Geneviève Pratviel

DNA And RNA Cleavage by Metal Complexes

Publisher Summary This chapter discusses DNA and RNA cleavage by metal complexes. DNA and RNA cleavage, a very active field of research, has been developed in two main and complementary directions within the past decade: oxidative cleavage and hydrolysis. In general, the difference between the two different approaches are (1) the preparation of new chemical tools to study genomic DNA. The recognition sites of most of the restriction enzymes are often limited to palindromic sequences, and it is useful to have artificial nucleases able to cleave DNA at any desired sequence, and (2), the synthesis of compounds that cleave nucleic acids should help in the design of potential therapeutic agents for the treatment of cancer and viral diseases. The challenging development of new, efficient DNA and RNA cleavage agents can require a strong cooperation between chemists, biochemists, and molecular biologists.

Porphyrin Derivatives for Telomere Binding and Telomerase Inhibition

The capacity of G‐quadruplex ligands to stabilize four‐stranded DNA makes them able to inhibit telomerase, which is involved in tumour cell proliferation. A series of cationic metalloporphyrin derivatives was prepared by making variations on a meso‐tetrakis(4‐N‐methyl‐pyridiniumyl)porphyrin skeleton (TMPyP). The DNA binding properties of nickel(II) and manganese(III) porphyrins were studied by surface plasmon resonance, and the capacity of the nickel porphyrins to inhibit telomerase was tested in a TRAP assay. The nature of the metal influences the kinetics (the process is faster for Ni than for Mn) and the mode of interaction (stacking or external binding). The chemical alterations did not lead to increased telomerase inhibition. The best selectivity for G‐quadruplex DNA was observed for Mn‐TMPyP, which has a tenfold preference for quadruplex over duplex.

Dendrislides, dendrichips: a simple chemical functionalization of glass slides with phosphorus dendrimers as an effective means for the preparation of biochips

Materials with tailored surface properties are useful for diverse applications, in particular for adsorption and/or covalent immobilization of various biomolecules such as nucleic acids and proteins. One of the main challenges is to design a reactive surface chemistry for stable binding of biomolecules on the support, which in addition keeps away the biomolecules from the support to reduce interference. We report here a novel support functionalization that is based on the formation of a reactive layer, covalently grafted on glass slides onto which amino-modified DNA probes were covalently fixed. The layer is composed of spherical home-made neutral phosphorus dendrimers containing a large number of aldehyde functions at their periphery. The method of preparation of these dendrimer-activated glass slides (so-called dendrislides) is performed in a few steps and results in stable activated supports, since aldehyde dendrimers are stable compounds. After immobilization of nucleic acids the so-called dendrichip products were investigated by means of hybridization experiments using complementary fluorescent labelled-oligonucleotide targets. Our results indicate that this novel grafting technology leads to surfaces with a high binding capacity for amino-modified oligonucleotides compared to commercially available aldehyde slides and with a detection limit of 1 pM labelled targets. Since links between the surface, the dendrimers and the nucleic acids are covalent, the dendrichips could be re-used up to 10 times without significant change in the fluorescence signal intensity. Finally, the performance of dendrichips in detection of a single base mutation was demonstrated.

DNA Oxidation by Copper and Manganese Complexes

Publisher Summary This chapter discusses DNA oxidation by copper and manganese complexes. Although, DNA proves to be highly resistant to hydrolysis, its sensitivity toward oxidation is now well documented. DNA appears to be the critical target for many oxidation processes because of the reactive oxygen species, ionizing radiations, ultraviolet (UV) light, photosensitizers, or dyes. Redox-active metal complexes have been known for decades to be able to mediate oxidative degradation of DNA. Because of the large literature on iron complexes, the most famous representatives of which are Fe-bleomycin and Fe-edta, this chapter is restricted on copper and manganese complexes. The mechanisms of oxidative DNA damage reported in vitro with these two chemical nucleases illustrate the diversity of the processes that one may consider for the study of in vivo DNA damage. Oxygen rebound, reaction of hydroxyl radicals, or the attack of a molecule of water at a carbon cation, give rise to an alcohol at the oxidized carbon of the deoxyribose.

Magnesium monoperoxophtalate: an efficient single oxygen atom donor in DNA cleavage catalyzed by metalloporphyrin.

The peracid compound magnesium monoperoxophtalate (MMPP), water-soluble at physiological pH, behaves as a promising oxygen donor in the oxidative cleavage of DNA catalyzed by meso-tetrakis-(4-N-methylpyridyl)porphyrinatomanganese(III) pentaacetate. Shown on cleavage of supercoiled phi X174 DNA, MMPP is significantly more efficient than KHSO5, another recently developed single oxygen atom donor (Fouquet et al., J. Chem. Soc., Chem. Commun., 1987, 1169).

