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.
Highlights
DNA is a molecule that has the outstanding molecular recognition ability, through which each strand hybridizes with its counterpart in a sequence-specific manner
Metallo-base pairs, consisting of two ligand-bearing nucleosides and a bridging metal ion, have unique and fascinating characteristics as noted below. (i) Metal-mediated base pairing leads to a significant thermal stabilization of DNA duplexes; (ii) the formation of the base pairs can be regulated by the addition and the removal of specific metal ions; (iii) the consecutive incorporation of metallo-base pairs provides multi-metal assembly within DNA helical structures
We investigated the effects of a metal cofactor, MgII ion, on the Terminal deoxynucleotidyl transferase (TdT)-catalyzed polymerization of H nucleotides
Summary
DNA is a molecule that has the outstanding molecular recognition ability, through which each strand hybridizes with its counterpart in a sequence-specific manner. An adenine base (A) on one strand and a thymine base (T) on the other strand form a base pair, and so do a guanine (G) and a cytosine (C). This complementary base pairing is achieved by hydrogen bonding between the nucleobases. Owing to the high molecular recognition ability and programmability, DNA self-assembly has been extensively utilized to nanostructures and nanomaterials [1,2,3]. Metal coordination is one of the most employed interactions for designing the self-assembly of molecules [6] and has been utilized for the construction of DNA-based materials [7,8,9]. Metal-mediated base pairing systems have attracted increasing attention in the field of nucleic acid chemInist.tJr.yM, oel.sSpcie. c20ia16l,ly17,in906DNA-based materials science and nanotechnology [10,11]
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