Circularly-Permuted Pistol Ribozyme: A Synthetic Ribozyme Scaffold for Mammalian Riboswitches
Circularly-Permuted Pistol Ribozyme: A Synthetic Ribozyme Scaffold for Mammalian Riboswitches
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DOI：
10.1021/acssynbio.1c00213
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发表时间：
2021-08-10
影响因子：
4.7
通讯作者：
Yokobayashi, Yohei
中科院分区：
文献类型：
--
作者：
Mustafina, Kamila;Nomura, Yoko;Yokobayashi, Yohei
嵌入 mRNA 非翻译区的小分子响应自裂解核酶（适体酶）充当核糖开关，允许对哺乳动物细胞中的基因表达进行化学调节。适体酶是通过将自裂解核酶与识别小分子的RNA适体融合而设计的，以便核酶在小分子存在的情况下被激活或被抑制。然而，适合哺乳动物核糖开关应用的适体、核酶和适体酶设计策略的多样性仍然有限。这项工作的重点是一种新的核酶支架，用于工程化在哺乳动物细胞中发挥作用的适体酶和核糖开关。我们研究了手枪核酶类（CPP）的循环排列变体作为哺乳动物核糖开关应用的合成核酶支架。通过半理性设计和高通量筛选，我们基于三种不同的适体酶结构设计了鸟嘌呤和四环素激活的核糖开关，得到的核糖开关的ON/OFF比高达8.6。我们的工作将 CPP 添加到用于哺乳动物合成生物学应用的有限核酶支架工具箱中，并强调了探索天然基序之外的核酶的机会。
A small molecule-responsive self-cleaving ribozyme (aptazyme) embedded in the untranslated region of an mRNA functions as a riboswitch that allows chemical regulation of gene expression in mammalian cells. Aptazymes are engineered by fusing a self-cleaving ribozyme with an RNA aptamer that recognizes a small molecule so that the ribozyme is either activated or inhibited in the presence of the small molecule. However, the variety of aptamers, ribozymes, and aptazyme design strategies suitable for mammalian riboswitch applications is still limited. This work focuses on a new ribozyme scaffold for engineering aptazymes and riboswitches that function in mammalian cells. We investigated circularly permuted variants of the pistol ribozyme class (CPP) as a synthetic ribozyme scaffold for mammalian riboswitch applications. Through semirational design and high-throughput screening, we designed guanine and tetracycline activated riboswitches based on three distinct aptazyme architectures, resulting in riboswitches with ON/OFF ratios as high as 8.6. Our work adds CPP to the limited ribozyme scaffold toolbox for mammalian synthetic biology applications and highlights the opportunities in exploring ribozymes beyond natural motifs.