Engineering of ribozyme-based riboswitches for mammalian cells

Artificial RNA riboswitches – apart from protein-based gene regulation systems, which have been known about for a long time – have become increasingly important in biotechnology and synthetic biology. Aptamer-controlled hammerhead ribozymes (so-called aptazymes) have been shown to be a versatile platform for the engineering of novel gene regulators. Since aptazymes are cis-acting elements that are located in the untranslated regions of a gene of interest, their application does not need any further protein co-factor. This presents the opportunity to simplify complex gene networks while simultaneously expanding the repertoire of available parts. Nevertheless, the generation of novel aptazymes requires a functional aptamer–ribozyme connection, which can be difficult to engineer. This article describes a novel approach for using fluorescence activated cell sorting (FACS) in order to identify functional aptazymes in bacteria and their subsequent transfer into mammalian cells.

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► Description of a novel theophylline-responsive aptazyme.
► FACS-mediated screening for functional variants in bacteria.
► Introduction of aptazymes into mammalian cells.