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An in vitro translation, selection and amplification system for peptide nucleic acids

Nature Chemical Biology volume 6, pages 148–155 (2010)Cite this article

Abstract

Methods to evolve synthetic, rather than biological, polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include (i) sequence-specific polymerization of synthetic building blocks on an amplifiable template, (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures, (iii) selection of synthetic polymer libraries for desired binding or catalytic properties and (iv) amplification of template sequences that survive selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) using a set of 12 PNA pentamer building blocks. We validated the system by performing six iterated cycles of translation, selection and amplification on a library of 4.3 × 108 PNA-encoding DNA templates and observed >1,000,000-fold overall enrichment of a template encoding a biotinylated (streptavidin-binding) PNA. These results collectively provide an experimental foundation for PNA evolution in the laboratory.

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Figure 1: Strategies to evolve biological and synthetic polymers.
Figure 2: A PNA genetic code that exhibits uniform DNA-templated polymerization.
Figure 3: Sequence-specific DNA-templated PNA polymerization using all 12 building blocks in a variety of contexts.
Figure 4: Translation of a DNA template into PNA and displacement of the resulting PNA strand.
Figure 5: A full cycle of translation, displacement, simulated selection and PCR amplification for a single DNA template encoding a PNA 40-mer.
Figure 6: Model selection of a DNA-templated PNA 40-mer library.

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Acknowledgements

This research was supported by the US Office of Naval Research (N00014-03-1-0749), the National Institutes of Health (R01GM065865) and the Howard Hughes Medical Institute. Y.B. gratefully acknowledges the support of an NSF Graduate Research Fellowship.

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Authors and Affiliations

  1. Department of Chemistry and Chemical Biology and the Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    Yevgeny Brudno, Michael E Birnbaum, Ralph E Kleiner & David R Liu

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  1. Yevgeny Brudno
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  2. Michael E Birnbaum
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  3. Ralph E Kleiner
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  4. David R Liu
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Contributions

Y.B. and D.R.L. designed the research. Y.B., M.E.B., and R.E.K. performed the experiments. All authors contributed to data analysis and manuscript preparation.

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Correspondence to David R Liu.

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Competing interests

Y.B. and D.R.L. are co-inventors on a Harvard University patent describing DNA-templated polymerization.

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Brudno, Y., Birnbaum, M., Kleiner, R. et al. An in vitro translation, selection and amplification system for peptide nucleic acids. Nat Chem Biol 6, 148–155 (2010). https://doi.org/10.1038/nchembio.280

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