In this study the position of the thiazole orange derivative in triplex-forming oligonucleotides (TFOs) is varied and the fluorescence of the resulting complexes with DNA duplexes, single-stranded DNAs and RNAs are evaluated. Under similar conditions single attachment of the TO-dye to 2′-O-propargyl nucleotides in the TFOs (assembly dependent fluorescence enhancing nucleic acids, AFENA) led to probes with low fluorescent intensity in the single-stranded state with fluorescence quantum yield (ΦF) of 0.9 %–1.5 %. Significant increase in fluorescence intensity was detected after formation of DNA triplexes (ΦF=23.5 %–34.9 %). Under similar conditions, Watson–Crick-type duplexes formed by the probes with single stranded (ss) RNA and ssDNA showed lower fluorescence intensities. Bugle insertions of twisted intercalating nucleic acid (TINA) monomers were shown to improve the fluorescent characteristics of GT/GA-containing antiparallel AFENA–TFOs. Self-aggregation of TFOs caused by guanosines was eliminated by TINA insertion which also promoted DNA triplex formation at pH 7.2. Importantly these AFENA–TINA–TFOs can bind to the duplex in the presence of complementary RNA at 37 °C.