Том 45, № 6 (2019)

The discovery of RNA-guided nucleases have enabled to leap forward in genome editing of cells and organisms. These nucleases can be delivered into cells as plasmid DNA, mRNA or ribonucleoprotein complexes (RNPs). The delivery in the form of RNP has some advantages because the target gene editing begins immediately without the process of intracellular synthesis of components and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated protein 9) system assembly. This strategy makes it possible to directly control RNP concentration and to decrease the number of off-targets due to rapid degradation of the complex in the cell. However, the task to develop RNP delivery systems remains unsolved. This review is devoted to RNP delivery into cells and tissues using physical approaches and different carriers. Special attention is paid to novel approaches that improve the RNP delivery efficiency.

Modern data are presented on the pharmaceutical application of natural polysaccharides, such as pullulan, alginates, hyaluronic acid, dextrans, pectin, chitosan, and arabinogalactan, which are already actively used or can be used in the future for optimizing different drug forms, water solubility and improving bioavailability. The emphasis on natural polysaccharides is made in relation to their good biocompatibility, biodegradation, hydrophilicity, good stability, safety, lack of toxicity, and adhesive properties. The indicated parameters are important and necessary for agents used for drug delivery into the human body.

Biogenic polyamines spermine and spermidine are present in eukaryotic cells in micro- and millimolar concentrations, that determines the multiplicity of their functions and the necessity to support normal cell growth. Analogs and derivatives of spermine and spermidine are widely used in biochemistry of polyamines, a number of fundamentally significant results for the field were obtained with their help. C-methylated analogs of polyamines are unique, since among these compounds functionally active in vitro and in vivo spermidine and spermine mimetics were found. Biochemical properties of the compounds of this family can be regulated by moving the methyl group along the backbone of a polyamine and/or changing the configuration of the chiral center. The peculiarities of the interaction of C-methylated analogs of polyamines with the enzymes of their metabolism, the activity in the cell culture and the methods of synthesizing these compounds are discussed.

The misuses of antimicrobials in infectious diseases have led to the progress of extensive resistance in the infectious organisms. The failure of accessible antimicrobials to control infections makes it essential to discover alternatives to currently existing drugs. Their connection to infectious diseases and their natural ability to increase antimicrobial resistance in microbes, has led to platforms for research focused on new techniques to control them. Pathogenicity in many bacteria is regulated by quorum sensing (QS). Inhibition of QS system may cause reduction of virulence and protect against bacterial infections. These bacteria rely on chemical communication (or QS) to coordinate activities necessary for their survival in groups by some course of action. Their dependence on QS has made those signaling systems within bacteria an attractive target for the design of new anti-infective agents. Compounds that can interrupt these processes are known as QS inhibitors. The QS is the key regulator of virulence in various bacteria. Various plant extracts and their chemical constituents exhibited their effects on bacterial virulence factors by inhibiting QS genes and QS-controlled factors and effects on bacterial growth. The anti-QS approach is a promising one in the fight against infections pathogens, thereby making the bacteria more susceptible to traditional antimicrobials. The QS inhibitors (QSI) may provide the newest weapon against infections involving drug-resistant bacterial strains. These QSIs come from a variety of sources and have a wide array of structures.

Pharmacokinetics of the promising antitumor peptide HLDF-6-AA (Ac-ThrGlyGluAsnHisArg-NH₂) was studied using its uniformly tritiated derivative. Experiments were performed on male Wistar rats, Balb/c mice and Chinchilla rabbits. The tritium labeled peptide [³H]HLDF-6-AA with the molar radioactivity of 50 Ci/mmol was obtained by the reaction of high-temperature solid-state catalytic isotope exchange (HSCIE). Under intravenous bolus administration of HLDF-6-AA peptide to rats and rabbits, the characteristics of its pharmacokinetic profile in the blood were obtained and the values of the main pharmacokinetic parameters of HLDF-6-AA peptide, and its active metabolite HisArg-NH₂ were calculated. It was shown that parameters of the retention time in the body and the rate of elimination for peptides HLDF-6-AA and HisArg-NH₂ in rats and rabbits are close and in rats they are about 7 and 21 min, respectively. It was shown that repeated administration of the drug does not lead to a change in its regular cumulation and does not cause a change in its pharmacokinetics compared with a single administration. The linearity of the dependence of pharmacokinetic parameters on the amount of administered peptide in the range of 2–22 mg/kg was proved in experiments in rats. As a result of the study of the distribution of HLDF-6-AA peptide and its metabolite HisArg-NH₂ between the blood and peripheral tissues of mice, it was shown that the maximum concentration of HLDF-6-AA peptide is observed in the kidney tissues and a somewhat smaller concentration in the omentum. It was found that 15–20 min after intraperitoneal administration of HLDF-6-AA to mice, the concentration of HisArg-NH₂ peptide begins to exceed the concentration of HLDF-6-AA peptide, which is caused by its greater resistance to proteolytic hydrolysis. The highest concentration of HisArg-NH₂ peptide is observed in the kidney and liver tissues.

