Development of Novel Nucleic Acid Probes Based on the Template-directed Formation and Interaction of Metal Complexes

Metal complexes or chelators were covalently attached to the end of oligodeoxyribonucleotide (ODN) to prepare several ODN conjugates (probes). The sequences of the probes were designed so as to bind neighboring sites on the target. The interaction between functional metal complexes in the probes would generate a specific signal, depending on the sequence of the target serving as a template. The general concept of "DNA labeling by functional metal complexes" was demonstrated by the discrimination of the SNP (single nucleotide polymorphism) base and the recognition of a repetitive sequence. For the former system, a set of DNA conjugates, EDTA-modified and phen-modified ODN (phen = 1,10-phenanthroline), were prepared for the DNA-templated formation of luminescent lanthanide complexes [Ln: Tb (III) or Eu(III)]. When a ternary duplex consisting of the target and the two probes EDTA and phen moieties of the probes face each other and are expected to function as dehydration/capturing units for lanthanide ion, respectively. The characteristic emissions of Tb (III) and Eu(III) ions were clearly observed only in the presence of a fully matched target. Their luminescence lifetimes were 1.26 and 1.34 ms for Tb(III) and Eu(III); they are long enough to be subjected to time-resolved luminescence measurements. Biallelic polymorphism in the thiopurine S-methyltransferase (TPMT) gene, wt/wt (G/G), mut/mut (C/C), and wt/mut (G/C), were distinguished as emissions in green [Tb(III)], red [Tb(III)], and yellow [Tb (III) + Eu(III)], respectively; the colors were identified even by the naked eye. The concept of the cooperative formation of luminescent lanthanide complexes could also be applied to the biomolecular sensor by the concomitant use of an aptamer and a stem-loop structured ODN caring EDTA and phen at its both ends. For the latter system, the [Ru(phen)2(dppz)](2+) (dppz = dipyrido[3,2-a:2',3'-c]phenazine) complex was attached to the 5' end of the short ODN to form conjugates. The sequence is complementary to one unit of the repetitive sequence of human telomere. The complex expands the planar aromatic ring of dppz for an intercalation with the Ru(phen)2 moiety remaining in a groove. The preference of the tethered [Ru(phen)(2)(dppz)](2+) complex to the duplex structure should promote the binding of another probe to an adjacent site. That is, a cooperative behavior is expected for probe hybridization when it binds with repetitive sequences. The Delta-isomer of the probe showed a high cooperativity during the recognition of the human telomere repeats, while the A-isomer did not. By the addition of a target, the emission intensity of the A-isomer modified probe increased to be 6-times higher than before addition. This is the first example of a pair of diastereomeric fluorescent DNA probes that have been studied regarding their cooperativity during hybridization to adjacent sites.