Marina A. Zenkova

Isolation of ribosomal subunits containing intact rRNA from human placenta: Estimation of functional activity of 80S ribosomes

We have elaborated a method for the isolation of ribosomal subunits from fresh unfrozen human placenta containing intact rRNA and a complete set of ribosomal proteins. Activity of 80S ribosomes obtained by reassociation of 40S and 60S subunits in nonenzymatic poly(U)-dependent binding of Phe-tRNA(Phe) was equal to 80% (above 1.5 mol [14C]Phe-tRNA(Phe) is coupled to 1 mol of ribosomes). The activity of 80S ribosomes in poly(U)-directed synthesis of polyphenylalanine was tested in a polysome-free protein-synthesizing system from rabbit reticulocytes. About 100 mol of phenylalanine residue was polymerized by a mole of ribosomes at a rate of 0.83 residues per minute in this system (2 h, 37 degrees C).

Novel Cholesterol-Based Cationic Lipids for Gene Delivery

Gene therapy based on gene delivery is a promising strategy for the treatment of human disease. Here we present data on structure/biological activity of new biodegradable cholesterol-based cationic lipids with various heterocyclic cationic head groups and linker types. Enhanced accumulation of nucleic acids in the cells mediated by the lipids was demonstrated by fluorescent microscopy and flow cytometry. Light scattering and atomic force microscopy were used to find structure/transfection activity correlations for the lipids. We found that the ability of the lipids to stimulate intracellular accumulation of the oligodeoxyribonucleotides and plasmid DNA correlates well with their ability to form in solution lipid/NA complexes of sizes that do not exceed 100 nm. Screening of the lipids revealed the most promising transfection agents both in terms of low toxicity and efficient delivery: cholesterol-based lipids with positively charged pyridine and methyl imidazole head groups and either the ester or carbamate linker.

Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group

The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5′-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5′-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.

Artificial ribonucleases: synthesis and RNA cleaving properties of cationic conjugates bearing imidazole residues

Abstract Small mimics of the ribonuclease active centre were synthesized by conjugating imidazole residues to a dicationic compound. These compounds were shown to cleave tRNA under physiological conditions. The compounds provide new probes for the investigation of RNA structure in solution and potential catalytic RNA cleaving groups for antisense oligonucleotide derivatives.

Nucleic acids in exosomes: Disease markers and intercellular communication molecules

The term “exosomes” is currently used to describe specific vesicular structures of endosomal origin produced by the majority of eukaryotic cells. These natural vesicles have been under study for more than two decades. Nevertheless, a real splash of scientific interest in studies on exosomes took place only during recent years, when the concept of the role and functions of exosomes in multicellular organisms was essentially reconsidered. The major role in this was played by the discovery of exosomal mRNA and miRNA in 2007, which stimulated the idea of regulatory and communicative role of exosomes in the organism and also encouraged considering exosomes and other vesicles as potential biomarkers. The present review summarizes the up to date knowledge on the composition and probable physiological functions of nucleic acids released by different cells as components of exosomes. We also touch upon the problem of using these data in clinical diagnosis.

Novel cationic liposomes provide highly efficient delivery of DNA and RNA into dendritic cell progenitors and their immature offsets

Here we report on the application of cationic liposomes formed by new cationic lipids and the lipid-helper DOPE (dioleoylphosphatidylethanolamine) for the transfection of plasmid DNA and mRNA into dendritic cells (DCs) progenitors and immature DCs of bone-marrow origin in vitro and the use of these DCs to induce the suppression of B16 melanoma metastases in vivo. The cationic lipids contain one (X2, S1, S2 and S3) or two (2X3) cholesterol residues or long-chain hydrocarbon substituent (2D3) linked with spermine. Data show that liposomes 2X3-DOPE, 2D3-DOPE, X2-DOPE and S2-DOPE display high transfection efficiency in respect to DNA (30-47% of DC progenitors and up to 57% of immature DC were transfected) and RNA (up to 57% of cells were transfected). The studied lipids exhibited an efficiency of DNA and RNA delivery in DCs several times higher in comparison with Lipofectamine 2000. Observed ex vivo the higher transfection efficiencies of DCs with mRNAs encoding of a set of tumor-associated antigens provided by cationic liposomes 2X3-DOPE and 2X2-DOPE corresponded to a 3-5 fold suppression of metastasis number in a model of murine B16 melanoma in vivo. The injection into mice of these pulsed DCs resulted in a slight pro-inflammatory response which was balanced by the positive effect of the antitumor cytokine production induced by the DCs. The obtained data show that the novel spermine-based polycationic lipids can be applied in the preparation of antitumor DC-based vaccine.