Porphyromonas gingivalis is the main pathogen of periodontitis. The purpose of this study was to analyze the effect of p62 protein on P. gingivalis-induced autophagy in human periodontal ligament fibroblast (HPDLF). HPDLF cells were first infected with P. gingivalis; autophagy and p62 expression levels were observed at different time points (0, 12, 18, and 24 h) to detect the relationship between p62 and autophagy over time. Autophagy and apoptosis of HPDLF cells infected with P. gingivalis were detected using siRNA specific interference with p62 expression. Fluorescence microscopy and electron microscopy showed autophagic vacuoles in HPDLF cells infected with P. gingivalis. Western blotting showed that p62 expression peaked at 12 h and began to decrease at 18 h. The expression of p62 was silenced by siRNA compared with the control group; the ratio of LC3-II/LC3-I decreased significantly, while the expression level of caspase-3 increased significantly. The the CCK-8 cell viability assay showed that the silencing of p62 reduced HPDLF cell viability, and the plate coating method showed that the silencing of p62 reduced the survival rate of P. gingivalis in the HPDLF cells. Therefore, in vitro studies showed that the presence of p62 is beneficial for maintaining the level of autophagy in HPDLF cells induced by P. gingivalis and inhibiting apoptosis. p62 facilitates the removal of P. gingivalis that are invading cells during this process.

A series of diterpenes that contain the morpholine, pyrrolidine, benzyl, nitrophenyl, and 1,2,3-tetrazole heterocyclic fragments have been synthesized from acetylene derivatives of abietic, 7-formylabietic, dehydroabietic, maleopimaric, and dihydroquinopimaric acids. The antibacterial activity of the synthesized compounds against microorganisms and pathogenic fungi has been studied, and the primary assessment of their cytotoxicity and hemolytic activity has have been carried out. It has been found that the acetylene derivatives of abietic and dehydroabietic acids that contain the pyrrolidine substituent can effectively inhibit the growth of fungi Candida albicans and Cryptococcus neoformans, and their low hemolytic ability has been shown.

The regioselective synthesis of betulin 3,28-bis- and 28-monoacylates (nicotinate and isonicotinates) as well as of 3β-hemisuccinyl dipterocarpol was carried out using the dicyclohexylcarbodiimide method. The morphological and biochemical blood tests on a model of CCl₄-induced rat hepatitis showed that the activities of the betulin bisacylates 3β,28-bis-О-nicotinate and 3β,28-bis-О-isonicotinate were higher than that of monoacylate 28-О-isonicotinate. 3β-Hydroxydipterocarpol was found to be the most active among triterpenoids of the dammarane type.

In this study, a new series of succinimide hybrid molecules containing isothiocyanate, coumarin, isatin, and furan moieties was synthesized and screened for α-glucosidase and antioxidant activities. Preliminary results revealed that all molecules showed good α-glucosidase inhibition effectiveness. Antioxidant activities of the hybrid molecules were determined using cupric reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP) assays. Also, the radical scavenging activities of the synthesized compounds were assayed by using \({\text{ABT}}{{{\text{S}}}^{{\centerdot {\text{ + }}}}}\) and \({\text{DPP}}{{{\text{H}}}^{\centerdot }}\) methods. The results showed that all compounds exhibited moderated to high scavenging activity.

DNA-substrates containing fluorescent DNA base analogs are widely used to study protein–nucleic acid interactions. In the case of DNA-recognizing enzymes, this approach allows one to register conformational changes in DNA during the formation of enzyme–substrate complexes. An important part of such research is the design of model DNA substrates, which includes both the photophysical properties of the fluorescent groups and their location relative to a specific recognition site, namely, in the same chain on the 5′-, 3′-side or in the complementary chain opposite the specific site. In this work, we report a comparative study of the sensitivity of various fluorescent DNA base analogs, such as 2-aminopurine (aPu), pyrrolocytosine (C^Py), 1,3-diaza-2-oxophenoxazine (tC^O) and 3-hydroxychromone (3HC), to conformational transformations of DNA in the process of interaction with formamidopyrimidine-DNA glycosylase (Fpg) from Escherichia coli.

