Antisense Oligonucleotides Immobilized on Silicon-Organic Nanoparticles as a Tool for Prolonged Correction of Hypertensive States.

We propose an original method for controlling BP by administration of Si~ODN nanocomposites containing antisense oligonucleotides fixed on silicon-organic nanoparticles. ODN in nanocomposites are targeted to mRNA of the genes encoding angiotensin-converting enzyme (ACE1) and type 1 angiotensin-II receptor (AT1A). The experiments were performed on hypertensive ISIAH rats, a genetic model of hypertension. Single inhalation or intraperitoneal administration of the nanocomposites targeted to ACE1 mRNA or ATA1 mRNA, respectively, led to a pronounced decrease (by ~30 mm Hg) in systolic BP in ISIAH rats over a week. The use of scrambled ODN in the nanocomposites had no effect. A decrease in the expression of ACE1 and AT1A genes under the effect of the corresponding antisense ODN was demonstrated, which attested to directed effect of the test preparations.

[Impact of Delivery Method on Antiviral Activity of Phosphodiester, Phosphorothioate, and Phosphoryl Guanidine Oligonucleotides in MDCK Cells Infected with H5N1 Bird Flu Virus].

We have previously described nanocomposites containing conjugates or complexes of native oligodeoxyribonucleotides with poly-L-lysine and TiO2 nanoparticles. We have shown that these nanocomposites efficiently suppressed influenza A virus reproduction in MDCK cells. Here, we have synthesized previously undescribed nanocomposites that consist of TiO2 nanoparticles and polylysine conjugates with oligonucleotides that contain phosphoryl guanidine or phosphorothioate internucleotide groups. These nanocomposites have been shown to exhibit antiviral activity in MDCK cells infected with H5N1 influenza A virus. The nanocomposites containing phosphorothioate oligonucleotides inhibited virus replication ~130-fold. More potent inhibition, i.e., ~5000-fold or ~4600-fold, has been demonstrated by nanocomposites that contain phosphoryl guanidine or phosphodiester oligonucleotides, respectively. Free oligonucleotides have been nearly inactive. The antiviral activity of oligonucleotides of all three types, when delivered by Lipofectamine, has been significantly lower compared to the oligonucleotides delivered in the nanocomposites. In the former case, the phosphoryl guanidine oligonucleotide has appeared to be the most efficient; it has inhibited the virus replication by a factor of 400. The results make it possible to consider phosphoryl guanidine oligonucleotides, along with other oligonucleotide derivatives, as potential antiviral agents against H5N1 avian flu virus.

Toward Gene Therapy of Hypertension: Experimental Study on Hypertensive ISIAH Rats.

TiO2-based nanocomposites were prepared to deliver oligonucleotides into cells. The nanocomposites were designed by the immobilization of polylysine-containing oligonucleotides on TiO2-nanoparticles (TiO2·PL-DNA). We showed for the first time the possibility of using the proposed nanocomposites for treatment of hypertensive disease by introducing them into hypertensive ISIAH rats developed as a model of stress-sensitive arterial hypertension. The mRNA of the gene encoding angiotensin I-converting enzyme (ACE1) involved in the synthesis of angiotensin II was chosen as a target. Administration (intraperitoneal injection and inhalation) of the nanocomposite showed a significant (by 20-30 mm Hg) decrease in systolic blood pressure when the nanocomposite contained the ACE1 gene-targeted oligonucleotide. When using the oligonucleotide with a random sequence, no effect was observed. Further development and improvement of the inhalation nanocomposite drug delivery to systemic hypertensive disease treatment promises new possibilities for clinical practice.

[Composites of Peptide Nucleic Acids with Titanium Dioxide Nanoparticles. IV+. Antiviral Activity of Nanocomposites Containing DNA/PNA Duplexes].

Antiviral activity of TiO2 · PL · DNA/PNA nanobiocomposites was studied on the MDCK cell culture infected with influenza A virus (subtype H3N2). PNA fragment in nanocomposites as a DNA/PNA heteroduplex is electrostatically bound to titanium dioxide nanoparticles precovered with polylysine (TiO2 · PL). It was shown that TiO2 · PL · DNA1/PNA1 nanobiocomposit bearing PNA1 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%. It was determined that the 50% cytotoxic concentration (TC50) of the TiO2 · PL · DNA1/PNA1 nanocomposite is more than 1200 mg/mL. And 50% effective inhibitory concentration (IC50) is less than 0.003 mg/mL. Based on these data, the selectivity index (SI) for TiO2 · PL · DNA1/PNA1 nanobiocomposite defined as the ratio TC50/LC50, is more than 400. Thus TiO2 · PL · DNA/PNA nanobiocomposites can not only penatrate through cell membrane, but and are able to exhibit a high specific antisense activity, without causing toxic effects on the living cells.

