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  [in Japanese] DNA Recognition and Analysis Through Cooperative Metal-ion Complex Formation of Split Probes  [in Japanese]

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Author(s)

    • 井原 敏博 IHARA Toshihiro
    • 熊本大学大学院自然科学研究科産業創造工学専攻 Department of Applied Chemistry and Biochemistry, Graduate School of Science and Technology, Kumamoto University
    • 北村 裕介 KITAMURA Yusuke
    • 熊本大学大学院自然科学研究科産業創造工学専攻 Department of Applied Chemistry and Biochemistry, Graduate School of Science and Technology, Kumamoto University

Abstract

DNAに金属配位基を共有結合で導入した種々のDNAコンジュゲートを調製した.これらDNAコンジュゲートは,共存する特定の金属イオンと錯生成し,その分子(DNA)認識能,及びその他の様々な性質を可逆的に変化させることができる.コンプレキサン型の配位子であるグルタミン酸,及びハードな配位子であるイミノ二酢酸を有するDNAコンジュゲートは,それぞれ銅イオン,及びハードなルイス酸である希土類金属イオン共存下二量体を形成し,C<sub>2</sub>対称な塩基配列を有する標的DNAにハイブリダイズして安定な三本鎖構造を形成することが分かった.EDTAと1,10-フェナンスロリンを導入したDNAコンジュゲートは,標的DNAにタンデムにハイブリダイズし,両配位子は互いに向かい合い,協同的に希土類金属の配位環境をつくる.この系にEu<sup>3+</sup>やTb<sup>3+</sup>を添加すると,発光性の錯体を形成することが明らかになった.発光強度は標的DNAの塩基配列に依存し,両金属を同時に用いると遺伝子混合物の同時多色アッセイが可能であることが示された.

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 Cu<sup>2+</sup>, respectively. The cooperativity (ω, the ratio of the equilibrium constants of the second binding to the first one) was estimated to be <i>ca</i>. 165 for the later conjugate. The techniques were applied to colorimetric SNP (single nucleotide polymorphism) analysis using a luminescent lanthanide (Ln<sup>3+</sup> : Tb<sup>3+</sup> or Eu<sup>3+</sup>) 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 Tb<sup>3+</sup> and Eu<sup>3+</sup>, respectively, in the presence of the target. Their luminescence lifetimes were 1.26 and 1.34 ms for Tb<sup>3+</sup> and Eu<sup>3+</sup> ; 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, 2012-03-05

    The Japan Society for Analytical Chemistry

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Codes

  • NII Article ID (NAID)
    10030135293
  • NII NACSIS-CAT ID (NCID)
    AN00222633
  • Text Lang
    JPN
  • Article Type
    ART
  • ISSN
    05251931
  • NDL Article ID
    023598338
  • NDL Call No.
    Z17-9
  • Data Source
    CJP  NDL  J-STAGE 
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