title: Evolution of thermophilic DNA polymerases for the recognition and amplification of C2'-modified DNA.
abstract: The PCR amplification of oligonucleotides enables the evolution of sequences called aptamers that bind specific targets with antibody-like affinity. However, in many applications the use of these aptamers is limited by nuclease-mediated degradation. In contrast, oligonucleotides that are modified at their sugar C2' positions with methoxy or fluorine substituents are stable to nucleases, but they cannot be synthesized by natural polymerases. Here we report the development of a polymerase-evolution system and its use to evolve thermostable polymerases that efficiently interconvert C2'-OMe-modified oligonucleotides and their DNA counterparts via 'transcription' and 'reverse transcription' or, more importantly, that PCR-amplify partially C2'-OMe- or C2'-F-modified oligonucleotides. A mechanistic analysis demonstrates that the ability to amplify the modified oligonucleotides evolved by optimizing interdomain interactions that stabilize the catalytically competent closed conformation of the polymerase. The evolved polymerases should find practical applications and the developed evolution system should be a powerful tool for tailoring polymerases to have other types of novel function.
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Tingjian
authors.identifier: 0000-0002-4458-4269
authors.initials: T
authors.lastname: Chen
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Narupat
authors.initials: N
authors.lastname: Hongdilokkul
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Zhixia
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Ramkrishna
authors.initials: R
authors.lastname: Adhikary
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Shujian S
authors.initials: SS
authors.lastname: Tsuen
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Floyd E
authors.initials: FE
authors.lastname: Romesberg
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004261:DNA Replication / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D019143:Evolution, Molecular; D006358:Hot Temperature; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis
chemical_list: D052157:Aptamers, Nucleotide; D009841:Oligonucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase