Asya S. Levina

Photomodification of RNA and DNA fragments by oligonucleotide reagents bearing arylazide groups

Photomodification of ribo- and deoxyribo-octanucleotides by oligonucleotide reagents (6- and 7-mers) bearing p-azidotetrafluorobenzamido and 2-nitro-5-azidobenzamido groups has been investigated. It is shown that the oligonucleotides with a perfluoroarylazide group were effective modifiers both of deoxyribo- and ribo-targets. Maximum extent of cross-linked product formation (70%) was obtained when the deoxyribo-octanucleotide was modified by a heptanucleotide reagent with a perfluoroarylazide group. Selectivity of the photomodification was also high (50% on the G-residue at a certain position).

N-(2-Hydroxyethyl)phenazinium derivatives of oligonucleotides as effectors of the sequence-specific modification of nucleic acids with reactive oligonucleotide derivatives

It has been found that mono‐ and especially diphenazinium derivatives of oligonucleotides complementary to the DNA sequence adjacent to the target sequence of the addressed alkylation of DNA, significantly enhance the extent and specificity of alkylation with p‐(N‐2‐chloroethyl‐N‐methylamino)benzylamide derivatives of the addressing oligonucleotides, thus playing the role of effector of the sequence‐specific (complementary addressed) modification.

The mechanism of recognition of templates by DNA polymerases from pro- and eukaryotes as revealed by affinity modification data.

Pt(2+)-containing derivatives of oligodeoxyribonucleotides were used to evaluate the ligand affinity to the template sites of Klenow fragment of DNA polymerase I from E. coli and DNA polymerase alpha from human placenta. The values of Kd and Gibbs energy (delta G degree) for the complexes of oligodeoxyribonucleotides and their derivatives with the template sites of these enzymes were determined from the effects protecting the enzyme from inactivation by Pt(2+)-containing oligonucleotides. Kd and delta G degree values of the complexes made by DNA polymerases and orthophosphate, triethylphosphate, d(pC)n, d(pT)n, d(pG)n, d(pA)n (where n = 1-25), heterooligonucleotides of various length and structure, and oligothymidylates with partially and completely ethylated internucleotide phosphates were evaluated. The obtained data enabled us to suggest 19-20 mononucleotide units of the template to interact with the protein. Only one template internucleotide phosphate forms a Me(2+)-dependent electrostatic contact (delta G = -1.1...-1.7 kcal/mol) and a hydrogen bond (delta G = -4.4...-4.9 kcal/mol) with the enzyme. It is likely that the mononucleoside units of the template form hydrophobic contacts with the enzymes. The efficiency of such interaction changes with the hydrophobicity of the bases: C less than T less than G approximately A. For both homo- and heterooligonucleotides the contributions of nucleoside units to the affinity of the templates to the enzymes is due to the complementary interactions with the primers. A hypothetical model for the template-primer interaction with DNA polymerases is suggested.

DNA polymerase I (Klenow fragment): Role of the structure and length of a template in enzyme recognition

The values of K d and Gibbs energy (ΔG°) have been measured for complexes of the template site of DNA polymerase I Klenow fragment with the homo‐oligonucleotides d(pC) n , d(pT) n , d(pG) n and d(pA) n and hetero‐oligonucleotides of various structures and lengths. These parameters were evaluated from the protective effect of the oligonucleotide on enzyme inactivation by the affinity reagents d(Tp)2C[Pt2+(NH3)2OH](pT)7 and d[(Tp)2C(Pt2+(NH3)2OH)p]3T of the template site. The present results and previously reported data [(1985) Biorg. Khim. 13, 357–369] indicate that the nucleoside components of the template form complexes as a result of their hydrophobic interactions with the enzyme. Only one template internucleotide phosphate forms an Me2+‐dependent electrostatic contact and a hydrogen bond with the enzyme. The 19–20‐nucleotide fragments of the template appear to interact with the protein molecule.

High-performance method for specific effect on nucleic acids in cells using TiO2~DNA nanocomposites

Nanoparticles are used to solve the current drug delivery problem. We present a high-performance method for efficient and selective action on nucleic acid target in cells using unique TiO2·PL-DNA nanocomposites (polylysine-containing DNA fragments noncovalently immobilized onto TiO2 nanoparticles capable of transferring DNA). These nanocomposites were used for inhibition of human influenza A (H3N2) virus replication in infected MDCK cells. They showed a low toxicity (TC50 ≈ 1800 μg/ml) and a high antiviral activity (>99.9% inhibition of the virus replication). The specificity factor (antisense effect) appeared to depend on the delivery system of DNA fragments. This factor for nanocomposites is ten-times higher than for DNA in the presence of lipofectamine. IC50 for nanocomposites was estimated to be 1.5 μg/ml (30 nM for DNA), so its selectivity index was calculated as ~1200. Thus, the proposed nanocomposites are prospective for therapeutic application.

