Substantial Antiviral Potential of Deoxyribozymes Fixed on Anatase Nanoparticles Against Influenza A Viruses in vitro and in vivo.

Influenza A viruses (IAV) are a high threat to humanity because of a lack of proper effective antiviral drugs and resistance of viruses to existing vaccines. We describe the sufficient anti-IAV effect of Ans/PL-Dz nanocomposites that contain deoxyribozymes (Dz) immobilized on anatase TiO2 nanoparticles (Ans) through polylysine linker (PL). The Dz-containing nanocomposites appear to be more efficient than the Ans/PL-ODN nanocomposites that contain common oligodeoxyribonucleotides (ODN) targeted to the same RNA regions of the viral genome. The simultaneous use of nanocomposites that contain Dz and ODN, which are targeted to different sites of viral RNA provides a higher overall effect than the independent action of each of them (synergism). The inhibition of IAV with the proposed nanocomposites was shown to be effective, sequence-specific, and dose-dependent. The most efficient Ans/PL-Dz nanocomposite exhibited a high antiviral effect in vivo on mice models. The efficiency of IAV inhibition with this nanocomposite in vitro and in vivo is higher than that for the approved antiflu drug oseltamivir. The results open the prospect of creating a unique antiviral agent suitable for IAV suppression.

Non-agglomerated silicon-organic nanoparticles and their nanocomplexes with oligonucleotides: synthesis and properties.

The development of efficient and convenient systems for the delivery of nucleic-acid-based drugs into cells is an urgent task. А promising approach is the use of various nanoparticles. Silica nanoparticles can be used as vehicles to deliver nucleic acid fragments into cells. In this work, we developed a method for the synthesis of silicon-organic (Si-NH2) non-agglomerated nanoparticles by the hydrolysis of aminopropyltriethoxysilane (APTES). The resulting product forms a clear solution containing nanoparticles in the form of low molecular weight polymer chains with [─Si(OH)(C3H6NH2)O─] monomer units. Oligonucleotides (ODN) were conjugated to the prepared Si-NH2 nanoparticles using the electrostatic interaction between positively charged amino groups of nanoparticles and negatively charged internucleotide phosphate groups in oligonucleotides. The Si-NH2 nanoparticles and Si-NH2·ODN nanocomplexes were characterized by transmission electron microscopy, atomic force microscopy and IR and electron spectroscopy. The size and zeta potential values of the prepared nanoparticles and nanocomplexes were evaluated. Oligonucleotides in Si-NH2·ODN complexes retain their ability to form complementary duplexes. The Si-NH2 Flu nanoparticles and Si-NH2·ODNFlu nanocomplexes were shown by fluorescence microscopy to penetrate into human cells. The Si-NH2 Flu nanoparticles predominantly accumulated in the cytoplasm whereas ODNFlu complexes were predominantly detected in the cellular nuclei. The Si-NH2·ODN nanocomplexes demonstrated a high antisense activity against the influenza A virus in a cell culture at a concentration that was lower than their 50% toxic concentration by three orders of magnitude.

High antiviral effect of TiO2·PL-DNA nanocomposites targeted to conservative regions of (-)RNA and (+)RNA of influenza A virus in cell culture.

Background: The development of new antiviral drugs based on nucleic acids is under scrutiny. An important problem in this aspect is to find the most vulnerable conservative regions in the viral genome as targets for the action of these agents. Another challenge is the development of an efficient system for their delivery into cells. To solve this problem, we proposed a TiO2·PL-DNA nanocomposite consisting of titanium dioxide nanoparticles and polylysine (PL)-containing oligonucleotides. Results: The TiO2·PL-DNA nanocomposites bearing the DNA fragments targeted to different conservative regions of (-)RNA and (+)RNA of segment 5 of influenza A virus (IAV) were studied for their antiviral activity in MDCK cells infected with the H1N1, H5N1, and H3N2 virus subtypes. Within the negative strand of each of the studied strains, the efficiency of DNA fragments increased in the direction of its 3'-end. Thus, the DNA fragment aimed at the 3'-noncoding region of (-)RNA was the most efficient and inhibited the reproduction of different IAV subtypes by 3-4 orders of magnitude. Although to a lesser extent, the DNA fragments targeted at the AUG region of (+)RNA and the corresponding region of (-)RNA were also active. For all studied viral subtypes, the nanocomposites bearing the DNA fragments targeted to (-)RNA appeared to be more efficient than those containing fragments aimed at the corresponding (+)RNA regions. Conclusion: The proposed TiO2·PL-DNA nanocomposites can be successfully used for highly efficient and site-specific inhibition of influenza A virus of different subtypes. Some patterns of localization of the most vulnerable regions in IAV segment 5 for the action of DNA-based drugs were found. The (-)RNA strand of IAV segment 5 appeared to be more sensitive as compared to (+)RNA.

