Discrimination of single-nucleotide alterations by G-specific fluorescence quenching
Discrimination of single-nucleotide alterations by G-specific fluorescence quenching
复制标题
DOI：
10.1002/cbic.200400325
复制
发表时间：
2005-06-01
期刊：
影响因子：
3.2
通讯作者：
Saito, I
中科院分区：
文献类型：
--
作者：
Dohno, C;Saito, I
描述了一种通过鸟嘌呤Q荧光猝灭检测单碱基变化的新策略。我们设计了一种新的碱基识别荧光（BDF）核苷，4‘PyT，在胸腺嘧啶糖的C4’位置含有一个芘环甲酰胺荧光基团。含有4' pyt的寡脱氧核苷酸仅在互补腺嘌呤碱基存在时表现出增强的荧光。相比之下，含有4'PyT/ N碱基对（N = C， G或T）的错配双工的荧光弱得多。这种高a选择性荧光是鸟嘌呤特异性猝灭效率的产物；当与4'PyT互补的碱基不匹配时，芘甲酰胺荧光团能够与邻近的G碱基密切相互作用（在插入的情况下最可能的相互作用），因此G碱基在不匹配的双相体中发生了有效的猝灭。相比之下，含有4'PyT/A碱基对的双链化合物表现出强烈的发射，因为在这种情况下，荧光团位于较小的凹槽中，能够逃脱G碱基的荧光猝灭。
A new strategy for the detection of single-base alterations through fluorescence quenching by guanine Q is described. We have devised a novel base-discriminating fluorescent (BDF) nucleoside, 4'PyT that contains a pyrenecorboxamide fluorophore at the thymidine sugar's C4'-position. 4'PyT-containing oligodeoxynucleotides only exhibited enhanced fluorescence in response to the presence of a complementary adenine base. In contrast, the fluorescence of mismatched duplexes containing 4'PyT/ N base pairs (N = C, G, or T) was considerably weaker. This highly A-selective fluorescence was a product of guanine-specific quenching efficiency; when the complementary base to 4'PyT was a mismatch, the pyrenecarboxamide fluorophore was able to interact intimately with neighboring G bases (the most likely interaction in the case of intercalation), so effective quenching by the G bases occurred in the mismatched duplexes. In contrast, duplexes containing 4'PyT/A base pairs exhibited strong emission, since in this case the fluorophores were positioned in the minor groove and able to escape fluorescence quenching by the G bases.