Selection of an improved RNA polymerase ribozyme with superior extension and fidelity.
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 approximately 9 x 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.
Zaher
Hani S
HS
Department of Molecular Biology and Biochemistry, Simon Fraser University, BC, Canada.
Unrau
Peter J
PJ
D001483:Base Sequence; D002384:Catalysis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D004526:Efficiency; D005240:Feasibility Studies; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering / Q000379:methods*; D011786:Quality Control; D016337:RNA, Catalytic / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013698:Templates, Genetic
D016337:RNA, Catalytic; D012321:DNA-Directed RNA Polymerases