Engelhardt Institute of Molecular Biology, The Russian Academy of Sciences, 119991 Moscow, Russia.
Institute of Higher Nervous Activity and Neurophysiology, The Russian Academy of Sciences, 117485 Moscow, Russia.
Int J Mol Sci. 2021 May 14;22(10):5186. doi: 10.3390/ijms22105186.
Pairs of unnatural nucleotides are used to expand the genetic code and create artificial DNA or RNA templates. In general, an approach is used to engineer orthogonal systems capable of reading codons comprising artificial nucleotides; however, DNA and RNA polymerases capable of recognizing unnatural nucleotides are required for amplification and transcription of templates. Under favorable conditions, in the presence of modified nucleotide triphosphates, DNA polymerases are able to synthesize unnatural DNA with high efficiency; however, the currently available RNA polymerases reveal high specificity to the natural nucleotides and may not easily recognize the unnatural nucleotides. Due to the absence of simple and rapid methods for testing the activity of mutant RNA polymerases, the development of RNA polymerase recognizing unnatural nucleotides is limited. To fill this gap, we developed a method for rapid analysis of mutant RNA polymerase activity on templates containing unnatural nucleotides. Herein, we optimized a coupled cell-free translation system and tested the ability of three unnatural nucleotides to be transcribed by different T7 RNA polymerase mutants, by demonstrating high sensitivity and simplicity of the developed method. This approach can be applied to various unnatural nucleotides and can be simultaneously scaled up to determine the activity of numerous polymerases on different templates. Due to the simplicity and small amounts of material required, the developed cell-free system provides a highly scalable and versatile tool to study RNA polymerase activity.
成双的非天然核苷酸被用于扩展遗传密码并创建人工 DNA 或 RNA 模板。一般来说，人们采用一种方法来设计能够读取包含非天然核苷酸的密码子的正交系统；但是，需要能够识别非天然核苷酸的 DNA 和 RNA 聚合酶来扩增和转录模板。在有利条件下，在修饰的核苷酸三磷酸存在的情况下，DNA 聚合酶能够高效地合成非天然 DNA；然而，目前可用的 RNA 聚合酶对天然核苷酸具有高度特异性，可能不容易识别非天然核苷酸。由于缺乏测试突变 RNA 聚合酶活性的简单快速方法，识别非天然核苷酸的 RNA 聚合酶的开发受到限制。为了填补这一空白，我们开发了一种用于快速分析含有非天然核苷酸的模板上突变 RNA 聚合酶活性的方法。在这里，我们优化了一种偶联的无细胞翻译系统，并通过证明开发方法的高灵敏度和简单性，测试了三种非天然核苷酸被不同 T7 RNA 聚合酶突变体转录的能力。该方法可应用于各种非天然核苷酸，并可同时扩展到确定不同模板上许多聚合酶的活性。由于所需的材料简单且数量少，开发的无细胞系统提供了一种高度可扩展且多功能的工具来研究 RNA 聚合酶活性。
