Biophysical studies of a ruthenium(II) polypyridyl complex binding to DNA and RNA prove that nucleic acid structure has significant effects on binding behaviors

Abstract

The interactions of a metal complex [Ru(phen)(2)PMIP](2+) {Ru=ruthenium, phen=1,10-phenanthroline, PMIP=2-(4-methylphenyl)imidazo[4,5-f]1,10-phenanthroline} with yeast tRNA and calf thymus DNA (CT DNA) have been investigated comparatively by UV-vis spectroscopy, fluorescence spectroscopy, viscosity measurements, isothermal titration calorimetry (ITC), as well as equilibrium dialysis and circular dichroism (CD). Spectroscopic studies together with ITC and viscosity measurements indicate that both binding modes of the Ru(II) polypyridyl complex to yeast tRNA and CT DNA are intercalation and yeast tRNA binding of the complex is stronger than CT DNA binding. ITC experiments show that the interaction of the complex with yeast tRNA is driven by a moderately favorable enthalpy decrease in combination with a moderately favorable entropy increase, while the binding of the complex to CT DNA is driven by a large favorable enthalpy decrease with a less favorable entropy increase. The results from equilibrium dialysis and CD suggest that both interactions are enantioselective and the Delta enantiomer of the complex may bind more favorably to both yeast tRNA and CT DNA than the Lambda enantiomer does, and that the complex is a better candidate for an enantioselective binder to yeast tRNA than to CT DNA. Taken together, these results indicate that the structures of nucleic acids have significant effects on the binding behaviors of metal complexes.

Fig. 1

Structure of complex [Ru(phen)₂PMIP]²⁺

Fig. 2

UV and visible spectra of complex [Ru(phen)₂PMIP]²⁺ upon the addition of yeast tRNA (a) and CT DNA (b). a, Arrow represented the concentration of yeast tRNA increased gradually from 0 (top) to 25.0 µM (bottom). Inset: plot of ΔA versus [RNA] for the titration of the Ru(II) polypyridyl complex with yeast tRNA. b Arrow represented the concentration of CT DNA increased gradually from 0 (top) to 30.0 µM (bottom). Inset: plot of ΔA at 263 nm versus [DNA] for the titration of the Ru(II) polypyridyl complex with CT DNA. The filled squares were the experimental data and the solid lines represented the best fit. The concentrations of the Ru(II) complex was 10.0 µM. Experiments were performed at 25°C in 5 mM Tris–HCl buffer at pH 7.2. Data are expressed as mean ± SD. (n = 3)

Fig. 3

Fluorescence emission spectra of complex [Ru(phen)₂PMIP]²⁺ in the presence of yeast tRNA (a) and CT DNA (b). a, Arrow represented the concentration of yeast tRNA increased gradually from 0 (top) to 20.0 µM (bottom). The concentrations of the Ru(II) polypyridyl complex was 5.0 µM, and the slit width of 3.0 nm was used for both the excitation and emission beams. Inset: Stern–Volmer plot of the quenching of fluorescence of the Ru(II) complex by yeast tRNA. b, Arrow represents the concentration of CT DNA increased gradually from 0 (bottom) to 200 µM (top). The concentrations of the Ru(II) complex was 10.0 µM, and the slit widths of 1.5 and 3.0 nm were used for the excitation and emission beams respectively. Inset: plot of relative fluorescence intensity against the molar ratio of CT DNA to the Ru(II) polypyridyl complex. The filled squares represented the experimental data. Measurements were carried out at 25°C in 5 mM Tris–HCl buffer at pH 7.2. Data are expressed as mean ± SD. (n = 3)

Fig. 4

Stern–Volmer plots of the quenching of fluorescence of complex [Ru(phen)₂PMIP]²⁺ by [Fe(CN)₆]⁴⁻ in the absence (filled square) and presence (open square) of CT DNA. The concentrations of CT DNA and the Ru(II) polypyridyl complex were 160 and 4 µM, respectively. Measurements were carried out at 25°C in 5 mM Tris–HCl buffer at pH 7.2. Data are expressed as mean ± SD. (n=3)

Fig. 5

Effects of increasing concentrations of complex [Ru(phen)₂PMIP]²⁺ on the relative viscosities of CT DNA at 28°C in 5 mM Tris–HCl buffer at pH 7.2. The concentrations of CT DNA was 0.50 mM, and the molar ratios of the Ru(II) polypyridyl complex to CT DNA were 0.02, 0.04, 0.06, and 0.08, respectively. Data are expressed as mean ± SD. (n = 3)

Fig. 6

ITC profiles for the binding of complex [Ru(phen)₂PMIP]²⁺ to yeast tRNA and CT DNA at 25°C in 30 mM HEPES buffer at pH 7.2. The top panels (a and c) represent the raw data for sequential 10- μL injections of the Ru(II) polypyridyl complex (100 µM) into yeast tRNA (25.0 µM) and CT DNA (25.0 µM), respectively. The bottom panels (b and d) show the plot of the heat evolved (kcal) per mole of the Ru(II) complex added, corrected for the heat of Ru(II) complex dilution, against the molar ratio of the Ru(II) complex to yeast tRNA and CT DNA, respectively. The data (filled square) were fitted to a single set of identical sites model and the solid line represented the best fit

Fig. 7

CD spectra of the dialysates of complex [Ru(phen)₂PMIP]²⁺ after dialysis against yeast tRNA (red) and CT DNA (blue) for 24 h. The concentrations of yeast tRNA, CT DNA, and the Ru(II) polypyridyl complex were 1.0, 1.0, and 0.10 mM, respectively. Experiments were performed at 25°C