Generation of supercoils in nicked and gapped DNA drives DNA unknotting and postreplicative decatenation

Due to the helical structure of DNA the process of DNA replication is topologically complex. Freshly replicated DNA molecules are catenated with each other and are frequently knotted. For proper functioning of DNA it is necessary to remove all of these entanglements. This is done by DNA topoisomerases that pass DNA segments through each other. However, it has been a riddle how DNA topoisomerases select the sites of their action. In highly crowded DNA in living cells random passages between contacting segments would only increase the extent of entanglement. Using molecular dynamics simulations we observed that in actively supercoiled DNA molecules the entanglements resulting from DNA knotting or catenation spontaneously approach sites of nicks and gaps in the DNA. Type I topoisomerases, that preferentially act at sites of nick and gaps, are thus naturally provided with DNA–DNA juxtapositions where a passage results in an error-free DNA unknotting or DNA decatenation.     

Innovative instrumentation for microarray scanning and analysis: application for characterization of oligonucleotide duplexes behavior.

Accuracy in microarray technology requires new approaches to microarray reader development. A microarray reader system (optical scanning array or OSA reader) based on automated microscopy with large field of view, high speed 3 axis scanning at multiple narrow-band spectra of excitation light has been developed. It allows fast capture of high-resolution, multi-fluorescence images and is characterized by a linear dynamic range and sensitivity comparable to commonly used photo-multiplier tube (PMT)-based laser scanner. Controlled by high performance software, the instrument can be used for scanning and quantitative analysis of any type of dry microarray. Studies implying temperature-controlled hybridization chamber containing a microarray can also be performed. This enables the registration of kinetics and melting curves. This feature is required in a wide range of on-chip chemical and enzymatic reactions including on-chip PCR amplification. We used the OSA reader for the characterization of hybridization and melting behaviour of oligonucleotide:oligonucleotide duplexes on three-dimensional Code Link slides.     

Design of synthetic oligonucleotide duplexes for site-directed mutagenesis with recombinant plasmids

Screening for site-mutated plasmids may be greatly facilitated by the different occurrence of the restriction sites distinguishing the altered sequence from the original one. Synthetic oligonucleotides can be so designed that they, apart from defining the mutation, also create a new restriction site recognizable in the restriction pattern of the mutant plasmid.     

Cartridge-based high-throughput purification of oligonucleotides for reliable oligonucleotide arrays

A novel, cartridge-based procedure for the efficient and irreversible detritylation of oligonucleotides is reported. This method, combined with a process for the elimination of depurinated fragments produces, in a highly parallel fashion, oligonucleotides with better purity than those traditionally obtained using reversed-phase high-performance liquid chromotography purification. Our combined detritylation and purification methodology compares favorably with commercial cartridge-based purification systems. The benefits of working with pure oligonucleotides, with regard to higher signal and better signal linearity, are shown in array-based hybridization experiments.     

A new peptide vector for efficient delivery of oligonucleotides into mammalian cells.

The development of antisense and gene therapy has focused mainly on improving methods for oligonucleotide and gene delivery into cells. In the present work, we describe a potent new strategy for oligonucleotide delivery based on the use of a short peptide vector, termed MPG (27 residues), which contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen. The formation of peptide vector/oligonucleotide complexes was investigated by measuring changes in intrinsic tryptophan fluorescence of peptide and of mansyl-labelled oligonucleotides. MPG exhibits relatively high affinity for both single- and double-stranded DNA in a nanomolar range. Based on both intrinsic and extrinsic fluorescence titrations, it appears that the main binding between MPG and oligonucleotides occurs through electrostatic interactions, which involve the basic-residues of the peptide vector. Further peptide/peptide interactions also occur, leading to a higher MPG/oligonucleotide ratio (in the region of 20/1), which suggests that oligonucleotides are most likely coated with several molecules of MPG. Premixed complexes of peptide vector with single or double stranded oligonucleotides are delivered into cultured mammalian cells in less than 1 h with relatively high efficiency (90%). This new strategy of oligonucleotide delivery into cultured cells based on a peptide vector offers several advantages compared to other commonly used approaches of delivery including efficiency, stability and absence of cytotoxicity. The interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and crossing of the plasma membrane. The mechanism of cell delivery of oligonucleotides by MPG does not follow the endosomal pathway, which explains the rapid and efficient delivery of oligonucleotides in the nucleus. As such, we propose this peptide vector as a powerful tool for potential development in gene and antisense therapy.     

