Magnitude Of Linear Dichroism Research Articles

Effects of the Ligand Structure of Cu(II) Complexes on Oxidative DNA Cleavage

Cu complexes were synthesized by substituting the hydrogen of the amine group of basic ligand 2,2′‐dipicoylamine (dpca) (complex 2) with CH3CO (complex 1), phenyl (complex 3), and methyl (complex 4), respectively, and their DNA cleavage activity was investigated using linear dichroism (LD) and electrophoresis. The DNA cleavage efficiencies of Cu complexes 3 and 4 with phenyl and methyl, which are electron‐donating functional groups, turned out to be the highest, and LD magnitudes rapidly decreased at 260 nm. In particular, Cu complex 3 showed a rapid LD magnitude reduction to 63% of the total for 90 min, and to 50% of the total at 12 min. DNA cleavage efficiencies were high in the order of phenyl > methyl > HCH3CO, and the highest DNA cleavage efficiency was observed in the presence of electron‐donating groups. The electrophoresis results are also consistent with the changes in LD spectra over time. The Cu complexes (1–4) were found to cleave DNA through oxidative pathways, and the major reaction oxygen species involved in DNA cleavage were the superoxide radical (·O2−), singlet oxygen (1O2), and hydroxyl radical (·OH).

Binding mode of proflavine to DNA probed by polarized light spectroscopy

It is well known that proflavine binds to DNA. Here we investigate the binding mode of proflavine to native DNA using absorption, circular dichroism (CD), and linear dichroism (LD) spectroscopy as well as by fluorescence techniques. The observed changes of proflavine upon complexation with DNA can be summarized as a red shift and hypochromism in the absorption spectrum. The negative LDr in the proflavine absorption region has a magnitude comparable to or larger than that of the DNA absorption region, confirming the intercalative binding mode of proflavine to DNA. Saturation of the LD spectrum in the proflavine absorption region at R = 0.25 and a decrease in the fluorescence intensity provide further evidence of intercalation. Furthermore, the coupling of electric transition of intercalated proflavine is observed. Although proflavine has been reported to position itself along the DNA stem at high [proflavine]/[DNA base] ratios, the spectral characteristics, which include a clear isosbestic point in the absorption spectrum and proportionality in the LD magnitude in the proflavine absorption region, do not show any possibility of exterior binding.

Dependence of the base sequence on the [Cu(2,2′-bipyridine)2(NO3)](NO3)-induced oxidative DNA cleavage probed by linear dichroism

The oxidative DNA cleavage induced by the [Cu(2,2′-bipyridine)2(NO3)]NO3 (Cu(bpy)2) complex was examined using the linear dichroism (LD) technique. Using this method, the oxidative DNA cleavage by the Fenton mechanism was reported to occur through two sequential first-order reactions. As the Cu(bpy)2 concentration increased, the rate constant of both first order reactions increased, as expected. The activation energy of the second step was estimated to be 0.190 kJ mol−1. A similar method was applied for various synthetic polynucleotides. Poly[d(G-C)2], poly(dG)·poly(dC), and poly(dA)·poly(dT) produced a time-dependent decrease in LD, which could be elucidated by a single component exponential decay. This observation is in contrast to Fenton-type oxidative DNA cleavage. On the other hand, poly[d(A-T)2] produced a time-dependent decrease in the LD magnitude, which could be elucidated by two sequential first order reactions.

DNA cleavage induced by [Cu(L)x(NO3)2] (L = 2,2′-dipyridylamine, 2,2′-bipyridine, dipicolylamine, x = 1 or 2): Effect of the ligand structure

The efficiency of [Cu(2,2′-bipyridine)2(NO3)]NO3, [Cu(2,2′-dipyridylamine)2(NO3)2], and [Cu(dipicolylamine)2(NO3)2] complexes (complex 1, 2 and 3, respectively) in oxidative DNA cleavage was examined by electrophoresis and linear dichroism (LD). Among the three Cu complexes, complex 1 showed the highest efficiency in super-coiled DNA (scDNA) cleavage in electrophoresis. The presence of tiron, a superoxide radical scavenger, suppressed the reaction almost completely. The LD signal at 260nm decreased gradually as the time passed. The decrease in LD magnitude was explained best by the sum of the two single exponential curves. This suggests that the cleavage reaction involves two first order kinetic processes; an increase in flexibility due to scission of one of the strands and a shortening in the DNA stem due to cut of both strands of double stranded DNA (dsDNA). In agreement with the electrophoresis data, complex 1 exhibited the highest efficiency with the superoxide radical found to be the essential reactive oxygen species. The order of efficiency in both scDNA and dsDNA was as follows: complex 1>complex 2>complex 3. The electrochemical properties alone were insufficient to explain the observed efficiencies, even though reduction of the central Cu ion is essential for the oxidative DNA cleavage. This highlights the importance of an ability to ligate the molecular oxygen (or hydrogen peroxide) to the central Cu ion to produce the superoxide radical, in addition to the reduction of Cu ion, in oxidative DNA cleavage.

