Ribozyme-Based Aminoglycoside Switches of Gene Expression Engineered by Genetic Selection in S-cerevisiae
Ribozyme-Based Aminoglycoside Switches of Gene Expression Engineered by Genetic Selection in S-cerevisiae
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DOI：
10.1021/sb500062p
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发表时间：
2015-05-01
影响因子：
4.7
通讯作者：
Hartig, Joerg S.
中科院分区：
文献类型：
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作者：
Klauser, Benedikt;Atanasov, Janina;Hartig, Joerg S.
条件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，设计在不同环境下可靠运行的正交遗传开关非常重要。基于RNA的开关的优点是效应分子可以立即与插入靶RNA的调节模块相互作用，从而无需通常介导遗传控制的转录因子。人工核糖开关的特点是简单、尺寸小以及高度模块化。我们最近报道了一系列基于锤头核酶的人工核糖开关，它们可以通过切换 mRNA、tRNA 或 rRNA 功能来实现基因表达的转录后调控。迄今为止，更广泛的应用受到中等开关性能和有限的可用效应分子的阻碍。在这里，我们报告了锤头核酶的重新设计，以便有效地响应氨基糖苷类抗生素。我们首先在酿酒酵母中建立了体内选择方案，使我们能够搜索大序列空间以寻找优化的开关。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，增加了使核酶配体依赖性的设计选项。这些创新使得新霉素依赖性 RNA 模块的开发成为可能，将基因表达量提高了 25 倍。所提出的氨基糖苷响应核糖开关属于迄今为止报道的性能最好的基于 RNA 的遗传调节因子。开发的体内选择方案应允许对大序列空间进行采样，以进一步优化核糖开关的工程。
Systems for conditional gene expression are I 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 ribozyrne-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.