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Discrimination of Single‐Nucleotide Alterations by G‐Specific Fluorescence Quenching
Author(s) -
Dohno Chikara,
Saito Isao
Publication year - 2005
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200400325
Subject(s) - fluorescence , quenching (fluorescence) , guanine , fluorophore , chemistry , intercalation (chemistry) , fluorescence anisotropy , photochemistry , base pair , nucleotide , base (topology) , stereochemistry , dna , biochemistry , organic chemistry , mathematical analysis , physics , mathematics , quantum mechanics , membrane , gene
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.