Binding of AR-1-144, a tri-imidazole DNA minor groove binder, to CCGG sequence analyzed by NMR spectroscopy

The interactions of N-[2-(dimethylamino)ethyl]-1-methyl-4-[1-methyl-4-[4-fo rmamido-1-methylimidazole-2-carboxamido]imidazole-2-carboxamido]imidazole-2 -carboxamide (AR-1-144), a tri-imidazole polyamide minor groove binder, wit h DNA have been investigated by NMR and CD spectroscopy. A series of DNA ol igonucleotides with a C/G-containing four-bp core, i.e. CCGG, COCO, GGCC, a nd GCGC, have been titrated with AR-1-144 at different ratios. AR-1-144 fav ors the CCGG sequence. The flanking sequence of the CCGG core also influenc es the binding preference, with a C or T being favored on the 3'-side of th e CCGG core. The three-dimensional structure of the symmetric 2:1 side-by-s ide complex of AR-1-144 and GAACCGGTTC, determined by NOE-constrained NMR r efinement, 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 bo nds with DNA. The same DNA binding preference of AR-1-144 was also confirme d by circular dichroism spectroscopy, indicating that the DNA binding prefe rence of AR-1-144 is independent of concentration. The cooperative binding of an AR-1-144 homodimer to the (purine)CCGG(pyrimidine) core sequence appe ars 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 ex plain the binding pattern found in the crystal structure of a di-imidazole drug bound to CATGGCCATG.