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

Nature Chemistry volume 11, pages 533–542 (2019)Cite this article

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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).

References

  1. Westheimer, F. H. Why nature chose phosphates. Science 235, 1173–1178 (1987).

    Article  CAS  Google Scholar 

  2. Benner, S. A. Understanding nucleic acids using synthetic chemistry. Acc. Chem. Res. 37, 784–797 (2004).

    Article  CAS  Google Scholar 

  3. Benner, S. A. & Hutter, D. Phosphates, DNA, and the search for nonterrean life: a second generation model for genetic molecules. Bioorg. Chem. 30, 62–80 (2002).

    Article  CAS  Google Scholar 

  4. Malyshev, D. A. & Romesberg, F. E. The expanded genetic alphabet. Angew. Chem. Int. Ed. 54, 11930–11944 (2015).

    Article  CAS  Google Scholar 

  5. Pinheiro, V. B. & Holliger, P. The XNA world: progress towards replication and evolution of synthetic genetic polymers. Curr. Opin. Chem. Biol. 16, 245–252 (2012).

    Article  CAS  Google Scholar 

  6. Pinheiro, V. B. et al. Synthetic genetic polymers capable of heredity and evolution. Science 336, 341–344 (2012).

    Article  CAS  Google Scholar 

  7. Taylor, A. I. et al. Catalysts from synthetic genetic polymers. Nature 518, 427–430 (2015).

    Article  CAS  Google Scholar 

  8. Malyshev, D. A. et al. A semi-synthetic organism with an expanded genetic alphabet. Nature 509, 385–388 (2014).

    Article  CAS  Google Scholar 

  9. Liu, C. et al. Phosphonomethyl oligonucleotides as backbone-modified artificial genetic polymers. J. Am. Chem. Soc. 140, 6690–6699 (2018).

    Article  CAS  Google Scholar 

  10. Zhang, S. L., Blain, J. C., Zielinska, D., Gryaznov, S. M. & Szostak, J. W. Fast and accurate nonenzymatic copying of an RNA-like synthetic genetic polymer. Proc. Natl Acad. Sci. USA 110, 17732–17737 (2013).

    Article  CAS  Google Scholar 

  11. Ghadessy, F. J. et al. Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution. Nat. Biotechnol. 22, 755–759 (2004).

    Article  CAS  Google Scholar 

  12. Shaw, B. R. et al. Reading, writing, and modulating genetic information with boranophosphate mimics of nucleotides, DNA, and RNA. Ann. NY Acad. Sci. 1002, 12–29 (2003).

    Article  CAS  Google Scholar 

  13. King, D. J., Ventura, D. A., Brasier, A. R. & Gorenstein, D. G. Novel combinatorial selection of phosphorothioate oligonucleotide aptamers. Biochemistry 37, 16489–16493 (1998).

    Article  CAS  Google Scholar 

  14. Meng, M. & Ducho, C. Oligonucleotide analogues with cationic backbone linkages. Beilstein J. Org. Chem. 14, 1293–1308 (2018).

    Article  Google Scholar 

  15. Nielsen, P. E. DNA analogues with nonphosphodiester backbones. Annu. Rev. Biophys. Biomol. Struct. 24, 167–183 (1995).

    Article  CAS  Google Scholar 

  16. Steinbeck, C. & Richert, C. The role of ionic backbones in RNA structure: an unusually stable non-Watson–Crick duplex of a nonionic analog in an apolar medium. J. Am. Chem. Soc. 120, 11576–11580 (1998).

    Article  CAS  Google Scholar 

  17. Micklefield, J. Backbone modification of nucleic acids: synthesis, structure and therapeutic applications. Curr. Med. Chem. 8, 1157–1179 (2001).

    Article  CAS  Google Scholar 

  18. Summerton, J. Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim. Biophys. Acta 1489, 141–158 (1999).

    Article  CAS  Google Scholar 

  19. Nielsen, P. E. & Egholm, M. An introduction to peptide nucleic acid. Curr. Issues Mol. Biol. 1, 89–104 (1999).

    CAS  PubMed  Google Scholar 

  20. Dineva, M. A., Chakurov, S., Bratovanova, E. K., Devedjiev, I. & Petkov, D. D. Complete template-directed enzymatic synthesis of a potential antisense DNA containing 42 methylphosphonodiester bonds. Bioorgan. Med. Chem. 1, 411–414 (1993).

