Unusually strong binding to the DNA minor groove by a highly twisted benzimidazole diphenylether: induced fit and bound water

Abstract

RT29 is a dicationic diamidine derivative that does not obey the classical "rules" for shape and functional group placement that are expected to result in strong binding and specific recognition of the DNA minor groove. The compound contains a benzimidazole diphenyl ether core that is flanked by the amidine cations. The diphenyl ether is highly twisted and gives the entire compound too much curvature to fit well to the shape of the minor groove. DNase I footprinting, fluorescence intercalator displacement studies, and circular dichroism spectra, however, indicate that the compound is an AT specific minor groove binding agent. Even more surprisingly, quantitative biosensor-surface plasmon resonance and isothermal titration calorimetric results indicate that the compound binds with exceptional strength to certain AT sequences in DNA with a large negative enthalpy of binding. Crystallographic results for the DNA complex of RT29 compared to calculated results for the free compound show that the compound undergoes significant conformational changes to enhance its minor groove interactions. In addition, a water molecule is incorporated directly into the complex to complete the compound-DNA interface, and it forms an essential link between the compound and base pair edges at the floor of the minor groove. The calculated DeltaCp value for complex formation is substantially less than the experimentally observed value, which supports the idea of water being an intrinsic part of the complex with a major contribution to the DeltaCp value. Both the induced fit conformational changes of the compound and the bound water are essential for strong binding to DNA by RT29.

Figure 1

Structures for RT29 with other diamidines and DNA hairpin and self-complementary duplex sequences.

Figure 2

(A). DNase I footprinting for RT29 bound to the 3′-end radiolabeled 117-bp restriction fragment from pBS and (B) 176-bp restriction fragment from pTUC. The cleavage products from the DNase I digestion were resolved on an 8% polyacrylamide gel containing 8M urea. The concentration (μM) of the drug is shown at the top of the appropriate gel lanes. Control tracks labeled “Ct” contained no drug. The track labeled G represents dimethylsulphate-piperidine markers specific for guanine. Numbering of the sequence is shown at the left side of the gels. Densitometer traces with sequence numbering are shown below the gel results.

Figure 2

(A). DNase I footprinting for RT29 bound to the 3′-end radiolabeled 117-bp restriction fragment from pBS and (B) 176-bp restriction fragment from pTUC. The cleavage products from the DNase I digestion were resolved on an 8% polyacrylamide gel containing 8M urea. The concentration (μM) of the drug is shown at the top of the appropriate gel lanes. Control tracks labeled “Ct” contained no drug. The track labeled G represents dimethylsulphate-piperidine markers specific for guanine. Numbering of the sequence is shown at the left side of the gels. Densitometer traces with sequence numbering are shown below the gel results.

Figure 3

(A). FID analysis of RT −29 (0.75 μM) binding to all possible four base-pair DNA sequences (upper panel) emphasizing the overall rank and relative rank order of sites containing three contiguous A/T base pairs flanked by one G/C base pair [5′-(A/T)₃-G/C sites]. Also illustrated is a comparison of the binding of RT-29 to the four possible 5′-ATTC-N sites (lower panel). The merged-bar FID analysis histogram in the upper panel is color-coded such that four base pair A/T-only DNA cassettes are blue [5′-(A/T)₄ sites] while those containing 5′-(A/T)₃-G/C sites are red; all other sites are black. (B) The plot shown illustrates the change in F (decrease) that occurs as ethidium bromide is displaced from oligonucleotides containing 5′-ATTC-N sites as a function of increasing RT-29 concentration; these data suggest that RT-29 exhibits a slight preference for 5′-ATTC-T sites (T > A > G > C).

Figure 4

Induced CD signals for RT29 with the d(CGCGAATTCGCG)₂ duplex in MES 10 buffer at 25°C. Molar ratios of compound to DNA duplex are 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.5 from the bottom to the top at the induced CD wavelength maximum near 320nm.

