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阵列制造的强大新方法，并在三个重要且有趣的应用中演示其实用性。这些阵列是对高密度DNA阵列的RNA类似物，在过去的二十年中，这些阵列已证明是生物分子分析的强大工具，尤其是在全球转录组学和全基因组全基因组遗传变异分析领域。在我们的方法中，高密度DNA阵列是通过使用光保护型核苷磷酰胺的标准光刻志方法制造的，将表面结合的DNA分子酶上酶学复制到其RNA互补中（也已构成了为表面），并且DNA模板被酶促销毁，留下所需的RNA Array Array。 我们的战略始于技术开发和优化。 RNA阵列的效用在很大程度上取决于阵列质量。两个关键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。感兴趣的其他参数是RNA链的表面密度，用于阵列制造的底物的性质以及采用各种类型的修饰核苷的能力。测量这些参数所需的工具将被开发并用于指导用于阵列制造的化学和酶学的优化（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，底物类型以及包括各种修饰的核苷。技术开发和优化将在生物分子分析中开放新可能性的三个独立应用的背景下进行：1。）荧光RNA模拟GFP的识别和表征； 2.）测定3'-和5'- UTR mRNA序列中的microRNA结合位点； 3.）确定RNA结合蛋白的结合特异性。综上所述，上述针对RNA阵列技术的有针对性优化和应用程序的程序将产生一种强大而多才多艺的新工具，用于生物分子分析，该工具将为正常和疾病生物学研究开辟许多新的领域。