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

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Abstract

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).

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Acknowledgements

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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  1. Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

    Alexander I Taylor & Philipp Holliger

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  1. Alexander I Taylor
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  2. Philipp Holliger
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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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Taylor, A., Holliger, P. Directed evolution of artificial enzymes (XNAzymes) from diverse repertoires of synthetic genetic polymers. Nat Protoc 10, 1625–1642 (2015). https://doi.org/10.1038/nprot.2015.104

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