Expanding the toolbox of synthetic riboswitches with guanine-dependent aptazymes
Expanding the toolbox of synthetic riboswitches with guanine-dependent aptazymes
复制标题
用鸟嘌呤依赖性适体扩展合成核糖开关的工具箱
作者：
J. Stifel;Maike Spöring;J. Hartig
摘要以核酶为基础的人工核糖开关是研究配体依赖性基因表达调控的多功能工具。这些所谓的适体酶的优点是它们的模块化结构和它们所需的非常小的编码空间。在过去的20年里，人们构建了各种各样的核酸适体-核酶组合，所得到的核酸适体酶在原核和真核系统中应用广泛。大多数体内功能性适体酶是OFF开关，而ON开关在例如基因治疗载体中的潜在应用方面更有利。我们在模式生物大肠杆菌和哺乳动物细胞培养物中使用深入研究的鸟嘌呤敏感xpt适体开发了新的ON开关适体酶。利用基于细菌中荧光激活细胞分选的高通量筛选，我们鉴定了高达9.2倍的ON开关和OFF开关，动态范围高达32.7倍。为了在HeLa细胞中构建ON开关，我们使用了基于现有四环素敏感的ON开关的合理设计方法。我们发现，四环素反应的通信模块也在鸟嘌呤适体酶的背景下发挥作用，表现出高度的模块化。在这里，设计了具有四倍动态范围的鸟嘌呤响应ON开关。总之，我们介绍了一系列新的鸟嘌呤依赖性核酶开关在细菌和人类细胞培养，显着拓宽现有的工具箱。
Abstract Artificial riboswitches based on ribozymes serve as versatile tools for ligand-dependent gene expression regulation. Advantages of these so-called aptazymes are their modular architecture and the comparably little coding space they require. A variety of aptamer-ribozyme combinations were constructed in the past 20 years and the resulting aptazymes were applied in diverse contexts in prokaryotic and eukaryotic systems. Most in vivo functional aptazymes are OFF-switches, while ON-switches are more advantageous regarding potential applications in e.g. gene therapy vectors. We developed new ON-switching aptazymes in the model organism Escherichia coli and in mammalian cell culture using the intensely studied guanine-sensing xpt aptamer. Utilizing a high-throughput screening based on fluorescence-activated cell sorting in bacteria we identified up to 9.2-fold ON-switches and OFF-switches with a dynamic range up to 32.7-fold. For constructing ON-switches in HeLa cells, we used a rational design approach based on existing tetracycline-sensitive ON-switches. We discovered that communication modules responding to tetracycline are also functional in the context of guanine aptazymes, demonstrating a high degree of modularity. Here, guanine-responsive ON-switches with a four-fold dynamic range were designed. Summarizing, we introduce a series of novel guanine-dependent ribozyme switches operative in bacteria and human cell culture that significantly broaden the existing toolbox.
登录
查看更多内容
利用体内筛选方法工程改造适体酶开关以在哺乳动物细胞中进行条件基因表达
影响因子：
--
作者：
Rehm C;Klauser B;Hartig JS
通讯作者：
Hartig JS
影响因子：
4.7
作者：
Beilstein, Kim;Wittmann, Alexander;Suess, Beatrix
通讯作者：
Suess, Beatrix
影响因子：
16.6
作者：
Felletti, Michele;Stifel, Julia;Wurmthaler, Lena A.;Geiger, Sophie;Hartig, Joerg S.
通讯作者：
Hartig, Joerg S.
影响因子：
4.7
作者：
Ceres, Pablo;Garst, Andrew D.;Marcano-Velazquez, Joan G.;Batey, Robert T.
通讯作者：
Batey, Robert T.
影响因子：
4.7
作者：
Klauser, Benedikt;Atanasov, Janina;Hartig, Joerg S.
通讯作者：
Hartig, Joerg S.