Rationalising sequence selection by ligand assemblies in the DNA minor groove: the case for thiazotropsin A

Abstract

DNA-sequence and structure dependence on the formation of minor groove complexes at 50-XCYRGZ-30, where Y ¼ T and R ¼ A, by the short lexitropsin thiazotropsin A are explored based on NMR spectroscopy, isothermal titration calorimetry (ITC), circular dichroism (CD) and qualitative molecular modelling. The structure and solution behaviour of the complexes are similar whether X ¼ A, T, C or G and Z ¼ T, A, I (inosine) or C, 50-CCTAGI-30 being thermodynamically the most favoured (DG¼ 11.1 0.1 kcal mol 1). Binding site selectivity observed by NMR for 50-ACTAGT-30 in the presence of 50- TCTAGA-30 when both accessible sequences are concatenated in a 15-mer DNA duplex construct is consistent with thermodynamic parameters (|DG|ACTAGT > |DG|TCTAGA) measured separately for the binding sites and with predictions from modelling studies. Steric bulk in the minor groove for Z ¼ G causes unfavourable ligand–DNA interactions reflected in lower Gibbs free energy of binding (DG ¼ 8.5 0.01 kcal mol 1). ITC and CD data establish that thiazotropsin A binds the ODNs with binding constants between 106 and 108 M 1 and reveal that binding is driven enthalpically through hydrogen bond formation and van der Waals interactions. The consequences of these findings are considered with respect to ligand self-association and the energetics responsible for driving DNA recognition by small molecules in the DNA minor groove.