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Directed evolution of artificial enzymes (XNAzymes) from diverse repertoires of synthetic genetic polymers


This protocol describes the directed evolution of artificial endonuclease and ligase enzymes composed of synthetic genetic polymers (XNAzymes), using 'cross-chemistry selective enrichment by exponential amplification' (X-SELEX). The protocol is analogous to (deoxy)ribozyme selections, but it enables the development of fully substituted catalysts. X-SELEX is initiated by the synthesis of diverse repertoires (here 1014 different sequences), using xeno nucleic acid (XNA) polymerases, on DNA templates primed with DNA, RNA or XNA oligonucleotides that double as substrates, allowing selection for XNA-catalyzed cleavage or ligation. XNAzymes are reverse-transcribed into cDNA using XNA-dependent DNA polymerases, and then PCR-amplified to generate templates for subsequent rounds or deep sequencing. We describe methods developed for four XNA chemistries, arabino nucleic acids (ANAs), 2′-fluoroarabino nucleic acids (FANAs), hexitol nucleic acids (HNAs) and cyclohexene nucleic acids (CeNAs), which require 1 week per round, and typically 10–20 rounds; in principle, these methods are scalable and applicable to a wide range of novel XNAzyme chemistries, substrates and reactions.

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Figure 1: Overview of X-SELEX.
Figure 2: Synthesis and preparation of XNAzyme libraries (Steps 1–20).
Figure 3: Reaction, selection and reverse transcription of XNAzyme libraries (Steps 26–34).
Figure 4: Amplification and generation of XNAzyme cDNA templates (Steps 35–46).


  1. 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 

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

    Article  CAS  Google Scholar 

  3. 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 

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

    Article  CAS  Google Scholar 

  5. Silverman, S.K. In vitro selection, characterization, and application of deoxyribozymes that cleave RNA. Nucleic Acids Res. 33, 6151–6163 (2005).

    Article  CAS  Google Scholar 

  6. Keefe, A.D. & Cload, S.T. SELEX with modified nucleotides. Curr. Opin. Chem. Biol. 12, 448–456 (2008).

    Article  CAS  Google Scholar 

  7. Vaught, J.D. et al. Expanding the chemistry of DNA for in vitro selection. J. Am. Chem. Soc. 132, 4141–4151 (2010).

    Article  CAS  Google Scholar 

  8. Lin, Y., Qiu, Q., Gill, S.C. & Jayasena, S.D. Modified RNA sequence pools for in vitro selection. Nucleic Acids Res. 22, 5229–5234 (1994).

    Article  CAS  Google Scholar 

  9. Hollenstein, M., Hipolito, C.J., Lam, C.H. & Perrin, D.M. Toward the combinatorial selection of chemically modified DNAzyme RNase A mimics active against all-RNA substrates. ACS Comb. Sci. 15, 174–182 (2013).

    Article  CAS  Google Scholar 

  10. Renders, M., Miller, E., Hollenstein, M. & Perrin, D. A method for selecting modified DNAzymes without the use of modified DNA as a template in PCR. Chem. Commun. 51, 1360–1362 (2015).

    Article  CAS  Google Scholar 

  11. Burnett, J.C. & Rossi, J.J. RNA-based therapeutics: current progress and future prospects. Chem. Biol. 19, 60–71 (2012).

    Article  CAS  Google Scholar 

  12. Taylor, A.I., Arangundy-Franklin, S. & Holliger, P. Towards applications of synthetic genetic polymers in diagnosis and therapy. Curr. Opin. Chem. Biol. 22, 79–84 (2014).

    Article  CAS  Google Scholar 

  13. Santoro, S.W. & Joyce, G.F. A general purpose RNA-cleaving DNA enzyme. Proc. Natl. Acad. Sci. USA 94, 4262–4266 (1997).

    Article  CAS  Google Scholar 

  14. Wu, Y. et al. Inhibition of bcr-abl oncogene expression by novel deoxyribozymes (DNAzymes). Hum. Gene Ther. 10, 2847–2857 (1999).

    Article  CAS  Google Scholar 

  15. Lan, N., Howrey, R.P., Lee, S.W., Smith, C.A. & Sullenger, B.A. Ribozyme-mediated repair of sickle β-globin mRNAs in erythrocyte precursors. Science 280, 1593–1596 (1998).

    Article  CAS  Google Scholar 

  16. Breaker, R.R. & Joyce, G.F. The expanding view of RNA and DNA function. Chem. Biol. 21, 1059–1065 (2014).

    Article  CAS  Google Scholar 

  17. Herdewijn, P. & Marliere, P. Toward safe genetically modified organisms through the chemical diversification of nucleic acids. Chem. Biodivers. 6, 791–808 (2009).

