An error-prone T7 RNA polymerase mutant generated by directed evolution.
Viruses replicate their genomes at exceptionally high mutation rates. Their offspring evolve rapidly and therefore, are able to evade common immunological and chemical antiviral agents. In parallel, virus genomes cannot tolerate a further increase in mutation rate: Experimental evidence exists that even few additional mutations are sufficient for the extinction of a viral population. A future antiviral strategy might therefore aim at increasing the error-producing capacity of viral replication enzymes. We employed the principles of directed evolution and developed a scheme for the stringent positive selection of error-prone polymerase activity. A mutant T7 RNA polymerase with a nucleotide substitution error rate at least 20-fold greater than that of the wild-type was selected. This enzyme synthesized highly heterogeneous RNA products in vitro or in vivo and also decreased the replication efficiency of wild-type bacteriophage T7 during infection.
Brakmann
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Evotec Biosystems AG, Schnackenburgallee 114, 22525 Hamburg, Germany. sbrakma@gwdg.de
Grzeszik
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D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics
D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases