Klauser Benedikt, Atanasov Janina, Siewert Lena K, Hartig Jörg S
Department of Chemistry, Konstanz Research School Chemical Biology (KoRSCB), University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany.
ACS Synth Biol. 2015 May 15;4(5):516-25. doi: 10.1021/sb500062p. Epub 2014 May 28.
Systems for conditional gene expression are powerful tools in basic research as well as in biotechnology. For future applications, it is of great importance to engineer orthogonal genetic switches that function reliably in diverse contexts. RNA-based switches have the advantage that effector molecules interact immediately with regulatory modules inserted into the target RNAs, getting rid of the need of transcription factors usually mediating genetic control. Artificial riboswitches are characterized by their simplicity and small size accompanied by a high degree of modularity. We have recently reported a series of hammerhead ribozyme-based artificial riboswitches that allow for post-transcriptional regulation of gene expression via switching mRNA, tRNA, or rRNA functions. A more widespread application was so far hampered by moderate switching performances and a limited set of effector molecules available. Here, we report the re-engineering of hammerhead ribozymes in order to respond efficiently to aminoglycoside antibiotics. We first established an in vivo selection protocol in Saccharomyces cerevisiae that enabled us to search large sequence spaces for optimized switches. We then envisioned and characterized a novel strategy of attaching the aptamer to the ribozyme catalytic core, increasing the design options for rendering the ribozyme ligand-dependent. These innovations enabled the development of neomycin-dependent RNA modules that switch gene expression up to 25-fold. The presented aminoglycoside-responsive riboswitches belong to the best-performing RNA-based genetic regulators reported so far. The developed in vivo selection protocol should allow for sampling of large sequence spaces for engineering of further optimized riboswitches.
条件性基因表达系统是基础研究和生物技术中的强大工具。对于未来的应用而言，设计出能在不同环境中可靠发挥作用的正交遗传开关至关重要。基于RNA的开关具有这样的优势，即效应分子能直接与插入到目标RNA中的调控模块相互作用，无需通常介导基因调控的转录因子。人工核糖开关的特点是简单、体积小且具有高度模块化。我们最近报道了一系列基于锤头状核酶的人工核糖开关，它们能够通过切换mRNA、tRNA或rRNA的功能对基因表达进行转录后调控。然而，到目前为止，其应用范围较窄，原因在于开关性能一般以及可用的效应分子种类有限。在此，我们报道了对锤头状核酶进行重新设计，使其能有效响应氨基糖苷类抗生素。我们首先在酿酒酵母中建立了一种体内筛选方案，这使我们能够在大片段序列空间中搜索优化的开关。然后，我们设想并表征了一种将适体连接到核酶催化核心的新策略，增加了使核酶依赖配体的设计选项。这些创新成果促成了新霉素依赖型RNA模块的开发，该模块可使基因表达上调达25倍。所展示的氨基糖苷类响应核糖开关属于目前报道的性能最佳的基于RNA的遗传调控因子。所开发的体内筛选方案应能用于在大片段序列空间中进行采样，以设计出进一步优化的核糖开关。
