title: Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae.
abstract: 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.
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology (KoRSCB), University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Klauser
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology (KoRSCB), University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany.
authors.forename: Janina
authors.initials: J
authors.lastname: Atanasov
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology (KoRSCB), University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany.
authors.forename: Lena K
authors.initials: LK
authors.lastname: Siewert
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology (KoRSCB), University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany.
authors.forename: Jörg S
authors.initials: JS
authors.lastname: Hartig
mesh_terms: D000617:Aminoglycosides / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D009690:Nucleic Acid Conformation; D012313:RNA / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics*; D058928:Riboswitch / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D000617:Aminoglycosides; D016337:RNA, Catalytic; D058928:Riboswitch; C427204:hammerhead ribozyme; D012313:RNA
keywords: RNA switch; gene regulation; genetic selection; hammerhead ribozyme; neomycin; riboswitch