The interactions of N‐[2‐(dimethylamino)ethyl]‐1‐methyl‐4‐[1‐methyl‐4‐[4‐formamido‐1‐methylimidazole‐2‐carboxamido]imidazole‐2‐carboxamido]imidazole‐2‐carboxamide (AR‐1‐144), a tri‐imidazole polyamide minor groove binder, with DNA have been investigated by NMR and CD spectroscopy. A series of DNA oligonucleotides with a C/G‐containing four‐bp core, i.e. CCGG, CGCG, GGCC, and GCGC, have been titrated with AR‐1‐144 at different ratios. AR‐1‐144 favors the CCGG sequence. The flanking sequence of the CCGG core also influences the binding preference, with a C or T being favored on the 3′‐side of the CCGG core. The three‐dimensional structure of the symmetric 2 : 1 side‐by‐side complex of AR‐1‐144 and GAACCGGTTC, determined by NOE‐constrained NMR refinement, reveals that each AR‐1‐144 binds to four base pairs, i.e. at C5‐G6‐G7‐T8, with every amide‐imidazole unit forming two potential hydrogen bonds with DNA. The same DNA binding preference of AR‐1‐144 was also confirmed by circular dichroism spectroscopy, indicating that the DNA binding preference of AR‐1‐144 is independent of concentration. The cooperative binding of an AR‐1‐144 homodimer to the (purine)CCGG(pyrimidine) core sequence appears to be weaker than that of the distamycin A homodimer to A/T sequences, most likely due to the diminished hydrophobic interactions between AR‐1‐144 and DNA. Our results are consistent with previous footprinting data and explain the binding pattern found in the crystal structure of a di‐imidazole drug bound to CATGGCCATG.

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