荧光小分子2-氨基-5,6,7-三甲基-1,8-萘啶识别dsDNA中胞嘧啶凸出  

Fluorescence recognition of a single cytosine bulge using a small molecule 2-amino-5,6,7-trimethyl-1,8-naphthyridine

作　　者：胡亮亮[1] 薄红艳[1] 高强[1] 漆红兰[1] 张成孝[1] 

机构地区：[1]陕西省生命分析化学重点实验室,陕西师范大学化学与材料科学学院,西安710062

出　　处：《中国科学：化学》2011年第11期1732-1739,共8页SCIENTIA SINICA Chimica

基　　金：国家自然科学基金(20805029);中央高校科研业务费(GK201002035)资助

摘　　要：双链DNA(dsDNA)中单碱基凸出结构(bulge structure)具有重要生物学意义,这种结构也是DNA靶向药物的目标部位之一.荧光小分子2-氨基-5,6,7-三甲基-1,8-萘啶(ATMND)能够通过氢键识别胞嘧啶(cytosine),因而对dsDNA中凸出的胞嘧啶表现出明显的特异性结合.与其余三种凸出的碱基相比,ATMND与凸出部位胞嘧啶的结合伴随着ATMND荧光的明显猝灭,因而可以用于胞嘧啶凸出结构的识别.利用解旋温度测量、荧光、圆二色光谱对ATMND和存在胞嘧啶凸出结构的dsDNA相互作用进行了研究.荧光滴定结果表明ATMND和dsDNA中凸出部位未配对的胞嘧啶的结合常数K11=4.8×105M?1.通过对含胞嘧啶凸出结构的dsDNA与ATMND结合前后的解旋温度曲线进行解析,发现胞嘧啶凸出结构相邻碱基对凸出的胞嘧啶与ATMND的结合有较大的影响.荧光测量结果也表明ATMND荧光的猝灭效率与凸出结构相邻碱基的类型有关,当相邻碱基为鸟嘌呤(guanine,G)时,荧光猝灭效率最高.基于dsDNA中凸出的碱基对ATMND荧光猝灭效率存在明显差异这一现象,设计了探针DNA实现了乳腺癌相关基因(PGR gene rs3740753)中单核苷酸多态性(G/C变异)的荧光分型.DNA bulge structures are of general biological significance and are potential targets for therapeutic drugs. A bulge binding agent, 2-amino-5,6,7-trimethyl-1,8-naphthyridine （ATMND） possess hydrogen-bonding sites complementary with cytosine, showed a selective binding affinity to a single cytosine bulge in duplex DNA. Interactions between ATMND and bulge DNA were investigated using UV melting experiments, circular dichroism and binding constants was quantitated by fluorescence titration with K11 of4.8×10^5 M^-1. The free energy changes for the binding of ATMND to a cytosine bulge with different flanking bases were determined by a curve fitting of the thermal denaturation profile of DNA in the presence and absence of ATMND. The fluorescence of ATMND was quenched efficiently when it was bind to a single cytosine bulge in duplex DNA, the change of ATMND fluorescence intensity was utilized to type the cytosine related single base mutation with the naked eye by intentional construction of a duplex DNA with a bulge opposite the target base.

关 键 词：荧光 胞嘧啶凸出 2-氨基-5 6 7-三甲基-1 8-萘啶 氢键 识别 

分 类 号：R737.9[医药卫生—肿瘤]