Single base interrogation by a fluorescent nucleotide: each of the four DNA bases identified by fluorescence spectroscopyw

Single nucleotide polymorphisms (SNPs) are single nucleotide gene variations. SNPs in a protein-encoding region can result in a mutant protein and subsequently predispose human beings to common diseases and disorders. Thus, disease diagnosis and identification of targets for drug discovery by SNP detection is an active area of research. Most methods that have been developed for detection of SNPs rely on monitoring hybridization of an oligonucleotide probe to the sequence of interest; the probe binds to the wild-type sequence but not to DNAs containing an SNP. Hybridization is usually detected by change in the fluorescence emission of a fluorophore that is linked to the oligonucleotide probe. Hybridization assays require a careful selection of the sequence of the oligonucleotide probe and the annealing conditions, especially the temperature. It would be advantageous if, instead of monitoring hybridization, fluorescent DNA probes that signal single-base mismatches within duplexes could be used. A promising class of compounds for SNP detection are modified nucleosides having fluorophores that are sensitive to their proximal environment. One strategy for the design of fluorescent nucleotides for SNP detection is conjugation of a fluorophore to the nucleobase through a linker. For example, Saito and coworkers have conjugated pyrene to pyrimidine nucleosides and called them ‘‘base-discriminating fluorescent nucleosides’’ (BDFs) because they can detect and distinguish between mismatches. A different strategy is to extend the aromatic ring system of the bases in natural nucleosides and use the fluorescence of the base itself as a reporter group. However, most of the fluorescent nucleobase analogs that have been reported to date are limited in their ability to differentiate between all the possible base-pairing partners. In this communication we report a nucleoside containing a fluorescent base that is able to discriminate between the four bases of DNA. We have recently described nucleoside Ç (‘‘C-spin’’), an analog of cytidine that contains a rigid spin label fused to the nucleobase, for the study of nucleic acid structure and dynamics by EPR spectroscopy (Fig. 1). Ç is structurally related to the fluorescent nucleosides tC and tC. Reduction of the nitroxide to the corresponding hydroxyl amine with DTT yielded a fluorescent nucleoside which made nucleoside Ç an attractive spectroscopic probe, because it can be used for biophysical studies of nucleic acids by both fluorescence and EPR spectroscopies. However, when studying the fluorescent properties of oligomers containing this modification we found that the hydroxyl amine was readily oxidized back to the nitroxide upon exposure to oxygen, thereby quenching the fluorescence. In the search for a more stable fluorescent analog of Ç, we sought to reduce the nitroxide to the corresponding amine. Nitroxides have been reduced to amines by sodium sulfide in DMSO and DMF. Limited solubility of nucleic acids in polar aprotic solvents prompted us to investigate this reaction in H2O. Reduction of nucleoside Ç with Na2S in water at 45 1C did indeed proceed to give the highly fluorescent amine Ç (FF = 0.31) after 8 h and in nearly quantitative yield (Scheme 1). To study the reduction of Ç in DNA, an aqueous solution of the oligodeoxyribonucleotide 50-d(GACCTCGÇATCGTG) was treated with Na2S at 45 1C for 14 h. Analysis of the crude reaction mixture by EPR spectroscopy revealed the disappearance of the nitroxide (data not shown). Furthermore, Fig. 1 Structure and base pair properties of Ç.