T7 RNA polymerase engineering and RNA amplification

描述(由申请人提供)：微阵列是强大的工具，可以影响新的诊断程序并加快药物开发过程。一个重要但在很大程度上尚未实现的应用是从RNA有限的样本中进行基因表达分析，例如血液、针刺活检、激光捕获显微切割(LCM)样本，甚至是单个细胞。目前最广泛使用的基于T7RNA聚合酶的扩增方法缺乏必要的复杂性来满足这些新出现的需求。为了克服这些限制，我们建议使用一种创新的蛋白质工程方法来提高T7RNA扩增的效率。通过合理设计和随机诱变、突变体文库选择和筛选相结合的方法，我们的目标是鉴定一种高活性的T7RNA聚合酶，其动力学熟练程度是现有酶的4-5倍。我们将在第二阶段扩展这一新方法，以包括发现突变的T7聚合酶，这些酶更耐热(从而延长高效转录更长时间，创造更多产品)，并可以更有效地整合生物素化和Cy修饰的核苷酸5-10倍。总而言之，这些改进将使对输入RNA的需求减少约20倍，并将修饰核苷酸的费用减少多达一个数量级。

DESCRIPTION (provided by applicant): Microarrays are powerful tools that can impact new diagnostic procedures and expedite the drug development process. A significant but largely unrealized application is gene expression analysis from RNAlimited samples, such as blood, needle biopsies, laser capture microdissection (LCM) samples, and even single cells. The most widely used T7 RNA polymerase-based amplification method currenly lacks the necessary sophistication to meet these emerging needs. To overcome these limitations, we propose to increase the efficiency of T7 RNA amplification using an innovative protein engineering approach. Through combined rational design and random mutagenesis, mutant library selection and screening, we aim to identify a hyperactive T7 RNA polymerase that is 4-5 times more kinetically proficient than the current enzyme. We will expand this novel approach in phase II to include the discovery of mutant T7 polymerase enzymes that are both more thermostable (and thus extend high efficiency transcription for longer times, creating more product) and can incorporate biotinylated and Cy-modified nucleotides 5- to 10-fold more efficiently. Taken together, these improvements will lower the demand for input RNA by approximately 20-fold, and reduce the expense of modified nucleotides by as much as an order of magnitude.