G-rich sequence-specific recognition and scission of human genome by PNA/DNA hybrid G-quadruplex formation

Hole in one: A single peptide nucleic acid (PNA) effectively targets the G-rich region in double-stranded DNA through formation of a PNA/DNA hybrid G-quadruplex. Only one target site in the whole human genome was selectively cleaved by the hybrid G-quadruplex. Such site-selective scission of DNA is central to gene manipulation for molecular biology, biotechnology, and therapy.     

Direct visualization of enzymatic cleavage and oxidative damage by hydroxyl radicals of single-stranded DNA with a cationic polythiophene derivative

A new method has been developed for the label-free, convenient, and real-time monitoring of the cleavage of single-stranded DNA by single-strand-specific S1 nuclease and hydroxyl radical based on cationic water-soluble poly[3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride](PMNT). The PMNT can form an interpolyelectrolyte complex with ssDNA (duplex) through electrostatic interactions, in which PMNT takes a highly conjugated and planar conformation, and thus PMNT exhibits a relatively red-shifted absorption wavelength. When ssDNA is hydrolyzed by S1 nuclease or hydroxyl radical into small fragments, the PMNT/ssDNA duplex cannot form. In this case, the PMNT remains in random-coil conformation and exhibits a relatively short absorption wavelength. The nuclease digestion or oxidative damage by hydroxyl radical of DNA can be monitored by absorption spectra or just visualized by the "naked-eye" in view of the observed PMNT color changes in aqueous solutions. This assay is simple and rapid, and there is no need to label DNA substrates. The most important characteristic of the assay is direct visualization of the DNA cleavage by the "naked-eye", which makes it more convenient than other methods that rely on instrumentation. The assay also provides a promising application in drug screening based on the inhibition of oxidative damage of DNA.     

Modulation of photo-oxidative DNA damage by cationic surfactant complexation

The natural packaging of DNA in the cell by histones provides a particular environment affecting its sensitivity to oxidative damage. In this work, we used the complexation of DNA by cationic surfactants to modulate the conformation, the dynamics, and the environment of the double helix. Photo-oxidative damage initiated by benzophenone as the photosensitizer on a plasmid DNA complexed by dodecyltrimethylammonium chloride (DTAC), tetradecyltrimethylammonium chloride (TTAC), cetyltrimethyammonium chloride (CTAC) and bromide (CTAB) was detected by agarose gel electrophoresis. By fluorescent titration in the presence of ethidium bromide (EB) and agarose gel electrophoresis, we experimentally confirmed the complexation diagrams with a critical aggregation concentration on DNA matrix (CAC DNA) delimiting two regions of complexation, according to the DNA-phosphate concentration. The study of the photo-oxidative damage shows, for the first time, a direct correlation between the DNA complexation by these surfactants and the efficiency of DNA cleavage, with a maximum corresponding to the CAC DNA for DTAC and CTAC, and to DNA neutralization for CTAC and CTAB. The localization of a photosensitizer having low water solubility, such as benzophenone, inside the hydrophobic domains formed by the surfactant aggregated on DNA, locally increases the photoinduced cleavage by the free radical oxygen species generated. The inefficiency of a water-soluble quencher of hydroxyl radicals, such as mannitol, confirmed this phenomenon. The detection of photo-oxidative damage constitutes a new tool for investigating DNA complexation by cationic surfactants. Moreover, highlighting the drastically increased sensitivity of a complexed DNA to photo-oxidative damage is of crucial importance for the biological use of surfactants as nonviral gene delivery systems.     

Design of a new fluorescent cofactor for DNA methyltransferases and sequence-specific labeling of DNA

