title: Pyrene-labeled base-discriminating fluorescent DNA probes for homogeneous SNP typing.
abstract: This paper describes the design of novel base-discriminating fluorescent (BDF) nucleobases and their application to single nucleotide polymorphism (SNP) typing. We devised novel BDF nucleosides, (Py)U and (Py)C, which contain a pyrenecarboxamide chromophore connected by a propargyl linker. The fluorescence spectrum of the duplex containing a (Py)U/A base pair showed a strong emission at 397 nm on 327 nm excitation. In contrast, the fluorescence of duplexes containing (Py)U/N base pairs (N = C, G, or T) was considerably weaker. The proposed structure of the duplex containing a matched (Py)U/A base pair suggests that the high polarity near the pyrenecarboxamide group is responsible for the strong A-selective fluorescence emission. Moreover, the fluorescence of the duplex containing a (Py)U/A base pair was not quenched by a flanking C/G base pair. The fluorescence properties are quite different from previous BDF nucleobases, where fluorescence is quenchable by flanking C/G base pairs. The duplex containing the C derivative, (Py)C, selectively emitted fluorescence when the base opposite (Py)C was G. The drastic change of fluorescence intensity by the nature of the complementary base is extremely useful for SNP typing. (Py)U- and (Py)C-containing oligodeoxynucleotides acted as effective reporter probes for homogeneous SNP typing of DNA samples containing c-Ha-ras and BRCA2 SNP sites.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University and SORST, Japan Science and Technology Corporation, Kyoto 615-8510, Japan.
authors.forename: Akimitsu
authors.initials: A
authors.lastname: Okamoto
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Kanatani
authors.forename: Isao
authors.initials: I
authors.lastname: Saito
mesh_terms: D000577:Amides / Q000737:chemistry; D015342:DNA Probes / Q000138:chemical synthesis* / Q000737:chemistry; D003841:Deoxycytidine / Q000737:chemistry*; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D020641:Polymorphism, Single Nucleotide; D011721:Pyrenes / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
chemical_list: C520208:5-(3-(1-pyrenecarboxamido)propynyl)-2'-deoxyuridine; D000577:Amides; D015342:DNA Probes; D005456:Fluorescent Dyes; D011721:Pyrenes; D003841:Deoxycytidine; D003857:Deoxyuridine