DNA Recognition and Analysis Through Cooperative Metal-ion Complex Formation of Split Probes

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  • IHARA Toshihiro
    Department of Applied Chemistry and Biochemistry, Graduate School of Science and Technology, Kumamoto University
  • KITAMURA Yusuke
    Department of Applied Chemistry and Biochemistry, Graduate School of Science and Technology, Kumamoto University

Bibliographic Information

Other Title
  • スプリット型プローブの協同的金属錯体形成を利用するDNAの認識及び検出
  • ストリット型プローブの協同的金属錯体形成を利用するDNA の認識及び検出
  • ストリットガタ プローブ ノ キョウドウテキ キンゾク サクタイ ケイセイ オ リヨウ スル DNA ノ ニンシキ オヨビ ケンシュツ

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Abstract

Metal chelators were covalently attached to the end of synthetic oligodeoxyribonucleotide (ODN) to prepare several ODN conjugates. The sequences of a pair of the conjugates were designed so as to form a ternary duplex or triplex with the targets, where their auxiliary units face each other, providing a microenvironment to accommodate a metal ion. The thermal stability of the ternary complexes is significantly stabilized by the addition of appropriate metal ions. That is, the two conjugates formed a dimer with an appropriate metal ion on the target DNA to form a stable complex. The triplex DNA structures containing convergently bound idaODN (the conjugate with iminodiacetic acid) and gluODN (the conjugate with glutamic acid) conjugates were stabilized by lanthanide ions and Cu2+, respectively. The cooperativity (ω, the ratio of the equilibrium constants of the second binding to the first one) was estimated to be ca. 165 for the later conjugate. The techniques were applied to colorimetric SNP (single nucleotide polymorphism) analysis using a luminescent lanthanide (Ln3+ : Tb3+ or Eu3+) as metal ions. Among the library of the conjugates with complexane-type chelators and heterocyclic aromatic ligands, only the combination of EDTA (ethylenediaminetetraacetic acid) conjugates (edtaODN) and phen (1,10-phenanthroline) conjugates (phenODN) provided significant emissions with quantum yields of 12 and 5.3% for Tb3+ and Eu3+, respectively, in the presence of the target. Their luminescence lifetimes were 1.26 and 1.34 ms for Tb3+ and Eu3+ ; they are long enough to be subjected to time-resolved luminescence measurements. Biallelic polymorphism in the TPMT (thiopurine S-methyltransferase) gene, wt/wt (G/G), mut/mut (C/C), and wt/mut (G/C), were distinguished as emissions in green, red, and yellow, respectively ; the colors were identified even by the naked eye.

Journal

  • BUNSEKI KAGAKU

    BUNSEKI KAGAKU 61 (3), 193-206, 2012

    The Japan Society for Analytical Chemistry

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