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

TY - JOUR

T1 - Directed evolution of artificial enzymes (XNAzymes) from diverse repertoires of synthetic genetic polymers

AU - Taylor, Alexander I.

AU - Holliger, Philipp

N1 - Publisher Copyright: © 2015 Nature America, Inc. All rights reserved.

PY - 2015/10/29

Y1 - 2015/10/29

N2 - 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 10 14 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 â 1/41 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.

AB - 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 10 14 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 â 1/41 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.

UR - http://www.scopus.com/inward/record.url?scp=84942520548&partnerID=8YFLogxK

U2 - 10.1038/nprot.2015.104

DO - 10.1038/nprot.2015.104

M3 - Article

C2 - 26401917

AN - SCOPUS:84942520548

SN - 1754-2189

VL - 10

SP - 1625

EP - 1642

JO - Nature Protocols

JF - Nature Protocols

IS - 10

ER -