Discrimination of single-nucleotide alterations by G-specific fluorescence quenching

A new strategy for the detection of single-base alterations through fluorescence quenching by guanine (G) is described. We have devised a novel base-discriminating fluorescent (BDF) nucleoside, 4'PyT, that contains a pyrenecarboxamide 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.