Highly selective fluorescent nucleobases for designing base-discriminating fluorescent probes*

There is increasing interest in single nucleotide polymorphism (SNP) typing since they can be used as markers to identify the genes that underlie complex diseases and to realize the full potential of pharmacogenomics in analyzing variable response to drugs. Among the different methodologies for SNP genotyping, the homogenous assay is more amenable than the heterogeneous one. In this article, we will describe some of our most recently developed novel base-discriminating fluorescent (BDF) nucleosides useful for homogenous SNP typing. Our novel concept led to the investigation of a new type of pyrene-labeled BDF nucleosides PyU, PyC, 8pyA, and MePydA, which emitted strong fluorescence only when the bases opposite the BDF bases are A, G, T, and C, respectively. The DNA probes containing four different BDF bases enabled us to distinguish single-base alterations by simply mixing with a sample solution of target DNA. An example of SNP typing of c-Ha-ras SNP sequence has also been demonstrated. Detection of base insertion in insertion/deletion (indel) polymorphisms using pyrene excimer fluorescent probe has also been explored.