Emergence of diverse ligase reactivities from a single RNA evolution experiment

ABSTRACT RNA enzymes that assemble RNA from shorter pieces would have been important for primordial RNA-based biology. To explore the scope of RNA-catalyzed RNA assembly, we used directed evolution to re-engineer a ligase ribozyme that uses 5′-phosphorimidazolide RNA substrates into a ribozyme that ligates RNA substrates carrying the biologically relevant 5′-triphosphate group. Unexpectedly, this single RNA evolution experiment generated, in addition to the desired triphosphate ligase, four other distinct classes of ligase ribozymes. Three of these four ligase classes were isolated at extremely low abundances, collectively covering