    Article  CAS  Google Scholar 

  18. Schmidt, M. Xenobiology: a new form of life as the ultimate biosafety tool. Bioessays 32, 322–331 (2010).

    Article  CAS  Google Scholar 

  19. Lagoja, I.M., Marchand, A., Van Aerschot, A. & Herdewijn, P. Synthesis of 1,5-anhydrohexitol building blocks for oligonucleotide synthesis. Curr. Protoc. Nucleic Acid Chem. 14, 1.9.1–1.9.22 (2003).

    Article  Google Scholar 

  20. Zlatev, I., Manoharan, M., Vasseur, J.-J. & Morvan, F. Solid-phase chemical synthesis of 5-triphosphate DNA, RNA, and chemically modified oligonucleotides. Curr. Protoc. Nucleic Acid Chem. 50, 1.28.1–1.28.16 (2012).

    Article  Google Scholar 

  21. Deleavey, G.F. et al. Synergistic effects between analogs of DNA and RNA improve the potency of siRNA-mediated gene silencing. Nucleic Acids Res. 38, 4547–4557 (2010).

    Article  CAS  Google Scholar 

  22. Chu, B.C., Wahl, G.M. & Orgel, L.E. Derivatization of unprotected polynucleotides. Nucleic Acids Res. 11, 6513–6529 (1983).

    Article  CAS  Google Scholar 

  23. Breaker, R.R. & Joyce, G.F. A DNA enzyme that cleaves RNA. Chem. Biol. 1, 223–229 (1994).

    Article  CAS  Google Scholar 

  24. Breaker, R.R. & Joyce, G.F. A DNA enzyme with Mg2+-dependent RNA phosphoesterase activity. Chem. Biol. 2, 655–660 (1995).

    Article  CAS  Google Scholar 

  25. Elshire, R.J., Glaubitz, J.C., Sun, Q. & Poland, J.A. A robust, simple genotyping-by-sequencing (GBS) approach for high-diversity species. PLoS ONE 6, e19379 (2011).

    Article  CAS  Google Scholar 

  26. Brody, J.R. & Kern, S.E. Sodium boric acid: a tris-free, cooler conductive medium for DNA electrophoresis. Biotechniques 36, 214–216 (2004).

    Article  CAS  Google Scholar 

  27. Tolle, F., Wilke, J., Wengel, J. & Mayer, G. By-product formation in repetitive PCR amplification of DNA libraries during SELEX. PLoS ONE 9, e114693 (2014).

    Article  Google Scholar 

  28. Shao, K. et al. Emulsion PCR: a high efficient way of PCR amplification of random DNA libraries in aptamer selection. PLoS ONE 6, e24910 (2011).

    Article  CAS  Google Scholar 

  29. Matochko, W.L., Cory Li, S., Tang, S.K.Y. & Derda, R. Prospective identification of parasitic sequences in phage display screens. Nucleic Acids Res. 42, 1784–1798 (2014).

    Article  CAS  Google Scholar 

  30. Goecks, J., Nekrutenko, A., Taylor, J. & Galaxy Team Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 11, R86 (2010).

    Article  Google Scholar 

  31. Blankenberg, D. et al. Galaxy: a web-based genome analysis tool for experimentalists. Curr. Protoc. Mol. Biol. 89, 19.10.1 19.10. 21 (2010).

    Google Scholar 

  32. Giardine, B. et al. Galaxy: a platform for interactive large-scale genome analysis. Genome Res. 15, 1451–1455 (2005).

    Article  CAS  Google Scholar 

  33. Alam, K.K., Chang, J.L. & Burke, D.H. FASTAptamer: a bioinformatic toolkit for high-throughput sequence analysis of combinatorial selections. Mol. Ther. Nucleic Acids 4, e230 (2015).

    Article  CAS  Google Scholar 

  34. Elle, I.C. et al. Selection of LNA-containing DNA aptamers against recombinant human CD73. Mol. Biosyst. 11, 1260–1270 (2015).

    Article  CAS  Google Scholar 

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The authors thank J. Sutherland for helpful discussions. This work was supported by the UK Medical Research Council (MRC) programme grant U105178804 and by grants from the European Science Foundation (ESF) and the UK Biotechnology and Biological Sciences Research Council (BBSRC; 09-EuroSYNBIO-OP-013).

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A.I.T. and P.H. conceived and designed the protocols, and wrote the manuscript.

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Correspondence to Alexander I Taylor.

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The authors declare no competing financial interests.

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Taylor, A., Holliger, P. Directed evolution of artificial enzymes (XNAzymes) from diverse repertoires of synthetic genetic polymers. Nat Protoc 10, 1625–1642 (2015).

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