 这些应用所需的热力学数据将通过以下方式获得 使用微量热计(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- 特殊设计的寡聚核酸中的螺旋转化 其序列将被系统地改变的分子。这 这种方法使我们能够将测量到的热力学参数 具有特定结构和/或构象特征，由UV和 CD波谱以及高场核磁共振波谱。事实上，在之前的 预算期间，我们使用了光谱和 大范围热力学表征的量热技术 具有生物学意义的DNA二级结构形式。在.期间 在下一个预算期，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方面 具有生物和生物医学意义的核酸结构 还没有或没有得到足够的热力学表征。 具体地说，在所要求的预算期间，我们提议 确定为碱基序列、碱基修饰和溶液的函数 条件相对稳定性(DeltaGo)，依赖于温度的 跃迁(deltaHo，deltaCp)和熔化协作性 (deltaHvH/deltaHcal)以下核酸系统：DNA双链 致突变病变；DNA三联体；DNA和RNA四联体；DNA/RNA 杂交双链和弯曲的DNA。所产生的数据将实质上 扩展现有的热力学程序库。最终，我们打算 建立定义相对分子质量的核酸相图 稳定性和MAP的温度和溶剂诱导的相互转化 序列特定的构象状态。 考虑碱基修饰和/或 选择性局部控制机制中的构象异质性 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中依赖的局部构象偏好和转换 在DNA/RNA杂交体中是最重要的。量热计 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 有利于特定结构形式的序列可以被识别并 与特定的职能角色相关。简而言之，热力学 我们计划获得的数据对解释人类基因组将是重要的 结构-功能关系方面的序列数据，以及 为了开发一种合理的方法来设计有效的第三方 用于治疗和诊断方案的链状低聚物。

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.