HPLC photofingerprinting of conformational peculiarities and transitions in oligonucleotide duplexes.

Two self-complementary sequence-isomeric decadeoxyribonucleotides were exposed to UV light under conditions in which they assume duplex structures. After that they were analyzed in the denatured state by reversed-phase high-performance liquid chromatography (HPLC). Characterization of the separated photoproducts allowed localization of cyclobutane pyrimidine dimers in the sequences of the modified oligonucleotides. For [d(GGAAATTTCC)]2, which is known to contain in its central part a stretch of rigid B'-conformation with decreased mobility of constituent bases, lower yields of thymine dimers, as compared with that for ordinary B-form [d(CCTTTAAAGG)]2, were found. On the contrary, mixed thymine-cytosine heterodimers generated in the former oligonucleotide demonstrate the increase in photoreactivity of these residues at the B'-B junction. This is probably due to the peculiar conformation adopted by this decanucleotide. Stimulation of B'-B transition, by increasing the temperature before melting, reduced an inhibition of thymine photodimer formation. During the melting of both oligonucleotides yields of all identified photoinduced cyclobutadipyrimidines were reduced. Possible influences of some metal cations on the stability of the B'-form were also studied by this photoprobing technique. The present study demonstrates the feasibility of HPLC photofingerprinting as a new approach for structural analysis of nucleic acids.     

Polyion complex micelles with protein-modified corona for receptor-mediated delivery of oligonucleotides into cells.

Graft-copolymers, containing poly(ethylene glycol) (PEG) and polyethyleneimine (PEI) chains have been proposed as carriers for delivery of phosphorothioate oligonucleotides (SODNs). Complexes of such copolymers with SODN self-assemble into particles having a core of neutralized PEI and SODN and a corona of PEG. Transferrin molecules are attached to the PEG corona using avidin/biotin construct. For this purpose, biotin moieties are covalently linked to the free ends of the PEG chains in the PEG-g-PEI copolymer. SODNs are reacted with mixtures of biotinylated and biotin-free PEG-g-PEI copolymers of various compositions to adjust the number of the biotin moieties in the complex. Resulting complexes have small size (ca. 40 nm) and do not aggregate in aqueous solutions for at least several days. To attach transferrin, they are supplemented first with avidin and then with biotin-transferrin conjugate. This increases the effective diameter of the particles to ca. 75-103 nm, depending on the composition of the complex. Cellular accumulation and fluorescence microscopy studies characterize the effects of these modifications on interaction of fluorescently labeled SODNs with KBv cell monolayers. The data suggest significant enhancement of SODN association with cells resulting from modification of the complex with transferrin. SODN complimentary to the site 546-565 of human mdr 1-mRNA was used to inhibit expression of the drug efflux transporter, P-glycoprotein (P-gp), in multiple drug resistant (MDR) cancer cells (KBv, MCF-7 ADR). Accumulation of a P-gp specific probe, rhodamine 123, in the cell monolayers is used to characterize the effects on P-gp efflux system following the treatment of the cells with antisense SODN or its complexes. This study suggests that antisense SODN incorporated in the complexes retain the ability to inhibit P-gp efflux system, while complexes of the randomized control SODN are inactive. Therefore, the antisense SODN is released from the complex and interacts with its intracellular target upon interaction of the complexes with the cells. Furthermore, modification of the complexes with transferrin leads to a significant increase of the effects of the antisense SODN on the P-gp efflux system in the cells. Overall, this study suggests that polyion complex micelles with protein-modified corona are promising tools for the delivery of antisense SODN.     

Spongelike alginate nanoparticles as a new potential system for the delivery of antisense oligonucleotides.