[Eficient inhibition of human influenza A virus by oligonucleotides electrostatically fixed on polylysine-containing TiO2 nanoparticles].

Antiviral activity of 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 pre-covered 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% (or by factors of~400 and 1000) at a low concentration of DNA(v3') in nanocomposite (0.1 and 0.2 µM, respectively). The TiO2 * PL * DNA(r) nanocomposite containing oligonucleotide noncomplementary to viral RNA or the oligonucleotide unbound to the nanoparticles show very low antiviral activity (inhibition by factors of~3.5 and 1.3, respectively).

[Composites of peptide nucleic acids with titanium dioxide nanoparticles. III. Kinetics of PNA dissociation from nanocomposites containing DNA/PNA duplexes].

When delivering peptide nucleic acids (PNA) to the cells in the nanocomposites TiO2 · PL · DNA/PNA, containing titanium dioxide nanoparticles coated with polylysine (PL) and immobilized DNA/PNA duplexes, it is important not only to transport them to the cell, but also ability to control the release rate of the PNA-drug from the carrier. PNA desorption from TiO2 · PL · DNA/PNA nanocomposite in time has been shown. Desorption is caused by dissociation of immobilized DNA/PNA duplex while the DNA remains on the carrier and PNA goes away in solution. It has been found that the half-retention times of PNA on TiO2 · PL · DNA/PNA nanocomposites containing DNA/PNA duplexes with overlapping complementary base pairs equal to 10, 12, 14, and 16 are 10, 14, 22 and 70 minutes, respectively. Thus, it has been shown that the release rate of the PNA-drug from nanocomposites can be adjusted by varying the overlap of complementary base pairs in the immobilized DNA/PNA duplex. This method of PNA immobilization may be used for designing of nanocomposites with optimum release time of the PNA-drugs. Created TiO2 · PL · DNA/PNA nanocomposites can be used to efficiently deliver therapeutically significant drug PNA and their selective effect on the pathogenic nucleic acid in the cell.

[TiO2-DNA nanocomposites capable of penetrating into cells].

Methods of noncovalent immobilization of DNA fragments onto titanium dioxide nanoparticles (TiO2) were developed, which led to TiO2-DNA nanocomposites capable of penetrating through cell membranes. TiO2 nanoparticles of different forms (amorphous, anatase, brookit) 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 cetavlon. All three methods provide an efficient and stable immobilization of DNA fragments onto nanoparticles, which leads to nanocomposites with a density for an oligonucleotide up to 40 nmol/mg. It is shown that DNA fragments in nanocomposites retain their ability to form complementary complexes and can be delivered into cells without transfection agents and other methods of exposure.

[Affinity capture of specific DNA fragments with the use of short synthetic sequences].

The ability of short peptide nucleic acid (PNA) oligomers and oligonucleotides containing modified residues of 5-methylcitidine, 2-aminoadenosine and 5-propynyl-2'-deoxyuridine (strong binding oligonucleotides, SBO) to affinity capture the target double-stranded DNA fragment from mixture by means of the end invasion was compared. Both types of probes were highly effective at the conditions used. The SBO-based probes may represent a handy and easily prepared alternative to PNA for selection of target DNA fragments from mixtures.

[Design of deoxyribozymes for inhibition of influenza A virus].

Influenza A viruses take a significant place in human and animal pathology causing epidemics and epizootics. Therefore, the development of new antiflu drugs has become more and more urgent. Deoxyribozymes can be considered as promising antiviral agents due to their ability to efficiently and highly specifically cleave RNA molecules. In this study, a number ofgenomic sequences of the most relevant influenza A virus subtypes, H5N1, H3N2, and H1N1, were analyzed. Conservative regions were revealed in five the least variable segments of the fragmented viral RNA genome, and potential sites of their cleavage with "10-23" deoxyribozymes were determined. 46 virus-specific 33-mer deoxyribozymes with the general structure of 5'N8AGGCTAGCTACAACGAN9 were designed and synthesized. Screening of the antiviral activity of these agents in conjugation with lipofectin on the Madin-Darby Canine Kidney cells infected with highly pathogenic avian influenza virus A/chicken/Kurgan/05/2005 (H5N1) revealed 17 deoxyribozymes, which suppressed the titer of virus cytopathicity by more than 2.5 IgTCID50/mL (i.e. the virus neutralization index was more than 300), with five of them suppressing the virus titer by a factor of 1000 and more. The most active deoxyribozymes appeared to be specific to segment 5 of the influenza A virus genome, which encoded nucleoprotein (NP).

