title: The Effects of Magnesium Ions on the Enzymatic Synthesis of Ligand-Bearing Artificial DNA by Template-Independent Polymerase.
abstract: A metal-mediated base pair, composed of two ligand-bearing nucleotides and a bridging metal ion, is one of the most promising components for developing DNA-based functional molecules. We have recently reported an enzymatic method to synthesize hydroxypyridone (H)-type ligand-bearing artificial DNA strands. Terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase, was found to oligomerize H nucleotides to afford ligand-bearing DNAs, which were subsequently hybridized through copper-mediated base pairing (H-Cu(II)-H). In this study, we investigated the effects of a metal cofactor, Mg(II) ion, on the TdT-catalyzed polymerization of H nucleotides. At a high Mg(II) concentration (10 mM), the reaction was halted after several H nucleotides were appended. In contrast, at lower Mg(II) concentrations, H nucleotides were further appended to the H-tailed product to afford longer ligand-bearing DNA strands. An electrophoresis mobility shift assay revealed that the binding affinity of TdT to the H-tailed DNAs depends on the Mg(II) concentration. In the presence of excess Mg(II) ions, TdT did not bind to the H-tailed strands; thus, further elongation was impeded. This is possibly because the interaction with Mg(II) ions caused folding of the H-tailed strands into unfavorable secondary structures. This finding provides an insight into the enzymatic synthesis of longer ligand-bearing DNA strands.
authors.affiliation: Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. takezawa@chem.s.u-tokyo.ac.jp.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Takezawa
authors.affiliation: Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. tkobayashi@chem.s.u-tokyo.ac.jp.
authors.forename: Teruki
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. shionoya@chem.s.u-tokyo.ac.jp.
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Shionoya
mesh_terms: D002384:Catalysis; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000494:pharmacology*; D009711:Nucleotides / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet
chemical_list: D008024:Ligands; D009711:Nucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium
keywords: DNA polymerase; artificial DNA; bioinorganic chemistry; enzymatic synthesis; metal-mediated base pair