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The review describes the fundamental electrochemical properties of nucleic acids manifested on solid electrodes, with an emphasis on the spatial structure of macromolecules. The formation of the double helix impedes the contact of the electroactive groups of the nitrogenous bases with the electrode surface, resulting in the disappearance of the analytical signal of deoxyribonucleic acid (DNA). The insufficient electroactivity of the double-stranded DNA is overcome by introducing electrochemically active fragments into the nucleic acid sequence through the polymerase incorporation of chemically modified nucleotides. Currently, an extensive range of artificial nucleotides has been synthesized, which contain various electroactive groups capable of both oxidation and reduction on electrode surfaces at different potentials. Artificial modified nucleotides must exhibit high electrochemical activity while also serving as good substrates for enzymes (polymerases) involved in nucleic acid amplification reactions. Introducing modified nucleotides instead of natural ones into polymerase reactions represents a compromise between the number of labels inserted in one amplicon and the length and quantity of the resulting products. Modified nucleotides find application in the detection of gene mutations and single-nucleotide polymorphisms, nucleic acid sequencing, determination of protein and peptide concentrations, and the detection of pathogenic viruses and bacteria. With the advancement of isothermal amplification methods, the development, synthesis, and investigation of artificial nucleotides have become highly relevant for creating new off-laboratory electrochemical nucleic acid analyzers. [ABSTRACT FROM AUTHOR]

The work demonstrates that recombinant CRISPR-nuclease Cas12a, purified after heterologous expression with a simplified method using single-stage metal-chelate chromatography, can be successfully utilized in DETECTR technology. The combination of CRISPR-nuclease Cas12a obtained in such a way with recombinase polymerase amplification (RPA) allowed one to ensure the selectivity of detection of Dickeya solani—the dangerous bacterial phytopathogen causing the potato disease known as "blackleg"—against closely related and unrelated bacterial phytopathogens with a limit of detection of one copy of the bacterial genome per amplification reaction. The result can be determined visually, without the use of complex instrumental methods, by changing the color of the reaction sample when illuminated with blue light that creates the basis for development of field DNA diagnostics of D. solani. The use of simplified chromatographic purification will significantly reduce the time and resources required to obtain a functionally active CRISPR-nuclease Cas12a for development and production of DNA diagnostics based on DETECTR technology. [ABSTRACT FROM AUTHOR]

Recombinase polymerase and loop-mediated isothermal amplification can be conducted in nonlaboratory conditions, making these methods promising for the development of rapid tests for the DNA diagnosis of infectious diseases in humans, agricultural animals, and plants in the "point-of-care" testing or "in-field" detection format. The review discusses the fundamental principles underlying these methods and describes their current status, with a focus on noninstrumental methods for the detection of results in isothermal amplification using colorimetry and lateral flow assays. Approaches to enhancing the selectivity of isothermal amplification through its combination with CRISPR/Cas detection or by combining two methods based on the "nested amplification" principle are thoroughly examined. [ABSTRACT FROM AUTHOR]

The analysis of cytochrome P450 transcripts was carried out by the nanopore sequencing in liver tissue samples of three donors and HepG2 line cells. It was demonstrated that direct mRNA sequencing with a MinION nanopore sequencer (Oxford Nanopore Technologies) makes it possible to obtain quantitative profiles for transcripts (and their splice variants) of cytochrome P450 superfamily genes encoding isoforms involved in metabolism of the majority (~80%) of drugs. The splice variant profiles substantially differ for donors. The cytochrome P450 gene expression at the transcript level is significantly weaker in cells of the HepG2 line compared with that in the normal liver tissue. This limits the capability of the direct mRNA nanopore sequencing for studying alternative splicing of cytochrome P450 transcripts in HepG2 cells. Both quantitative and qualitative profiles of the cytochrome P450 gene expression at the transcript level notably differ in human liver tissue and HepG2 cells. [ABSTRACT FROM AUTHOR]