[Composites of peptide nucleic acids with titanium dioxide nanoparticles. II+. Dissociation of DNA/PNA duplexes within TiO2 x polylysine x DNA/PNA nanocomposites and in solution. Effect of polylysine].

When creating effective drugs it is important not only to transport them into the cells, but also important to have the possibility of release them from the "transporter" after delivery into the cell. It was shown that peptide nucleic acids (PNA) in nanocomposite TiO2 x PL x DNA/PNA dissociate with typical shape of the thermal denaturation curve, and polylysine (PL) in the nanocomposite has practically no effect on the dissociation of the DNA/PNA duplexes. These data suggest that the PNA in the nanocomposite TiO2 x PL x DNA/PNA have been immobilized reversible and able to dissociate and be released from TiO2-carrier into solution. In contrast that, the dissociation of DNA/DNA and DNA/PNA duplexes in physiological solution at the presence of PL--was not observed. PL in solution abnormally strong influences on the nature of the optical density dependence on temperature and time for D-duplexes and in a less degree--for P-duplexes. It has been suggested, that PL with DNA/DNA duplexes in physiological solution forms triple polycomplexes (-DNA/DNA x PL)m, consisting of several (m) chains of PL connected with DNA/DNA duplexes. And such polycomplexes able to aggregate and precipitate. PL in solution can interact with DNA/PNA duplexes to form monocomplexes PL x (DNA/PNA)n consisting of one chain PL and one or more (n) DNA/PNA duplexes that do not precipitate, however the dissociation of DNA/PNA duplexes from such monocomplexes is difficult.

[Composites of peptide nucleic acids with titanium dioxide nanoparticles. I. Construction of nanocomposites containing DNA/PNA duplexes and their delivery into HeLa cells].

In order to investigate the possibility of using titanium dioxide (TiO2) nanoparticles to transport peptide nucleic acids (PNA) in eukaryotic cells, a PNA oligomer has been synthesized, and method of PNA immobilization in the form of hybrid DNA/PNA duplexes on the surface of TiO2 nanoparticles covered with polylysine (PL) has been designed. Attaching of DNA/PNA duplex on TiO2 x PL nanoparticles occurred due to electrostatic interactions between the negatively charged DNA chain and the positively charged amino groups of PL. Binding of the PNA with the nanocomposite achieved through noncovalent Watson-Crick interactions between the PNA and complementary DNA. The capacity of obtained TiO2 x PL x DNA/PNA nanocomposites depending on immobilization conditions was 10-30 nmol PNA per 1 mg of TiO2 particles, which corresponds to -1-3 PNA molecules per one TiO2 particle with size of 4-6 nm. By method ofconfocal laser scanning microscopy on the example of the fluorescein labeled PNA oligomer (Flu)PNA it has been shown that the PNA molecules in composition of TiO2 x PL x DNA/(Flu)PNA nanocomposites effectively penetrate and accumulate in HeLa cells without the use oftransfection agents, electroporation, or other auxiliary procedures has been shown.

[Direct site-specific cleavage of double-stranded DNA by conjugates of bleomycin A5 with triplex-forming oligonucleotide].

Monofunctional conjugates of 15-mer triplex-forming oligonucleotide (TFO) with covalently attached bleomycin A5 residue at the 5'-end (Blm-p15) were synthesized. Bifunctional conjugates of TFO containing, in addition to Blm, the residues of intercalator 6-chloro-2-methoxy-9-aminoacridine (Acr) or (N-(2-hydroxyethyl)phenazinium (Phn) were obtained for the first time. The Acr and Phn residues were attached to the 3'-phosphate group of TFO through L1 and L2 linkers, respectively, resulting in the compounds Blm-p15pL1-Acr and Blm-p15pL2-Phn. The values of dissociation constants of the corresponding triplexes were evaluated using the gel retardation method. The Acr residue in Blm-p15pL1-Acr was shown to enhance the stability of the formed triplex by one order of magnitude. It was demonstrated that all synthesized conjugates are capable of specifically and nonspecifically damaging a target DNA, forming direct breaks and alkaline-labile sites. The extent of the specific cleavage of the target DNA was 15% in the case of a fivefold excess of the conjugates over the DNA duplex. The site-specific triplex-mediated cleavage of a target DNA was shown for the first time to occur predominantly (> 90%) with the formation of the direct breaks of both DNA strands. The results show the availability of bleomycin-containing oligonucleotides as antigene compounds.