Recently, three-dimensional nucleic acid nanostructures have attracted great interest, which have been made available through the DNA origami technique. We have proposed a different way of constructing nucleic acid nanoobjects, namely, template-directed assembly employing branched oligonucleotides as templates and building blocks, which include nonnucleotidic linkers, particularly, branching units for connecting three or more oligonucleotide chains. For the design and 3D modelling of such nanostructures as DNA tetrahedron, DNA cube and DNA fullerene C24, we have used Blender software. As we found, Blender not only allows one to visualize complex DNA and RNA nanostructures, but also helps to choose the parameters for their synthesis.

Only a limited number of tools are available to study cytosine methylation in DNA. One of the representatives of the recently discovered methyl-dependent DNA endonucleases is an enzyme GlaI. It is of great interest for determining the methylation status of eukaryotic genomic DNA due to its ability to cleave only methylated DNA. However, the ability of the GlaI endonuclease to recognize oxidized derivatives of 5-methylcytosine (^mC), in particular another epigenetic base, 5-hydroxymethylcytosine (^hmC), has not yet been characterized. It is not possible to fully use the potential of GlaI in the analysis of methylation due to the notable occurrence of the latter in the DNA of mammals. In this study, the efficiency of cleavage of DNA substrates with various combinations of ^mC and ^hmC by methyl-dependent DNA-endonuclease GlaI was compared; the kinetic parameters of cleavage reactions for fully methylated and fully hydroxymethylated recognition site were determined. It was shown that in most cases GlaI recognized substrates containing ^mC better than substrates containing ^hmC in the same positions. The most effective hydrolysis of substrates containing modifications in the sequence 5'-GCGC-3'/3'-CGCG-5' required the presence of ^hmC not only in the central but also in the edge positions in both DNA chains as in the case of ^mC.

Platinum complexes are among the most commonly prescribed drugs in cancer therapy but show significant toxicity to healthy tissues as a side effect. Platinum polyoxometalates are platinum complexes liganded with cluster anions consisting of oxygen atoms and transition metals. Their properties related to a potential use as anticancer drugs have never been studied. In this paper, we have investigated the effect of platinum (IV) polyoxoniobate of the [Nb₆O₁₉{Pt(OH)₂}]₂ structure containing two platinum centers and two polynuclear Lindqvist type anions on the activity of a number of DNA polymerases belonging to different families (Klenow fragment of Escherichia coli DNA polymerase I, bacteriophage RB69 DNA polymerase, human DNA polymerases β and κ, DNA polymerase IV from Sulfolobus solfataricus). On its own, platinum polyoxoniobate did not act as an inhibitor of DNA polymerases, but was capable of forming adducts with DNA.

Aptamers are DNA or RNA molecules that specifically bind certain targets with high affinity due to the formation of unique spatial structures. Aptamers with specified properties could be obtained from combinatorial libraries of nucleic acids by in vitro selection procedure. Here, we optimized conditions of chemical synthesis of DNA library on the automated ASM-800 DNA/RNA synthesizer. Enzymatic hydrolysis and high-throughput sequencing methods were used to analyze compositions of synthesized combinatorial libraries. The DNA library generated under the chosen conditions is characterized by the sufficiently uniform randomization and can be applied for the in vitro selection.

One of the promising strategies in the development of efficient antibacterial drugs is the use of antisense oligonucleotides specifically interacting with mRNAs of genes controlling the key functions of the bacterial cell such as survival and reproduction. The present paper reports the development of an algorithm for searching and testing the antibacterial activity of antisense oligonucleotides using mRNA of the rpoD gene encoding the sigma factor of the Staphylococcus aureus RNA polymerase. Based on the calculated thermodynamic parameters and the rpoD mRNA 5'-fragment (1–377 nt region) secondary structure model, 11 antisense oligonucleotides 18–20 nt in length complementary to the rpoD mRNA regions with unstable secondary structure have been selected. Hybridization of the selected oligonucleotides with the total S. aureus RNA followed by selective in vitro digestion of antisense oligonucleotide/mRNA rpoD heteroduplexes with RNase H and assessment of rpoD mRNA digestion rate using PCR revealed six candidate sequences which reduced the rpoD mRNA level by 2.2–4 times. Phosphorothioate oligonucleotides targeted to the regions 1–20, 30–47, and 82–101 of the rpoD mRNA synthesized on the basis of these sequences efficiently suppressed the growth of S. aureus bacteria in vitro. It has been demonstrated that the efficiency of penetration of these oligonucleotides into the bacterial cell may be significantly increased by introducing arginine-rich peptide into the oligonucleotide structure. As a result of this work, we have developed a simple and efficient algorithm for selection of antisense oligonucleotides against bacterial RNAs encompassing all stages starting from the binding stage and ending with the analysis of biological activity.

