Cytosine Detection by a Fluorescein‐Labeled Probe Containing Base‐Discriminating Fluorescent Nucleobase

We report on a new method for the detection of a base at a specific site in a DNA sequence by monitoring the fluorescence emission of fluorescein. To achieve this goal, we developed a new base‐discriminating fluorescent (BDF) nucleobase, naphthodeazaadenine ( ND A). The fluorescence spectrum of the duplex possessing a cytosine base as a complementary base of ND A showed a fluorescence peak at 383 nm when using an excitation wavelength of 350 nm. When the complementary base of ND A was one of the other bases, the fluorescence intensity was very low. The fluorescence emission spectrum of ND A overlapped with the fluorescence excitation spectrum of fluorescein in the wavelength range of 400–500 nm. Thus, we designed FRET‐BDF probes containing ND A as the FRET donor and fluorescein as the acceptor. The interaction of these two fluorophores, which are separated by defined base pairs, allowed an efficient energy transfer that resulted in a dominant fluorescence emission of fluorescein at 520 nm when using an excitation wavelength of 350 nm. Fluorescence emission from FRET‐BDF probes was observed only when the complementary base of ND A is C, thus achieving a clear distinction of a C base on the complementary DNA strand. However, the general utility of our method is limited due to the quenching of the ND A fluorescence by a G/C base pair flanking ND A.