Development of tetracycline inducible self-cleaving RNA switches

A. Wittmann and B. Suess, Institute for Molecular Biosciences, Goethe Universität Frankfurt am Main, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany.

Gene regulation on the level of RNA has been a field of intense research in the past few years. Here, allosteric ribozymes (aptazymes) constitute a powerful tool. They comprise a catalytic ribozyme domain linked to an aptamer domain that regulates ribozyme activity.

We developed aptazymes that are composed of the self-cleaving hammerhead ribozyme N79 of Schistosoma mansoni and a tetracycline binding aptamer. We randomized the linker between ribozyme and aptamer domain and applied in vitro selection under physiologic conditions in order to select for sequences that permit allosteric ribozyme regulation. After eleven rounds of selection, we isolated individual molecules with most of them showing tetracycline inducible hammerhead cleavage. Interestingly, we observed no significant consensus sequence but rather similarities in the base pairing pattern of the linker. The aptazymes cleave in presence of 1 µM tetracycline as fast as the parental hammerhead ribozyme domain without aptamer whereas cleavage is inhibited up to 333 fold in absence of tetracycline. Reporter gene assays revealed that some switches reduce gene expression in yeast in presence of tetracycline; however, gene expression was not affected in mammalian cells indicating that further screening in vivo may be required for the identification of functional allosteric ribozymes in higher eukaryotes.¹ We have established a 96-well screening assay and are currently testing the in vitro enriched aptazyme pool in HeLa cells.