ABSTRACT
The binding of bis-MGB to target DNA was studied by DNase footprint, native gel shift, circular dichroism, thermal dissociation, electrospray mass-spectrometry, and molecular modelling methods. A new method for the determination of the relative affinity of ligands against various dsDNA sequences was elaborated by using ESI-QTOF mass spectrometry. Information about affinity, sequence preferences, conformation and mode of interaction between bis-MGB and target DNA was obtained. Our experiments demonstrated that MGB have different affinity for similar cognate target sequences depending on the sequence context of the target region and other structural factors.
Subject(s)
DNA/chemistry , Nylons/chemistry , Base Sequence , Binding Sites , Ligands , Nucleic Acid Conformation , Spectrometry, Mass, Electrospray Ionization/methodsABSTRACT
Design, synthesis, physico-chemical and in vitro biological studies of new pyrimidine oligo(2'-O-methylribonucleotide) conjugates with oligocarboxamide minor groove binders (MGB) and benzoindoloquinoline intercalator (BIQ) are described. These conjugates formed stable triple helices with the target double-stranded DNA and inhibited its in vitro transcription upon binding.
Subject(s)
DNA/metabolism , Ribonucleotides/metabolism , Base Sequence , DNA/chemistry , Spectrophotometry, UltravioletABSTRACT
This report examines the organization properties of new fluorescent DNA-lipids, either alone in water or in interaction with 1-octyl-beta-d-glucopyranoside micelles or egg phosphatidylcholine vesicles. We first describe the design and the syntheses of the conjugates. Then, we use UV-Vis absorption, steady-state fluorescence emission, electron microscopy, and fluorescence correlation spectroscopy after two-photon excitation to show that these DNA-lipids form spherical micelles in aqueous solution and incorporate much better in micelles than in vesicles. We also investigate the significance of the lipophilic chains of these DNA-lipids on the melting behavior of the double-stranded hybrids: in water melting curves are broadened whereas in amphiphilic assemblies duplexes melt as the unconjugated controls. This work is expected to be useful for improving the rational design of antisense medicines.
ABSTRACT
Design, synthesis and physico-chemical studies of new pyrimidine oligo(2'-O-methylribonucleotide) conjugates with one or two oligo(pyrrolecarboxamide) minor groove binders (MGB) are described.
Subject(s)
DNA/chemistry , Oligoribonucleotides/chemical synthesis , Pyrimidines , Base Sequence , Binding Sites , Indicators and Reagents , Nucleic Acid Conformation , Nucleic Acid Hybridization , Oligoribonucleotides/chemistryABSTRACT
New conjugates containing two parallel or antiparallel carboxamide minor groove binders (MGB) attached to the same terminal phosphate of one oligonucleotide strand were synthesized. The conjugates interact with their target DNA stronger than the individual components. Effect of conjugated MGB on DNA duplex and triplex stability and their sequence specificity was demonstrated on the short oligonucleotide duplexes and on the triplex formed by model 16-mer oligonucleotide with HIV polypurine tract.
Subject(s)
DNA/chemistry , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Base Pairing , Binding Sites , Cytosine , DNA, Viral/chemistry , Guanine , HIV/genetics , Indicators and ReagentsABSTRACT
Amino-p-quinacridine compounds (PQs) have been shown to stabilize strongly and specifically triple-helical DNA. Moreover, these derivatives display photoactive properties that make them efficient DNA cleavage agents. We exploited these two properties (triplex-specific binding and photoactivity) to selectively cleave a double-stranded (ds)DNA sequence present in the HIV-1 genome. Cleavage was first carried out on a linearized plasmid (3300 bp) containing the HIV polypurine tract (PPT) that allowed targeting by a triplex-forming oligonucleotide (TFO). PQ(3)(), the most active compound of the series, efficiently cleaved double-stranded DNA in the vicinity of the PPT when this sequence had formed a triplex with a 16-mer TFO. Investigation of the cleavage at the molecular level was addressed on a short DNA fragment (56 bp); the photoinduced cleavage by PQ(3)() occurred only in the presence of the triple helix. Nevertheless, unusual cleavage patterns were observed: damage was observed at guanines located 6-9 bp away from the end of the triple helical site. This cleavage is very efficient (up to 60%), does not require alkaline treatment, and is observed on both strands. A quinacridine-TFO conjugate produced the same cleavage pattern. This observation, along with others, excludes the hypothesis of a triplex-induced allosteric binding site of PQ(3 )()adjacent to the damaged sequence and indicates that PQ(3 )()preferentially binds in the vicinity of the 5'-triplex junction. Irradiation in the presence of TFO-conjugates with acridine (an intercalative agent) and with the tripeptide lys-tryp-lys led to a complete inhibition of the photocleavage reaction. These results are interpreted in terms of competitive binding and of electron-transfer quenching. Together with the findings of simple mechanistic investigations, they led to the conclusion that the photoinduced damage proceeds through a direct electron transfer between the quinacridine and the guanines. This study addresses the chemical mechanism leading to strand breakage and characterizes the particular photosensitivity of the HIV-DNA target sequence which could be an oxidative hot spot for addressed photoinduced strand scission by photosensitizers.
