Sequence-dependent variation in DNA minor groove width dictates orientational preference of Hoechst 33258 in A-tract recognition: Solution NMR structure of the 2:1 complex with d(CTTTTGCAAAAG)₂

The solution structure of the dodecamer duplex d(CTTTTGCAAAAG) ₂ and its 2:1 complex with the bis-benzimidazole Hoechst 33258 has been investigated by NMR and NOE-restrained molecular dynamics (rMD) simulations. Drug molecules are bound in each of the two A-tracts with the bulky N-methylpiperazine ring of each drug located close to the central TG (CA) step, binding essentially to the narrow minor groove of each A-tract, MD simulations over 1 ns, using an explicit solvation model, reveal time-averaged sequence-dependent narrowing of the minor groove from the 3'-end towards the 5'-end of each TTTT sequence. Distinct junctions at the TpG (CpA) steps, characterised by large positive roll, low helical and propeller twists and rapid AT base pair opening rates, add to the widening of the groove at these sites and appear to account for the bound orientation of the two drug molecules with the N-methylpiperazine ring binding in the wider part of the groove close to the junctions. Comparisons between the free DNA structure and the 2:1 complex (heavy atom RMSD 1.55 ) reveal that these sequence-dependent features persist in both structures. NMR studies of the sequence d(GAAAAGCTTTTC)₂, in which the A-tracts have been inverted with the elimination of the TpG junctions, results in loss of orientational specificity of Hoechst 33258 and formation of multiple bound species in solution, consistent with the drug binding in a number of different orientations.