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

一个选择性地与错配位点结合的小分子配体可以取代错配结合蛋白，并为异源双链分析带来创新。配体-萘啶二聚体(NPT-NPT)与双链DNA中的鸟嘌呤-鸟嘌呤错配有很强的选择性结合。5‘-CGG-3’/5‘-CGG-3’序列中G-G错配的结合常数为1.9×107M-1。NPT-NPT由两个2-氨基-1，8-萘啶(NPT)发色团和连接发色团的Tinker组成，其设计使每个NPT在G-G错配中与每个鸟嘌呤产生三个氢键，并通过与侧翼碱基对堆叠来稳定得到的萘啶-鸟嘌呤对。针对双链DNA中的G-X(X=G、A和T)错配，我们合成了一系列由2-氨基-1，8-萘啶和杂环组成的杂化配体。在与G-G错配强烈结合而与G-A错配适度结合的萘啶二聚体中取代一个萘啶发色团，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-氨基喹啉显著减少了不仅与G-G结合，而且与G-A和G-T错配的结合。与之形成鲜明对比的是，由2-氨基-1，8-萘啶和8-氮喹诺酮组成的杂化配体对G-A错配的稳定作用比萘啶二聚体要强得多。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。通过紫外滴定、圆二色谱、电喷雾飞行时间和等温滴定量热法分析了萘啶-氮喹诺酮杂化产物的结合情况。结果表明，3个配体分子结合在一个G-A错配位点上，结合常数为8.8±0.74×105M-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.