Selection of an improved RNA polymerase ribozyme with superior extension and fidelity

Abstract

Our current understanding of biology suggests that early life relied predominantly on RNA for catalysis and replication. Here, we report the isolation of an RNA polymerase ribozyme called B6.61 that exhibits superior extension and fidelity relative to its progenitor, the Round-18 polymerase. The B6.61 polymerase was selected from a mutagenized pool containing ; 9 3 10 14 sequence variants through the use of a novel large-scale in vitro compartmentalization system. B6.61 polymerized all tested primer-template (PT) complexes faster than the Round-18 variant. For one PT, B6.61 exhibited dramatically faster elongation past one full helical turn and incorporated at least 20 nucleotides of sequence, setting a new extension record for an RNA polymerase ribozyme. The increased efficiency of the B6.61 construct was related to improvements in fidelity, with the new variant incorporating less incorrect wobble base pairs than its parent. This new polymerase demonstrates the feasibility of evolving an artificial RNA replicase ribozyme in the foreseeable future.

The Persistent Contributions of RNA to Eukaryotic Gen(om)e Architecture and Cellular Function

“…In any event, by applying in vitro synthesis and selection procedures (Ellington and Szostak 1990;Tuerk and Gold 1990;Gold et al 2012), several laboratories are making great strides toward generating RNA molecules with the ability to self-replicate (Doudna and Szostak 1989;Johnston et al 2001;Zaher and Unrau 2007;Lincoln and Joyce 2009;Shechner et al 2009;Wochner et al 2011;Attwater et al 2013;Mast et al 2013), although these RNA polymerase ribozymes still fail to completely self-replicate (Deamer 2005). Cooperation of two or more RNA enzymes in hypercycles (Eigen and Schuster 1977) may be a solution to this problem (Vaidya et al 2012) (see also below).…”

The RNA Worlds in Context

“…Yarus (2010), an unabashed enthusiast for an RNA world, argues for a closely related replicative precursor. In vitro evolution studies directed towards an RNA replicase ribozyme continue apace and are of great importance in establishing the biochemical plausibility of RNA-catalyzed RNA replication (Johnston et al 2001;Zaher and Unrau 2007;Lincoln and Joyce 2009;Shechner et al 2009). …”

Entropy Production and the Origin of Life

“…Indications that RNA might have played a part in its own replication are various; 1) the observed ability of RNA to self-splice [6], 2) to act as a rybozyme; i.e. as a polymerase catalyst in template assisted polymerization of itself [7,8], and 3) its ability to act as a catalyst for the formation of peptide bonds between amino acids [9].…”