Subject(s)
Aminoacridines/chemistry , DNA Damage , DNA, Viral/chemistry , Guanine/analogs & derivatives , HIV-1/genetics , Aminoacridines/metabolism , Base Sequence , Binding Sites , Binding, Competitive , DNA, Viral/genetics , DNA, Viral/metabolism , Electrophoresis, Polyacrylamide Gel , Guanine/chemistry , Molecular Sequence Data , Nucleic Acid Conformation , Photochemistry , Photosensitizing Agents/chemistry , Photosensitizing Agents/metabolism , Plasmids/chemistry , Plasmids/metabolismABSTRACT
This review describes recent advances in the development of DNA-photocleavage agents. Major mechanisms of photosensitized DNA photocleavage are presented and the most popular categories of compounds are considered, which include metal complexes and many organic functional derivatives. DNA-targeted conjugates of photosensitizers are also described and discussed.
Subject(s)
DNA Damage , DNA/metabolism , Photosensitizing Agents/metabolism , Photosensitizing Agents/pharmacology , Base Sequence/drug effects , Binding Sites/drug effects , DNA Fragmentation/drug effects , PhotochemistryABSTRACT
A series of oligonucleotides conjugated to intercalators, as well as fluorescent and lipophilic substances, minor groove binders and photoactive molecules were synthesized for studies of their ability to form a stable triple helix. Purine-rich short double stranded DNA fragments from HIV-1 genome and pyrimidine 16-mer oligodeoxyribonucleotide were used as models. A conjugate of a dipyrido[3,2-a:2',3'-c]phenazine-ruthenium (II) complex and a triple helix-forming oligonucleotide was constructed. Upon sequence-specific duplex and triplex formation of the conjugate, the ruthenium complex becomes highly fluorescent. The attached ruthenium complex induces a stabilization of the DNA triple helix and a significant increase of the time of residence of the third strand on the duplex.
Subject(s)
DNA, Viral/chemistry , DNA/chemistry , Intercalating Agents/chemical synthesis , Oligonucleotides/chemical synthesis , HIV-1/genetics , Nucleic Acid ConformationABSTRACT
Topoisomerase I is an ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins as well as for indolocarbazole antibiotics such as rebeccamycin. To achieve a sequence-specific cleavage of DNA by topoisomerase I, a triple helix-forming oligonucleotide was covalently linked to indolocarbazole-type topoisomerase I poisons. The three indolocarbazole-oligonucleotide conjugates investigated were able to direct topoisomerase I cleavage at a specific site based upon sequence recognition by triplex formation. The efficacy of topoisomerase I-mediated DNA cleavage depends markedly on the intrinsic potency of the drug. We show that DNA cleavage depends also upon the length of the linker arm between the triplex-forming oligonucleotide and the drug. Based on a known structure of the DNA-topoisomerase I complex, a molecular model of the oligonucleotide conjugates bound to the DNA-topoisomerase I complex was elaborated to facilitate the design of a potent topoisomerase I inhibitor-oligonucleotide conjugate with an optimized linker between the two moieties. The resulting oligonucleotide-indolocarbazole conjugate at 10 nM induced cleavage at the triple helix site 2-fold more efficiently than 5 microM of free indolocarbazole, while the other drug-sensitive sites were not cleaved. The rational design of drug-oligonucleotide conjugates carrying a DNA topoisomerase poison may be exploited to improve the efficacy and selectivity of chemotherapeutic cancer treatments by targeting specific genes and reducing drug toxicity.
