Development and Applications of High Density RNA Arrays

产品说明：该项目的总体目标是完善和优化我们开发的用于制造高密度RNA阵列的强大新方法，并在三个重要且有趣的应用中展示其实用性。这些阵列是高密度DNA阵列的RNA类似物，在过去的二十年中，高密度DNA阵列被证明是生物分子分析的非常强大的工具，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，高密度DNA阵列通过标准的PCR方法使用光保护的核苷亚磷酰胺来制造，表面结合的DNA分子被酶促复制到它们的RNA互补物中（也被束缚到表面），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。其他感兴趣的参数是RNA链的表面密度，用于阵列制造的底物的性质，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，并用于指导优化（最大化序列保真度和长度）的化学和酶学用于阵列制造。优化的其他关键方面包括链密度、底物类型和包含各种修饰的核苷。技术开发和优化将在三个独立的应用背景下进行，这些应用为生物分子分析开辟了新的可能性：1。GFP的荧光RNA模拟物的鉴定和表征; 2.）确定3 '-和5'-UTR mRNA序列中的微小RNA结合位点;和3.）确定RNA结合蛋白的结合特异性。综上所述，上述RNA阵列技术的靶向优化和应用程序将产生一个强大而多功能的生物分子分析新工具，这将为正常和疾病生物学的研究开辟许多新的前沿。

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.