In this work, the possibility of precise physicochemical analysis of formation of nucleic acid complexes has been demonstrated using thermal cyclers with fluorescence signal detection. Individual features of heating blocks of various thermal cyclers have been identified. A nonlinear correlation between fluorescence signal intensity and concentration of fluorescent dyes (5,6)-carboxyfluorescein and Rhodamine B has been identified. Using UV and fluorescence melting, the comparison of melting curves at various concentration of full-complementary and mismatched (A/A and C/A) complexes has been conducted. The experimental values of thermodynamic parameters of complex formation for full-complementary and mismatched complexes have been obtained.

This work describes the basic physicochemical properties of phosphoryl guanidine oligonucleotides (PGOs)—a new type of DNA analog with a partially or totally uncharged backbone. Replacement of the negatively charged oxygen atom with an electroneutral 1,3-dimethylimidazolidine-2-imine (DMI) group is shown to influence the hydrophobicity of oligonucleotide, its electrophoretic mobility, and UV absorption spectra. Thermal stability of DMI-containing DNA-duplexes was studied in aqueous solutions with different ionic strength. It is determined that, in the case of low ionic strength, the presence of DMI groups significantly enhances the thermal stability of the duplex. In the case of nearly physiological condition (0.1 M NaCl, 10 mM MgCl₂) or higher ionic strength, DMI groups do not affect the duplex stability or even slightly reduce it (melting temperature changes, on average, by –1.2°C per one DMI group). An important feature of totally uncharged PGO is its ability to form the duplex effectively regardless the ionic strength, even in deionized water. In addition, according to CD data, DMI groups do not disturb the double helix structure of DNA, maintaining it in the B form.

In 2018, three scientists shared the Nobel Prize in Chemistry: Frances H. Arnold, “for the directed evolution of enzymes”; George P. Smith and Sir Gregory P. Winter, “for the phage display of peptides and antibodies”. All authors are associated with the development of methods for obtaining the useful proteins and peptides, which are based on imitation of the natural “method” of biological evolution, namely, on a combination of random variability and nonrandom selection. In this review, we consider modern methods for designing engineering polymerases, which determine the progress in the enzymatic synthesis and evolution of unnatural nucleic acids. The development of these methods provides an opportunity to obtain and study a huge set of new biopolymers inaccessible to nature, as well as various ligands, catalysts, and materials based thereon.

Interferon-regulated factors play a central role in the activation of the innate immune response. The interferon-regulatory factor 7 (IRF7) is one of the factors that are quickly activated and are involved in a cellular response to a viral infection. In this work, monoclonal lines, based on HEK293FT cells defective in the IRF7 gene, were obtained using a CRISPR-Cas9 genome-editing method. These lines differed in the viability, proliferation rate, and susceptibility to infection with influenza A virus. Transcriptomic analysis of the most susceptible cell clone revealed differential expression of IRF7 factor as well as the other interferon-regulated genes.

The attachment of lipophilic molecules of natural origin, which have natural means for cell internalization, to small interfering RNA (siRNA) is an effective way of delivering siRNA to cells for biomedical purposes in vitro and in vivo. Earlier, we showed that the attachment of cholesterol to the 5'-end of the sense strand of nuclease-resistant siRNA through the optimized linker allows it to penetrate the cells and suppress the expression of the target gene. However, the effectiveness of the conjugates is different for cells of different origin, and in hematopoietic cells, they are not active, despite effective accumulation. In this work, we investigated the accumulation of fluorescently labeled cholesterol conjugates of siRNA using endocytosis inhibitors and showed that fluorescently labeled 5'-cholesterol conjugate of siRNAs penetrate KB-3-1 and K562 cells in several ways whose contribution differs depending on cell type and the presence of serum. In a serum-free medium, it was found that macropinocytosis and clathrin-dependent endocytosis contribute to the accumulation of the conjugate in KB-3-1 cells, while clathrin-dependent endocytosis makes the main contribution in K562 cells, while inhibitors of different types of endocytosis do not reduce the biological activity of the conjugate without a fluorescent label.

