DAPI(4’,6-diamidino-2-phenylindole)is an important DNA-binding drugs that interferes with the activity of number of DNA transcription processing enzymes involved in regulatory and structural functions. Strong inhibitory activity of DAPI has been reported for RNA polymeraseⅡ and attributed to the high binding affinity of DAPI in the minor groove of AT-rich sequences. The strong DAPI-DNA interaction interferes with the binding of TBP (TATA-binding protein) to its consensus TATA box sequence, preventing formation of the transcription factors-DNA complex. This complex structure is required for RNA polymerase Ⅱ recognizes to initiate gene transcription. Traditionally, DAPI is used in biochemical and cytochemical studies as a specific fluorescent dye of DNA and chromosome. The special properties of DAPI–DNA can be used as a DNA probe of highly fluorescent complexes. It has been shown in footprinting experiments that DAPI will bind to two or more contiguous AT base pairs is covered when DAPI is bound. DAPI binds our d(CGCAAATTTGCG)2 in the minor groove of the dodecamer by forming a bifurcated hydrogen bond between its indole NH (imino proton) group and the two O2 of T7 in the central AAATTT region,where as two terminal amidinos form conventional hydrogen bonds with A6. Bifurcated or three-centered hydrogen bonds, in which the hydrogen atom is within bonding distance of two heteroatoms have been suggested to be present in selected small molecule structures with minor groove binding drugs. The detailed features of DAPI-d(CGCAAATTTGCG)2 complex revealed in this study can be used in designing a new drug of better selectivity.