Comparison of Binding Stoichiometry of [Ru(1,10-phenanthroline)2dipyrido [3,2-a:2',3'-c]phenazine]2+and its Bis-derivative to DNA

A new bis-Ru(II) complex, in which two [Ru(1,10-phenanthroline)<TEX>$_2$</TEX>dipyrido[3,2-a:2',3'-c]phenazine]<TEX>$^{2+}$</TEX> were tethered by a 1,3-bis(4-pyridyl)propane linker, was synthesized and its binding mode and stoichiometry to DNA was investigated by optical spectroscopy including linear dichroism (LD) and fluorescence intensity measurement. The magnitude of the negatively reduced LD signal of the bis-Ru(II) complex in the dipyrido[3,2-a:2',3'-c]phenazine (DPPZ) ligand absorption region appeared to be similar compared to that in the DNA absorption region, which is considered to be a diagnostic for DPPZ ligand intercalation. The binding stoichiometry measured from its LD magnitude and enhanced fluorescence intensity corresponds to one ligand per three DNA bases, effectively violating the nearest neighbouring site exclusion model for classical DNA intercalation. This observation is in contrast with monomer analogue [Ru(1,10-phenanthroline)<TEX>$_2$</TEX>dipyrido[3,2-a:2',3'-c]phenazine]<TEX>$^{2+}$</TEX>, which is saturated at the DPPZ ligand to DNA base ratio of 0.25, or one DPPZ ligand per four nucleobases.

Comparison of the Binding Modes of [Ru(2,2'-bipyridine)3]2+

The <TEX>$[Ru(tpy)_2]Cl_2$</TEX> (tpy:2,2':6',2"-terpyridine) complex was synthesized and its structure was confirmed by <TEX>$^1H$</TEX>-NMR and elemental analysis. Its binding mode toward DNA was compared with the well-known <TEX>$[Ru(bpy)_3]Cl_2$</TEX> (bpy:2,2-bipyridyl), using isotropic absorption, linear dichroism(LD) spectroscopy, and an energy minimization study. Compared to <TEX>$[Ru(bpy)_3]^{2+}$</TEX>, the <TEX>$[Ru(tpy)_2]^{2+}$</TEX> complex exhibited very little change in its absorption pattern, especially in the MLCT band, upon binding to DNA. Furthermore, upon DNA binding, both Ru(II) complexes induced a decrease in the LD magnitude in the DNA absorption region. The <TEX>$[Ru(tpy)_2]^{2+}$</TEX> complex produced a strong positive LD signal in the ligand absorption region, which is in contrast with the <TEX>$[Ru(bpy)_3]^{2+}$</TEX> complex. Observed spectral properties led to the conclusion that the interaction between the ligands and DNA bases is negligible for the <TEX>$[Ru(tpy)_2]^{2+}$</TEX> complex, although it formed an adduct with DNA. This conclusion implies that both complexes bind to the surface of DNA, most likely to negatively charged phosphate groups via a simple electrostatic interaction, thereby orienting to exhibit the LD signal. The energy minimization calculation also supported this conclusion.

Amine terminated G-6 PAMAM dendrimer and its interaction with DNA probed by Hoechst 33258

Fluorescence characteristics of Hoechst 33258 bound to G-6 dendrimer, to the DNA–G-6 dendrimer complex, and to DNA were compared with that in an aqueous solution. The spectral properties including fluorescence emission spectrum, accessibility of anionic quencher, as well as the fluorescence decay time of the Hoechst 33258 are different for all three conditions, indicating that the environments in these conditions are different. Close analysis of the fluorescence properties led us to suggest that Hoechst 33258 located at or near the contact area of the dendrimer and DNA in the DNA–G-6 complex. In the complex, in the absence of Hoechst 33258, the shape of the circular dichroism in the DNA absorption region remained, indicating that DNA is in B form in the complex. On the other hand, the magnitude of linear dichroism (LD) decreased upon DNA–G-6 dendrimer complex formation. The decrease in LD magnitude reflects the shortening of the DNA contour length, which is expected from the fact that a large part of linear DNA is required to wrap the surface of G-6 dendrimer.