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

选择性结合错配位点的小分子配体可以取代错配结合蛋白，为异双工分析带来创新。配体萘啶二聚体（Npt-Npt）在双链DNA中与鸟嘌呤错配体强而选择性结合。5′-CGG-3′/5′-CGG-3′序列G-G错配的结合常数为1.9 × 107 M-1。Npt-Npt由两个2-氨基- 1,8 -萘啶（Npt）发色团和一个连接发色团的Tinker组成，使得每个Npt与G-G错配中的每个鸟嘌呤产生三个氢键，并且所得到的萘-鸟嘌呤对通过与侧翼碱基对堆叠而稳定。我们针对双链DNA中的G-X （X = G, a， T）错配，合成了一系列由2-氨基1,8 -萘啶和杂环组成的杂化配体。用2,8-二氨基萘啶、2-氨基吡啶、2,6 -二氨基吡啶、2-氨基6-甲基吡啶和2-氨基喹啉取代萘啶二聚体中的一个萘啶发色团，不仅能显著降低与G-G的结合，还能显著降低与G-A和G-T的结合。与此形成鲜明对比的是，由2-氨基- 1,8 -萘啶和8-氮喹诺酮组成的杂化配体比萘啶二聚体更能稳定G-A错配。结构-活性研究清楚地表明，两个杂环的氢键表面对于G-A错配的强结合和特异性是必不可少的。采用紫外滴定法、CD光谱法、ESI-TOF法和等温滴定量热法对萘啶-阿扎喹诺酮复合物的结合进行了分析。结果表明，3个配体分子与G-A错配位点结合，结合常数为8.8±0.74 × 105 M-1。

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.