Studying functional significance of the sequence 980–1061 in the central domain of human 18S rRNA using complementary DNA probes

Region 980-1061 in human 18S rRNA has been chosen on the basis of our previous results, indicating that cross-linking sites of the alkylating mRNA analogs are located within this region. In the present study, we have used 10 DNA 15-mers complementary to various overlapping sequences within the 18S rRNA positions 980-1061. Their abilities to bind selectively to the target rRNA sequences were proved by hydrolysis of 18S rRNA within heteroduplexes with the corresponding probes by RNase H. Four sequences (980-994, 987-1001, 1025-1039 and 1032-1046) were found to be well accessible for binding of the respective cDNA probes within 40S subunits. None of the oligomers inhibited tRNA(Phe)-dependent binding of oligo(U) messenger to 40S subunits and binding of Met-tRNA(imet) to 40S subunits in the presence of eIF-2 and nonhydrolysable GTP analog. Nevertheless, two probes (complementary to the 18S rRNA sequences 987-1001 and 1025-1039) being covalently attached to 40S subunits, inhibited translation of poly(U) by human 80S ribosomes in a cell-free system. The same oligomers revealed the most pronounced inhibitory action on the binding of messenger trinucleotide in the complex pAUG.40S.Met-tRNA(imet).eIF-2.GTP. Results of these functional assays demonstrate the importance of the 18S rRNA sequences 987-1001 and 1025-1039 for translation process on human ribosomes, most probably at the initiation step.

Inactivation of a non-enveloped RNA virus by artificial ribonucleases: Honey bees and Acute bee paralysis virus as a new experimental model for in vivo antiviral activity assessment

RNA-containing viruses represent a global threat to the health and wellbeing of humans and animals. Hence, the discovery of new approaches for the design of novel vaccines and antiviral compounds attains high attention. Here we describe the potential of artificial ribonucleases (aRNases), low molecular weight compounds capable to cleave phosphodiester bonds in RNA under mild conditions, to act as antiviral compounds via destroying the genome of non-enveloped RNA viruses, and the potential of utilizing honey bee larvae and adult bees (Apis mellifera) as a novel experimental system for the screening of new antiviral compounds. Pre-incubation of an Acute bee paralysis virus (ABPV) suspension with aRNases D3-12, K-D-1 or Dp12F6 in a concentration-dependent manner increased the survival rate of bee larvae and adult bees subsequently infected with these preparations, whereas incubation of the virus with aRNases ABL3C3 or L2-3 had no effect at all. The results of RT-PCR analysis of viral RNA isolated from aRNase-treated virus particles confirmed that virus inactivation occurs via degradation of viral genomic RNA: dose-dependent inactivation of ABPV correlates well with the cleavage of viral RNA. Electron microscopy analysis revealed that the morphology of ABPV particles inactivated by aRNases remains unaffected as compared to control virus preparations. Altogether the obtained results clearly demonstrate the potential of aRNases as a new virus inactivation agents and bee larvae/ABPV as a new in vivo system for the screening of antiviral compounds.

Cleavage of yeast tRNAPhe with complementary oligonucleotide conjugated to a small ribonuclease mimic.

An oligonucleotide conjugate bearing a chemical construct mimicking the catalytic center of ribonuclease A has been designed and studied. The conjugate efficiently cleaves yeast tRNAPhe at a single site adjacent to the target complementary sequence.

Synthesis and Pro-Apoptotic Activity of Novel Glycyrrhetinic Acid Derivatives

Triterpenoids are used for medicinal purposes in many countries. Some, such as oleanolic and glycyrrhetinic acids, are known to be anti‐inflammatory and anticarcinogenic. However, the biological activities of these naturally occurring molecules against their particular targets are weak, so the synthesis of new synthetic analogues with enhanced potency is needed. By combining modifications to both the A and C rings of 18βH‐glycyrrhetinic acid, the novel synthetic derivative methyl 2‐cyano‐3,12‐dioxo‐18βH‐olean‐9(11),1(2)‐dien‐30‐oate was obtained. This derivative displays high antiproliferative activity in cancer cells, including a cell line with a multidrug‐resistance phenotype. It causes cell death by inducing the intrinsic caspase‐dependent apoptotic pathway.