Design of Mammalian ON-Riboswitches Based on Tandemly Fused Aptamer and Ribozyme

Self-cleaving ribozymes engineered to be activated or inhibited by a small molecule binding to an RNA aptamer inserted within a ribozyme (aptazymes) have proven to be useful for controlling gene expression in living cells. In mammalian cells, an aptazyme embedded in the 5' or 3' untranslated region of an mRNA functions as a synthetic riboswitch to chemically regulate gene expression. However, the variety of aptazyme architectures and the ribozyme scaffolds that have been used for mammalian riboswitches has been limited. In particular, fewer synthetic riboswitches that activate gene expression in response to a small molecule (ON-switches) in mammalian cells have been reported compared to OFF-switches. In this work, we developed mammalian riboswitches that function as guanine-activated ON-switches based on a novel aptazyme architecture in which an aptamer and a ribozyme are fused in tandem. The riboswitch performance was optimized by fine-tuning the stability of a critical stem that controls the ribozyme structure and function, yielding switches with ON/OFF ratios greater than 6.0. Our new aptazyme architecture expands the RNA device toolbox for controlling gene expression in mammalian cells.