T7 RNA polymerase engineering and RNA amplification

扩大微阵列系统在全球基因表达谱分析中的应用是领先的生物学研究， 为医生提供诊断和预后价值，并促进药物治疗期间的靶标发现， 疫苗研发。人们对将这种微阵列能力扩展到更多的RNA限制性基因表达有着浓厚的兴趣。 材料到可以有信心地描绘单个细胞的程度。的最广泛使用的方法 制备用于微阵列分析的样品，该微阵列分析使用从mRNA合成cDNA，然后是T7 RNA 基于聚合酶的扩增目前缺乏满足这些新兴需求所必需的灵敏度。到 克服这些方法上的局限性，我们建议增加最终扩增的RNA产量1） 使用基于结构的工程和定向进化提高T7 RNA聚合酶的效率 功能，2）通过优化启动子-模板构建体的设计以满足特异性扩增需要， 3)通过重新标准化cDNA合成以降低RNA输入。综合改进包括 与目前商业上使用的RNA样品相比， 可用的套件。这将使微阵列基因分析与少至1 ng总RNA与一轮 在两轮扩增后，从单个细胞（~ 10 pg总RNA）扩增或微阵列分析 放大

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.