Interaction of Cationic Manganese Porphyrin with G‐Quadruplex Nucleic Acids Probed by Differential Labeling of the Two Faces of the Porphyrin

G-quadruplex nucleic acids have attracted much interest in recent years because they appear to be involved in the regulation of fundamental processes of life, in particular, those connected to the development of cancer. Several in vitro structures are now available. G-quadruplex nucleic acids consist of four strands of nucleic acids that form in guanine-rich DNA/RNA sequences with stretches of tandem guanine residues. The four strands are held together by a hydrogen-bonded arrangement of four guanines (the Gquartet unit). Stacking of planar consecutive G-quartets further stabilizes the G-quadruplex structures. G-quadruplex nucleic acids have four external grooves, and for those formed by intermolecular folding, they have also single-stranded loops connecting the guanine tracts. A central ionic channel with K (and/or Na) ions exists in the core of the superimposed G-quartets. Their presence in human cells has recently been shown. With the aim of interfering with their biological functions, a number of small ligands targeting G-quadruplex nucleic acids have emerged. meso-Tetrakis(4-N-methylpyridiniumyl)porphyrin (H2-TMPyP4) is a long known G-quadruplex ligand and is often used as a reference compound in the field. H2-TMPyP4, like other large aromatic ligands, should exhibit p–p stacking interactions with an external guanine quartet of the G-quadruplex structure. However, this type of binding is still under debate. Two structures of the complex between H2-TMPyP4 and G-quadruplex DNA were reported. The first structure (c-myc oncogene promoter sequence) shows the porphyrin interacting with the top Gquartet, although at a relatively long distance, not in perfect accordance with known p–p stacking interactions. The second structure (telomeric sequence) showed a porphyrin involved in a stacking interaction with a base pair in the loop at the top of the G-quardruplex but not with the uppermost G-quartet itself. Porphyrin H2-TMPyP4 interacts with telomeric Gquadruplex models with a relatively high binding constant, on the order of Ka 10–10m . The binding of H2-TMPyP4 to the c-myc promoter parallel quadruplex is tighter with a binding constant of Ka 10–10m . The variations in the binding constant arise from different ionic strengths and experimental conditions. The introduction of a metal ion such as Mn or Co into the center of the porphyrin macrocycle gives two molecules of water as axial ligands. This does not preclude the interaction of the porphyrin with the G-quadruplex but does lower the affinity compared to the non-metalated porphyrin. These metalated porphyrins could in principle interact with the Gquadruplex by external p–p stacking with the top G-quartet, provided the axial water ligand fits within the central ionic channel of the quadruplex, or alternatively, interact with the grooves (or loops) of the quadruplex. These two binding modes should differ by the accessibility of the two faces of the porphyrin macrocycle. External p–p stacking would have only one accessible face from the bulk solvent, while binding to the quadruplex grooves would leave two faces of the porphyrin accessible. In the absence of any structural data on the interaction of the metalated porphyrins containing axial water ligands with G-quadruplexes, and considering that the mode of interaction of H2-TMPyP4 with the top G-quartet of these structures is unknown, we determined the accessibility of the two faces of the manganese derivative of H2-TMPyP4, MnTMPyP4, while bound to a G-quadruplex using labeling experiments. The water-soluble and dissymmetric peroxide KHSO5 can coordinate with the manganese ion of the porphyrin by displacing the water molecule from one face of the porphyrin (Scheme 1). Heterolytic cleavage of the O O bond leads to the formation of a high-valent metal-oxo species. It is an oxomanganese(IV) porphyrin radical cation, (P)Mn=O, formally referred to as Mn=O, which is able to transfer its “oxo” oxygen atom onto a substrate by a mechanism similar to the cytochrome P450 enzymes. The activation of Mn-

Oxidative DNA Damage Mediated by Transition Metal Ions and Their Complexes

DNA damage by redox-active metal complexes depends on the interaction of the metal complex with DNA together with the mechanism of oxygen activation. Weak interaction, tight binding, and direct involvement of DNA in the coordination sphere of the metal are described. Metal complexes acting through the production of diffusing radicals and metal complexes oxidizing DNA by metal-centered active species are compared. Metal complexes able to form high-valent metal-oxo species in close contact with DNA and perform DNA oxidation in a way reminiscent of enzymatic chemistry are the most elegant systems.

Guanine Oxidation by Electron Transfer: One- versus Two-Electron Oxidation Mechanism

The degeneracy of the guanine radical cation, which is formed in DNA by oxidation of guanine by electron transfer, was studied by a detailed analysis of the oxidation products of guanine on oligonucleotide duplexes and by labeling experiments. It was shown that imidazolone, the major product of guanine oxidation, is formed through a one‐electron oxidation process and incorporates one oxygen atom from O2. The formation of 8‐oxo‐7,8‐dihydroguanine by a two‐electron oxidation process was a minor pathway. The two‐electron oxidation mechanism was also evidenced by the formation of a tris(hydroxymethyl)aminomethane adduct.

Concept for simultaneous and specific in situ monitoring of amyloid oligomers and fibrils via Förster resonance energy transfer.

Oligomeric species of amyloidogenic peptides or proteins are often considered as the most toxic species in several amyloid disorders, like Alzheimer or Parkinsons diseases, and hence came into the focus of research interest and as a therapeutic target. An easy and specific monitoring of oligomeric species would be of high utility in the field, as it is the case for thioflavin T fluorescence for the fibrillar aggregates. Here, we show proof of concept for a new sensitive method to increase specific detection of oligomers by two extrinsic fluorophores. This is achieved by exploiting a Förster resonance energy transfer (FRET) between the two fluorophores. Thus, a mixture of two extrinsic fluorophores, bis-ANS and a styrylquinoxalin derivative, enabled one to monitor simultaneously and in situ the presence of oligomers and fibrils of amyloidogenic peptides. Thereby, the formation of oligomers and their transformation into fibrils can be followed.

DNA CLEAVAGE BY A 'METALLOPORPHYRIN-SPERMINE-OLIGONUCLEOTIDE' MOLECULE

A ‘manganese porphyrin-spermine-oligonucleotide’ molecule, forms a stable triple helix structure (Tm= 42 °C), and cleaves a double-stranded DNA sequence of HIV-1.