title: Directed evolution of DNA polymerase, RNA polymerase and reverse transcriptase activity in a single polypeptide.
abstract: DNA polymerases enable key technologies in modern biology but for many applications, native polymerases are limited by their stringent substrate recognition. Here we describe short-patch compartmentalized self-replication (spCSR), a novel strategy to expand the substrate spectrum of polymerases in a targeted way. spCSR is based on the previously described CSR, but unlike CSR only a short region (a "patch") of the gene under investigation is diversified and replicated. This allows the selection of polymerases under conditions where catalytic activity and processivity are compromised to the extent that full self-replication is inefficient. We targeted two specific motifs involved in substrate recognition in the active site of DNA polymerase I from Thermus aquaticus (Taq) and selected for incorporation of both ribonucleotide- (NTP) and deoxyribonucleotide-triphosphates (dNTPs) using spCSR. This allowed the isolation of multiple variants of Taq with apparent dual substrate specificity. They were able to synthesize RNA, while still retaining essentially wild-type (wt) DNA polymerase activity as judged by PCR. One such mutant (AA40: E602V, A608V, I614M, E615G) was able to incorporate both NTPs and dNTPs with the same catalytic efficiency as the wt enzyme incorporates dNTPs. AA40 allowed the generation of mixed RNA-DNA amplification products in PCR demonstrating DNA polymerase, RNA polymerase as well as reverse transcriptase activity within the same polypeptide. Furthermore, AA40 displayed an expanded substrate spectrum towards other 2'-substituted nucleotides and was able to synthesize nucleic acid polymers in which each base bore a different 2'-substituent. Our results suggest that spCSR will be a powerful strategy for the generation of polymerases with altered substrate specificity for applications in nano- and biotechnology and in the enzymatic synthesis of antisense and RNAi probes.
authors.affiliation: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Ong
authors.forename: David
authors.initials: D
authors.lastname: Loakes
authors.forename: Szymon
authors.initials: S
authors.lastname: Jaroslawski
authors.forename: Kathleen
authors.initials: K
authors.lastname: Too
authors.forename: Philipp
authors.initials: P
authors.lastname: Holliger
mesh_terms: D000595:Amino Acid Sequence; D004247:DNA / Q000096:biosynthesis; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D019020:Directed Molecular Evolution; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D012313:RNA / Q000096:biosynthesis; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism; D013379:Substrate Specificity; D019914:Taq Polymerase / Q000235:genetics / Q000378:metabolism*
chemical_list: D003854:Deoxyribonucleotides; D012265:Ribonucleotides; D012313:RNA; D004247:DNA; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases; D004256:DNA Polymerase I