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

描述（由申请人提供）：研究的长期目标是显着推进我们对核酸结构，热力学和动力学的理解。拟议研究的直接目标是显着提高RNA折叠动力学研究的实验和理论能力，这与RNA功能有内在联系。尽管RNA在许多生物学功能如催化（核酶）和基因表达沉默（RNA干扰，RNAi）中起着关键作用，但RNA折叠还没有得到很好的理解。RNA具有直接的医学相关性，如目前在针对眼部疾病（年龄相关性黄斑变性或AMD）的III期临床试验和针对病毒感染（呼吸道合胞病毒或RSV）的II期试验中的RNAi疗法所示。 拟议的研究涉及多光谱，位点特异性（MS 3）探针的开发（合成，表征）和应用，以研究DNA和RNA结构中的局部环境。探针是腈和叠氮化物衍生的核苷。腈（CN）和叠氮（N3）基团代表对核苷结构的小扰动，同时提供局部核酸环境的灵敏IR探针。在CN和N3的末端氮原子的15 N标记后，15 N NMR光谱可用于探测局部核酸环境。总的来说，MS 3探针具有独特的能力，通过两种强大的光谱技术：红外（IR）和15 N NMR光谱，提供有关核酸中所有三个主要环境的位点特异性信息：大沟，小沟和糖/主链区域。将通过使用文献程序或通过利用偶氮转移反应的新合成路线来合成MS 3探针。将研究CN和N3伸缩频率和15 N NMR化学位移对溶剂（水合）、碱基对形成和温度的光谱依赖性。随后，将MS 3探针掺入12个碱基对DNA寡聚体和8个核苷酸RNA发夹中的各个位置。这些核酸结构将通过平衡温度依赖性IR和15 N NMR光谱技术进行研究，以探测熔融后这些结构中存在的各种局部环境。还将使用MS 3探针在DNA寡聚体中探索药物结合诱导的构象变化。这些结果将允许我们的合作者（Corcelli和MacKerell教授）进行适合核酸的力场的参数化和验证。这项与健康有关的研究的另一个目标是培养八名本科生的有机合成和光谱数据采集和分析技术。 公共卫生相关性：拟议的研究中心通过开发多光谱、位点特异性（MS 3）探针，显着提高研究RNA折叠动力学的能力，RNA折叠动力学与RNA功能有内在联系。尽管RNA在许多生物学功能如催化（核酶）和基因表达沉默（RNA干扰，RNAi）中起着关键作用，但RNA折叠还没有得到很好的理解。RNA具有直接的医学相关性，如目前在针对眼部疾病（年龄相关性黄斑变性或AMD）的III期临床试验和针对病毒感染（呼吸道合胞病毒或RSV）的II期试验中的RNAi疗法所示。

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).