Structure-function relationships of phenoxazine nucleosides for identification of mismatches in duplex DNA by fluorescence spectroscopy.

The effects of the flanking sequence on the mismatch-detection capabilities of the fluorescent nucleoside phenoxazine (tC(O)) were examined in a systematic fashion, and compared to the previously reported fluorescent, phenoxazine-based nucleoside Ç(f) . We see some similarities for the two fluorescent nucleosides, for example, the emission intensity of the C-mismatched duplex is always the highest, and a three-peak pattern in the spectrum emerges when the fluorosides are base-paired with A. However, phenoxazine was only able to distinguish a mismatch from the fully base-paired duplex in 11 out of 16 flanking sequences, and was able to identify each of the mismatches in six of those sequences. Therefore, tC(O) shows poorer discrimination of mismatches than was previously reported for Ç(f) , which could be used to identify all base-pairing partners in all immediately flanking sequences, albeit in some cases by using mercuric ions to selectively quench the emission of the T-mismatched duplex. The mercuric titration might resolve the overlap of fluorescence curves of tC(O) in some flanking sequences, but not for 5'-d(CtC(O) G) and 5'-d(TtC(O) A) due to overlap of A-mismatch and G-match fluorescence curves. A pH titration was performed on Ç(f) , tC(O) and a N5-methylated derivative of tC(O) , which showed that the emergence of the three-peak pattern is associated with the de-protonation of N5 in the fluorosides. We also show that neither the α- nor β-anomer of the phenothiazine nucleoside (tC) was able to detect a mismatch in any of the flanking sequences examined.