Using human chromosome 18 (Ch18) genes as an example, a PCR analysis of the interindividual variability of expression of 244 genes was performed in the liver tissue. Although the quantitative profiles of the Ch18 transcriptome, expressed as the number of cDNA copies per single cell, showed a high degree of correlation between donors (Pearson correlation coefficient rp ranged from 0.963 to 0.966), the expression of the significant number of genes (from 13% to 19%, depending on the method of experimental data normalization) varied by more than 4-fold when comparing donors pairwise. At the same time, the proportion of differentially expressed genes increased with a decrease in the level of their expression. It is shown that the higher quantitative variability of low-abundance transcripts has mainly biological rather than technical nature. Bioinformatic analysis of the interindividual variability of the differential expression of Ch18 genes in the human liver tissue did not reveal any statistically significant groups of genes related to certain biological processes. This obviously indicates a rather transient nature of the interindividual variability of Ch18 gene expression, probably reflecting the response of cells of an individual to specific external stimuli. [ABSTRACT FROM AUTHOR]

CRISPR/Cas13a nucleases are currently considered to be the basis for the development of a new generation of biosensors for the ultrasensitive, in-field detection of bacterial and viral pathogens. A recombinant Cas13a nuclease with functional affinity was obtained as a result of heterologous expression in E. coli with a single-step purification process via metal-chelating chromatography with the N-terminal polyhistidine tag. The simplified procedure of Cas13a nuclease purification broadens the possibilities for the development and practical application of diagnostic biosensing systems based on it. Moreover, our results indicate that the currently uncharacterized protein U2PWF1 of Leptotrichia wadei represents Cas13a nuclease. [ABSTRACT FROM AUTHOR]

A method of thermal dissociation of DNA–protein conjugates was developed for the selection of aptamers to human alpha-fetoprotein. The method is based on the identification of the DNA oligonucleotides from the combinatorial DNA libraries with the highest specificity to targets, which remained within the conjugate after two stages: a) washing of unbound sequences; and b) removal of the sequences bound to the DNA but displaying a low affinity to it. The undestroyed most stable complexes were subjected to the thermal dissociation with the involvement of the corresponding oligonucleotides in the next rounds of selection. A comparison of the proposed method with the standard washing demonstrated a more intensive enrichment of the library. Due to the tighter conditions for washing of the target-bound DNA oligonucleotides the use of thermal dissociation can increase the efficiency of selection of highly specific aptamers to their molecular targets. [ABSTRACT FROM AUTHOR]

Using random (combinatorial) DNA-libraries with various degrees of diversity, it was shown that their amplification by polymerase chain reaction in real time resulted in appearance of a maximum on amplification curves. The relative decrease of fluorescence after passing the maximum was directly proportional to the logarithm of the number of oligonucleotide sequence variants in the random DNA-library provided that this number was within the interval from 1 to 104 and remained practically unaltered when the number of variants was in the interval from 105 to 108. The obtained dependence was used in the course of the SELEX procedure to evaluate changes in the diversity of random DNA-libraries from round to round in selection of DNA-aptamers to the recombinant SMAD4 protein. As a result, oligonucleotides containing sequences able to form a site of SMAD4-DNA interactions known as SBE (SMAD-binding element) have been selected thus indicating that the SMAD4-SBE interaction dominates the aptamer selection. [ABSTRACT FROM AUTHOR]

Zinc ions and glycosaminoglycans (GAGs) are found in amyloid deposits and are known to modulate the β-amyloid peptide (Аβ) aggregation, which is thought to be a key event in the pathogenesis of Alzheimer's disease (AD). Correlation spectroscopy was used to study how the H6R and D7H mutations of the metal-binding domain (MBD) of Аβ42 affect the modulation of its zinc-induced aggregation by the model GAG heparin. The H6R mutation was shown to decrease and the D7H mutation to increase the Аβ42 propensity to aggregate in the presence of zinc ions. In addition, H6R diminished and D7H enhanced the modulating effect of heparin. The difference in the heparin-dependent modulation was associated with coordination of zinc ions within the MBDs of the mutant peptides. The findings indicate that anion-binding sites formed by complexes of zinc ions with the Аβ MBD play an essential role in the interaction of zinc-induced Аβ aggregates with heparin. [ABSTRACT FROM AUTHOR]

The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP–template–polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature–time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX). [ABSTRACT FROM AUTHOR]

An effect of primer strand slippage was observed in the primer extension reaction due to replacement of thymidine triphosphate for modified analogs of dUTP. The effect was detected when using Vent (exo–) DNA polymerase. It is notably suppressed by adding to reaction mixture the natural nucleotides dATP, dCTP, and dGTP. [ABSTRACT FROM AUTHOR]

