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Evolution of thermophilic DNA polymerases for the recognition and amplification of C2ʹ-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 C2ʹ 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 C2ʹ-OMe-modified oligonucleotides and their DNA counterparts via ‘transcription’ and ‘reverse transcription’ or, more importantly, that PCR-amplify partially C2ʹ-OMe- or C2ʹ-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.

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Figure 1: Preparation of phage particles and their use in the polymerase evolution system.
Figure 2: Distribution of mutations selected in evolved polymerases.
Figure 3: Characterization of evolved polymerases and the nuclease stability of their transcribed products.
Figure 4: Characterization of the SFM4-3-mediated amplification of C2ʹ-F oligonucleotides and analysis of the stability of the resulting PCR products.

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Acknowledgements

We thank the National Institutes of Health (GM097489) and the Defense Advanced Research Projects Agency (DARPA; N66001-14-2-4052) for supporting this work. We thank P. Schultz for the plasmid pEVOL-pAzF.

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Contributions

T.C. and F.E.R. conceived and designed the experiments. T.C., N.H., Z.L, R.A. and S.S.T. performed experiments. T.C., N.H., Z.L. and F.E.R. analysed the data. T.C. and F.E.R. co-wrote the paper. F.E.R. supervised the project.

Corresponding author

Correspondence to Floyd E. Romesberg.

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The authors declare no competing financial interests.

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Chen, T., Hongdilokkul, N., Liu, Z. et al. Evolution of thermophilic DNA polymerases for the recognition and amplification of C2ʹ-modified DNA. Nature Chem 8, 556–562 (2016). https://doi.org/10.1038/nchem.2493

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