PARALLEL SYNTHESIS OF RNA OLIGONUCLEOTIDE MICROARRAYS

本课题的目的是开发一种在固体表面上平行合成RNA微阵列（RNA芯片）的新技术。尽管dna芯片已经成为一种高效、全面的癌症基因分析、药物发现和各种生物检测的工具，但RNA芯片的并行开发一直不可能，主要是因为没有实用的方法可以实现高效、经济的RNA微阵列并行合成。在本研究中，我们将开发一种利用新型光化学反应与传统RNA合成化学相结合的RNA芯片合成方法。该方法使用光生成试剂，允许在一系列反应中对单个反应步骤进行光学控制，例如对阻断基团的去保护。使用微阵列反应器和可编程数字光学投影仪，可以以高效率和低成本实现数千种不同RNA序列的并行合成。我们第一阶段的目标是使用提出的光定向平行合成方法获得原型RNA寡核苷酸阵列，并展示合成的RNA芯片的杂交模式。利用我们的芯片技术，一种新的组合策略将被应用于筛选数百种光反应条件。探讨光控rna芯片合成的新应用。我们的II期目标是生产含有20-40个残基组成的寡核苷酸的下一代高质量RNA芯片，并建立RNA癌症基因芯片和RNA文库芯片在反义寡核苷酸和潜在抗肿瘤药物鉴定RNA靶点方面的应用。RNA芯片提供了明确的序列信息，是在癌症分析和药物开发以及许多人类疾病研究中直接研究RNA与各种配体分子相互作用不可缺少的有力工具。预计随着RNA基因组学信息的快速增长，将会有一个升级的努力来识别RNA序列作为特定的诊断和治疗靶点。我们提出的研究是提供一种高效、灵活和易于获取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.