Shape readout of AT-rich DNA by carbohydrates

Abstract

Gene expression can be altered by small molecules that target DNA; sequence as well as shape selectivities are both extremely important for DNA recognition by intercalating and groove-binding ligands. We have characterized a carbohydrate scaffold (1) exhibiting DNA "shape readout" properties. Thermodynamic studies with 1 and model duplex DNAs demonstrate the molecule's high affinity and selectivity towards B* form (continuous AT-rich) DNA. Isothermal titration calorimetry (ITC), circular dichroism (CD) titration, ultraviolet (UV) thermal denaturation, and Differential Scanning Calorimetry were used to characterize the binding of 1 with a B* form AT-rich DNA duplex d[5'-G2 A6 T6 C2 -3']. The binding constant was determined using ITC at various temperatures, salt concentrations, and pH. ITC titrations were fit using a two-binding site model. The first binding event was shown to have a 1:1 binding stoichiometry and was predominantly entropy-driven with a binding constant of approximately 10(8) M(-1) . ITC-derived binding enthalpies were used to obtain the binding-induced change in heat capacity (ΔCp ) of -225 ± 19 cal/mol·K. The ionic strength dependence of the binding constant indicated a significant electrolytic contribution in ligand:DNA binding, with approximately four to five ion pairs involved in binding. Ligand 1 displayed a significantly higher affinity towards AT-tract DNA over sequences containing GC inserts, and binding experiments revealed the order of binding affinity for 1 with DNA duplexes: contiguous B* form AT-rich DNA (d[5'-G2 A6 T6 C2 -3']) >B form alternate AT-rich DNA (d[5'-G2 (AT)6 C2- 3']) > A form GC-rich DNA (d[5'-A2 G6 C6 T2 -3']), demonstrating the preference of ligand 1 for B* form DNA.

Keywords: DNA major groove; carbohydrate scaffold; shape recognition.

Figure 1

Structure of the ligands used in this study.

Figure 2

(A) CD titration of d[5′-G₂A₆T₆C₂-3′] in the presence of 1. From top to bottom at 280 nm the CD intensity decreased with an increasing amount of 1. (B) The plot between changes in CD intensity (at 280 nm) as a function of the molar ratio of 1 to the DNA duplex. The continuous lines in the plot reflect the linear least squares fit of each apparent linear domain of the experimental data (filled squares) before and after the apparent inflection point. The inflection point corresponds to the binding site size. (B1) CD scans display a change in the CD intensity at stoichiometry ratio (drug/DNA) of 0, 0.5, 1.0, and 2.0. (C) CD titration of d[5′-G₂A₆T₆C₂-3′] in the presence of 1. From bottom to top at 265 nm the CD intensity increased with an increasing amount of 1. (D) The plot between changes in CD intensity (at 265 nm) as a function of the molar ratio of 1 to the DNA duplex. The continuous lines in the plot reflect the linear least squares fit of each apparent linear domain of the experimental data (filled squares) before and after the apparent inflection point. The inflection point corresponds to the binding site size. (D1) CD scans display a change in the CD intensity at stoichiometry ratio (drug/DNA) of 0, 0.5, 1.0, and 2.0. Buffer condition: 100 mM KCl, 10 mM SC, 0.5 mM EDTA, and pH =5.5. T = 25°C. [DNA] = 4 :M/duplex.

Figure 3

Overlay of the normalized UV thermal denaturation profiles of poly(dA).poly(dT) in the presence of “1” at indicated rbd’s (ratio of base pair to drug) using experiment (thick line) and theoritical (McGhee fit, dotted line) data.

Figure 4

ITC profile (top) and thermodynamic contribution (bottom) of binding interaction between 1 and d[5′-G₂A₆T₆C₂-3′]. The ITC profile was fit using the two sets of sites binding model where first binding site was predominately entropy driven while second binding site was enthalpy driven.

Figure 5

Temperature dependence of the binding enthalpy of 1-DNA interactions. The data points were determined experimentally (derived by direct ITC titration between 1 and DNA) within a temperature range of 20-35°C at 100 mM KCl, 10 mM SC, 0.5 mM EDTA, and pH 5.5. The slope of the linear least-square fit of the data gives a δ C_p value of −225±19 cal mol⁻¹ K⁻¹.

Figure 6

Salt dependence of direct ITC derived binding constants at 25°C. The experimental data points were fit by using linear regression yielding a slope of −3.25, indicating the participation of approximately ∼4-5 NH₃ ⁺ groups of 1 in electrostatic interactions with the host DNA. Buffer conditions: 100 mM KCl, 10 mM SC, 0.5 mM EDTA, and pH 5.5.

Figure 7

Dissection of the components of free energy of 1 binding with d[5′-G₂A₆T₆C₂-3′] at pH = 5.5 (in the absence of drug protonation during the binding event) at 25 °C. The contribution from +G_conf is considered to be zero for the interaction of 1 with DNA. The +G_hyd is the average, without considering the error value (±10).

Figure 8

The energy compensation plot of DNA-drug interaction. The filled squares illustrate the energy compensation plot of intercalators and the open squares represent the data for groove binders. 1 is represented by the red square for comparison. The energy compensation data of 1 with the DNA duplex d[5′-G₂A₆T₆C₂-3′] compared at both pH readings. The figure is adapted and modified from the work of Chaires (59).