Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.
Department of Chemistry, University of Konstanz, Konstanz, Germany.
Nat Methods. 2014 Nov;11(11):1154-60. doi: 10.1038/nmeth.3136. Epub 2014 Oct 5.
RNAs are ideal for the design of gene switches that can monitor and program cellular behavior because of their high modularity and predictable structure-function relationship. We have assembled an expression platform with an embedded modular ribozyme scaffold that correlates self-cleavage activity of designer ribozymes with transgene translation in bacteria and mammalian cells. A design approach devised to screen ribozyme libraries in bacteria and validate variants with functional tertiary stem-loop structures in mammalian cells resulted in a designer ribozyme with a protein-binding nutR-boxB stem II and a selected matching stem I. In a mammalian expression context, this designer ribozyme exhibited dose-dependent translation control by the N-peptide, had rapid induction kinetics and could be combined with classic small molecule-responsive transcription control modalities to construct complex, programmable genetic circuits.
RNAs 非常适合设计基因开关，因为它们具有高度的模块化和可预测的结构-功能关系，可以监测和编程细胞行为。我们已经组装了一个表达平台，其中嵌入了一个模块化的核酶支架，该支架将设计核酶的自我切割活性与细菌和哺乳动物细胞中转基因的翻译相关联。设计了一种筛选细菌中核酶文库的方法，并在哺乳动物细胞中验证具有功能三级茎环结构的变体，从而得到了一个具有蛋白结合 nutR-boxB 茎 II 和选定匹配茎 I 的设计核酶。在哺乳动物表达环境中，这种设计核酶通过 N 肽表现出剂量依赖性的翻译控制，具有快速诱导动力学，并且可以与经典的小分子响应转录控制模式结合，构建复杂的、可编程的遗传回路。
