[RNA polymerase ribozymes]

Abstract

The RNA World is a hypothetical ancient evolutionary era during which RNA was both genome and catalyst. During that time, RNA was the only kind of enzyme yet in existence, and one of its chief duties was the replication of RNA. This scenario presupposes that among all possible RNA sequences, there exist RNA replicase ribozymes, capable of synthesizing RNA using the information in an RNA template. The goal of the present work is to provide experimental evidence in support of this conjecture, by isolating such ribozymes in the laboratory. We created a large pool of RNA molecules each containing a previously isolated RNA ligase ribozyme and a large stretch of random RNA. Applying in vitro evolution to select for molecules that could extend a tethered RNA primer using nucleoside triphosphates, we isolated nine distinct classes of polymerase ribozymes. Two of these rudimentary polymerases were further evolved to the point that they each could add 14 nucleotides to an untethered primer-template. One of them was subjected to a detailed further characterization. The polymerization it catalyzes was shown to be accurate, with an average fidelity of nearly 97%. It was shown to be general, with primer-templates of all sequences and lengths being accepted as substrates. Finally, it was shown to be partially processive, with the polymerase achieving processivity as high as 90% in a few instances. The polymerase is currently limited by its low affinity for the primer-template. Future work will focus on improving primer- template binding, in order to produce a polymerase that can synthesize longer RNA.