Design of Mammalian ON-Riboswitches Based on Tandemly Fused Aptamer and Ribozyme
Design of Mammalian ON-Riboswitches Based on Tandemly Fused Aptamer and Ribozyme
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DOI：
10.1021/acssynbio.9b00371
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发表时间：
2020-01-01
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;Fukunaga, Keisuke;Yokobayashi, Yohei
经过改造的自裂解核酶可通过与插入核酶（适配酶）内的 RNA 适体结合的小分子来激活或抑制，已被证明可用于控制活细胞中的基因表达。在哺乳动物细胞中，嵌入 mRNA 5' 或 3' 非翻译区的适体酶充当合成核糖开关，以化学方式调节基因表达。然而，用于哺乳动物核糖开关的适体酶结构和核酶支架的种类有限。特别是，与关闭开关相比，在哺乳动物细胞中响应小分子（开启开关）激活基因表达的合成核糖开关较少。在这项工作中，我们开发了哺乳动物核糖开关，其功能作为鸟嘌呤激活的ON开关，基于一种新型的适体酶结构，其中适体和核酶串联融合。通过微调控制核酶结构和功能的关键茎的稳定性，优化了核糖开关的性能，产生开/关比大于 6.0 的开关。我们的新适体酶架构扩展了用于控制哺乳动物细胞基因表达的 RNA 装置工具箱。
Self-cleaving ribozymes engineered to be activated or inhibited by a small molecule binding to an RNA aptamer inserted within a ribozyme (aptazymes) have proven to be useful for controlling gene expression in living cells. In mammalian cells, an aptazyme embedded in the 5' or 3' untranslated region of an mRNA functions as a synthetic riboswitch to chemically regulate gene expression. However, the variety of aptazyme architectures and the ribozyme scaffolds that have been used for mammalian riboswitches has been limited. In particular, fewer synthetic riboswitches that activate gene expression in response to a small molecule (ON-switches) in mammalian cells have been reported compared to OFF-switches. In this work, we developed mammalian riboswitches that function as guanine-activated ON-switches based on a novel aptazyme architecture in which an aptamer and a ribozyme are fused in tandem. The riboswitch performance was optimized by fine-tuning the stability of a critical stem that controls the ribozyme structure and function, yielding switches with ON/OFF ratios greater than 6.0. Our new aptazyme architecture expands the RNA device toolbox for controlling gene expression in mammalian cells.