Abstract—: A modification of the enzymatic method for the preparation of combinatorial random DNA libraries, which combines amplification in isolated microvolumes with the simultaneous incorporation of modified nucleotides and subsequent separation of DNA strands, was developed. Deoxyuridine triphosphate with hydrophobic substituents such as structural analogues of amino acid side chains in the C5 position of the pyrimidine ring was used to introduce modifications into DNA. To prevent competitive amplification, which reduces the representativeness of combinatorial libraries, PCR in inverse emulsion was used. The separation of the strands of PCR products was carried out. There were six single-stranded DNA libraries with complete substitution of deoxythymidine via modified analogues with various functional groups. These DNA libraries are suitable for generating aptamers to protein targets through additional hydrophobic interactions from the introductions of appropriate modifications, and are completely compatible with the SELEX aptamer selection methodology. [ABSTRACT FROM AUTHOR]

Large inrush currents can be harmful to equipment subjected to it and can also disturb other devices through voltage dips. Traditional ways of dealing with large inrush currents include the usage of parasitic elements in the supply, negative temperature coefficient (NTC) resistances and start-up relays. These solutions have several disadvantages among which are a non-negligible standby consumption, using a relay or a compromise between inrush current and load losses using an NTC. This paper proposes a solution which solves these disadvantages. The proposed circuit uses a semi-controlled bridge rectifier requiring a very low standby power consumption and is capable of withstanding grid interruptions. Moreover, it is designed to power on only when the load is not too heavy. This is accomplished by the use of positive temperature coefficient thermistors. The presented solution can be used for DC-link start-up circuits in applications with a power range from several watts up to a few kilowatts and it is particularly suitable for on-board battery chargers designed for electrical vehicles. [ABSTRACT FROM AUTHOR]

The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of the target protein with aptamers immobilized on a solid phase has been investigated. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as a model target protein. The affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads resulted in an approximately 10-fold increase of the concentration of the target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM was possible without interference from other peptides present in the tryptic digest. [ABSTRACT FROM AUTHOR]

Using reverse transcription in conjunction with the quantitative real-time PCR or digital droplet PCR, the transcriptome profiling of human chromosome 18 has been carried out in liver hepatocytes and hepatoblastoma cells (HepG2 cell line) in terms of the absolute number of each transcript per cell. The transcript abundance varied within the range of 0.006 to 9635 and 0.011 to 4819 copies per cell for the HepG2 cell line and hepatocytes, respectively. There was significant correlation between expression profiles of chromosome 18 genes in hepatocytes and HepG2 cells: the Spearman's correlation coefficient was 0.81. The frequency distribution of transcripts by their abundance was bimodal for HepG2 cells and unimodal for liver hepatocytes. Bioinformatic analysis of the differential gene expression has shown that chromosome 18 genes, overexpressed in HepG2 cells compared to hepatocytes, are associated with cell division and cell adhesion processes. It is suggested that enhanced expression of these genes is related to proliferative activity of cultured HepG2 cells. The differences in transcriptome profiles should be taken into consideration when cultured HepG2 cells are used as a model of liver hepatocytes. [ABSTRACT FROM AUTHOR]

Thermodynamic analysis of plasma gasification of various renewable carbon-bearing materials was carried out using various oxygen-containing oxidants (oxygen, air, water). The possibility of obtaining calorific synthesis gas suitable for the needs of heat power engineering was confirmed. [ABSTRACT FROM AUTHOR]

Here, we describe the synthesis and purification of six deoxyuridine triphosphate derivatives that contain protein-like functional groups and alkene linkers of various lengths. Using KOD XL and Deep Vent polymerases, these derivatives have been incorporated into single-stranded DNA, achieving a high degree of DNA modification. These polymerases are able to utilize highly modified DNA strands as templates for synthesizing unmodified DNA. The synthesized deoxyuridine triphosphate derivatives are promising as substrates for producing modified aptamers to various target proteins using, e.g., the systematic evolution of ligands by exponential enrichment (SELEX) methodology. [ABSTRACT FROM AUTHOR]

