Studies on a rapid detection system of SNPs using mismatch-binding molecules

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A small molecular ligand that selectively binds to a mismatched site could replace mismatch-binding proteins and bring an innovation to heteroduplex analyses. The ligand naphthyridine dimer (Npt-Npt) strongly and selectively binds to guanine-guanine mismatches in duplex DNA. The binding constant to a G-G mismatch in the 5' -CGG-3' /5' -CGG-3' sequence is 1.9 × 107 M-1. Npt-Npt consisting of two 2-amino-1, 8-naphthyridine (Npt) chromophores and a Tinker connecting the chromophores is designed so that each Npt produces three hydrogen bonds to each one of the guanines in the G-G mismatch and the resultant naphthyridine -guanine pair is stabilized by stacking with the flanking base pairs. We have synthesized a series of hybrid ligands consisting of 2-amino 1, 8-naphthyridine and heterocycles targeting the G-X (X = G, A, and T) mismatches in duplex DNA. Substitution of one naphthyridine chromophore in the naphthyridine dimer, which binds strongly to the G-G mismatch and moderately to the G-A mismatch, with 2,8-diaminonaphthyridine, 2-aminopyridine, 2, 6 diaminopyridine, 2-amino 6-methylpyridine, and 2-aminoquinoline dramatically decreased binding not only to the G-G but also to G-A and G-T mismatches. In marked contrast, a hybrid ligand consisting of 2-amino-1, 8-naphthyridine and 8-azquinolone stabilized the G-A mismatch much more strongly than the naphthyridine dimer. Structure-activity studies clearly demonstrated that the hydrogen-bonding surfaces of two heterocycles are essential for strong binding and specificity to the G-A mismatch. The binding of the naphthyridine-azaquinolone hybrid was analyzed by UV-titration, CD spectrum, ESI-TOF, and isothermal titration calorimetry. The data showed that three ligand molecules bound to a G-A mismatch site with the binding constant of 8.8 ± 0.74 × 105 M-1.