The aim of this study was to design a new antisense oligonucleotide (ON) carrier system based on alginate nanoparticles and to investigate its ability to protect ON from degradation in the presence of serum. Pharmacokinetics and tissue distribution of ON-loaded nanoparticles have been determined after intravenous administration. An original and dynamic process for ON loading into polymeric nanoparticles has been applied. It is based on the diffusion of ON or ON/polylysine complex into the nanoparticle or the alginate gel, respectively. Indeed, the single coincubation of ON with nanoparticles led, within a few days, to an extremely efficient association. The diffusion kinetic of ON was shown to be dependent on several parameters, incubation temperature, ON concentration, presence or absence of polylysine, polylysine molecular weight, and nanoparticle preparation procedure. This new alginate-based system was found to be able to protect [33P]-radiolabeled ON from degradation in bovine serum medium and to modify their biodistribution, as an important accumulation of radioactivity was observed in the lungs, in the liver, and in the spleen after intravenous administration into mice. ON may be associated efficiently with calcium alginate in a colloidal state. Such nanosponges are promising carriers for specific delivery of ON to lungs, liver, and spleen.     

Invasion of complementary oligonucleotides into (CA/TG)31 repetitive region of linear and circular DNA duplexes

(CA/TG)_n repeats belong to microsatellite DNA. They are the most abundant among the other dinucleotide repeats in mammals, constituting approximately 0.25% of the entire genome. These repeats are recombination hot spots; however, the corresponding mechanisms are yet vague. We postulated that one of the reasons underlying an increase in the recombination frequency in the repetitive region could be the con-formational characteristics of duplex resulting from a specific geometry of base-stacking contacts, providing for initiation of a single-stranded DNA invasion in th e duplex homologous regions. This work for the first time demonstrates a DNA-DNA interaction of the d(CA)₁₀ and d(TG)₁₀ oligonucleotides with linear and circular duplexes containing (CA/TG)₃₁ repeats during their coincubation in a protein-free water solution at 37°C. Using radioactively labeled oligonucleotides, we demonstrated that the duplex—oligonucleotide interaction intensity depended on the molar ratio of duplex-to-oligonucleotide at a duplex concentration of 30 nM. A decrease in this concentration to 3 nM had no effect on the intensity of oligonucleotide invasion. It was demonstrated that over 1% of the duplexes yet much less than 10% were involved in the interaction with oligonucleotides assuming that one oligonucleotide molecule interacted with one molecule of the duplex. Analysis of the kinetics showed that d(CA)₁₀ invasion commenced from the first minute of incubation with duplexes, while d(TG)₁₀ interacted with the duplex even at a higher rate. The role of conformational plasticity of CA/TG repeats in the discovered interaction is discussed as well as its biological significance, in particular, the role of CA microsatellites in the initiation of homologous recombination.     

Preparation of photoreactive oligonucleotide duplexes and their application for photoaffinity modification of DNA-binding proteins]

To introduce photoreactive dNTP residues to the 3'-end of a mononucleotide gap, base-substituted photoreactive deoxynucleoside triphosphate derivatives, (5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-trans-3-aminopropenyl-1]- and 5-(N-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]- trans-3-aminopropenyl-1)-2'-deoxyuridine 5'-triphosphates, were used as substrates in the DNA polymerase beta-catalyzed reaction. The resulting nick, containing a modified base at the 3'-end, was sealed by T4 phage DNA ligase. This approach enables the preparation of DNA duplexes bearing photoreactive groups at predetermined position(s) of the nucleotide chain. Using the generated photoreactive DNA duplexes, the photoaffinity modifications of DNA polymerase beta and human replicative protein A (hRPA) were carried out. It was shown that DNA polymerase beta and hRPA subunits were modified with the photoreactive double-stranded DNA considerably less effectively than by the nicked DNA. In the case of double-stranded DNA, the hRPA p70 subunit was preferentially labeled, implying a crucial role of this subunit in the protein-DNA interaction.     

Progress in the delivery of therapeutic oligonucleotides: organ/cellular distribution and targeted delivery of oligonucleotides in vivo

Oligonucleotide (ODN) therapy is a powerful tool for modulation of gene expression in vivo. With advances in ODN chemistry and progress in formulation development, ODNs are becoming widely acceptable drugs. This review summarizes the current status and future trend of the in vivo application of ODN therapeutics, especially antisense ODNs. Here, we review the current understanding of the tissue/organ distribution and cellular uptake of ODN drugs administered parenterally or nonparenterally to intact animals. The problems and advantages inherent in the use of different delivery methods for the treatment of particular diseases are discussed in detail. Emphasis is placed on the most widely studied ODN analogs, the phosphorothioates (PS). Lessons learned from antisense PS studies have broad implications for ODN therapeutics in general.     

