title: Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2'-5' Linkages.
abstract: Information-bearing nucleic acids display universal 3'-5' linkages, but regioisomeric 2'-5' linkages occur sporadically in non-enzymatic RNA synthesis and may have aided prebiotic RNA replication. Herein we report on the enzymatic synthesis of both DNA and RNA with site-specific 2'-5' linkages by an engineered polymerase using 3'-deoxy- or 3'-O-methyl-NTPs as substrates. We also report the reverse transcription of the resulting modified nucleic acids back to 3'-5' linked DNA with good fidelity. This enables a fast and simple method for "structural mutagenesis" by the position-selective incorporation of 2'-5' linkages, whereby nucleic acid structure and function may be probed through local distortion by regioisomeric linkages while maintaining the wild-type base sequence as we demonstrate for the 10-23 RNA endonuclease DNAzyme.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH (UK).
authors.forename: Christopher
authors.initials: C
authors.lastname: Cozens
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH (UK).
authors.forename: Hannes
authors.initials: H
authors.lastname: Mutschler
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH (UK).
authors.forename: Geoffrey M
authors.initials: GM
authors.lastname: Nelson
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH (UK).
authors.forename: Gillian
authors.initials: G
authors.lastname: Houlihan
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH (UK).
authors.forename: Alexander I
authors.initials: AI
authors.lastname: Taylor
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH (UK). ph1@mrc-lmb.cam.ac.uk.
authors.forename: Philipp
authors.initials: P
authors.lastname: Holliger
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004798:Enzymes / Q000737:chemistry*; D009696:Nucleic Acids / Q000138:chemical synthesis*
chemical_list: D004798:Enzymes; D009696:Nucleic Acids
keywords: DNAzyme; nucleic acids; nucleotides; polymerase; regioselectivity