Knockdown of different influenza A virus subtypes in cell culture by a single antisense oligodeoxyribonucleotide.

Influenza is a heavy socially significant viral infection that affects humans, birds, and wild and domestic animals. The threat of existing and new highly pathogenic subtypes of influenza A virus (IAV) makes it necessary to develop an effective drug that may affect different IAV strains. For this purpose, oligodeoxynucleotides (DNA fragments) attached to titanium dioxide (TiO2) nanoparticles through a polylysine linker, forming TiO2·PL-DNA nanocomposites, that penetrated into cells without transfection agents were used. For the first time, efficient (≥99.9%) suppression of the reproduction of different subtypes of IAV, including highly pathogenic H5N1 and H1N1, was achieved. These results were obtained using the TiO2·PL-DNA nanocomposite bearing a single antisense oligodeoxynucleotide (0.1µM) targeted to the conserved 3'-noncoding region of RNA segment 5, which is common to all tested strains. Very efficient suppression of the reproduction of different subtypes of IAV was probably achieved due to the use of the proposed delivery system for oligonucleotides in the form of the TiO2·PL-DNA nanocomposites. These results indicate the possibility of creating an efficient drug to affect existing and newly emerging pathogenic IAV strains.

SiO2 nanoparticles as platform for delivery of 3'-triazole analogues of AZT-triphosphate into cells.

A system for delivery of analogues of AZT-triphosphates (AZT*TP) based on SiO2 nanoparticles was proposed. For this purpose, a simple and versatile method was developed for the preparation of SiO2∼dNTP conjugates using the 'click'-reaction between AZTTP and premodified nanoparticles containing the alkyne groups. The substrate properties of SiO2∼AZT*TP were tested using Klenow fragment and HIV reverse transcriptase. The 3'-triazole derivatives of thymidine triphosphate being a part of the SiO2∼AZT*TP nanocomposites were shown to be incorporated into the growing DNA chain. It was shown by confocal microscopy that the proposed SiO2∼AZT*TP nanocomposites penetrate into cells. These nanocomposites were shown to inhibit the reproduction of POX and Herpes viruses at nontoxic concentrations.

SiO2 nanoparticles as platform for delivery of nucleoside triphosphate analogues into cells.

A system for delivery of analogues of 2'-deoxyribonucleoside triphosphate (dNTP) based on SiO(2) nanoparticles was proposed. A simple and versatile method was developed for the preparation of SiO(2)-dNTP conjugates using the 'click'-reaction between premodified nanoparticles containing the azido groups and dNTP containing the alkyne-modified γ-phosphate group. The substrate properties of SiO(2)-dNTP were tested using Klenow fragment and HIV reverse transcriptase. Nucleoside triphosphates being a part of the SiO(2)-dNTP nanocomposites were shown to be incorporated into the growing DNA chain. The rate of polymerization with the use of SiO(2)-dNTP or common dNTP in case of HIV reverse transcriptase differed insignificantly. It was shown by confocal microscopy that the proposed SiO(2)-dNTP nanocomposites bearing the fluorescent label penetrate into cells and even into cellular nuclei.

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 TiO(2)·PL-DNA nanocomposites (polylysine-containing DNA fragments noncovalently immobilized onto TiO(2) 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 (TC(50) ≈ 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. IC(50) 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.

Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in 80S ribosomes.

To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive mRNA analogs were applied. Derivatives of the UUCUAAA heptaribonucleotide containing the UUC codon for Phe and the stop signal UAAA, which bore a perfluoroaryl azido group at either the fourth nucleotide or the 3'-terminal phosphate, were synthesized. The UUC codon was directed to the ribosomal P site by the cognate tRNA(Phe), targeting the UAA stop codon to the A site. Mild UV irradiation of the ternary complexes consisting of the 80S ribosome, the mRNA analog and tRNA resulted in tRNA-dependent crosslinking of the mRNA analogs to the 40S ribosomal proteins and the 18S rRNA. mRNA analogs with the photoreactive group at the fourth uridine (the first base of the stop codon) crosslinked mainly to protein S15 (and much less to S2). For the 3'-modified mRNA analog, the major crosslinking target was protein S2, while protein S15 was much less crosslinked. Crosslinking of eukaryotic (e) RF1 was entirely dependent on the presence of a stop signal in the mRNA analog. eRF3 in the presence of eRF1 did not crosslink, but decreased the yield of eRF1 crosslinking. We conclude that (i) proteins S15 and S2 of the 40S ribosomal subunit are located near the A site-bound codon; (ii) eRF1 can induce spatial rearrangement of the 80S ribosome leading to movement of protein L4 of the 60S ribosomal subunit closer to the codon located at the A site; (iii) within the 80S ribosome, eRF3 in the presence of eRF1 does not contact the stop codon at the A site and is probably located mostly (if not entirely) on the 60S subunit.