Proteo-Science Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
ACS Synth Biol. 2020 Oct 16;9(10):2648-2655. doi: 10.1021/acssynbio.0c00384. Epub 2020 Oct 5.
We here designed an selection scheme for obtaining an aptamer with which to rationally construct an artificial riboswitch as its component part. In fact, a nanosized DNA-binding aptamer obtained through this scheme allowed us to easily and successfully create eukaryotic riboswitches that upregulate internal ribosome entry site-mediated translation in response to the ligand (nanosized DNA) in wheat germ extract, a eukaryotic cell-free expression system. The induction ratio of the best riboswitch ligand-dose-dependently increased to 21 at 300 μM ligand. This switching efficiency is much higher than that of the same type of riboswitch with a widely used theophylline-binding aptamer, which was selected without considering its utility for constructing riboswitches. The selection scheme described here would facilitate obtaining various ligand/aptamer pairs suitable for constructing artificial riboswitches, which could serve as elements of synthetic gene circuits in synthetic biology.
我们在这里设计了一种选择方案，用于获得适体，以便合理构建作为其组成部分的人工核糖开关。实际上，通过该方案获得的纳米大小的 DNA 结合适体使我们能够轻松，成功地创建真核核糖体进入位点介导的翻译在上调响应于配体（纳米 DNA）的真核无细胞表达系统中，在小麦胚提取物中的人工核糖开关。最佳核糖开关配体的诱导比随配体浓度的增加而呈剂量依赖性地增加到 300μM 配体的 21。这种切换效率远高于不考虑其构建核糖开关实用性而选择的具有广泛使用的茶碱结合适体的同类型核糖开关。这里描述的选择方案将有助于获得各种适合构建人工核糖开关的配体/适体对，它们可以作为合成生物学中合成基因电路的元件。
