Nature Chemistry: Directed evolution of DNA polymerase

已有 2306 次阅读 2016-6-9 09:04 |系统分类:论文交流

Evolution of thermophilic DNA polymerases for the recognition and amplification of C2ʹ-modified DNA

Tingjian Chen, Narupat Hongdilokkul, Zhixia Liu, Ramkrishna Adhikary, Shujian S. Tsuen and Floyd E. Romesberg

Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

The PCR amplification of oligonucleotides enables the evolution of sequences called aptamers that bind specific targetswith antibody-like affinity. However, in many applications the use of these aptamers is limited by nuclease-mediateddegradation. In contrast, oligonucleotides that are modified at their sugar C2positions with methoxy or fluorinesubstituents are stable to nucleases, but they cannot be synthesized by natural polymerases. Here we report thedevelopment of a polymerase-evolution system and its use to evolve thermostable polymerases that efficientlyinterconvert C2-OMe-modified oligonucleotides and their DNA counterparts via transcriptionand reverse transcriptionor, more importantly, that PCR-amplify partially C2-OMe- or C2-F-modified oligonucleotides. A mechanistic analysisdemonstrates that the ability to amplify the modified oligonucleotides evolved by optimizing interdomain interactions thatstabilize the catalytically competent closed conformation of the polymerase. The evolved polymerases should find practicalapplications and the developed evolution system should be a powerful tool for tailoring polymerases to have other typesof novel function.

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