We herein demonstrated for the first time the direct recognition of duplex DNA bearing the 5-methyl-2′-deoxycytosine and 2′-deoxyguanosine base pair by triplex DNA formation. Triplex-forming oligonucleotides contained the novel artificial nucleoside analogues 2-amino-2′-deoxy-nebularine derivatives, and their molecular design, synthesis, and functional evaluation are described.