An examination of the ability of titanium dioxide nanoparticles and its conjugates with oligonucleotides to penetrate into eucariotis cells.

In this study we examine the possibility that TiO2 nanoparticles and their conjugates can penetrate into cultivated cells without any special transfection procedures. Oligonucleotides and their derivates were conjugated with the TiO2 nanoparticles, which were obtained as colloidal solutions at a concentration of TiO2 0.3M by TiCl4 hydrolysis. The electronic microscopy of various cell cultures (KCT, Vero, and MDCK) treated with nanoparticle solutions (20 µg/µl) showed that nanoparticles could enter the cells and accumulate in the vacuoles and phagosomes and form inclusions in cytoplasm. Thus, we demonstrated the penetration of TiO2 nanoparticles and their oligonucleotide conjugates into intracellular space without any auxiliary operations. Most other researches used electroporation techniques for similar purposes [1, 2, 5].

[Synthesis of polyamine-containing oligonucleotides].

A simple and efficient method of synthesis of polyamine-oligonucleotide conjugates 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.

[Enhancement of hybridization analysis efficacy by means of controlled fragmentation of analyzed DNA].

The influence of fragmentation of analyzed DNA amplicon on the efficacy of specific sequence detection by means of heterophase hybridization analysis was investigated. The detection of DNA sequence was carried out colorimetrically after introduction of biotin label into the oligonucleotide probe immobilized on a solid support upon its limited elongation in the complex with the analyzed DNA using Taq polymerase. Two simple and reproducible approaches to DNA analyte fragmentation were suggested. They are based on the formation apurinic/apyrimidinic sites in DNA followed by their degradation upon the thermal treatment. Apurinization of DNA was achieved with a mild acidic treatment. The apyrimidinic sites were formed when DNA fragment containing dTMP and dUMP residues in various ratios was treated with uracil-DNA-glycosylase (UDG). DNA analytes pretreated by one of these approaches can be used without additional purification for hybridization analysis of DNA with the use of Taq polymerase. The efficacy of hybridization analysis is shown to be higher in the case of the fragmented DNA in comparison with the native DNA amplicon. The use of fragmented DNA analytes allows utilizing bridged oligonucleotides as highly selective probes with reduced hybridization properties.

[Functionalized nanocomposite coating of a glass surface for oligonucleotide immobilization].

A new type of coating for manufacturing DNA chips was constructed of the basis of an organic-inorganic nanocomposite based on the polyvinylbutyral-tetraethoxysilane copolymer. The organosilicon composite was functionalized by introduction of ethanolamine vinyl ether copolymers, which contain amino groups and anchor vinyloxide units capable of reacting with silanol groups of the nanocomposite. The resulting coatings form a film on glass slides with a high surface density of amino groups (up to 700 groups/nm2) suitable for three-dimensional immobilization of oligonucleotides. The use of bifunctional reagents (e.g., phenylene diisothiocyanate) for the attachment of oligonucleotides bearing amino linkers to the amino-containing surface provides an immobilization density of 0.5-1.6 pmol/mm2. Immobilization with a higher density (10-12 pmol/mm2) was achieved for attachment to amino-containing glass slides upon the use of oligonucleotides containing selectively activated terminal phosphate groups. The activation of oligonucleotides was carried out with the triphenylphosphine-dithiodipyridine pair in the presence of dimethylaminopyridine N-oxide. The resulting DNA chips were shown to be useful in principle for DNA detection.

[Detection of the DeltaF508 trinucleotide deletion in the human CFTR gene by UV immobilization on nylon of a complex of a PCR fragment with a highly specific probe]. / Vyiavlenie trinukleotidnoi deletsii DeltaF508 v gene CFTR cheloveka putem UF-immobilizasii na kaprone kompleksa PTsR-fragmenta s vysokospetsifichnym zondom.