Figure 5

(A). SPR sensorgrams for binding of RT29 with AATT in Tris50 buffer at 25°C. The smooth black lines are best fit curves by global kinetics fitting as described in the text. For RT29, the concentrations from bottom to top are 0, 0.25, 0.75, 1.5, 2.5, 3.5, 5.5, 7.5, 10 and 20nM. (B) Binding results for RT29 with the AATT hairpin sequence (Figure 1) at different salt concentrations 0.2 (▲), 0.5 (•), and 1.0 (■) M NaCl) at 25 ºC. The inset shows the linear relation of log K to –log [Na⁺] which gives a slope of 1.8. (C) SPR sensorgrams for the binding kinetics of RT29 with AATT in Tris100 buffer at 25 ºC and a flow rate of 100μl/min on a low density C4-streptavidin chip. The black lines are best fit curves by global kinetics fitting. The RT29 concentrations from bottom to top are 0, 2.5, 5.5 10, 20, 30, and 40 nM.

Figure 5

(A). SPR sensorgrams for binding of RT29 with AATT in Tris50 buffer at 25°C. The smooth black lines are best fit curves by global kinetics fitting as described in the text. For RT29, the concentrations from bottom to top are 0, 0.25, 0.75, 1.5, 2.5, 3.5, 5.5, 7.5, 10 and 20nM. (B) Binding results for RT29 with the AATT hairpin sequence (Figure 1) at different salt concentrations 0.2 (▲), 0.5 (•), and 1.0 (■) M NaCl) at 25 ºC. The inset shows the linear relation of log K to –log [Na⁺] which gives a slope of 1.8. (C) SPR sensorgrams for the binding kinetics of RT29 with AATT in Tris100 buffer at 25 ºC and a flow rate of 100μl/min on a low density C4-streptavidin chip. The black lines are best fit curves by global kinetics fitting. The RT29 concentrations from bottom to top are 0, 2.5, 5.5 10, 20, 30, and 40 nM.

Figure 5

(A). SPR sensorgrams for binding of RT29 with AATT in Tris50 buffer at 25°C. The smooth black lines are best fit curves by global kinetics fitting as described in the text. For RT29, the concentrations from bottom to top are 0, 0.25, 0.75, 1.5, 2.5, 3.5, 5.5, 7.5, 10 and 20nM. (B) Binding results for RT29 with the AATT hairpin sequence (Figure 1) at different salt concentrations 0.2 (▲), 0.5 (•), and 1.0 (■) M NaCl) at 25 ºC. The inset shows the linear relation of log K to –log [Na⁺] which gives a slope of 1.8. (C) SPR sensorgrams for the binding kinetics of RT29 with AATT in Tris100 buffer at 25 ºC and a flow rate of 100μl/min on a low density C4-streptavidin chip. The black lines are best fit curves by global kinetics fitting. The RT29 concentrations from bottom to top are 0, 2.5, 5.5 10, 20, 30, and 40 nM.

Figure 6

Thermodynamics results, ΔG from SPR, ΔH from ITC, and –TΔS (calculated from ΔG = ΔH –TΔS), for binding of RT29 to the AATT site (Figure 1) at different temperatures.

Figure 7

The upper panel is an ITC curve for titration of RT29 (0.050mM) into 0.005mM d(GCGAATTCGC)₂ duplex (CAC20 buffer with 200mM NaCl) at 35 ºC. The integrated, blank subtracted heat are plotted versus molar ratio in the lower panel.

Figure 8

Equilibrium geometries of RT29 (top) and DB75 (bottom) were calculated by the density functional method at the 631G(p, d) level. Tube models are shown at right with space filling electrostatic potential molecular surfaces on the left. The middle structure is RT29 in the conformation observed in the crystal complex. The crystal conformation was frozen in the calculations for comparison to the unconstrained minimum energy conformations of RT29 and DB75. A can be seen, there is a very significant difference between the equilibrium, unbound and bound conformations of RT29. Note that the bound conformation of RT29 is much closer to that for DB75 and other similar minor groove binding compounds.

Figure 9

Crystal structures for RT 29 bound in the minor groove of an AATT (left) and ATTC site (right) are shown. The water molecules that complete the complexes are shown as red spheres. The water is at the bottom, phenylamidine group, in the AATT complex and at the top, benzimidazole amidine group, in the ATTC complex.

Figure 10

Comparison of binding thermodynamics for DB75, DB921 and RT29 with an AATT site, and RT29 with an ATTC site at 25 ºC in buffer with 0.2M NaCl.