Harnessing natural RNA triplex elements found in lncRNAs MALAT1 and NEAT1 for engineering of novel riboswitch platforms.

Synthetic riboswitches are efficient tools to render gene expression inducible by small molecules. Here, we present a novel riboswitch platform based on RNA triplexes stabilizing non-polyadenylated transcripts while still allowing translation of the respective messenger RNA. By incorporating a tetracycline aptamer, we rendered triplex formation dependent upon the presence of the ligand. The new riboswitch platform was able to induce gene expression up to 25-fold as a standalone switch. Combination of the triplex system with an aptazyme placed into the 5'-UTR achieved a 42-fold induction of gene expression. Further, we were able to exchange the triplex, the aptamer, the maturation platform cleaving off the poly-A tail of the switch platform, and the regulated transgene, demonstrating a high degree of modularity. The new riboswitch platform is designed to function independently of nuclear processes such as transcription and RNA processing, potentially expanding the application of these synthetic riboswitches to strictly cytoplasmic therapeutic RNA vectors.