Expanding Synthetic Biology's Toolkit By in Vitro Evolution: Characterization of a Fast and Uniquely Unnatural Hammerhead Ribozyme

Ribozymes, and more specifically hammerhead ribozymes, have been successfully used in synthetic biology approaches to artificially control gene expression via trans- or cis-acting mechanisms. A typical Hammerhead ribozyme consists of a highly conserved catalytic core of around 15 nucleotides flanked by three based-paired stems and two loops. This structural arrangement includes specific non- Watson-Crick interactions that prime the ribozyme for activity. Furthermore, kissing-loop interaction of loop II with loop I, which often occurs in naturally occurring ribozymes, mediates enhanced catalytic activity in natural instances of these ribozymes. In this study, we describe characterization of an atypical hammerhead ribozyme obtained through in vitro evolution that features an unusually short stem-loop II structure and a high catalytic activity. The minimal stem-loop II structure and the retaining of full functionality of this structure motivated us to characterize this molecule further. Extensive mutational analyses and biochemical assays evaluating the effects of point mutations on catalysis were done for this purpose. Our analyses showed sensitivity of catalytic rates to specific sequences of stem-loop II and stem I structure.  We carried out de novo in silico simulations to obtain plausible 3D structural models that can explain our observed biochemical experiments, providing a deeper insight on the structural arrangement as well as a plausible three-dimensional folding pattern that would account for the high catalytic activity, or lack of, in certain mutants. Molecular dynamics simulations to further refine a plausible structure for this ribozyme are ongoing. This study has further been complemented by multiple genome-wide scans for naturally occurring RNA molecules sharing this specific RNA motif. These scans have yielded no significant hits in 108 eukaryotic and prokaryotic genomes searched so far, suggesting a lack of natural occurrences of this ribozyme. This apparent under-representation in extant genomes, suggest that this specific hammerhead ribozyme might have been under negative selective pressures throughout evolution. These characteristics underline a positive potential in using this specific ribozyme motif in synthetic biology approaches. The ease of activating/deactivating this ribozyme through its peripheral structures sets forth a solid, promising ground, to generation of an effective tool for efficient modulation of gene expression.