    Article  CAS  Google Scholar 

  21. Higuchi, H., Endo, T. & Kaji, A. Enzymic synthesis of oligonucleotides containing methylphosphonate internucleotide linkages. Biochemistry 29, 8747–8753 (1990).

    Article  CAS  Google Scholar 

  22. Brudno, Y., Birnbaum, M. E., Kleiner, R. E. & Liu, D. R. An in vitro translation, selection and amplification system for peptide nucleic acids. Nat. Chem. Biol. 6, 148–155 (2010).

    Article  CAS  Google Scholar 

  23. Murakami, A., Blake, K. R. & Miller, P. S. Characterization of sequence-specific oligodeoxyribonucleoside methylphosphonates and their interaction with rabbit globin mRNA. Biochemistry 24, 4041–4046 (1985).

    Article  CAS  Google Scholar 

  24. Arzumanov, A. A. & Dyatkina, N. B. An alternative route for preparation of α-methylphosphonyl-β,γ-diphosphates of thymidine derivatives. Nucleos. Nucleot. 13, 1031–1037 (1994).

    Article  CAS  Google Scholar 

  25. Burgers, P. M. J. & Eckstein, F. Stereochemistry of internucleotide bond formation by polynucleotide phosphorylase from Micrococcus luteus. Biochemistry 18, 450–454 (1979).

    Article  CAS  Google Scholar 

  26. Xia, S. & Konigsberg, W. H. Mispairs with Watson–Crick base-pair geometry observed in ternary complexes of an RB69 DNA polymerase variant. Protein Sci. 23, 508–513 (2014).

    Article  CAS  Google Scholar 

  27. Genna, V., Gaspari, R., Dal Peraro, M. & De Vivo, M. Cooperative motion of a key positively charged residue and metal ions for DNA replication catalyzed by human DNA polymerase-η. Nucleic Acids Res. 44, 2827–2836 (2016).

    Article  Google Scholar 

  28. Genna, V., Donati, E. & De Vivo, M. The catalytic mechanism of DNA and RNA polymerases. ACS Catal. 8, 11103–11118 (2018).

    Article  CAS  Google Scholar 

  29. Genna, V., Carloni, P. & De Vivo, M. A strategically located Arg/Lys residue promotes correct base paring during nucleic acid biosynthesis in polymerases. J. Am. Chem. Soc. 140, 3312–3321 (2018).

    Article  CAS  Google Scholar 

  30. Genna, V., Colombo, M., De Vivo, M. & Marcia, M. Second-shell basic residues expand the two-metal-ion architecture of DNA and RNA processing enzymes. Structure 26, 40–50.e2 (2018).

    Article  CAS  Google Scholar 

  31. Cozens, C., Pinheiro, V. B., Vaisman, A., Woodgate, R. & Holliger, P. A short adaptive path from DNA to RNA polymerases. Proc. Natl Acad. Sci. USA 109, 8067–8072 (2012).

    Article  CAS  Google Scholar 

  32. Wynne, S. A., Pinheiro, V. B., Holliger, P. & Leslie, A. G. Structures of an apo and a binary complex of an evolved archeal B family DNA polymerase capable of synthesising highly Cy-dye labelled DNA. PLoS ONE 8, e70892 (2013).

    Article  CAS  Google Scholar 

  33. Bergen, K., Betz, K., Welte, W., Diederichs, K. & Marx, A. Structures of KOD and 9°N DNA polymerases complexed with primer template duplex. ChemBioChem 14, 1058–1062 (2013).

    Article  CAS  Google Scholar 

  34. Genna, V., Vidossich, P., Ippoliti, E., Carloni, P. & De Vivo, M. A self-activated mechanism for nucleic acid polymerization catalyzed by DNA/RNA polymerases. J. Am. Chem. Soc. 138, 14592–14598 (2016).

    Article  CAS  Google Scholar 

  35. Nakamura, T., Zhao, Y., Yamagata, Y., Hua, Y. J. & Yang, W. Watching DNA polymerase η make a phosphodiester bond. Nature 487, 196–201 (2012).

    Article  CAS  Google Scholar 

  36. Pinheiro, V. B., Loakes, D. & Holliger, P. Synthetic polymers and their potential as genetic materials. BioEssays 35, 113–122 (2013).

    Article  CAS  Google Scholar 

  37. Dunn, M. R. & Chaput, J. C. Reverse transcription of threose nucleic acid by a naturally occurring DNA polymerase. ChemBioChem 17, 1804–1808 (2016).

