DNA POLYMORPHISM IN SOLUTION--A THERMODYNAMIC STUDY

溶液中的 DNA 多态性——热力学研究

• 批准号: 2174106
• 负责人: KENNETH J. BRESLAUER
• 金额: $ 26.34万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2174106
• 关键词: DNA; DNA binding protein; DNA damage; RNA; biophysics; chemical stability; circular dichroism; conformation; gene mutation; intermolecular interaction; mathematical model; microcalorimetry; nucleic acid chemical synthesis; nucleic acid sequence; nucleic acid structure; solutions; stop flow technique; structural biology; synthetic nucleotide; thermodynamics; triple helix

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.

我们建议继续我们的热力学特征 控制稳定性和构象的分子力 溶液中核酸分子的偏好。 我们的最终 目的是建立一个全面的热力学库 提供评估序列特异性结构所需的数据库 - 特异性和溶剂特异性构象的偏好 自然核酸内部功能至关重要的结构域。 目前正在生成令人印象深刻的序列数据卷 人类基因组项目，这种能力越来越多 重要的。 最终，人们想评估本地序列是否 基因组中的域有利于特定的结构基序 到生物作用或控制的功能部位。 相同 解决方案的合理设计也需要热力学数据 条件和第三链寡核苷酸序列用于 反义/抗原治疗策略和基于核酸的酸性 诊断协议。 这些应用所需的热力学数据将由 使用微量钙化（均热混合和温度扫描） 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变换 分子具有系统变化的序列。 这 方法使我们能够关联测得的热力学参数 具有特定的结构和/或构象特征，由紫外线和/或 高场NMR也通过CD光谱法。 实际上，在上一个 预算期，我们已经使用了这种光谱和 量热的技术在热力学上表征广泛 DNA二级结构形式的生物学意义。 在 下一个预算期，我们建议通过 将我们的量热研究集中在最近发现或重新发现的 生物学和生物医学意义的核酸结构 尚未或没有足够的热力学表征。 具体而言，在要求的预算期内，我们建议 确定基本序列，基础修改和解决方案的函数 条件相对稳定性（Deltago），依赖温度 过渡（Deltaho，deltacp）和融化合作社 （deltahvh/deltahcal）以下核酸系统：DNA双链体 有诱变病变； DNA三倍体； DNA和RNA四副词； DNA/RNA 混合双链体；和弯曲的DNA。 最终的数据将大大 扩展现有的热力学库。 最终，我们打算 建立定义相对的核酸相图 稳定性并绘制温度和溶剂诱导的互连 序列特异性构象状态。 考虑基础修改和/或的潜在作用 选择性，局部控制机制的构象异质性 诸如蛋白核酸相互作用，药物-DNA结合等事件的事件 基因表达和DNA包装，一种预测序列的能力 - 依赖性的局部构象偏好和DNA中的转化 在DNA/RNA中，杂种至关重要。 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，以便 可以识别有利于特定结构形式的序列 与特定功能角色相关。 简而言之 我们建议获得的数据对于解释人类基因组很重要 结构功能关系的序列数据以及 用于开发有效设计的合理方法 用于治疗和诊断方案的链寡聚物。