Subject(s)
Aminoglycosides , Anti-Bacterial Agents/chemistry , Carbazoles , DNA Topoisomerases, Type I/metabolism , DNA/metabolism , Indoles , Oligonucleotides/metabolism , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Base Sequence , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Models, Molecular , Oligonucleotides/chemical synthesis , Substrate Specificity , Topoisomerase I InhibitorsABSTRACT
The covalent linkage of a hairpin polyamide, which binds in the minor groove, to camptothecin provides an efficient system to direct topoisomerase I mediated DNA cleavage to specific sites. These conjugates are equally as potent at targeting the enzyme to a single site in a DNA fragment as camptothecin conjugates of ligands that bind in the major groove (triplex-forming oligonucleotides).
ABSTRACT
Amsacrine-4-carboxamide-oligonucleotide conjugates were synthesized and studied for their capacity to form DNA triple helices and to alter human topoisomerase II binding and cleavage properties. The intercalating agent was attached to the 3'- or the 5'-end of a 24 nt triple helix-forming oligonucleotide via linkers of different lengths. The stability of these DNA triple helices was investigated by gel retardation and melting temperature studies using a synthetic 70 bp DNA duplex target. The effect of the conjugates on DNA cleavage by topoisomerase II was evaluated using the 70 bp duplex and a 311 bp restriction fragment containing the same triple helix site. The conjugate with the amsacrine derivative linked to the 3' end of the TFO via a hexaethylene glycol linker modulates the extent of DNA cleavage by topoisomerase II at specific sites.
Subject(s)
Amsacrine/analogs & derivatives , Enzyme Inhibitors/chemistry , Intercalating Agents/chemistry , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Topoisomerase II Inhibitors , Amsacrine/chemistry , Base Sequence , Binding Sites , Chromatography, Gel , DNA/metabolism , DNA Footprinting , DNA Topoisomerases, Type II/metabolism , Etoposide/chemistry , Molecular Sequence Data , Molecular Structure , Nucleic Acid Denaturation , Substrate SpecificityABSTRACT
Indolocarbazole and benzopyridoquinoxaline derivatives have been shown to have anti-tumor activity and to stimulate DNA topoisomerase I-mediated cleavage. Two indolocarbazole compounds (R-6 and R-95) and one benzopyridoquinoxaline derivative (BPQ(1256)) were covalently attached to the 3'-end of a 16mer triple helix-forming oligonucleotide (TFO). These conjugates bind to DNA with a higher affinity than the unsubstituted oligonucleotides. Furthermore, they induce topoisomerase I-mediated and triplex-directed DNA cleavage in a sequence-specific manner.
Subject(s)
Aminoglycosides , Anti-Bacterial Agents/chemistry , Carbazoles , DNA/drug effects , Indoles , Nucleic Acid Conformation , Oligonucleotides/pharmacology , Pyridines/chemistry , Quinoxalines/chemistry , Base Sequence , DNA/chemistry , DNA/metabolism , DNA Footprinting , DNA Topoisomerases, Type I/metabolism , Hydrolysis , Oligonucleotides/chemistryABSTRACT
Optimized methods are described for post-synthetic conjugation of non-protected oligodeoxyribonucleotides to different ligands. Methods for the terminal functionalization of oligonucleotides by amino, sulfhydryl, thiophosphate or carboxyl groups using different chemical reactions and linkers in both organic and aqueous media are described and compared. Experimental conditions for subsequent coupling of ligands containing aliphatic and aromatic amines, aromatic alcohols, carboxylic, sulfhydryl, alkylating, aldehydic and other reactive nucleophilic and electrophilic groups to oligonucleotides were established, including covalent linkage to other oligonucleotides.
Subject(s)
Oligonucleotides/chemical synthesis , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Ligands , Oligonucleotides/chemistry , PhosphorylationABSTRACT
Topoisomerase I is an ubiquitous DNA cleaving enzyme and an important therapeutic target in cancer chemotherapy for the camptothecins as well as for indolocarbazole antibiotics such as rebeccamycin and its synthetic derivatives, which stabilize the cleaved DNA-topoisomerase I complex. The covalent linkage of a triple helixforming oligonucleotide to camptothecin or to the indolocarbazole derivative R-6 directs DNA cleavage by topoisomerase I to specific sequences. Sequence-specific recognition of DNA is achieved by the triple helix-forming oligonucleotide, which binds to the major groove of double-helical DNA and positions the drug at a specific site. The efficacy of topoisomerase I-induced DNA cleavage mediated by the rebeccamycin-conjugate and the camptothecin-conjugate was compared and related to the intrinsic potency of the isolated drugs.
