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A synthetic genetic polymer with an uncharged backbone chemistry based on alkyl phosphonate nucleic acids

Abstract

The physicochemical properties of nucleic acids are dominated by their highly charged phosphodiester backbone chemistry. This polyelectrolyte structure decouples information content (base sequence) from bulk properties, such as solubility, and has been proposed as a defining trait of all informational polymers. However, this conjecture has not been tested experimentally. Here, we describe the encoded synthesis of a genetic polymer with an uncharged backbone chemistry: alkyl phosphonate nucleic acids (phNAs) in which the canonical, negatively charged phosphodiester is replaced by an uncharged P-alkyl phosphonodiester backbone. Using synthetic chemistry and polymerase engineering, we describe the enzymatic, DNA-templated synthesis of P-methyl and P-ethyl phNAs, and the directed evolution of specific streptavidin-binding phNA aptamer ligands directly from random-sequence mixed P-methyl/P-ethyl phNA repertoires. Our results establish an example of the DNA-templated enzymatic synthesis and evolution of an uncharged genetic polymer and provide a foundational methodology for their exploration as a source of novel functional molecules.

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Fig. 1: Global and structural modelling of polymerase–P-Et-phATP diastereoisomer complexes.
Fig. 2: Surface electrostatic potential in DNA × DNA and phNA × DNA duplexes.
Fig. 3: Polymerase mutations that enable phNA synthesis.
Fig. 4: Structural context of (S)p P-alkyl-phNTP incorporation.
Fig. 5: MS analysis of phNA synthesis.
Fig. 6: Characterization of phNA aptamers.

Data availability

The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information files. The molecular modelling data and related settings for computations that support the findings of this study are available in the Zenodo database (https://zenodo.org/) with the following record 2579703 (https://doi.org/10.5281/zenodo.2579703).

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Acknowledgements

This work was supported by Trinity College Cambridge (S.A.,-F.), by the Medical Research Council (S.A.-F. A.I.T., S.P.-C., P.H., program no. MC_U105178804), by the Biotechnology and Biological Sciences Research Council (B.T.P., BBSRC grant no BB/N01023x/1), by the NICHD/ NIH Intramural Research Program (A.V. and R.W.) and by a European Molecular Biology Organization (EMBO) Long-Term Fellowship (V.G., ALTF 103-2018).

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S.A.-F. and P.H. conceived and designed the experiments. S.A.-F. performed all the experiments except the SPR measurements (A.T. and B.T.P.), MS (S.P.-C.) and steady-state kinetics (A.V. and R.W.) and Modelling and MD simulations (V.G. and M.O.). All the authors discussed the results, and jointly wrote the manuscript.

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Correspondence to Philipp Holliger.

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Arangundy-Franklin, S., Taylor, A.I., Porebski, B.T. et al. A synthetic genetic polymer with an uncharged backbone chemistry based on alkyl phosphonate nucleic acids. Nat. Chem. 11, 533–542 (2019). https://doi.org/10.1038/s41557-019-0255-4

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