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–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.

Modified Nucleotides as Substrates of Terminal Deoxynucleotidyl Transferase

“…The same result would also be observed if the 3’-modified primer fails to interact with the active center of TdT or it folds into unfavourable secondary structures as in the case of pyrene-2’-deoxynucleotide [ 26 ] or 2-methyl-3-hydroxypyridin-4(1 H )-one 2’-deoxynucleotide (dH) [ 74 ]. In our hands TdT does not extend the 4-thio-dU-elongated primer in the presence of deoxythymidine triphosphate (dTTP) (data not shown); a similar effect has been observed with dH [ 70 ].…”

“…However in the presence of Co 2+ TdT uses the former as substrate for the incorporation and subsequent elongation while only a single incorporation of dPyr 4OH is observed ( Figure 2 , lanes 5 and 6). Recently, it was demonstrated that binding affinity of TdT to the 3’-end of ON modified with 3-hydroxy-4-pyridone-based nucleotides depended on the Mg 2+ concentration and caused formation of unfavourable secondary structure of 3’-terminus [ 70 ]. As a consequence after incorporation of single nucleotide further elongation was impeded.…”

Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids

“…[51] The choice of the metal ion for the TdT-mediated polymerization of modified nucleotides also varies from case to case. For instance, Mg 2 + is the preferred cofactor if hydroxypyridone-or nitroindole-modifiedn ucleotides are considered, [45,52] but Mn 2 + is preferred with dImTP 4 ( Figure 4) as as ubstrate [53] and Co 2 + is often sufficient for the incorporation of base-modified nucleotides. [46][47]…”

“…Indeed, the TdT accepts ah ydroxypyridonemodified nucleotide as as ubstrate and incorporates up to five modifications on both ssDNA primers and on DNA duplexes containing 3'-end overhangs.I nterestingly,t he efficiency of the tailing reaction can be improved by decreasing the concentration of the Mg 2 + cofactor, as this cofactor is assumed to cause unfavorable folding of the modified nucleotides,w hich impedes further polymerization. [52] The combination of Cu 2 + and modifiedd sDNA leads to the assembly of two duplexes throughm etal-base pair formation,w hereast he addition of ethylenediaminetetraacetic acid (EDTA) triggers the disassembly of these constructs ( Figure 5B). [119] So far,o nly the hydroxypyridone-modified nucleotide [119] as well as imidazole-containing nucleotides [53,120] have been evaluated as substrates for the TdT in the context of metal-base pair formation, but this approachi sc ertainly expected to gain momentum in the near future.…”

Artificial DNA Base Pairing Mediated by Diverse Metal Ions

“…Very recently, hydroxypyridone-bearing nucleotides were consecutively incorporated into DNA strands by utilizing a template-independent DNA polymerase. 74,75 The enzymatically synthesized ligandbearing oligonucleotides formed metallo-DNA duplexes through the formation of HCu II H base pairs. Toward future applications, the regulation of electrical conductivity of DNA duplexes was demonstrated by utilizing the HCu II H base pairing.…”