The synthesis of deoxyuridine triphosphate derivatives with alkyl groups of various chain length attached to the pyrimidine base by the trans-alkene linker was performed. The efficiency of the incorporation of modified nucleotides during the synthesis of DNA with a high degree of modification by the primer extension reaction with Taq DNA polymerase was studied. [ABSTRACT FROM AUTHOR]

MicroRNA is a special type of regulatory molecules modulating gene expression. Circulating microRNAs found in blood and other biological body fluids are now considered as potential biomarkers of human pathology. Quantitative changes of particular microRNAs have been recognized in many oncological diseases and other disorders. A recently developed method of droplet digital PCR (ddPCR) possesses a number of advantages making this method the most suitable for verification and validation of perspective microRNA markers of various human pathologies. These advantages include high accuracy and reproducibility of microRNA quantification as well as possibility of direct high-throughput determination of the absolute number of microRNA copies within a wide dynamic range. The present review considers microRNA biogenesis, the origin of circulating microRNAs, and methods used for their quantification. The special technical features of ddPCR, which make this method especially attractive for studying microRNAs as biomarkers of human pathologies and for basic research devoted to aspects of gene regulation by microRNA molecules, are also discussed. [ABSTRACT FROM AUTHOR]

Alzheimer's disease is the most prevalent neurodegenerative pathology. According to the amyloid cascade hypothesis, transition of the amyloid-β peptide (Aβ) from the monomeric form to the aggregated state is a key event in pathogenesis of the Alzheimer's disease. The mechanism of Aβ aggregation is intensively studied in vitro, by means of synthetic peptides and various physico-chemical methods allowing evaluation of size, molecular structure, and morphology of the formed aggregates. The review considers both the wellknown and recently introduced physico-chemical methods for analysis of Aβ aggregation, including microscopy, optical and fluorescent methods, electron paramagnetic resonance, electrochemical and electrophoretic methods, gel-filtration, and mass spectrometric methods. Advantages and disadvantages of these methods are considered. Special attention is paid to the unique possibility of simultaneous analysis of both Aβ monomers and its oligomers as well as large aggregates by means of atomic force microscopy or fluorescence correlation spectroscopy. The high detection sensitivity of the latter method provides opportunity for investigating the aggregation process in Aβ solutions of low peptide concentrations. Among mass spectrometric methods, the ion mobility mass spectrometry is considered as a method enabling to obtain information about both the spectrum of Aβ oligomers and their structure. Simultaneous employment of several methods providing complementary data about Aβ aggregates is the best experimental approach for studying the process of Aβ aggregation in vitro. [ABSTRACT FROM AUTHOR]

Interaction of intranuclear β-amyloid with DNA is considered to be a plausible mechanism of Alzheimer's disease pathogenesis. The interaction of single- and double-stranded DNA with synthetic peptides was analyzed using surface plasmon resonance. The peptides represent the metal-binding domain of β-amyloid (amino acids 1-16) and its variants with chemical modifications and point substitutions of amino acid residues which are associated with enhanced neurotoxicity of β-amyloid in cell tests. It has been shown that the presence of zinc ions is necessary for the interaction of the peptides with DNA in solution. H6R substitution has remarkably reduced the ability of domain 1-16 to bind DNA. This is in accordance with the supposition that the coordination of a zinc ion by amino acid residues His6, Glu11, His13, and His14 of the β-amyloid metal-binding domain results in the occurrence of an anion-binding site responsible for the interaction of the domain with DNA. Zinc-induced dimerization and oligomerization of domain 1-16 associated with phosphorylation of Ser8 and the presence of unblocked amino- and carboxy-terminal groups have resulted in a decrease of peptide concentrations required for detection of the peptide-DNA interaction. The presence of multiple anion-binding sites on the dimers and oligomers is responsible for the enhancement of the peptide-DNA interaction. A substitution of the negatively charged residue Asp7 for the neutral residue Asn in close proximity to the anion-binding site of the domain 1-16 of Aβ facilitates the electrostatic interaction between this site and phosphates of a polynucleotide chain, which enhances zinc-induced binding to DNA. [ABSTRACT FROM AUTHOR]

