Continuous In Vitro Evolution of a Ribozyme that Catalyzes Three Successive Nucleotidyl Addition Reactions Original Research Article

Variants of the class I ligase ribozyme, which catalyzes joining of the 3′ end of a template bound oligonucleotide to its own 5′ end, have been made to evolve in a continuous manner by a simple serial transfer procedure that can be carried out indefinitely. This process was expanded to allow the evolution of ribozymes that catalyze three successive nucleotidyl addition reactions, two template-directed mononucleotide additions followed by RNA ligation. During the development of this behavior, a population of ribozymes was maintained against an overall dilution of more than 10406. The resulting ribozymes were capable of catalyzing the three-step reaction pathway, with nucleotide addition occurring in either a 5′→3′ or a 3′→5′ direction. This purely chemical system provides a functional model of a multi-step reaction pathway that is undergoing Darwinian evolution.