PARALLEL SYNTHESIS OF RNA OLIGONUCLEOTIDE MICROARRAYS

该方案的目的是开发一种新的固体表面RNA微阵列(RNA芯片)的并行合成技术。尽管DNA芯片已经成为癌症基因分析、药物发现和各种生物检测的高效和全面的工具，但RNA芯片的并行开发一直是不可能的，主要是因为还没有实用的方法来实现高效和经济的RNA微阵列的并行合成。在这项建议中，将开发一种利用新颖的光化学反应与传统的RNA合成化学相结合的方法来合成RNA芯片。该方法使用光生试剂来允许在一系列反应中对单个反应步骤进行光学控制，例如解除封闭基团的保护。利用微阵列反应器和可编程数字光学投影仪，可以高效率、低成本地实现数千种不同RNA序列的并行合成。我们的第一阶段目标是利用所提出的光引导并行合成方法获得RNA寡核苷酸阵列的原型，并展示合成的RNA芯片的杂交模式。使用我们的芯片技术的一种新的组合策略将被应用于数百个光反应条件的筛选。还将探索光控RNA芯片合成的新应用。我们的第二阶段目标是生产包含20-40个残基的高质量寡核苷酸的下一代RNA芯片，并建立RNA癌基因芯片和RNA文库芯片在反义寡核苷酸和潜在抗肿瘤药物识别RNA靶点方面的应用。RNA芯片提供了明确的序列信息，在癌症分析和药物开发以及许多人类疾病的研究中，是直接研究RNA与各种配体分子相互作用不可或缺的强大工具。预计随着RNA基因组学中信息的快速增长，在确定RNA序列作为特定诊断和治疗靶点方面将会有更大的努力。我们提出的研究是为了提供一种高效、灵活和易于访问的技术来加速这些发现过程。

The objective of this proposal is to develop a novel technology for parallel synthesis of RNA microarray on solid surfaces (RNA-chips). Although DNA-chips have emerged as a high efficient and comprehensive tool for cancer gene analysis, drug discovery and a variety of bioassays, a parallel development of RNA-chips has not been possible, primarily because there is no practical method that can achieve efficient and economic parallel synthesis of RNA microarrays. In this proposal, a method of RNA-chip synthesis using novel photochemical reactions in combination with conventional chemistry of RNA synthesis will be developed. The method uses photogenerated reagents to allow optical control of a single reaction step, such as deprotection of blocking groups, in a sequence of reactions. Using a microarray reactor and a programmable digital optical projector, parallel synthesis of thousands of diverse RNA sequences can be achieved with high efficiency and low costs. Our Phase I goals are to obtain prototype RNA oligonucleotide arrays using the proposed method of light directed parallel synthesis and demonstrate hybridization patterns of the RNA-chips synthesized. A novel combinatory strategy using our chip technology will be applied to screen hundreds of photoreaction conditions. New applications of the light controlled RNA-chip synthesis will be explored. Our Phase II goals are to produce next generation of high quality RNA-chips containing oligonucleotides composed of 20-40 residues and establish the applications of RNA cancer gene-chips and RNA library-chips in identification of RNA targets by antisense oligonucleotides and potential antitumor agents. RNA-chips provide unambiguous sequence information and are indispensable and powerful tool for direct investigation of RNA interactions with a variety of ligand molecules in cancer analysis and drug development and in the study of many human diseases. It is expected that in keeping pace with rapidly mounting information in RNA genomics, there will be an escalated effort in identifying RNA sequences as specific diagnose and therapeutic targets. Our proposed research is to provide a highly efficient, flexible and easily accessible technology to accelerate these discovery processes.