T7 RNA polymerase engineering and RNA amplification

描述(由申请人提供)：扩大微阵列系统用于全球基因表达谱分析的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标发现。人们强烈希望将这种微阵列能力扩展到更多RNA受限的材料，以使单个细胞可以放心地进行分析。最广泛使用的用于微阵列分析的样品制备方法，使用从mRNA合成cDNA，然后基于T7RNA聚合酶扩增，目前缺乏必要的灵敏度来满足这些新出现的需求。为了克服这些方法学上的局限性，我们建议通过1)利用基于结构的工程和功能的定向进化来提高T7 RNA聚合酶的效率，2)通过针对特定的扩增需要优化启动子-模板结构的设计，以及3)通过为较低的RNA输入重新标准化cDNA合成来提高最终的扩增RNA产量。与目前商业上可用的试剂盒相比，这两项改进预计将使细胞限制的RNA样本需求降低100倍。这将使微阵列基因分析只需单轮扩增即可获得1 ng的总RNA，或在两轮扩增后从单个细胞(总RNA-10 pg)进行微阵列分析。

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.