Recombinant viral capsids as an efficient vehicle of oligonucleotide delivery into cells

Delivery of oligonucleotides (ON) into cells is a technical challenge. In this study, we utilized the capsid of the hepatitis B virus (HBV) to meet this goal. A single and short open reading frame of the virus programs efficient capsid production in bacteria. We show that these capsids can encapsulate ON in vitro and then mediate their delivery into cells with extreme efficiency. This process is cell type non-specific, rendering the recombinant HBV capsid a potentially valuable vehicle for ON delivery into a wide range of cultured cells.     

Raman study of potential "antisense" drugs: nonamer oligonucleotide duplexes with a central mismatch as a model system for the binding selectivity evaluation

A set of four 9-mer oligonucleotide duplexes formed between the 5'-GCATNTCAC-3', N=A,C,T,G, and the 5'-GTGATATGC-3' complement has been proposed as a model system for the investigation of novel oligonucleotide analogues (candidates for antisense use) binding selectivity. Raman measurements were carried out on a set of natural DNA 9-mer in order to verify suitability of the model and to obtain reference spectral data. Difference Raman spectra between the mismatch and match duplexes obtained at 15 degrees C exhibited numerous spectral features sensitively indicating the structural changes. All the three mismatches only very weakly disturb the overall B-form conformation of the duplex. Significant structural changes that occurred at the mismatch site are reflected mainly by the neighboring thymidine Raman bands at 1377, 1650, and 1675 cm(-1). The intensity change of the two latter bands is almost the same for the T:G and the T:T mismatch while in the case of the T:C mismatch it is just opposite, demonstrating a very different arrangement of the mismatched pair.     

The potential of oligonucleotides for therapeutic applications

Viral-derived particles have been widely used and described in gene therapy clinical trials. Although substantial results have been achieved, major safety issues have also arisen. For more than a decade, oligonucleotides have been seen as an alternative to gene complementation by viral vectors or DNA plasmids, either to correct the genetic defect or to silence gene expression. The development of RNA interference has strengthened the potential of this approach. Recent clinical trials have also tested the ability of aptamer molecules and decoy oligonucleotides to sequestrate pathogenic proteins. Here, we review the potential of oligonucleotides in gene therapy, outline what has already been accomplished, and consider what remains to be done.     

Comparison of the electrophoretic and hydrodynamic properties of DNA and RNA oligonucleotide duplexes.

The electrophoretic behavior of defined DNA and RNA oligonucleotide duplexes from 10 to 20 bp in length has been investigated as a function of salt conditions, gel concentration, and temperature. The RNA oligomers migrated much more slowly than the DNA oligomers of the same sequence under all conditions. From sedimentation equilibrium and velocity measurements, the apparent partial specific volume in 0.1 M KCI, 20 mM NaPi, pH 7, was determined as 0.56 +/- 0.015 ml g(-1) for DNA and 0.508 ml g(-1) for RNA. The translational friction coefficients were determined and compared with the values calculated for cylinders. Taking into account the shape factors, the solution density, and partial specific volumes, the effective degree of hydration was estimated as 0.8-1 g g(-1) DNA. There was no significant difference in the frictional coefficients of the DNA and RNA oligomers, indicating that the effective sizes of DNA and RNA are very similar in solution. The differential electrophoretic mobility of DNA and RNA must arise from the differences in interaction with counterions, which is probably a global property of the oligonucleotides.     

