Hammerhead ribozyme-based RNA switches have been proven to be powerful tools for conditional gene regulation in various organisms. We present neomycin-dependent hammerhead ribozymes (HHR) that influence gene expression in a ligand- and dose-dependent manner in S. cerevisiae. We utilized a novel design of fusing the aptamer domain to the HHR enabling for the first time the identification of genetic ON- and OFF-switches within the same library. For this purpose a neomycin aptamer was fused to stem 1 of a type 3 hammerhead ribozyme via an addressable three-way junction that shows high flexibility at the connection site. An in vivo screening approach identified sequences that allow to induce or repress gene expression 2- to 3-fold in response to neomycin addition. The ribozyme switches operate at neomycin concentrations that show no toxic effect on cell growth and pose powerful genetic tools to study and modulate cellular function in yeast.

中文翻译：

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用于直接控制酿酒酵母基因表达的新霉素依赖性锤头状核酶

基于锤头核酶的 RNA 开关已被证明是各种生物体条件基因调控的强大工具。我们提出了新霉素依赖的锤头状核酶 (HHR)，它以配体和剂量依赖的方式影响酿酒酵母中的基因表达。我们利用了一种将适体结构域融合到 HHR 的新颖设计，首次实现了同一文库中遗传 ON 和 OFF 开关的识别。为此，新霉素适体通过可寻址的三路连接点与 3 型锤头状核酶的茎 1 融合，该连接点在连接位点显示出高度的灵活性。一种体内筛选方法确定了响应新霉素添加可诱导或抑制基因表达 2 至 3 倍的序列。