Sequence-specific labeling of DNA is of immense interest for analytical and functional studies of DNA. We present a novel approach for sequence-specific labeling of DNA using a newly designed fluorescent cofactor for the DNA methyltransferase from Thermus aquaticus (M.TaqI). Naturally, M.TaqI catalyzes the nucleophilic attack of the exocyclic amino group of adenine within the double-stranded 5'-TCGA-3' DNA sequence onto the methyl group of the cofactor S-adenosyl-L-methionine (AdoMet) leading to methyl group transfer. The design of a new fluorescent cofactor for covalent labeling of DNA was based on three criteria: (1) Replacement of the methionine side chain of the natural cofactor AdoMet by an aziridinyl residue leads to M.TaqI-catalyzed nucleophilic ring opening and coupling of the whole nucleoside to DNA. (2) The adenosyl moiety is the molecular anchor for cofactor binding. (3) Attachment of a fluorophore via a flexible linker to the 8-position of the adenosyl moiety does not block cofactor binding. According to these criteria the new fluorescent cofactor 8-amino[1'-(N'-dansyl)-4'-aminobutyl]-5'-(1-aziridinyl)-5'-deoxyadenosine (3) was synthesized. 3 binds about 4-fold better than the natural cofactor AdoMet to M.TaqI and is coupled with a short duplex oligodeoxynucleotide by M.TaqI. The identity of the expected modified nucleoside was verified by electrospray ionization mass spectrometry after enzymatic fragmentation of the product duplex. In addition, the new cofactor 3 was used to sequence-specifically label plasmid DNA in a M.TaqI-catalyzed reaction.     

Metallodesferals as a new class of DNA cleavers: Specificity, mechanism and targetting of DNA scission reactions

Interaction of metal complexes with nucleic acids is currently attracting wide attention due to their potential utility as drugs, regulators of gene expression and tools for molecular biology. Many metal complexes exhibit nucleolytic activity, the most important examples being Cu(II)-OP, Fe(H)-BLM, Fe(II)-EDTA, metalloporphyrins, Ru and Co complexes of 4,7-diphenyl-l,10-phenanthroline and more recently by Ni(II) complexes. Desferal, a well known siderophore and a highly effective drug in chelation therapy of iron overload diseases, forms a stable octahedral co-ordination Fe(III) complex Eerrioxamine B. We have been interested in the DNA damaging properties of metallodesferals and this paper describes the DNA cleaving ability of metallodesferals, metal-dependent base selectively in DNA scission reactions, mechanistic studies on DNA cleavage by CuDFO and targetting of DNA cutting by covalent MDFO conjugates. This paper reports the synthesis of Cu(II), Co (III) and Ni(II) complexes of a siderophore chelating drug desferal, the studies on cleavage of plasmid DNA, the sequence preference of cleavage reactions, and C1’ as the primary site of hydroxyl radical attack in the reactions. Oligonucleotides covalently linked with this molecular scissor can direct the cleavage of either single or double strand DNA’s, mediated by duplex or triple helix structures respectively. Such targetting of DNA cleavage reactions, mediated by oligonucleotide-Cu(II)/Co(III) desferal conjugates has demonstrated reasonable site specificity and efficiency     

Photosensitization of isolated mitochondria by hematoporphyrin derivative (Photofrin): effects on bioenergetics.

Isolated rat liver mitochondria were incubated in the presence of 6 micrograms/ml of Photofrin and irradiated at the wavelength of 365 nm. After 45 s irradiation (30 W/m2), coupling defined as stimulation of respiration by externally added adenosine 5'-diphosphate (ADP) is totally lost. In contrast, membrane potential created by addition of succinate or adenosine 5'-triphosphate (ATP) is only slightly affected. Similarly, the ADP/O ratio is not modified after 20 s irradiation. These data suggest that modification of the mitochondrial membrane potential is not a primary event after irradiation.     

DNA alkylation by pyrrole-imidazole seco-CBI conjugates with an indole linker: sequence-specific DNA alkylation with 10-base-pair recognition through heterodimer formation

The sequence-specific DNA alkylation by conjugates 4 and 5, which consist of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides and 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) linked with an indole linker, was investigated in the absence or presence of partner Py-Im polyamide 6. High-resolution denaturing polyacrylamide gel electrophoresis revealed that conjugate 4 alkylates DNA at the sequences 5'-(A/T)GCCTA-3' through hairpin formation, and alkylates 5'-GGAAAGAAAA-3' through an extended binding mode. However, in the presence of partner Py-Im polyamide 6, conjugate 4 alkylates DNA at a completely different sequence, 5'-AGGTTGTCCA-3'. Alkylation of 4 in the presence of 6 was effectively inhibited by a competitor 7. Surface plasmon resonance (SPR) results indicated that conjugate 4 does not bind to 5'-AGGTTGTCCA-3', whereas 6 binds tightly to this sequence. The results suggest that alkylation proceeds through heterodimer formation, indicating that this is a general way to expand the recognition sequence for DNA alkylation by Py-Im seco-CBI conjugates.     

