Pyrene-Labeled Base-Discriminating Fluorescent DNA Probes for Homogeneous SNP Typing

TLDR

The study designs novel base‑discriminating fluorescent nucleobases for use in homogeneous SNP typing. The authors synthesized (Py)U and (Py)C nucleosides bearing a pyrenecarboxamide chromophore linked via a propargyl linker to serve as fluorescent probes. The probes exhibit strong, base‑specific fluorescence—(Py)U/A emits brightly at 397 nm while mismatched pairs show weak emission, and (Py)C fluoresces only opposite G—allowing accurate homogeneous SNP typing of c‑Ha‑ras and BRCA2 sites.

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.