Binding of the biogenic polyamines to deoxyribonucleic acids of varying base composition: base specificity and the associated energetics of the interaction

Abstract

Background: The thermodynamics of the base pair specificity of the binding of the polyamines spermine, spermidine, putrescine, and cadaverine with three genomic DNAs Clostridium perfringens, 27% GC, Escherichia coli, 50% GC and Micrococcus lysodeikticus, 72% GC have been studied using titration calorimetry and the data supplemented with melting studies, ethidium displacement and circular dichroism spectroscopy results.

Methodology/principal findings: Isothermal titration calorimetry, differential scanning calorimetry, optical melting studies, ethidium displacement, circular dichroism spectroscopy are the various techniques employed to characterize the interaction of four polyamines, spermine, spermidine, putersine and cadaverine with the DNAs. Polyamines bound stronger with AT rich DNA compared to the GC rich DNA and the binding varied depending on the charge on the polyamine as spermine>spermidine >putrescine>cadaverine. Thermodynamics of the interaction revealed that the binding was entropy driven with small enthalpy contribution. The binding was influenced by salt concentration suggesting the contribution from electrostatic forces to the Gibbs energy of binding to be the dominant contributor. Each system studied exhibited enthalpy-entropy compensation. The negative heat capacity changes suggested a role for hydrophobic interactions which may arise due to the non polar interactions between DNA and polyamines.

Conclusion/significance: From a thermodynamic analysis, the AT base specificity of polyamines to DNAs has been elucidated for the first time and supplemented by structural studies.

Figure 1. Chemical structure of polyamines.

Figure 2. ITC profiles for the titration of polyamines with DNAs.

Titration of spermine with (A) CP DNA (B) EC DNA (C) ML DNA and spermidine with (D) CP DNA (E) EC DNA (F) ML DNA at 293.15 K. The top panels represent the raw data for the sequential injection of polyamines into a solution of DNA and the bottom panels show the integrated heat data after correction of heat of dilution against molar ratio of DNA/[polyamine]. The data points were fitted to one site model and the solid line represent the best fit data.

Figure 3. Plot of variation of enthalpy of binding (ΔH^O) with temperature.

Plots for the binding of (A) spermine with CP DNA (▪), EC DNA (•), ML DNA (▴), (B) spermidine with CP DNA (□), EC DNA (○), ML DNA (▵), (C) putrescine with CP DNA (▪), EC DNA (•), ML DNA (▴) and (D) cadaverine with CP DNA (□), EC DNA (○), ML DNA (▵).

Figure 4. Plots of variationof thermodynamic parameters with entropy contribution.

Plot of ΔG^O and ΔH^O versus TΔS^O for the binding of (A) spermine with CP DNA (▪,•), EC DNA (▴,▾), ML DNA (♦,◂) (B) Plot of ΔG^O and ΔH^O versus TΔS^O of spermidine with CP DNA (□,○), CT DNA (▵,▿), ML DNA (⋄,⊲), (C) Plot of ΔG^O and ΔH^O versus TΔS^O of putrescine with CP DNA (▪,•), EC DNA (▴,▾), ML DNA (♦,◂) and (D) Plot of ΔG^O and ΔH^O versus TΔS^O of cadaverine with CP DNA (□,○), EC DNA (▵,▿), ML DNA (⋄,⊲).

Figure 5. Plots of variation of salt dependent thermodynamic parameters.

(A) Plot of ln K_a versus ln [Na⁺] for the binding of spermine with CP DNA (▪), EC DNA (•), MLDNA (▴). Bar diagram describing the variation of magnitude of thermodynamics parameters at three salt concentrations for (B) CP DNA, (C) EC DNA and (D) ML DNA at 293.15 K.

Figure 6. Melting profiles of DNA and DNA polyamine complexes.

Optical melting profiles (upper panels) of (A) CP DNA (□), spermine-CP DNA(▵), spermidine-CP DNA (O), (B) EC DNA (□), spermine-EC DNA(▵), spermidine-EC DNA(O), (C) ML DNA (□), spermine-ML DNA(▵), spermidine-ML DNA(O). DSC melting profiles (lower panels) of (D) CP DNA (solid lines) (E) EC DNA (solid lines), (F) ML DNA (solid lines) and respective DNA–spermine complex (- - - -) and DNA-spermidine complex (….).

Figure 7. Displacement plots of ethidium bromide-DNA complexes by polyamines.

Relative fluorescence intensity decrease of ethidium bromide (1.2 μM)-DNA(12.0 μM) complex induced by the binding of (A) spermine with CP DNA(-▪-), EC DNA (-•-), ML DNA (-▴-) and (B) spermidine with CP DNA(-▪-), EC DNA (-•-), ML DNA (-▴-) conducted in 10 mM SHE buffer pH 7.0 at 293.15 K (Inset: The values of IC₅₀ of CP, EC and ML DNA shown as a bar graph).

Figure 8. Circular dichroism spectral titration of DNA-poyamine complexes.

Intrinsic CD spectra of (A) CP DNA (30 µM) treated with 0–63 µM (curves 1 to 7) spermine (B) CP DNA (30 µM) treated with 0–135 µM (curves 1 to 7 ) spermidine (C) EC DNA (30 µM) treated with 0–95 µM (curves 1 to 7) spermine (D) EC DNA (30 µM) treated with 0–175 µM (curves 1 to 7) spermidine (E) ML DNA (30 µM) treated with 0–120 µM (curves 1 to 7) spermine and (F) ML DNA (30 µM) treated with 0–250 µM (curves 1 to 7 ) spermidine at 293.15 K.