Development and Application of Multi-Spectroscopic, Site-Specific (MS3) Probes of

多光谱、位点特异性 (MS3) 探针的开发和应用

• 批准号: 7881997
• 负责人: Scott H Brewer
• 金额: $ 19.59万
• 依托单位: FRANKLIN AND MARSHALL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7881997
• 关键词: Development; Site

DESCRIPTION (provided by applicant): The long-term objective of the research is to significantly advance our understanding of nucleic acid structure, thermodynamics, and kinetics. The immediate objective of the proposed research is to significantly enhance the experimental and theoretical capability for the study of RNA folding dynamics, which are inherently linked to RNA function. RNA folding is not well understood despite RNA<s pivotal role in numerous biological functions such as catalysivs (ribozymes) and gene expression silencing (RNA interference, RNAi). RNA has direct medical relevance as shown by RNAi therapies currently in Phase III clinical trials for eye disease (age-related macular degeneration or AMD) and in Phase II trials for viral infections (respiratory syncytial virus or RSV). The proposed research involves the development (synthesis, characterization) and application of multi-spectroscopic, site-specific (MS3) probes to the study of local environments in DNA and RNA structure. The probes are nitrile- and azide-derivatized nucleosides. The nitrile (CN) and azide (N3) groups represent small perturbations to the structure of the nucleosides, while providing sensitive IR probes of local nucleic acid environments. Upon 15N labeling of the terminal nitrogen atom of CN and N3, 15N NMR spectroscopy can be used to probe local nucleic acid environments. Overall, the MS3 probes have the unique ability to provide site-specific information about all three major environments in nucleic acids: the major groove, the minor groove, and the sugar/backbone region, via two powerful spectroscopic techniques: infrared (IR) and 15N NMR spectroscopy. The MS3 probes will be synthesized either by using either literature procedures or by new synthetic routes utilizing azo transfer reactions. The spectral dependence of the CN and N3 stretching frequencies and 15N NMR chemical shifts on solvent (hydration), base pair formation, and temperature will be investigated. Subsequently, the MS3 probes will be incorporated at various positions in a 12 base pair DNA oligomer and an 8 nucleotide RNA hairpin. These nucleic acid structures will be investigated by equilibrium temperature-dependent IR and 15N NMR spectroscopic techniques to probe the various local environments present in these structures upon melting. Conformational changes induced by drug binding will also be explored in the DNA oligomer using the MS3 probes. These results will allow the parameterization and validation of force fields suitable for nucleic acids by our collaborators (Professors Corcelli and MacKerell) to be performed. Another goal of this health related research is to train eight undergraduate students in organic synthesis and spectroscopic data acquisition and analysis techniques. PUBLIC HEALTH RELEVANCE: The proposed research centers on significantly enhancing the capability to study RNA folding dynamics, which is inherently linked to RNA function, through the development of multi-spectroscopic, site- specific (MS3) probes. RNA folding is not well understood despite RNA<s pivotal role in numerous biological functions such as catalysis (ribozymes) and gene expression silencing (RNA interference, RNAi). RNA has direct medical relevance as shown by RNAi therapies currently in Phase III clinical trials for eye disease (age-related macular degeneration or AMD) and in Phase II trials for viral infections (respiratory syncytial virus or RSV).

描述（由申请人提供）：该研究的长期目标是显著提高我们对核酸结构，热力学和动力学的理解。本研究的直接目的是显著提高RNA折叠动力学研究的实验和理论能力，RNA折叠动力学与RNA功能有着内在的联系。尽管RNA在许多生物学功能中起着关键作用，如催化（核酶）和基因表达沉默（RNA干扰，RNAi），但人们对RNA折叠的了解并不多。RNA具有直接的医学相关性，目前在眼部疾病（年龄相关性黄斑变性或AMD）的III期临床试验和病毒感染（呼吸道合胞病毒或RSV）的II期试验中显示了RNAi疗法。本研究涉及多光谱、位点特异性（MS3）探针在DNA和RNA结构局部环境研究中的开发（合成、表征）和应用。探针是腈和叠氮化物衍生的核苷。腈基（CN）和叠氮基（N3）对核苷的结构产生了微小的扰动，同时为局部核酸环境提供了敏感的红外探针。对CN和N3的末端氮原子进行15N标记后，可以利用15N核磁共振波谱法探测局部核酸环境。总的来说，MS3探针具有独特的能力，通过两种强大的光谱技术：红外（IR）和15N核磁共振光谱，提供有关核酸中所有三种主要环境的特定位点信息：主要凹槽，次要凹槽和糖/主干区域。MS3探针的合成将采用文献方法或利用偶氮转移反应的新合成路线。研究了CN和N3拉伸频率和15N NMR化学位移对溶剂（水化）、碱基对形成和温度的谱依赖性。随后，MS3探针将被整合到12碱基对DNA寡聚物和8核苷酸RNA发夹的不同位置。这些核酸结构将通过平衡温度依赖的IR和15N NMR光谱技术来研究，以探测这些结构中存在的各种局部环境。利用MS3探针还将探索药物结合引起的DNA寡聚物构象变化。这些结果将允许我们的合作者（Corcelli教授和MacKerell教授）进行适合核酸的力场的参数化和验证。这项健康相关研究的另一个目标是训练八名本科生掌握有机合成和光谱数据采集与分析技术。