Targeting RNA by small molecules: comparative structural and thermodynamic aspects of aristololactam-β-D-glucoside and daunomycin binding to tRNA(phe)

Abstract

Background: Interaction of aristololactam-β-D-glucoside and daunomycin with tRNA(phe) was investigated using various biophysical techniques.

Methodology/principal findings: Absorption and fluorescence studies revealed that both the compounds bind tRNA(phe) non-cooperatively. The binding of daunomycin was about one order of magnitude higher than that of aristololactam-β-D-glucoside. Stronger binding of the former was also inferred from fluorescence quenching data, quantum efficiency values and circular dichroic results. Results from isothermal titration calorimetry experiments suggested that the binding of both compounds was predominantly entropy driven with a smaller but favorable enthalpy term that increased with temperature. A large favorable electrostatic contribution to the binding of daunomycin to tRNA(phe) was revealed from salt dependence data and the dissection of the free energy values. The electrostatic component to the free energy change for aristololactam-β-D-glucoside-tRNA(phe) interaction was smaller than that of daunomycin. This was also inferred from the slope of log K versus [Na(+)] plots. Both compounds enhanced the thermal stability of tRNA(phe). The small heat capacity changes of -47 and -99 cal/mol K, respectively, observed for aristololactam-β-D-glucoside and daunomycin, and the observed enthalpy-entropy compensation phenomenon confirmed the involvement of multiple weak noncovalent interactions. Molecular aspects of the interaction have been revealed.

Conclusions/significance: This study presents the structural and energetic aspects of the binding of aristololactam-β-D-glucoside and daunomycin to tRNA(phe).

Figure 1. Chemical structures.

(A) aristololactam–β-D-glucoside and (B) daunomycin and (C) the cloverleaf structure of yeast tRNA^phe.

Figure 2. Absorption spectral titration of drugs with tRNA.

(A) ADG (11 µM) treated with 0, 55, 110, 165, 220, 330, 440, 550 and 660 µM (curves 1–9) of tRNA and (B) DAU (10.4 µM) treated with 0, 20.8, 52, 83.2, 124.8, 166.4, 208, 312 and 364 µM (curves 1–9) of tRNA. Inset: representative Scatchard plot of each complexation. The solid lines represent the non-linear least square best fit of the experimental points to the neighbour exclusion model.

Figure 3. Fluorescence spectral titration of drugs and tRNA.

(A) ADG (3.07 µM) treated with 0, 15.35, 30.7, 46.05, 61.4, 92.1, 122.8, 168.85 and 214.9 µM (curves 1–8) of tRNA and (B) DAU (3.0 µM) treated with 0, 15, 30, 45, 60, 75, 90, 105 and 120 µM (curves 1–8) of tRNA.

Figure 4. Stern-Volmer plots for the quenching of the fluorescence of drugs-tRNA complex by K₄[Fe(CN)₆].

(A) ADG and (B) DAU. Symbols are in absence (○) ADG, (□) DAU and, in presence (•) ADG and (▪) DAU of tRNA.

Figure 5. Job's plot for the binding of ADG and DAU with tRNA.

(A) ADG and (B) DAU.

Figure 6. Circular dichroic spectra of tRNA-drug complexes.

tRNA (60 µM) treated with (A) 30, 60, 90 and 108 µM (curves 1–4) of ADG and (B) 18, 36, 60 and 90 µM of DAU. Inset: Induced circular dichroic spectra of ADG and DAU, respectively.

Figure 7. ITC profiles for the binding of (A) ADG and (B) DAU with tRNA.

The top panels represent the raw data for sequential injection of tRNA into drug solution and the bottom panels show the integrated heat data after correction of heat of dilution against molar ratio of poly(A)/drug. The data points (closed circle) were fitted to a one site model, and the solid lines represent the best fit data.

Figure 8. Plot of variation of thermodynamic parameters.

ΔG (○, □) and ΔH (•, ▪) verses TΔS for the binding of ADG and DAU to tRNA.

Figure 9. Optical and DSC melting profiles of tRNA-drug complexes.

(A) optical melting of tRNA alone (▴), in presence of ADG (•) and in presence of DAU (▪), (B) DSC thermograms of tRNA, (C) tRNA-ADG complex and (D) tRNA-DAU complex.