Brain tumors are among the most intractable types of malignant neoplasms. Despite advances in the treatment of cancer, in particular, the development of new approaches in surgery, radiotherapy and chemotherapy, the incidence remains high with a low 5-year survival rate. Targeted therapy (TT) may be a solution to the problem of low efficacy of the applied cancer treatment methods. TT is based on the use of drugs that specifically affect specific types of tumors, which allows one to increase the effectiveness of treatment and minimize toxic effects on healthy tissues of the body. The combination of the unique properties of cancer cells allows one to find specific ligands that interact directly with the tumor, and to conduct TT for malignant tumors. Phage display technology is one of the promising approaches to the search for tissue- and/or organ-specific molecules [1]. Combinatorial phage peptide libraries make it possible to obtain highly specific peptides, including for various types of tumors. Currently, such tumor-targeting peptides are considered as means of targeted delivery of therapeutic genes, cytokines, imaging agents, pro-apoptotic peptides, and cytotoxic drugs. The purpose of this work was to obtain from the phage peptide library of bacteriophages exposing peptides, which ensure the accumulation of phage particles in human glioblastoma cells U-87 MG, and to assess the specificity of the selected peptides for cells of the primary cultures of human gliomas. During the research, the following tasks were solved: (1) Tumor-targeting peptides were selected using human glioblastoma cells U-87 MG in vitro and on a U-87 MG tumor in a xenograft model in vivo; amino acid sequences of selected peptides (SWTFGVQFALQH (26), HPSSGSA (92), PVSNKMS (83)) were determined; (2) primary cultures of human gliomas AS2, MG1, MG2, MG3, MG4 cells were obtained and characterized; (3) specificity of the binding of bacteriophages exposing selected tumor-targeting peptides to cells of primary cultures of human gliomas was evaluated using immunocytochemical analysis. The specific binding of selected tumor-targeting peptides with cells of the primary cultures AS2, MG1 was shown.

Nowadays, application of miRNases—artificial ribonucleases aimed at degradation of noncoding RNAs, in particular, miRNAs—represents one of the novel experimental approaches to inhibit tumorigenesis. miRNases integrate in their structure an addressing oligonucleotide, which provides specific binding with miRNA target, and a catalytic group, which promotes cleavage of the RNA substrate. Introduction of chemical modifications to the oligonucleotide domain of miRNases in the region that is complementary to miRNA may significantly increase the hybridization properties and nuclease resistance of this type of compound. However, the influence of such structural changes to the ribonuclease activity of miRNases remains unclear. In this work, to investigate the effect of 2'OMe modifications on the activity of miRNases, we synthesized two types of anti-miRNA-21 conjugates of the peptide [(ArgLeu)₂Gly]₂ and hairpin oligonucleotides in which 14-mer binding region to the miRNA target was fully or partially modified. It is shown that the introduction of 2'OMe modifications promotes a considerable increase in the affinity of miRNases to miRNA-21 but does not change significantly their nuclease resistance. Full modification of conjugates in the region that is complementary to miRNA negatively affects their ribonuclease activity, whereas partial introduction of 2'OMe nucleotides considerably enhances the cleavage activity of miRNases, which leads to a substantial decrease in the proliferation rate and migration potential of tumor cells, which are determined by the miRNA-21 expression.

Keywords: oligonucleotide–peptide conjugates, oncogenic miRNA, miRNA-21, 2'OMe modification, human epidermoid carcinoma KB-8-5.

A novel variant of the synthesis of 3'- and 5'-peptide conjugates of oligo(2'-O-methylribonucleotides) has been developed using thiol-maleimide chemistry. The method is based on the introduction of the maleimide group into an oligonucleotide using a novel bifunctional reagent, pentafluorophenyl ester of 3‑maleimidopropionic acid (MPPf), and the subsequent interaction of the resulting compound with an SH‑bearing peptide, which facilitates cell penetration. A series of RNase P-guiding 3'- and 5'-peptide conjugates of oligo(2'-O-methylribonucleotides) targeted to mRNA of the ftsZ and gyrA genes of Acinetobacter baumannii have been synthesized. The ability of these conjugates to guide the hydrolysis of model RNA targets by RNase P has been demonstrated. It has been shown that the introduction of the peptide molecule at the 5'-end of EGS oligo(2'-O-methylribonucleotides) practically does not reduce the efficiency of RNA hydrolysis by RNAse P.

The paper proposes a new variant for registering pathogens in the many-probes one-spot hybridization system. Using the example of Chlamydia trachomatis detection in blood serum samples, it has been demonstrated that hybridization of the analyzed DNA is characterized by a considerably stronger signal and higher specificity which may be advantageous for PointOfCare diagnostics (POC diagnostics).