Development and Applications of High Density RNA Arrays

高密度RNA阵列的开发及应用

• 批准号: 9130258
• 负责人: LLOYD M SMITH
• 金额: $ 27.98万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9130258
• 关键词: 5' Untranslated Regions; Affinity; Area; Binding; Binding Sites; Biological; Biology; Chemistry; Complement; Complex; DNA; DNA Binding; Development; Disease; Enzymatic Biochemistry; Genetic Variation; Goals; Health; Human Genome; Left; Length; Measures; Medical; Messenger RNA; Methods; MicroRNAs; Modeling; Monitor; Nature; Nucleic Acids; Nucleosides; Property; RNA; RNA-Binding Proteins; RNA-Induced Silencing Complex; Reporting; Research; Science; Specificity; Structure; Surface; Synthesis Chemistry; Technology; Therapeutic; Variant; analog; aptamer; chromophore; density; frontier; genome-wide; improved; interest; new technology; novel strategies; phosphoramidite; programs; technology development; tool; transcriptomics

DESCRIPTION: The overall goals of this project are to refine and optimize a powerful new approach that we developed for the fabrication of high density RNA arrays and to demonstrate its utility in three important and interesting applications. These arrays are RNA analogs to the high density DNA arrays that have proven over the past two decades to be tremendously powerful tools for biomolecular analysis, particularly in the areas of global transcriptomics and genome-wide genetic variation analysis. In our approach, a high density DNA array is fabricated by standard photolithographic methods using photoprotected nucleoside phosphoramidites, the surface-bound DNA molecules are enzymatically copied into their RNA complements (also tethered to the surface), and the DNA templates are enzymatically destroyed, leaving behind the desired RNA array. Our strategy begins with technology development and optimization. The utility of RNA arrays depends strongly upon the array quality. Two key interrelated quality parameters are the RNA strand sequence fidelity and the RNA lengths that can be obtained. Other parameters of interest are the surface density of the RNA strands, the nature of the substrates employed for array fabrication, and the ability to employ modified nucleosides of various types. Tools needed to measure these parameters will be developed and used to guide optimization (maximize sequence fidelity and length) of the chemistry and enzymology employed for array fabrication. Other key facets of optimization include strand density, substrate types, and inclusion of various modified nucleosides. Technology development and optimization will be performed in the context of three separate applications that open new possibilities in biomolecular analysis: 1.) Identification and characterization of fluorescent RNA mimics of GFP; 2.) Determination of microRNA binding sites in 3'- and 5'- UTR mRNA sequences; and 3.) Determining the binding specificity of RNA-binding proteins. Taken together, the above program of targeted optimization and application of RNA array technology will yield a powerful and versatile new tool for biomolecular analysis that will open many new frontiers for research in the study of normal and disease biology.

描述：该项目的总体目标是完善和优化我们为制造高密度 RNA 阵列而开发的强大新方法，并展示其在三个重要且有趣的应用中的实用性。这些阵列是高密度 DNA 阵列的 RNA 类似物，在过去二十年中已被证明是生物分子分析的极其强大的工具，特别是在全球转录组学和全基因组遗传变异分析领域。在我们的方法中，使用光保护核苷亚磷酰胺通过标准光刻方法制造高密度 DNA 阵列，表面结合的 DNA 分子被酶促复制到其 RNA 互补物（也束缚在表面）中，并且 DNA 模板被酶促破坏，留下所需的 RNA 阵列。 我们的战略始于技术开发和优化。 RNA 阵列的实用性在很大程度上取决于阵列的质量。两个关键的相互关联的质量参数是 RNA 链序列保真度和可以获得的 RNA 长度。其他感兴趣的参数包括 RNA 链的表面密度、用于阵列制造的基质的性质以及使用各种类型的修饰核苷的能力。将开发测量这些参数所需的工具，并用于指导阵列制造所用化学和酶学的优化（最大化序列保真度和长度）。优化的其他关键方面包括链密度、底物类型以及各种修饰核苷的包含。技术开发和优化将在三个独立应用的背景下进行，这为生物分子分析开辟了新的可能性：1.) GFP 荧光 RNA 模拟物的识别和表征； 2.) 3'-和5'-UTR mRNA序列中microRNA结合位点的测定； 3.) 确定RNA结合蛋白的结合特异性。总而言之，上述 RNA 阵列技术的针对性优化和应用计划将产生一种强大且多功能的生物分子分析新工具，为正常和疾病生物学研究开辟许多新领域。