Two nucleic acid building blocks were synthesized, consisting of two photostable green- and red-emitting cyanine–styryl dyes and (S)-3-amino-1,2-propanediol as a substitute for the ribofuranoside, and incorporated as base-pair surrogates by using automated phosphoramidte chemistry in the solid phase. The optical properties and, in particular, the energytransfer properties were screened in a range of DNA duplexes, in which the “counter bases” of the two dyes were varied and the distance between the two dyes was enlarged to up to three intervening adenosine–thymidine pairs. The DNA duplex with the best optical properties and the best red/green emission
ratio as the readout bore adenosine and thymidine opposite to the dyes, and the two dyes directly adjacent to each
other as the base surrogate pair. This structural arrangement can be transferred to RNA to obtain similarly fluorescent RNA probes. Representatively, the positively evaluated DNA duplex was applied to verify the fluorescence readout in living HeLa cells by using fluorescence confocal microscopy.