Subject(s)
Aminoglycosides , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/pharmacology , Camptothecin/pharmacology , DNA Topoisomerases, Type I/metabolism , Enzyme Inhibitors/pharmacology , Nucleic Acid Conformation , Oligodeoxyribonucleotides/metabolism , Anti-Bacterial Agents/chemistry , Antineoplastic Agents/chemistry , Base Sequence , Camptothecin/chemistry , Drug Design , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Substrate Specificity , Topoisomerase I InhibitorsABSTRACT
Some guanine-rich oligonucleotides inhibit HIV infectivity through interaction with the gp120 glycoprotein. Besides, photoinactivation of viruses attracts attention for blood decontamination. The feasibility of targeting a red light-absorbing chlorin-type photosensitizer to gp120 through covalent coupling with 8-mer phosphodiester oligodeoxynucleotides is investigated. Some conjugates inhibit binding of antibodies directed to gp120. Inhibition is significantly increased upon red light activation. The activity of the conjugates correlates with their ability to self-associate, a process strongly favored by the propensity of the hydrophobic chlorin moiety to dimerize. Thus, the photosensitizer moiety both promotes structures with a higher affinity for gp120 and, upon light activation, can induce site-directed damages to the protein.
Subject(s)
HIV Envelope Protein gp120/metabolism , Oligonucleotides/pharmacology , Photosensitivity Disorders , Photosensitizing Agents/pharmacology , Porphyrins/pharmacology , Amino Acid Sequence , Dimerization , Dose-Response Relationship, Radiation , Enzyme-Linked Immunosorbent Assay , HIV Envelope Protein gp120/drug effects , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp120/radiation effects , Light , Molecular Sequence Data , Oligonucleotides/metabolism , Porphyrins/physiologyABSTRACT
The stabilization of triple-helical DNA under physiological conditions is an important goal for the control of gene expression using the antigen strategy, an approach whereby an oligonucleotide binds to the major groove of double-helical DNA to fom a triple helix. To this end, triplex-specific intercalators, namely benzopyridoindole (BPI) and benzopyridoquinoxaline (BPQ) derivatives, have been conjugated to the 5' end or to an internucleotide position of a 15-mer oligonucleotide. These conjugates were then tested, using thermal denaturation experiments, for their ability to form and stabilize a triple-helical structure involving a 42-mer duplex target. All of the conjugates were found to do so. The B[h]PQ derivatives stabilized particularly well when attached to the 5' end with a delta Tm of 15 degrees C and -delta delta G degrees 37 of 3.4 kcal mol-1 (pH 6.9, 140 mM KCI, 15 mM sodium cacodylate, 2 mM MgCl2, 0.8 mM spermine). Though most of the derivatives when attached to the internucleotide position were not able to stabilize triple-helical DNA as well as when attached to the 5' end, one B[f]PQ derivative with an internucleotide attachment did so, with a delta Tm of 13 degrees C and -delta delta G degrees 37 of 2.8 kcal mol-1. To a lesser degree, these conjugates were also able to stabilize duplex structures with single-stranded targets. Results were compared to the stabilization obtained with acridine conjugates as well as to a similar study performed with a different sequence.
Subject(s)
DNA/metabolism , Intercalating Agents/metabolism , Interleukin-2/chemistry , Oligonucleotides/metabolism , Promoter Regions, Genetic , Hydrogen-Ion Concentration , Nucleic Acid Conformation , ThermodynamicsABSTRACT
Oligonucleotides covalently attached to a cholesteryl moiety are more stable in biological media and better taken up by eukaryotic cells. However, their anchoring in hydrophobic cellular membranes and endosomes after endocytosis restricts their access to cellular nucleic acids. New methods of cellular delivery and the biological activity of the conjugates were studied. The cholesteryl residue was conjugated via disulfide bond to the 5' or 3' terminal phosphate group of two oligodeoxyribonucleotide dodecamers complementary to the mutated region of Ha-ras oncogene mRNA. The conjugates were able to form complementary duplexes with the mutated 27-b target fragment of mRNA but not with the wild-type sequence. Efficient sequence-specific RNase H cleavage of complementary mRNA was induced with low (< or = 500 nM) concentrations of the conjugates. At higher concentrations, this cleavage was progressively inhibited, probably due to an interaction between RNase H and the cholesterol residue. The hydrophobic conjugates could be adsorbed onto poly(isohexylcyanoacrylate) nanoparticles via their cholesteryl moieties and delivered to eukaryotic cells. Cholesterol-conjugated oligonucleotides were able to sequence-specifically inhibit the proliferation of T24 human bladder carcinoma cells in culture.