Growth and scission energy of wormlike micelles formed by a cationic surfactant with long unsaturated tails

The structural and dynamic properties of micellar solutions of erucyl bis(hydroxyethyl)methylammonium chloride blended with 2-propanol, in the presence of KCl, have been investigated by means of light scattering and rheological experiments. In the dilute regime, the micellar growth is larger than expected from mean-field or scaling models. The results obtained in the vicinity of the overlap concentration suggest the presence of large aggregates, with size >100 nm, possibly micellar rings or microgels. In the semidilute regime, the relationship between the zero shear viscosity and the surfactant concentration is described by a power law with an exponent in agreement with the mean-field model of linear micelles. The methods based on the analysis of the temperature dependence of the complex shear modulus to provide a measure of the scission energy are discussed.     

DNA strand exchange stimulated by spontaneous complex formation with cationic comb-type copolymer

Cationic comb-type copolymers (CCCs) composed of a polycation backbone and water-soluble side chains accelerate by 4-5 orders the DNA strand exchange reaction (SER) between double helical DNA and its homologous single-strand DNA. The accelerating effect is considered due to alleviation of counterion association during transitional intermediate formation in sequential displacement pathway. CCCs stabilize not only matured hybrids but also the nucleation complex to accelerate hybridization.     

DNA-bleomycin interaction: Nucleotide sequence-specific binding and cleavage of DNA by bleomycin

Biosynthetic intermediates and synthetic analogues of bleomycin (BLM) have been investigated for their metal binding, dioxygen activation, and DNA cleavage. Molecular O₂ was activated by the Fe(II) complex of a synthetic model ligand. Nucleotide sequence specificities in DNA cleavage by the BLM-Fe(II) and deglyco-BLM-Fe(II) complexes were almost identical. It has been shown that (1) the β-aminoalanine-pyrimidine-β-hydroxyhistidine portion of BLM is essential for the metal binding and dioxygen activation and (2) the bithiazole moiety contributes to the specific binding to guanine base of DNA.     

Oxidative DNA strand scission induced by a trinuclear copper(II) complex

A novel trinuclear copper(II) complex, Cu3-L (L = N,N,N',N',N' ',N' '-hexakis(2-pyridyl)-1,3,5-tris(aminomethyl)benzene), exhibited efficient oxidative strand scission of plasmid DNA. The solution behavior of the complex has been studied by potentiometric titration, UV spectroscopy, and cyclic voltammetry. The data showed that there are three redox-active copper ions in the complex with three types of bound water. The complex demonstrated a moderate binding ability for DNA. Cu3-L readily cleaves plasmid DNA in the presence of ascorbate to give nicked (form II) and then linear (form III) products, while the cleavage efficiency using H2O2 is less than by ascorbate, suggesting that the cleavage mode of the trinuclear complex is somewhat different from the traditional Fenton-like catalysis. Meanwhile, Cu3-L is far more efficient than its mononuclear analogue Cu-DPA (DPA = 2,2'-dipyridylamine) at the same [Cu2+] concentration, which suggests a possible synergy between the three or at least two Cu(II) centers in Cu3-L that contributes to its relatively high nucleolytic efficiency. Furthermore, the presence of standard radical scavengers does not have clear effect on the cleavage efficiency, suggesting the reactive intermediates leading to DNA cleavage are not freely diffusible radicals.     

Specific formation of beads-on-a-chain structures on giant DNA using a designed polyamine derivative

Fluorescence microscopy was used to study the folding transition of giant DNAs, T4 DNA (ca. 166 kbp), and lambda DNA (ca. 48 kbp), which proceeds through intermediates with intramolecular segregation induced by pteridine-polyamine conjugates, i.e., 2-amino-6,7-dimethyl-4-(4,9,13-triazatridecylamino)pteridine and -4-(3-(aminopropyl)amino)pteridine. According to the results of DNA denaturation, UV and fluorescent spectroscopy, and transmission electron microscopic observations, it became clear that DNA folding induced by the polyamine derivative is not a continuous shrinking process but a combination of discontinuous processes.     