Thermodynamic Analysis of Stacking Hybridization of Oligonucleotides with DNA Template

Abstract Contiguous stacking hybridization of oligodeoxyribonucleotides with DNA as template was investigated using three types of complexes: oligonucleotide contiguously stacked with the stem of the preformed minihairpin (complexes I), oligonucleotide tandems containing two (complexes II) or three (complexes III) short oligomers with a common DNA template. Enthalpy ΔH° and entropy ΔS° of the coaxial stacking of adjacent duplexes were determined for GC/G*pC, GT/A*pC, AC/G*pT, AT/A*pT, CT/A*pG, AG/C*pT, AA/T*pT and TT/A*pA nicked (*) dinucleotide base pairs. The maximal efficiency of co-operative interaction was found for the GC/G*pC interface (ΔG°NN/N*pN=-2.7 kcal/mol) and the minimal one for the AA/T*pT interface (ΔG°/NN/N*pN=-1.2 kcal/mol) at 37 °C. As a whole, the efficiency of the base pairs interaction ΔG°NN/N*pN in the nick is not lower than that within the intact DNA helix ΔG°NN/NN). These observed the ΔG°NN/N*pN values are proposed may include the effect of the partial removal of fraying at the adjacent helix ends additionally to the effect of the direct stacking of the terminal base pairs in the duplex junction (ΔG°NN/NN. The thermodynamic parameters have been found to describe adequately the formation of all tandem complexes of the II and III types with oligonucleotides of various length and hybridization properties. The performed thermodynamic analysis reveals features of stacking oligonucleotide hybridization which allow one to predict the temperature dependence of association of oligonucleotides and the DNA template within tandem complexes as well as to determine optimal concentration for formation of these complexes characterized by high co-operativity level.

Efficient inhibition of human influenza a virus by oligonucleotides electrostatically fixed on polylysine-containing TiO2 nanoparticles

Antiviral activity of the TiO2·PL·DNA nanobiocomposites was studied on the MDCK cell culture infected with influenza A virus (subtype H3N2). DNA fragments in the nanocomposites are electrostatically bound to titanium dioxide nanoparticles precovered with polylysine. It was shown that TiO2·PL·DNA(v3′) nanocomposite bearing the DNA(v3′) fragment targeted to the 3′-end of the noncoding region of segment 5 of viral RNA specifically inhibited the virus reproduction with the efficiency of 99.8% and 99.9% (i.e., by factors of ∼400 and 1000, respectively) at a low concentration of DNA(v3′) in nanocomposite (0.1 and 0.2 μM, respectively). The TiO2·PL·DNA(r) nanocomposite containing an oligonucleotide noncomplementary to viral RNA or oligonucleotide DNA(v3′) unbound to the nanoparticles show very low antiviral activity (inhibition by factors of ∼3.5 and 1.3, respectively).

Design of TiO2~DNA nanocomposites for penetration into cells

Methods of noncovalent immobilization of DNA fragments on titanium dioxide nanoparticles (TiO2) were developed to design TiO2∼DNA nanocomposites, which were capable of penetrating through cell membranes. TiO2 nanoparticles of different forms (amorphous, anatase, brookite) with enhanced agglomeration stability were synthesized. The particles were characterized by X-ray diffraction, small-angle X-ray scattering, infrared spectroscopy and atomic force microscopy. Three approaches to the preparation of nanocomposites are described: 1) sorption of polylysine-containing oligonucleotides onto TiO2 nanoparticles, 2) the electrostatic binding of oligonucleotides to TiO2 nanoparticles bearing immobilized polylysine, and 3) sorption of oligonucleotides on TiO2 nanoparticles in the presence of cetyltrimethylammonium bromide (cetavlon). All three methods provide an efficient and stable immobilization of DNA fragments on nanoparticles that leads to nanocomposites with a capacity of up to 40 nmol/mg for an oligonucleotide. DNA fragments in nanocomposites were shown to retain their ability to form complementary complexes. It was demonstrated by confocal laser microscopy that the proposed nanocomposites penetrated into cells without transfection agents and other methods of exposure.

Synthesis of polyamine-containing oligonucleotides

A simple and efficient method of synthesis of polyamine-oligonucleotide conjugates (PA-oligos) in high yields (up to 95%) was suggested. The terminal phosphate group of deprotected oligonucleotides was selectively activated with the redox pair triphenylphosphine-dipyridyl disulfide in the presence of a nucleophilic catalyst, and the activated oligonucleotide derivative was subjected to the reaction with a polyamine.

Role of nucleoside components and internucleotide phosphate groups of oligodeoxyribonucleotide template in its binding to human DNA polymerase α

Affinity labelling of human placenta DNA polymerase α (EC 2.7.7.7) with the reactive oligodeoxyribonucleotide d(pT)2pC[Pt2+(NH3)2OH](pT)7 was used for quantitative analysis of enzyme interaction with oligodeoxyribonucleotides as templates. Dissociation constants and Gibbs energy values for different oligothymidylates d(pT) n T where n = 1–14 have been evaluated by competitive experiments of these ligands with Pt2+ reagent. The data obtained prove the formation of one Me2+‐dependent electrostatic contact and a hydrogen bond between the enzyme and one phosphate of these templates. One may suppose that the hydrophobic interaction of any other monomeric link of oligodeoxyribonucleotides with the enzyme template site takes place.