Deletion delta F508 has been revealed in PCR-amplified regions of human gene CFTR by color detection of the hybridization complex obtained by ligation of a tandem of short oligonucleotides on a DNA template followed by UV immobilization on nylon. The method allows reliable detection of the three-nucleotide deletion (insertion). The nonspecific signal depends on the nucleotide composition of the biotinylated tandem component. A significant level of the specific signal was achieved by using the PCR-amplified DNA fragments of different length (200-400 bp) irrespective of the position of the tandem-binding site in their sequences.

[Cleavage of RNA in a hybrid duplex by E. coli ribonuclease H. III. Substrate characteristics of hybrid duplexes of RNA and oligodeoxyribonucleotides containing a bleomycin A5 residue]. / Rasshcheplenie RNK v sostave gibridnykh dupleksov ribonukleazoi H E. coli. III. Substratnye svoistva gibridnykh dupleksov RNK i oligodezoksiribonukleotidov, soderzhashchikh ostatok bleomitsina A5.

The effect of the bleomycin A5 residue linked to four-, eight-, and twelve-mer oligodeoxyribonucleotides on the substrate properties of their tandem and continuous (with or without unmodified octanucleotide effectors) hybrid duplexes was studied using E. coli RNase H. The bleomycin derivatives of oligodeoxyribonucleotides were shown to form hybrid duplexes with practically the same thermostability as those formed by unmodified oligodeoxyribonucleotides. The RNA in the bleomycin-containing hybrid duplexes is cleaved by the E. coli RNase H; however, the initial hydrolysis rate (v0) is 2.6-3.4-fold reduced for the continuous duplexes. In the case of tandem hybrid complexes, the effect of bleomycin on v0 was less pronounced. We hypothesized that steric factors play a key role in the bleomycin inhibition and effectors probably determine the substrate properties of such hybrid complexes. Of all the tandem systems studied, the RNA duplex with the bleomycin-containing tetranucleotide flanked with two effectors displayed the best substrate properties. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

[A new approach to detect a particular DNA sequence by UV-immobilization of its hybridization complex with a highly specific probe resulting from ligation of a tandem of short oligonucleotides in solution]. / Novyi podkhod k vyiavleniiu analiziruemoi posledovatel'nosti DNK putem UF-immobilizatsii ee gibridizatsionnogo kompleksa s vysokospetsifichnym zondom, obrazuiushchimsia pri ligirovanii tandema korotkikh oligonukleotidov v rastvore.

A new approach was proposed for detecting amplified DNA fragments by hybridization with a highly selective oligonucleotide probe obtained by ligation of a tandem of three short oligonucleotides (pN8 + pN4 + pN8' Bio) in solution, with subsequent UV-immobilization of the hybridization product on a nylon membrane and its colorimetric detection with the streptavidin-alkaline phosphatase technique. Owing to the high selectivity of ligation, the 20-mer ligation product was detected on a membrane only when it was completely complementary to a template fragment. The results showed that any single-nucleotide substitution in the tetramer-binding site can be localized and identified with the use of all 12 possible tetramers.

Nuclease resistance and RNase H sensitivity of oligonucleotides bridged by oligomethylenediol and oligoethylene glycol linkers.

The properties of new chimeric oligodeoxynucleotides made of short sequences (tetramers, pentamers, octamers, and decamers) bridged by hexamethylenediol and hexaethylene glycol linkers have been investigated. These chimeric oligonucleotides showed an improved resistance toward snake venom 3'-phosphodiesterase, with an increased stability when a terminal 3'-3'-internucleotide phosphodiester bond is present. It also has been demonstrated that the hybrid complexes formed by bridged oligonucleotides and a complementary 20-mer RNA are able to elicit the activity of ribonuclease H (RNase H) from Escherichia coli. The substrate properties of chimeric oligonucleotides depend on the length of the oligonucleotide fragments bridged by linkers. Introduction of a nonnucleotide spacer into the native oligonucleotide only slightly hampers the extent of the RNA hydrolysis in the hybrid complexes, whereas a modification of the site of reaction is observed as a possible consequence of the steric disturbance due to the aliphatic linkers. Hence, these new chimeric oligonucleotides, namely, short oligonucleotide fragments bridged by nonnucleotide linkers, demonstrate a favorable combination of exonuclease resistance and high substrate activity toward RNase H. As a consequence, these chimeric oligonucleotides could be proposed as new, promising analogs to be used in the antisense strategy.