In Vitro Selection of RNA Aptamers Binding to Nanosized DNA for Constructing Artificial Riboswitches
In Vitro Selection of RNA Aptamers Binding to Nanosized DNA for Constructing Artificial Riboswitches
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体外选择与纳米 DNA 结合的 RNA 适体以构建人工核糖开关
DOI：
10.1021/acssynbio.0c00384
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发表时间：
2020
影响因子：
4.7
通讯作者：
Itoh Yu
中科院分区：
文献类型：
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作者：
Ogawa Atsushi;Itoh Yu
我们设计了一个体外筛选方案，以获得一个适体，从而合理地构建一个人工核糖开关作为其组成部分。事实上，通过该方案获得的纳米尺寸的DNA结合适体使我们能够容易且成功地创建真核核糖开关，其响应于小麦胚芽提取物（真核无细胞表达系统）中的配体（纳米尺寸的DNA）而上调内部核糖体进入位点介导的翻译。最佳核糖开关配体的诱导比在300 μM配体时剂量依赖性地增加至21。这种开关效率比具有广泛使用的茶碱结合适体的相同类型的核糖开关高得多，所述茶碱结合适体是在体外选择的，而没有考虑其用于构建核糖开关的效用。这里描述的选择方案将有助于获得适合于构建人工核糖开关的各种配体/适体对，其可以作为合成生物学中的合成基因电路的元件。
We here designed anin vitroselection 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 wasin vitroselected 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.