A highly efficient method for postsynthetic modification of unprotected oligonucleotides incorporating internal insertions of (R)-1-O-(4-ethynylbenzyl)glycerol has been developed through the application of click chemistry with water-insoluble pyren-1-yl azide and water-soluble benzyl azide and acceleration by microwave irradiation. The twisted intercalating nucleic acids (TINAs) obtained in these reactions, possessing bulged insertions of (R)-3-O-{4-[1-(pyren-1-yl)-1H-1,2,3-triazol-4-yl]benzyl}glycerol (7), formed parallel triplexes with thermal stabilities of 20.0, 34.0, and 40.0 degrees C at pH 7.2 in the cases of one, two, or three insertions of 7, respectively, separated by three nucleic bases. An oligonucleotide with four insertions of 7--each between three nucleic bases in the sequence--was unable to form complexes with complementary single- or double-stranded DNAs, as a result of self-aggregation of the pyrene moieties. This assumption was supported by the formation of a very strong excimer band at 460 nm in the fluorescence spectra. Molecular modeling of the parallel triplex with bulged insertion of the monomer 7 in the triplex-forming oligonucleotide (TFO) showed that only the pyrene moiety was stacking between the bases of the dsDNA, whereas 1,2,3-triazole did not participate in the triplex stabilization. Thermal denaturation studies of the duplexes and triplexes, as well as the fluorescence properties of TINA-triazole 7, are discussed and compared with previous studies on TINA.