    Article  CAS  Google Scholar 

  38. Thiviyanathan, V. et al. Structure of hybrid backbone methylphosphonate DNA heteroduplexes: effect of R and I stereochemistry. Biochemistry 41, 827–238 (2002).

    Article  CAS  Google Scholar 

  39. Vyazovkina, E. V. et al. Synthesis of specific diastereomers of a DNA methylphosphonate heptamer, d(CpCpApApApCpA), and stability of base pairing with the normal DNA octamer d(TpGpTpTpTpGpGpC). Nucleic Acids Res. 22, 2404–2409 (1994).

    Article  CAS  Google Scholar 

  40. Tsai, C. H., Chen, J. & Szostak, J. W. Enzymatic synthesis of DNA on glycerol nucleic acid templates without stable duplex formation between product and template. Proc. Natl Acad. Sci. USA 104, 14598–14603 (2007).

    Article  CAS  Google Scholar 

  41. Burmeister, P. E. et al. Direct in vitro selection of a 2′-O-methyl aptamer to VEGF. Chem. Biol. 12, 25–33 (2005).

    Article  CAS  Google Scholar 

  42. Alves Ferreira-Bravo, I., Cozens, C., Holliger, P. & DeStefano, J. J. Selection of 2′-deoxy-2′-fluoroarabinonucleotide (FANA) aptamers that bind HIV-1 reverse transcriptase with picomolar affinity. Nucleic Acids Res. 43, 9587–9599 (2015).

    PubMed  PubMed Central  Google Scholar 

  43. Yu, H., Zhang, S. & Chaput, J. C. Darwinian evolution of an alternative genetic system provides support for TNA as an RNA progenitor. Nat. Chem. 4, 183–187 (2012).

    Article  CAS  Google Scholar 

  44. Rangel, A. E., Chen, Z., Ayele, T. M. & Heemstra, J. M. In vitro selection of an XNA aptamer capable of small-molecule recognition. Nucleic Acids Res. 46, 8057–8068 (2018).

    Article  CAS  Google Scholar 

  45. Lee, E. J., Lim, H. K., Cho, Y. S. & Hah, S. S. Peptide nucleic acids are an additional class of aptamers. RSC Adv. 3, 5828–5831 (2013).

    Article  CAS  Google Scholar 

  46. Ichida, J. K. et al. An in vitro selection system for TNA. J. Am. Chem. Soc. 127, 2802–2803 (2005).

    Article  CAS  Google Scholar 

  47. Bing, T., Yang, X. J., Mei, H. C., Cao, Z. H. & Shangguan, D. H. Conservative secondary structure motif of streptavidin-binding aptamers generated by different laboratories. Bioorg. Med. Chem. 18, 1798–1805 (2010).

    Article  CAS  Google Scholar 

  48. Weber, P. C., Ohlendorf, D. H., Wendoloski, J. J. & Salemme, F. R. Structural origins of high-affinity biotin binding to streptavidin. Science 243, 85–88 (1989).

    Article  CAS  Google Scholar 

  49. Freitag, S., LeTrong, I., Klumb, L., Stayton, P. S. & Stenkamp, R. E. Structural studies of the streptavidin binding loop. Protein Sci. 6, 1157–1166 (1997).

    Article  CAS  Google Scholar 

  50. Houlihan, G., Arangundy-Franklin, S. & Holliger, P. Engineering and application of polymerases for synthetic genetics. Curr. Opin. Biotechnol. 48, 168–179 (2017).

    Article  CAS  Google Scholar 

  51. Krishna, H. & Caruthers, M. H. Alkynyl phosphonate DNA: a versatile ‘click’able backbone for DNA-based biological applications. J. Am. Chem. Soc. 134, 11618–11631 (2012).

    Article  CAS  Google Scholar 

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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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Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, UK

    Sebastian Arangundy-Franklin, Alexander I. Taylor, Benjamin T. Porebski, Sew Peak-Chew & Philipp Holliger

  2. Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain

    Vito Genna & Modesto Orozco

  3. Section on DNA Replication, Repair and Mutagenesis, Bethesda, MD, USA

    Alexandra Vaisman & Roger Woodgate

  4. Department of Biochemistry and Biomedicine, University of Barcelona, Barcelona, Spain

    Modesto Orozco

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  1. Sebastian Arangundy-Franklin
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Contributions

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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