Self-cleaving ribozymes: substrate specificity and synthetic biology applications

Abstract

Various self-cleaving ribozymes appearing in nature catalyze the sequence-specific intramolecular cleavage of RNA and can be engineered to catalyze cleavage of appropriate substrates in an intermolecular fashion, thus acting as true catalysts. The mechanisms of the small, self-cleaving ribozymes have been extensively studied and reviewed previously. Self-cleaving ribozymes can possess high catalytic activity and high substrate specificity; however, substrate specificity is also engineerable within the constraints of the ribozyme structure. While these ribozymes share a common fundamental catalytic mechanism, each ribozyme family has a unique overall architecture and active site organization, indicating that several distinct structures yield this chemical activity. The multitude of catalytic structures, combined with some flexibility in substrate specificity within each family, suggests that such catalytic RNAs, taken together, could access a wide variety of substrates. Here, we give an overview of 10 classes of self-cleaving ribozymes and capture what is understood about their substrate specificity and synthetic applications. Evolution of these ribozymes in an RNA world might be characterized by the emergence of a new ribozyme family followed by rapid adaptation or diversification for specific substrates.

Fig. 1. Mechanism of RNA self-scission and ligation by general acid–base catalysis.

Fig. 2. Aptazyme-based cleavage for mRNA degradation. The yellow regions indicate the aptamer domain, while the black areas indicate the associated ribozyme. The gray boxes indicate base-pairing and the red arrows are the sites of cleavage for each ribozyme. (A) HDV-based, (B) hammerhead-based, and (C) pistol-based aptazymes. (D) Aptazymes inserted in the 3′ untranslated regions of mRNA and cleaving after binding their respective ligands. (E) Cleavage of the ribozyme portion of the aptazyme leads to degradation of the mRNA.