Development of novel RNA-based regulators for mammalian synthetic biology

Mammalian regulatory devices use diverse mechanisms to allow flexible, precise, and comprehensive control over gene expression and cellular development. Fine-tuning of gene expression is critical for many synthetic biology applications. More recently, RNA-based regulatory devices have been explored for modulating mammalian gene expression. Due to their structural properties, riboregulators provide a convenient basis for the development of ligand-dependent controllable systems. In mammalian systems, insertion of self-cleaving ribozymes in the 5’ or 3’ untranslated region of the mRNA of the gene have been utilized for controlling gene expression on a post-transcriptional level. Ribozyme cleavage of the respective mRNA results in RNA degradation and hence significant downregulation of gene expression, whereas ribozyme inhibition upregulates the encoded protein expression. In this project, we are focused to develop novel aptazyme switches for the control of gene regulation with potential application in e.g. construction of mammalian synthetic circuits and gene therapy vectors. Aptazymes can be engineered by attaching ligand-sensing aptamer domain to the catalytically active ribozyme. Ligand-responsive ribozyme switches can regulate self-cleavage events in mRNAs to modulate the stability of the transcript in response to ligand levels. The utilization of aptazymes in synthetic biology and therapeutic applications has many advantages such as an increased robustness due to in cis regulation, small coding space and a high degree of modularity.