To develop a novel nucleoside analogue for the effective recognition of CG interruption in a homopurine–homopyrimidine tract of double-stranded DNA (dsDNA), we succeeded in the synthesis of a triplex-forming oligonucleotide (TFO) containing a novel 2′,4′-BNA (Q B ) bearing 1-isoquinolone as a nucleobase, and the triplex-forming ability and sequence-selectivity of the TFO (TFO-Q B ) were examined. On melting temperature (T m ) measurements, it was found that the TFO-Q B formed a stable triplex DNA in a highly sequence-selective manner under near physiological conditions.