T7 RNA polymerase engineering and RNA amplification

描述（由申请人提供）：微阵列系统用于全球基因表达谱分析的扩展使用正在引领生物学研究，为医生提供诊断和预后价值，并促进药物和疫苗开发过程中的靶点发现。有强烈的兴趣将这种微阵列的能力扩展到更多的rna有限的材料，以使单个细胞可以有信心地进行分析。目前使用最广泛的制备微阵列分析样品的方法是利用mRNA合成cDNA，然后进行基于T7 RNA聚合酶的扩增，但这种方法缺乏满足这些新需求所需的灵敏度。为了克服这些方法上的局限性，我们建议通过以下方法来提高最终扩增的RNA产量：1)利用基于结构的工程技术和定向功能进化来提高T7 RNA聚合酶的效率；2)优化启动子模板结构的设计以满足特定扩增需求；3)重新标准化低RNA输入的cDNA合成。与目前的市售试剂盒相比，这些综合改进有望将细胞限制性RNA样品需求降低100倍。这将使微阵列基因分析与少至1 ng的总RNA单轮扩增或微阵列分析，从单细胞（-10 pg总RNA）两轮扩增后。

DESCRIPTION (provided by applicant): Expanding use of microarray systems for global gene expression profiling is leading biological research, providing diagnostic and prognostic value to physicians and facilitating target discovery during drug and vaccine development. There is strong interest in extending this microarray capability to more RNA-limited material to the point that a single cell can be profiled with confidence. The most widely used method for preparing samples for microarray analysis, which uses cDNA synthesis from mRNA followed by T7 RNA polymerase-based amplification, currently lacks the necessary sensitivity to meet these emerging needs. To overcome these methodological limitations, we propose to increase the final amplified RNA yield by 1) improving the efficiency of T7 RNA polymerase using structure-based engineering with directed evolution of function, 2) by optimizing the design of the promoter-template construct for specific amplification needs and 3) by re-standardization of cDNA synthesis for lower RNA inputs. The combined improvements are expected to lower the cell-limited RNA sample requirement by 100-fold compared to current, commercially available kits. This will enable microarray gene analysis with as little as 1 ng of total RNA with a single round of amplification or microarray profiling from a single cell (-10 pg of total RNA) after two rounds of amplification.