Department of Research and Development, QIAGEN Beverly, 100 Cummings Center, Suite 407J, Beverly, MA 01915, USA.
Nucleic Acids Res. 2019 Apr 23;47(7):3619-3630. doi: 10.1093/nar/gkz104.
Reverse transcription is an essential initial step in the analysis of RNA for most PCR-based amplification and detection methods. Despite advancements in these technologies, efficient conversion of RNAs that form stable secondary structures and double-stranded RNA targets remains challenging as retroviral-derived reverse transcriptases are often not sufficiently thermostable to catalyze synthesis at temperatures high enough to completely relax these structures. Here we describe the engineering and improvement of a thermostable viral family A polymerase with inherent reverse transcriptase activity for use in RT-PCR. Using the 3173 PyroPhage polymerase, previously identified from hot spring metagenomic sampling, and additional thermostable orthologs as a source of natural diversity, we used gene shuffling for library generation and screened for novel variants that retain high thermostability and display elevated reverse transcriptase activity. We then created a fusion enzyme between a high-performing variant polymerase and the 5'→3' nuclease domain of Taq DNA polymerase that provided compatibility with probe-based detection chemistries and enabled highly sensitive detection of structured RNA targets. This technology enables a flexible single-enzyme RT-PCR system that has several advantages compared with standard heat-labile reverse transcription methods.
逆转录是大多数基于 PCR 的扩增和检测方法分析 RNA 的基本初始步骤。尽管这些技术取得了进步，但对于形成稳定二级结构和双链 RNA 靶标的 RNA 的有效转化仍然具有挑战性，因为逆转录酶通常不是足够热稳定以在足够高的温度下催化合成，从而完全松弛这些结构。在这里，我们描述了一种具有内在逆转录酶活性的耐热病毒 A 聚合酶的工程改造和改进，用于 RT-PCR。我们使用了先前从温泉宏基因组采样中鉴定出的 3173 PyroPhage 聚合酶和其他耐热同源物作为天然多样性的来源，使用基因改组进行文库生成，并筛选出保留高耐热性和显示出提高的逆转录酶活性的新型变体。然后，我们在一种表现出高活性的变体聚合酶和 Taq DNA 聚合酶的 5'→3'核酸酶结构域之间创建了一种融合酶，该酶与基于探针的检测化学兼容，并能够高度敏感地检测结构 RNA 靶标。与标准的不耐热逆转录方法相比，该技术实现了一种灵活的单酶 RT-PCR 系统，具有几个优点。