Covalent carcinogenic lesions in DNA: NMR studies of O6-methylguanosine containing oligonucleotide duplexes

We report on proton and phosphorus high resolution NMR investigations of the self-complementary dodecanucleotide d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6 meG.N 12-mers), N = C, T, A and G, which contain N3.O6meG10 interactions in the interior of the helix. These sequences containing a single modified O6meG per strand were prepared by phosphoamidite synthesis and provide an excellent model for probing the structural basis for covalent carcinogenic lesions in DNA. Distance dependent nuclear Overhauser effect (NOE) measurements and line widths of imino protons demonstrate that the N3 and O6meG.10 bases stack into the duplex and are flanked by stable Watson-Crick base pairs at low temperature for all four O6meG.N 12-mer duplexes. The imino proton of T3 in the O6meG.T 12-mer and G3 in the O6meG.N 12-mer helix, which are associated with the modification site, resonate at unusually high field (8.5 to 9.0 ppm) compared to imino protons in Watson-Crick base pairs (12.5 to 14.5 ppm). The nonexchangeable base and sugar protons have been assigned from two dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements on the O6meG.N 12-mer helices. The directionality of the distance dependent NOEs establish all O6meG.N duplexes to be right-handed helices in solution. The glycosidic torsion angles are in the anti range at the N3.O6meG10 modification site except for O6meG10 in the O6meG.G 12-mer duplex which adopts a syn configuration. This results in altered NOEs between the G3 (anti).O6meG10 (syn) pair and flanking G2.C11 and G4.C9 base pairs in the O6meG.G 12-mer duplex. We observe pattern reversal for cross peaks in the COSY spectrum linking the sugar H1' protons with the H2',2" protons at the G2 and O6meG10 residues in the O6meG.N 12-mer duplexes with the effect least pronounced for the O6meG.T 12-mer helix. The proton chemical shift and NOE data have been analyzed to identify regions of conformational perturbations associated with N3.O6meG10 modification sites in the O6meG.N 12-mer duplexes. The proton decoupled phosphorus spectrum of O6meG.T 12-mer duplex exhibits an unperturbed phosphodiester backbone in contrast to the phosphorus spectra of the O6meG.C 12-mer, O6meG.G 12-mer and O6meG.A 12-mer duplexes which exhibit phosphorus resonances dispersed over 2 ppm characteristic of altered phosphodiester backbones at the modification site. Tentative proposals are put forward for N3.O6meG10 pairing models based on the available NMR data and serve as a guide for the design of future experiments.     

13C NMR of the bases of three DNA oligonucleotide duplexes: assignment methods and structural features

Natural abundance 13C NMR spectra of three DNA oligomers have been obtained. Most of the base resonances are well resolved from one another. A combination of two independent methods was used in making assignments: a one-dimensional spectral comparison method and a two-dimensional proton-detected 1H-13C correlated experiment for the protonated carbons. There are large shielding changes (between 1.62 and -1.40 ppm) upon thermal dissociation of the duplex. The shapes of the chemical shift vs temperature curves are largely independent of sequence. The base carbon resonance frequencies are sensitive to hydrogen bonding, base stacking, sugar conformation, and changes in the glycosyl torsion angle.     

Equilenin-derived DNA adducts to cytosine in DNA duplexes: structures and thermodynamics

The drug Premarin is the most widely used formula for hormone replacement therapy. However, long-term exposure to estrogens from the Premarin drug increases the risk of breast cancer. Equilin and equilenin, major components of Premarin, are predominantly metabolized to 4-hydroxyequilenin (4-OHEN). The quinoids produced by 4-OHEN oxidation react with dG, dA, and dC to form unusual stable cyclic bulky adducts, with four stereoisomers identified for each base adduct. The 4-OHEN-dC adducts are most predominant. They are mutagenic in vitro and have been found in human tumor tissue. We have carried out molecular modeling and molecular dynamics simulations to investigate structures and thermodynamics of the four 4-OHEN-dC stereoisomeric adducts in DNA duplexes. Our results show that the structure of each stereoisomer adduct in duplex DNA is specifically governed by its unique stereochemistry. The bulky adducts, with an obstructed Watson-Crick edge and an equilenin ring system near perpendicular to the damaged cytosine, are located in the B-DNA major or minor groove, with the modified cytosine in the syn or anti conformation, respectively. The DNA duplex structures are distorted, in terms of Watson-Crick pairing at and near the lesion, stacking interactions, and groove dimensions. Stereochemistry determines the orientation of the equilenin rings with respect to the 5'- to 3'-direction of the modified strand, as well as the positioning of the equilenin moiety's methyl and hydroxyl groups for each stereoisomer. The unusual structures and the stereochemical effects underlie their biological processing as miscoding DNA lesions whose mutagenic properties may contribute to breast cancer.