Photosensitization by iodinated DNA minor groove binding ligands: Evaluation of DNA double-strand break induction and repair

Iodinated DNA minor groove binding bibenzimidazoles represent a unique class of UVA photosensitizer and their extreme photopotency has been previously characterized. Earlier studies have included a comparison of three isomers, referred to as ortho-, meta- and para-iodoHoechst, which differ only in the location of the iodine substituent in the phenyl ring of the bibenzimidazole. DNA breakage and clonogenic survival studies in human erythroleukemic K562 cells have highlighted the higher photo-efficiency of the ortho-isomer (subsequently designated UV(A)Sens) compared to the meta- and para-isomers. In this study, the aim was to compare the induction and repair of DNA double-strand breaks induced by the three isomers in K562 cells. Further, we examined the effects of the prototypical broad-spectrum histone deacetylase inhibitor, Trichostatin A, on ortho-iodoHoechst/UVA-induced double-strand breaks in K562 cells. Using γH2AX as a molecular marker of the DNA lesions, our findings indicate a disparity in the induction and particularly, in the repair kinetics of double-strand breaks for the three isomers. The accumulation of γH2AX foci induced by the meta- and para-isomers returned to background levels within 24 and 48 h, respectively; the number of γH2AX foci induced by ortho-iodoHoechst remained elevated even after incubation for 96 h post-irradiation. These findings provide further evidence that the extreme photopotency of ortho-iodoHoechst is due to not only to the high quantum yield of dehalogenation, but also to the severity of the DNA lesions which are not readily repaired. Finally, our findings which indicate that Trichostatin A has a remarkable potentiating effect on ortho-iodoHoechst/UVA-induced DNA lesions are encouraging, particularly in the context of cutaneous T-cell lymphoma, for which a histone deacetylase inhibitor is already approved for therapy. This finding prompts further evaluation of the potential of combination therapies.     

Targeted strand scission of DNA substrates by a tricopper(II) coordination complex

A trinuclear copper complex, [Cu(3)(II)(L)(H(2)O)(3)(NO(3))(2)](NO(3))(4).5H(2)O (1) (L = 2,2',2' '-tris(dipicolylamino)triethylamine), with pyridyl and alkylamine coordination exhibits a remarkable ability to promote specific strand scission at junctions between single- and double-stranded DNA. Strand scission occurs on the 3' overhang at the junction of a hairpin or frayed duplex structure and is not dependent on the identity of the base at which cleavage occurs. Target recognition minimally requires a purine at the first unpaired position and a guanine at the second unpaired position on the 5' strand. Incorporation of the necessary recognition elements into an otherwise unreactive junction resulted in specific strand scission at that new target and helped to confirm the predictive nature of this complex. Selective strand scission requires both a reductant and dioxygen, suggesting activation of O(2) by the reduced form of 1. The reaction utilizing the trinuclear complex does not appear to involve a diffusible radical species as suggested by its high specificity of target oxidation and its lack of sensitivity to radical quenching agents. Comparisons between the trinuclear copper complex, mononuclear analogues of 1, and [Cu(OP)(2)](2+) (OP = 1,10-phenanthroline) indicate that recognition and reactivity described in this report are dependent on the multiple metal ions within the same complex which together support its unique activity.     

Conformational conversion of DNA G-quadruplex induced by a cationic porphyrin

The interactions between cationic meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and the G-quadruplex (G4) of human telomeric single-strand oligonucleotide d(TTAGGG)₂ (S12) have been investigated by means of circular dichroism (CD), UV–visible absorption and fluorescence spectroscopies. It is found that TMPyP4 can preferentially induce the conformational conversion of the G4 structure from the parallel type to the parallel/antiparallel mixture in the presence of K⁺, and that it can directly induce the formation of antiparallel G4 structure from the single-strand oligonucleotide S12 in the absence of K⁺. Furthermore, the comparable experiments of TMPyP4 with two single-strand oligonucleotides S6 d(TTAGGG) and S24 d(TAGGG(TTAGGG)₃T) in the absence of K⁺ show that TMPyP4 can also induce the formation of antiparallel G4 from S24 but not from S6, indicating that the end-loops of the G4 structure are the key factors for the formation of G4 induced by TMPyP4.     

