Pyrene-labeled base-discriminating fluorescent DNA probes for homogeneous SNP typing

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, U-Py and C-Py, which contain a pyrenecarboxamide chromophore connected by a propargyl linker. The fluorescence spectrum of the duplex containing a U-Py/A base pair showed a strong emission at 397 nm on 327 nm excitation. In contrast, the fluorescence of duplexes containing U-Py/N base pairs (N = C, G, or T) was considerably weaker. The proposed structure of the duplex containing a matched U-Py/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 U-Py/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, C-Py, selectively emitted fluorescence when the base opposite C-Py was G. The drastic change of fluorescence intensity by the nature of the complementary base is extremely useful for SNP typing. U-Py- and C-Py-containing oligodeoxynucleotides acted as effective reporter probes for homogeneous SNP typing of DNA samples containing c-Ha-ras and BRCA2 SNP sites.