Sequence and length dependent thermodynamic differences in heterocyclic diamidine interactions at AT base pairs in the DNA minor groove

Abstract

With the goal of developing a better understanding of the antiparasitic biological action of DB75, we have evaluated its interaction with duplex alternating and nonalternating sequence AT polymers and oligomers. These DNAs provide an important pair of sequences in a detailed thermodynamic analysis of variations in interaction of DB75 with AT sites. The results for DB75 binding to the alternating and nonalternating AT sequences are quite different at the fundamental thermodynamic level. Although the Gibbs energies are similar, the enthalpies for DB75 binding with poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) are +3.1 and -4.5 kcal/mol, respectively, while the binding entropies are 41.7 and 15.2 cal/mol.K, respectively. The underlying thermodynamics of binding to AT sites in the minor groove plays a key role in the recognition process. It was also observed that DB75 binding with poly(dA).poly(dT) can induce T.A.T triplet formation and the compound binds strongly to the dT.dA.dT triplex.

Fig. 1

Structure of the compound and the DNA sequences used in this study.

Fig. 2

UV melting profiles at 260nm of the polymeric DNAs and DNA hairpins in the absence and presence of DB75. (A) Thermal melting curves of poly(dA)·poly(dT) and poly(dA-dT)·poly(dA-dT) at the indicated ratio. (B) Thermal melting curves of A15 and AT7 hairpins at the indicated ratio. (C) A phase diagram for T_m versus molar ratio for the thermal transitions of PolydA·PolydT (left) and DNA hairpins (right). The regions of triplex–duplex–single strand stability are labelled for the polymer. The oligomers have only duplex and single strand phase.

Fig. 3

CD titration spectra of the polymeric DNAs and DNA hairpins binding with DB75 at various mixing ratios. Insert: increase in CD magnitude at 391nm (○) and decrease in CD magnitude at 267nm (●).

Fig. 4

Temperature–dependent CD magnitude (relative to the CD at 10 °C, CD_T/CD₁₀°_C) of the polymeric DNAs and their DB75 complexes at 267 and 391 nm.

Fig. 5

DSC excess heat capacity (ΔC_p) versus temperature profiles for polymeric DNAs binding with DB75. Melting curves for poly(dA)·poly(dT) in the absence and presence of DB75 (A), and poly(dA-dT)·poly(dA-dT) in the absence and presence of DB75 (B).

Fig. 6

ITC curves for the binding of DB75 to the AT DNA polymers and hairpins in MES10 at 25 °C. Every peak represents the instrument response for injection of DB75 into DNA during the course of the titration (top). A binding isotherm from integration with respect to time, with appropriate dilution correction (bottom). For poly(dA)·poly(dT) (A) and poly(dA-dT)·poly(dA-dT) (B), the smooth lines show the fit to the results and best fit ΔH (Table 2) values for binding. For A₁₅ (C) and (AT)₇ (D) hairpins, a ‘model–free ITC’ protocol was used to obtain ΔH.

Fig. 7

Representative ITC titration and integrated heat data for DB75 binding to poly(dA)·poly(dT) (top) and poly(dA-dT)·poly(dA-dT) (bottom) at 10, 20, 30, 40 °C.

Fig. 8

Complete thermodynamic results for binding of DB75 to poly(dA)·poly(dT) (open) and poly(dA-dT)·poly(dA-dT) (solid) are shown as a function of temperature. Because the free energy changes much less with temperature than the enthalpy, there is significant enthalpy–entropy compensation as can be seen in the Figure. The ΔCp values in Table 2 are the slopes of the ΔH° plots and no curvature is apparent over this temperature range.

Fig. 9

SPR binding affinity: (a) SPR sensorgrams for the interaction of A₁₅ hairpin and (AT)₇ hairpin with DB75. The compound concentrations for DB75 from bottom to top are 0 to 1 μM. (b) RU values from the steady–state region of SPR sensorgrams were converted to r (r = RU/RU_max) and are plotted against the unbound compound concentration (flow solution) for A₁₅ (diamonds), (AT)₇ (circles) and (CG)₄ (squares) hairpins binding with DB75. The lines are the best fit values using appropriate binding models.

Fig. 10

Parsing the free energy (ΔG_obs) of polymeric DNAs binding with DB75 into the contributor energy terms. The two bars on the right represent ΔC_p*= ΔC_p/10 in unit of cal mol⁻¹K⁻¹.