DNA-based biosensor for the detection of strong damage to DNA by the quinazoline derivative as a potential anticancer agent

A disposable electrochemical DNA-based biosensor was developed and applied as a screening device to detect an effect of a synthetically prepared quinazoline derivative on the surface-attached double stranded calf thymus DNA. Screen-printed carbon electrodes without and with multi-walled carbon nanotubes interface served as the signal transducer. The quinazoline interaction with DNA was investigated voltammetrically using DNA-bound electrochemical indicators such as [Co(phen)₃]³⁺, [Ru(bpy)₃]²⁺, methylene blue, the K₃[Fe(CN)₆] complex present in the solution phase as well as by electrochemical impedance spectroscopy. A severe damage to DNA at the incubation of the biosensor in quinazoline solution was found which leads to the loss of DNA from the electrode surface. Agarose gel electrophoresis was used to verify the results.     

Photosensitization of DNA of defined sequence by furochromones, khellin and visnagin

The sequence specificity in the in vitro DNA photobinding of khellin and visnagin, two naturally occurring furochromones proposed for chemotherapy of vitiligo, was investigated by using DNA sequencing methodology. The 3'-5' exonuclease associated with the T4 DNA polymerase served as a tool for determining photoadducts distribution on DNA fragments of the lac I gene of Escherichia coli. The photoadduct distribution of psoralen is also studied for comparison. Upon UVA irradiation, visnagin mainly forms monoadducts with thymine and to a lower extent with cytosine. Alternating (A-T)n sequences are hot spots for visnagin photoaddition. This is a property shared with furocoumarins. TTT sites are also quite reactive to visnagin, as they are to methylated angelicins. In contrast, with psoralen derivatives, there is no preferential photobinding in 5'-TpA sites, and 5'-ApT sites react as well. Furthermore, many sites such as T in the GC context, and C in any context, react, although weakly. The visnagin photoadduct distribution resembles very much the photoadduct distribution of methylated angelicins as described by Miolo et al. The photoreaction of these two series of compounds is less sequence dependent than the photobinding of psoralen derivatives as described by Sage and Moustacchi and by Boyer et al. The sequence specificity in khellin-DNA photobinding is the same as for visnagin, even though it forms much fewer photoadducts. The absence of photo-oxidation of DNA after treatment with visnagin or khellin plus UVA suggests that furochromones do not present any photodynamic effect on DNA.     

Photosensitization by Norfloxacin is a Function of pH

Abstract— Norfloxacin is a fluoroquinolone (FQ) antibiotic that has been reported to cause cutaneous photosensitivity in animals and occasionally in humans. We have studied the fluorescence and singlet oxygen (¹O₂)-generating properties of norfloxacin. Upon UV excitation the drug fluoresces in water, and the relative intensities of two major fluorescence bands at ca 420 and 450 nm are affected by pH. The overall quantum yield of fluorescence (φ_F) is also strongly pH dependent: φ_F is low in 0.2 N HC1 solution (0.2), increasing steeply to 0.12 at pH 4, then gradually decreasing to 0.01 at pH 10. The changes in φ_F are accompanied by changes in fluorescence lifetime from 0.6 ns at pH 1 to 1.8 ns at pH 4. Norfloxacin exhibits phosphorescence in low temperature glasses. The formation of a triplet state at room temperature is also suggested by ¹O₂ phosphorescence in aerobic D₂O. This phosphorescence is “self-quenched” by norfloxacin itself with an efficiency that is pH dependent: k_q is 7.9 ×10⁶M^?1s^?1 at pD 4, decreases to 1.9 × 10⁶M^?1 s^?1 at pD 7.5 but then increases about 20-fold in alkaline D₂O solutions. This quenching causes the observed ¹O₂ production by norfloxacin (0.1 mM) to show a maximum at around pH 8–9. However, after correction for self-quenching, the quantum yield of ¹O₂ production (φ_so), measured by using perinaphthenone as a standard, yielded the following values: φ_so is about 0.07 in 0.2 N DCl solution, 0.08 at pH 7.5 and then increases smoothly to ～ 0.2 in 0.1 M NaOD solution. The relatively high, unquenched ¹O₂ production at physiological pH 7.4 (φ_so～ 0.08) suggests that ¹O₂ reactions may play an important role in the cutaneous phototoxicity of norfloxacin and other FQ antibiotics.