The Effects of Magnesium Ions on the Enzymatic Synthesis of Ligand-Bearing Artificial DNA by Template-Independent PolymeraseReport as inadecuate




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Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan





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Academic Editor: Eva Freisinger

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–CuII–H. In this study, we investigated the effects of a metal cofactor, MgII ion, on the TdT-catalyzed polymerization of H nucleotides. At a high MgII concentration 10 mM, the reaction was halted after several H nucleotides were appended. In contrast, at lower MgII 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 MgII concentration. In the presence of excess MgII ions, TdT did not bind to the H-tailed strands; thus, further elongation was impeded. This is possibly because the interaction with MgII 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. View Full-Text

Keywords: artificial DNA; metal-mediated base pair; enzymatic synthesis; DNA polymerase; bioinorganic chemistry artificial DNA; metal-mediated base pair; enzymatic synthesis; DNA polymerase; bioinorganic chemistry





Author: Yusuke Takezawa, Teruki Kobayashi and Mitsuhiko Shionoya *

Source: http://mdpi.com/



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