Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, 2500 Speedway, Austin, TX 78712, USA.
Science. 2016 Jun 24;352(6293):1590-3. doi: 10.1126/science.aaf5409.
Most reverse transcriptase (RT) enzymes belong to a single protein family of ancient evolutionary origin. These polymerases are inherently error prone, owing to their lack of a proofreading (3'- 5' exonuclease) domain. To determine if the lack of proofreading is a historical coincidence or a functional limitation of reverse transcription, we attempted to evolve a high-fidelity, thermostable DNA polymerase to use RNA templates efficiently. The evolutionarily distinct reverse transcription xenopolymerase (RTX) actively proofreads on DNA and RNA templates, which greatly improves RT fidelity. In addition, RTX enables applications such as single-enzyme reverse transcription-polymerase chain reaction and direct RNA sequencing without complementary DNA isolation. The creation of RTX confirms that proofreading is compatible with reverse transcription.
大多数逆转录酶 (RT) 属于具有古老进化起源的单一蛋白质家族。由于缺乏校对（3'-5' 外切核酸酶）结构域，这些聚合酶本质上容易出错。为了确定缺乏校对是逆转录的历史巧合还是功能限制，我们试图进化出一种具有高保真度和热稳定性的 DNA 聚合酶，以有效地利用 RNA 模板。具有进化差异的逆转录异聚酶 (RTX) 可在 DNA 和 RNA 模板上进行有效校对，从而极大地提高了 RT 的保真度。此外，RTX 使得单酶逆转录-聚合酶链式反应和直接 RNA 测序等应用成为可能，而无需分离互补 DNA。RTX 的创建证实了校对与逆转录是兼容的。
