Real-time detection of Fe.EDTA/H2O2-induced DNA cleavage by linear dichroism.

The conditions for the measurement of linear dichroism (LD) can be adjusted so as to solely reflect the length and the flexibility of DNA. The real-time detection of the EDTA.Fe(2+)-induced oxidative cleavage of double-stranded native and synthetic DNAs was performed using LD. The decrease in the magnitude of the LD at 260 nm, which reflects an increase in the flexibility and a decrease in the length of the DNA, can be described by the sum of two or three exponential curves in relation to the EDTA.Fe(2+) concentration. The fast component was assigned to the cleavage of one of the double strands, inducing an increase in the flexibility, while the other slower component was assigned to the cleavage of the double strand, resulting in the shortening of DNA. The decrease in the magnitude of the LD of poly[d(A-T)(2)] was similar to that of poly[d(I-C)(2)], while that of poly[d(G-C)(2)] was found to be the slowest, indicating that the resistance of poly[d(G-C)(2)] against the Fenton-type reagent was the strongest. This observation suggests that the amine group in the minor groove of the double helix may play an important role in slowing the EDTA.Fe(2+)-induced oxidative cleavage.

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.

Formation of Cu(II)-brazilin complex in the presence of DNA and its activities as chemical nuclease.

Brazilin, a traditional medicine for the treatment of pain and inflammation, forms a complex with Cu(II) in the presence as well as the absence of DNA. The Cu(II)-brazilin complex exhibited the strand cleavage activity for the pBR322 supercoiled DNA, converting supercoiled form to nicked form. The presence of various scavengers for the oxygen species suppresses or reduces the cleavage activity of the complex, indicating that the DNA cleavage is oxidative. The binding mode of the Cu(II)-brazilin complex was studied by absorption and CD spectroscopy. While a large metal-to-ligand charge transfer (MLCT) band was apparent when Cu(II) and brazilin was mixed in the presence and absence of DNA, the CD did not show any signal in the same region in the presence of DNA, suggesting a weak interaction between the Cu(II)-brazilin complex and DNA bases.

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 260 nm 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.

Real-time detection of DNA cleavage induced by [M(2,2'-bipyridine)2(NO3)](NO3) (M=Cu(II), Zn(II) and Cd(II)) complexes using linear dichroism technique.

The catalytic effect of [M(2,2'-bipyridine)2(NO3)](NO3) (M(bpy)2, M=Cu(II), Zn(II) and Cd(II)) on the super-coiled and double stranded DNA (scDNA and dsDNA) was examined by electrophoresis and a real-time detection linear dichroism (LD) technique. Although the Cu(bpy)2 complex effectively cleaved both types of DNA, the other two complexes were inactive. This was explained by the electrochemical properties of the metal complexes. The Cu(bpy)2 complex exhibited a redox potential at -0.222V with a peak to peak separation of 0.201V, whereas the other two metal complexes did not undergo any redox reaction. Both electrophoresis and LD measurements revealed the superoxide radical, ·O2(-), to be responsible for DNA cleavage. A kinetic study using the LD technique showed that the cleavage of dsDNA consisted of two first order reactions. The fast reaction is believed to reflect the cleavage of one strand, whereas the slow reaction involves the cleavage of the complementary strand at or near the first cleaved site.

Real-time detection of DNA cleavage induced by [M(2,2'-dipyridylamine)(2)(NO(3))(n)](x+) (M=Cd, Cu, Ni, Zn, n=1,2, x=0,1): effect of central metal ions.

[M(Hdpa)(2)(NO(3))(n)](x+) (M=Zn(II), Cd(II), Cu(II) and Ni(II), n=1,2, and x=0, 1) complexes were synthesized, and their activity as catalysts for DNA cleavage reactions were investigated using electrophoresis and linear dichroism technique (LD). All four metal complexes effectively cleaved pBR322 super-coiled DNA. The electrophoresis analysis showed that the [Zn(Hdpa)(2)(NO(3))](+) and [Cd(Hdpa)(2)(NO(3)) (2)] complexes most effectively cleaved the super-coiled DNA, whereas the [Ni(Hdpa)(2)(NO(3))](+) complex was least effective. The magnitude of LD in the DNA absorption region reflects the flexibility and length of DNA when the conditions for measurement are properly adjusted. The double stranded DNA cleavage increases the flexibility of DNA and reduces the length, reducing the magnitude of LD in DNA absorption region. Utilizing this LD property, the cleavage was detected in real-time by measuring the LD magnitude with respect to time. The decrease in the LD magnitude was described as the sum of two exponentials. The fast component was tentatively assigned to the cleavage of the single strand, reflecting the increase in the flexibility of DNA, and the slow component was assigned to the cut of the double strand which reduced the length of DNA. The average reaction time was the fastest for the Zn(II) complex and the slowest for the Ni(II) complex. The reaction time of the Cd(II) complex was as fast as that of the Zn(II) complex. Both the Zn(II) and Cd(II) belong to group 12, suggesting that [M(Hdpa)(2)(NO(3))(n)](x+) with central metal ions from group 12 most efficiently cleaved double stranded DNA.

Binding of meso-tetrakis(N-methylpyridium-4-yl)porphyrin to triplex oligonucleotides: evidence for the porphyrin stacking in the major groove.

Induced CD spectra of meso-tetrakis(N-methylpyridium-4-yl)porphyrin (TMPyP) complexed with d(A)12.d(T)12, d(G)12.d(C)12 duplex and d(A)12.[d(T)12]2, d(G)12.d(C)12.d(C)12+ triplex in the Soret band were compared in this study. When TMPyP is complexed with the duplex, a monomeric CD spectrum at a low [TMPyP]/[oligomer] ratio was apparent, while at a high mixing ratio, the excitonic CD was dominant. In contrast, when TMPyP was complexed with the triplex, the excitonic CD disappears at a relatively high mixing ratio, indicating the TMPyP exciton formation is inhibited by the third strand, which is located in the major groove. This observation indicates that the exciton is formed at the major groove of both AT- and GC-rich DNA, while the monomeric TMPyP binds at (or near) the minor groove of the AT site.