A new fluorescent nucleotide with desmethyl thiazole orange dyes, D′505, has been developed for expansion of the function of fluorescent probes for live-cell RNA imaging. The nucleoside unit of D′505 for DNA autosynthesis was soluble in organic solvents, which made the preparation of nucleoside units and the reactions in the cycles of DNA synthesis more efficient. The dyes of D′505-containing oligodeoxynucleotide were protonated below pH 7 and the oligodeoxynucleotide exhibited hybridization-sensitive fluorescence emission through the control of excitonic interactions of the dyes of D′505. The simplified procedure and effective hybridization-sensitive fluorescence emission produced multicolored hybridization-sensitive fluorescent probes, which were useful for live-cell RNA imaging. The acceptor-bleaching method gave us information on RNA in a specific cell among many living cells. © 2013 The Royal Society of Chemistry.