title: Discrimination of single-nucleotide alterations by G-specific fluorescence quenching.
abstract: 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.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University, 615-8510 Kyoto, Japan.
authors.forename: Chikara
authors.initials: C
authors.lastname: Dohno
authors.forename: Isao
authors.initials: I
authors.lastname: Saito
mesh_terms: D000225:Adenine / Q000737:chemistry; D020137:Base Pair Mismatch; D015374:Biosensing Techniques / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006147:Guanine / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D011721:Pyrenes / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013936:Thymidine / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D009711:Nucleotides; D009841:Oligonucleotides; D011721:Pyrenes; D006147:Guanine; D000225:Adenine; D013936:Thymidine