Abstract
Nature often combines independent functional domains to achieve complex function, but this approach has not been extensively explored with artificial enzymes. Here, a group I ribozyme, which can act as an endoribonuclease, was partnered with the R3C ribozyme, which catalyzes the ligation of RNA molecules. The conjoined ribozymes have the potential to perform successive RNA cleavage and joining reactions, resulting in their mutual integration into a target RNA substrate. When simply joined together, however, the ribozymes were unable to achieve this outcome because of inefficient transfer of the substrate between the two catalytic subunits. In vitro evolution was used to optimize the behavior of the conjoined ribozymes, resulting in bifunctional molecules with substantially improved integration activity. The ligase subunit of these molecules was unchanged, whereas the group I subunit acquired several mutations, mostly in peripheral regions. The generation and study of this bifunctional assembly helps shed light on the evolution of modular enzymes and the obstacles that must be overcome in bringing together independent functional domains. These molecules also may be useful as tools for the insertional mutagenesis of target mRNAs.