| 研究実績の概要 |
This study aims to develop suitable metal-mediated base pairs that can be enzymatically incorporated into DNA duplexes. During my last year’s research, I have made several progresses as listed below:
1.Several novel nucleobases were designed based on the criteria concluded from the previous researches. Some of the nucleoside derivatives were successfully synthesized. Oligonucleotides containing one or more such nucleotides were then synthesized. The 2-oxo-4-carboxyimidazole (ImOC) is one of the candidates.
2.Thermal melting analysis of DNA duplexes containing artificial nucleobases were conducted in the presence and absence of various transition metal ions. The results revealed that ImOC forms stable ImOC-Cu(II)-ImOC and ImOC-Hg(II)-ImOC base pairs. Besides, the previously reported 2-oxo-imidazole-4-carboxylate (ImOA) was found to form a novel Hg(II)-mediated ImOA-Hg(II)-ImOA base pair. The formation of these metal-mediated base pairs was further confirmed by ESI-TOF mass spectrometric measurements.
3.The mismatch behavior between ImOC and four natural nucleobases were probed. ImOC did not show Cu(II)-dependent stabilization with mismatch base pairs, confirming the base pair specificity of ImOC-Hg(II)-ImOC.
4.Quantitative formation of three consecutive Cu(II)-mediated base pairs was demonstrated within a duplex containing three ImOC-ImOC base pairs/ This result shows great potential of DNA-templated Cu(II) nanoarray.
|
| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
This study aims to develop suitable metal-mediated base pairs that can be enzymatically incorporated into DNA duplexes. So far, a series of novel metal-mediated base pairs have been designed, synthesized, and characterized. Among several candidates, the ImOC nucleobase showed promising results. The ImOC nucleobase was found to form stable ImOC-Cu(II)-ImOC and ImOC-Hg(II)-ImOC base pairs in the presence of equimolar Cu(II) and Hg(II) ions, respectively. The formation of these base pairs led to an increase in the duplex melting temperature by +20 °C and +11 °C, respectively. In addition, ImOA, possessing a carboxyamide group in place of the carboxylate, was found to form a Hg(II)-mediated base pair (ΔTm = +6 °C). The stabilization effects of these metal-mediated base pairs are large enough for future applications. Notably, ImOC did not show Cu(II)-dependent stabilization when paired with other natural nucleobases, confirming the base pair specificity. This finding meets a basic requirement for the selectivity and the fidelity of polymerase-mediated base-pair incorporation.
In summary, I succeed in the synthesis and characterization of ImOC base pairs. Although there are other nucleobase candidates waiting to be characterized, the progress in the last fiscal year is within expectation.
|
| 今後の研究の推進方策 |
The study of this fiscal year focuses on the enzymatic incorporation of the artificial metal-mediated base pairs, such as a Cu(II)-mediated 2-oxo-imidazole-4-carboxylate (ImOC) base pair, which were synthesized and characterized in the previous fiscal year. In particular, triphosphate derivatives of the artificial ligand-bearing nucleosides will be chemically synthesized. The template DNA strands containing the artificial nucleotides necessary for polymerase reactions will be synthesized. Polymerase-based single nucleotide incorporation will be examined in the absence and in the presence of the bridging metal ions. The reaction conditions, including pH, metal concentration, types of DNA polymerases, will be optimized to achieve efficient incorporation of metal-mediated base pairs. Specificity of the nucleotide incorporation will be investigated based on the kinetic analysis. The full-length primer extension as well as PCR amplification will be further studied.
In addition, as mentioned above, there are several nucleobase candidates that remain to be fully characterized. I will also investigate the remaining nucleobases with similar approach to that of ImOC.
|
