DNA minor groove induced dimerization of heterocyclic cations: compound structure, binding affinity, and specificity for a TTAA site

Abstract

With the increasing number and variations of genome sequences available, control of gene expression with synthetic, cell-permeable molecules is within reach. The variety of sequence-specific binding agents is, however, still quite limited. Many minor groove binding agents selectivity recognize AT over GC sequences but have less ability to distinguish among different AT sequences. The goal with this article is to develop compounds that can bind selectively to different AT sequences. A number of studies indicate that AATT and TTAA sequences have significantly different physical and interaction properties and different requirements for minor groove recognition. Although it has been difficult to get minor groove binding at TTAA, DB293, a phenyl-furan-benzimidazole diamidine, was found to bind as a strong, cooperative dimer at TTAA but with no selectivity over AATT. In order to improve selectivity, we made modifications to each unit of DB293. Binding affinities and stoichiometries obtained from biosensor-surface plasmon resonance experiments show that DB1003, a furan-furan-benzimidazole diamidine, binds strongly to TTAA as a dimer and has selectivity (K(TTAA)/K(AATT)=6). CD and DNase I footprinting studies confirmed the preference of this compound for TTAA. In summary, (i) a favorable stacking surface provided by the pi system, (ii) H-bond donors to interact with TA base pairs at the floor of the groove provided by a benzimidazole (or indole) -NH and amidines, and (iii) appropriate curvature of the dimer complex to match the curvature of the minor groove play important roles in differentiating the TTAA and AATT minor grooves.

Fig. 1

(a) Representative structures of compounds classified by their functional group modifications. Structures for all compounds are tabulated in Table 1. (b) DNA sequences used in this study.

Fig. 2

Equilibrium binding studies. SPR sensorgrams for binding of (a) DB293, (b) DB75, (c) DB828, and (d) DB270 to the TTAA at 25 °C. The unbound compound concentrations in the flow solutions range from 0.0001 μM in the lowest curve to 1 μM in the top curve. The RU values from the steady-state region of SPR sensorgrams were converted to r by r = RU/RUmax and are plotted versus the unbound compound concentration for DB293, DB75, DB809, and DB270 binding to (e) TTAA; Solid lines represent a one site fit for DB75, DB828 and a two site fit for DB293 and DB270 and (f) AATT; Solid lines represent a one site equation fit. Experiments were conducted in cacodylic acid buffer with 0.1 M NaCl added at 25 °C.

Fig. 3

Study of binding mode. CD titration for TTAA in (a) DB293, (b) DB832, (c) DB1003, and AATT in (d) DB1003. The concentration of duplex hairpin was 5 μM and the compound was added with 1 μM increment with total of 10 increments. In case of DB293 and DB1003 in TTAA, DNA saturates at 2:1 compound to DNA ratio confirming dimer formation for these compounds. DB832 in TTAA and DB1003 in AATT with similar compound to DNA ratio give weaker binding.

Fig. 4

Equilibrium binding studies. SPR sensorgrams for binding of (a) DB1003 and (b) DB832 to the TTAA at 25 °C. The unbound compound concentrations in the flow solutions range from 0.0001 μM in the lowest curve to 0.8 μM in the top curve. The RU values from the steady-state region of SPR sensorgrams were converted to r by r = RU/RUmax and are plotted versus the unbound compound concentration for DB1003 and DB832 binding to (c) TTAA and (d) AATT.

Fig. 5

Scatchard plots of the results for binding of DB1003 and DB832 to TTAA.

Fig. 6

Equilibrium binding studies. SPR sensorgrams for binding of (a) DB915 to the TTAA. The unbound compound concentrations in the flow solutions range from 0.0001 μM in the lowest curve to 0.8 μM in the top curve. (b) The binding plot for DB915 in the presence of TTAA and AATT.

Fig. 7

Equilibrium binding studies. SPR binding plots for DB1878 in the presence of TTAA and AATT.

Fig. 8

DNase I footprinting assays. (a) The radiolabeled MS1-198 bp (lanes “0”) or (c) TTAA-69 bp DNA fragments were incubated alone (lanes “DNA”) or in the presence of graded concentrations (μM) of the various compounds as indicated on the top of each lanes. Lanes “G” correspond to the G-track ladders that were used as markers for guanines positions to locate the protected sites. The respective densitometric analyses are presented in panels (b) and (d). Black boxes localize the sites that are protected by the compounds from DNase I cleavage.

Fig. 9

Comparative plot of selectivity (K_TTAA/K_AATT) of studied compounds against AATT and TTAA sequences. K_TTAA = (√ K₁×K₂); K_AATT is equilibrium binding constant for AATT site.