Subject(s)
Cholesterol/administration & dosage , Oligonucleotides, Antisense/administration & dosage , Adsorption , Base Sequence , Binding Sites , Cholesterol/metabolism , Cyanoacrylates , Drug Delivery Systems , Genes, ras , Humans , Molecular Sequence Data , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/metabolism , Particle Size , Pharmaceutical Vehicles , Polymers , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribonuclease H , Tumor Cells, CulturedABSTRACT
The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes.
Subject(s)
DNA/chemistry , Energy Transfer , Fluorescent Dyes , Aminoacridines , Base Sequence , Coumarins , Ethidium , Fluorescein , Fluoresceins , Molecular Sequence Data , Nucleic Acid Conformation , Nucleic Acid Hybridization , Oligodeoxyribonucleotides/chemistry , Rhodamines , Spectrometry, Fluorescence , Translocation, GeneticABSTRACT
Various derivatives of oligoribonucleotides were synthesized by the H-phosphonate method. Different modifications of the ribophosphate backbone were designed in order to protect the derivatives against nucleolytic enzymes present in the biological media. These modifications include coupling of fluorescein moiety to 3'-terminal ribose, 2'-O-methylation of ribose, introduction of phosphorothioate internucleotide bonds throughout the molecule, replacement of the two last 3'-terminal phosphodiester bonds by phosphoroamidates and coupling of the last 3'-terminal nucleotide via the 3'-3'-phosphodiester bond. All modifications were tested for their effect on the stability of the derivatives against phosphodiesterase from snake venom and nucleases of the cell culture media. 2'-O-methylated oligoribonucleotides containing either terminal 3'-3'-linkage or two 3'-terminal phosphoroamidate internucleotide bonds appeared to be the most stable under the most severe conditions used. The results demonstrate a possibility to use protected oligoribonucleotide derivatives for experiments in vivo when the use of deoxy-analogues might be ineffective. The uptake of 2'-O-methylated derivatives and their 5'-cholesterol conjugates (coupled via a disulfide bond) by human carcinoma cells did not differ from that of the corresponding oligodeoxyribonucleotides. 85% of the bound derivatives were found in the membrane-cytosolic fraction, while only 15% were found in the nuclear fraction. The oligonucleotide moiety of 2'-O-methyloligoribonucleotide-cholesterol conjugate was not translocated through the cellular membrane. After cleavage of the linkage between cholesterol and oligonucleotide by dithiothreitol the major portion of the oligonucleotide moiety was released into the media. The derivatives, as well as their 5'-cholesterol conjugates, which entered the cells, were stable and protected from action of dithiothreitol dissolved in culture media. These results demonstrate an endocytosis mechanism of penetration as observed in similar experiments using oligodeoxyribonucleotides.
Subject(s)
Neoplasms/metabolism , Oligoribonucleotides/metabolism , Animals , Base Sequence , Cholesterol/metabolism , Chromatography, High Pressure Liquid , Elapid Venoms/enzymology , Electrophoresis, Polyacrylamide Gel , Fluorescein , Fluoresceins , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides/metabolism , Oligoribonucleotides/chemical synthesis , Oligoribonucleotides/chemistry , Phosphoric Diester Hydrolases/metabolism , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
A method in which a cholesterol moiety was covalently attached to oligonucleotides via a disulfide bond has been proposed as a means for studying the penetration of oligonucleotides into living cells. This bond may be cleaved by a mild treatment with thiol-containing reagents during some stages of the uptake process. Attachment of the cholesterol moiety resulted in a 30-50-fold increase in uptake of the oligonucleotide derivative by T24 human carcinoma cells. However, more than 80% of the oligonucleotide derivative remained on the external surface of the cellular membrane. Within the cytoplasm, the oligonucleotide derivatives were found in endosome-like vesicles which were observed during the first 6 h following treatment. Oligonucleotide moieties never cross the membrane, and endocytosis, with or without receptors, is the principal mechanisms for cellular uptake. Only about 15% of the oligonucleotides that penetrated the cells were found in the nuclear fraction. Treatment of the cells with dithiothreitol resulted in a release of most of the cell-associated oligonucleotide derivatives from the external surface of the membrane, but did not change the chemical state or intracellular distribution of the penetrated oligonucleotide derivatives. Mechanisms of the binding of cholesterol-modified oligonucleotides to cellular membranes, non-receptor mediated endocytosis and the role oligonucleotide transportation mechanisms play in determining the fate of penetrated oligonucleotides within the cell are discussed.