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

DNA-sequence and structure dependence on the formation of minor groove complexes at 5′-XCYRGZ-3′, where Y = T and R = 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 = A, T, C or G and Z = T, A, I (inosine) or C, 5′-CCTAGI- 3′ being thermodynamically the most favoured (ΔG = -11.1 ± 0.1 kcal mol ^-1). Binding site selectivity observed by NMR for 5′-ACTAGT-3′ in the presence of 5′- TCTAGA-3′ when both accessible sequences are concatenated in a 15-mer DNA duplex construct is consistent with thermodynamic parameters (ΔG _ACTAGT > ΔG _TCTAGA) measured separately for the binding sites and with predictions from modelling studies. Steric bulk in the minor groove for Z = G causes unfavourable ligand-DNA interactions reflected in lower Gibbs free energy of binding (ΔG = -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.