Improved Aptazyme Design and In Vivo Screening Enable Riboswitching in Bacteria

摘要

The development of artificial RNA-based switches of gene expression is inspired by a wealth of recently discovered, naturally occurring riboswitches. Among these, only one example, the glmS riboswitch, operates by ribozyme-medi-ated cleavage of the RNA message upon activation by a small metabolite in bacteria. On the other hand, several examples of so-called aptazymes based on the hammerhead ribozyme (HHR) have been generated that can be controlled by ligands interacting with introduced aptamer domains. Recent results have shown that naturally occurring hammerheads comprise a tertiary interaction between stems I and II, thereby stabilizing the catalytically active conformation. Such extended hammerhead ribozymes display much higher activities, enabling efficient cleavage even when low magnesium concentrations are present such as inside cells. Here, we present a novel design strategy for ligand-controlled hammerhead ribozymes that enables the construction of artificial riboswitches that function in bacteria.