Synthetic riboswitches gain increasing interest for controlling transgene expression in diverse applications ranging from synthetic biology, functional genomics, and pharmaceutical target validation to potential therapeutic approaches. However, existing systems often lack the pharmaceutically suited ligands and dynamic responses needed for advanced applications. Here we present a series of synthetic riboswitches for controlling gene expression through the regulation of alternative splicing. Placing the 5′-splice site into a stem structure of a tetracycline-sensing aptamer allows us to regulate the accessibility of the splice site. In the presence of tetracycline, an exon with a premature termination codon is skipped and gene expression can occur, whereas in its absence the exon is included into the coding sequence, repressing functional protein expression. We were able to identify RNA switches controlling protein expression in human cells with high dynamic ranges and different levels of protein expression. We present minimalistic versions of this system that circumvent the need to insert an additional exon. Further, we demonstrate the robustness of our approach by transferring the devices into the important research model organism Caenorhabditis elegans, where high levels of functional protein with very low background expression could be achieved.
RNA-based gene control mechanisms pose an elegant and straightforward way to switch on, off, or fine-tune transgene expression without the need for expressing regulatory proteins. A small molecule effector binds directly to a ligand-binding aptamer RNA structure and thereby modulates expression of an associated target gene. We established genetic switches based on regulation of self-cleaving ribozymes and polyadenylation that allow for control of transgene expression in bacteria, yeast, human cell lines and Caenorhabditis elegans in a robust and dose-dependent manner.
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