Investigation results on the arc plasmatorch for water-steam heating are presented. The construction arrangement of steam plasma generator with copper electrodes of the stepped geometry was firstly implemented. The energy characteristics of plasmatorch and erosion of electrodes reflect the features of their behavior at arc glow in the plasma-forming environment of steam. The results of numerical study of the thermal state of the composite copper-steel electrodes had a significant influence on optimization of anode water-cooling aimed at improvement of its operation life. [ABSTRACT FROM AUTHOR]

A method of atomic force microscopy-based fishing (AFM fishing) has been developed for protein detection in the analyte solution using a chip with an immobilized aptamer. This method is based on the biospecific fishing of a target protein from a bulk solution onto the small AFM chip area with the immobilized aptamer to this protein used as the molecular probe. Such aptamer-based approach allows to increase an AFM image contrast compared to the antibody-based approach. Mass spectrometry analysis used after the biospecific fishing to identify the target protein on the AFM chip has proved complex formation. Use of the AFM chip with the immobilized aptamer avoids interference of the antibody and target protein peaks in a mass spectrum. [ABSTRACT FROM AUTHOR]

ptamers interacting selectively with the anion-binding exosites 1 and 2 of thrombin were merged into dimeric oligonucleotide constructs by means of a poly-(dT)-linker of 35 nucleotides (nt) in length. Complexes of thrombin with the aptamers and their hetero- and homodimeric constructs were measured using an optical biosensor Biacore-3000. The K values obtained for the hetero- and homodimeric constructs were correspondingly 25-30- and 2-3-fold lower than those for the primary aptamers. Analysis of temperature dependencies of the K values within the temperature interval of 10-40°C has shown that affinity increases with the temperature decrease. The values of the enthalpy change Δ H upon formation of complexes of thrombin with the aptamers and the heterodimeric construct were basically the same. The value of the entropy change Δ S upon complex formation of thrombin with the aptamer heterodimeric construct was 1.5-2-fold higher than the Δ S values for the complexes with the aptamers. The complex formation and dissociation rates increased with the elevation of temperature from 10 to 37°C. However, at both temperatures the dissociation rate for the complex of thrombin with the heterodimeric construct was evidently lower that for the complexes with the aptamers. [ABSTRACT FROM AUTHOR]

Zinc ions form complexes with β-amyloid peptides and play an important role in Alzheimer's disease pathogenesis. It has been demonstrated by turbidimetry and correlation spectroscopy that synthetic peptide Aβ16 representing the metal-binding domain of β-amyloid is able to interact with nucleic acids, chondroitin polysulfate, and dextran sulfates in the presence of zinc ions. The amino acid D7H substitution enhanced the peptide binding to polyanions, whereas the H6R and H6A-H13A substitutions abolished this interaction. It is suggested that the metal-binding domain may serve as a zinc-dependent site of β-amyloid interaction with biological polyanions including DNA, RNA, and glycosaminoglycans. [ABSTRACT FROM AUTHOR]

Recent achievements in the whole-genome sequencing especially viral and bacterial ones together with the development of methods of bioinformatics and molecular biology, have created preconditions for transition from synthesis of genes to assembly of the whole genomes based on chemically synthesized blocks, oligonucleotides. The creation of artificial genomes and artificial cells will undoubtedly render huge influence on a deepening of knowledge on mechanisms of functioning of living systems at a cellular level, on a way of origin and evolution of life, and also on biotechnology of the future, and will generate preconditions for the further development of synthetic biology and nanobiotechnology. [ABSTRACT FROM AUTHOR]

Application of proteomic results to scientific and medical practice will depend in many respects on progress of affinity microchips technologies. This determines continuous search for inexpensive and robust affinity reagents alternative to monoclonal antibodies. Among synthetic mimetics of antibodies, the oligonucleotide aptamers are of the greatest interest as the affinity reagents due to the possibility to automate their selection and due to the low cost of oligonucleotide synthesis. In the review we consider the problems related to the automation and optimization of aptamer selection and also to selection of photoaptamers capable to form photoinduced covalent complexes with the protein targets. The existing approaches to the post-selection modification of the aptamers to increase their affinity and selectivity to protein targets are discussed. [ABSTRACT FROM AUTHOR]

A construction arrangement of electric arc plasmatorch with copper tubular electrodes for steam plasma generation was developed and tested. Experimental data on energy curves of this plasmatorch with nominal power of 100 kW are presented. [ABSTRACT FROM AUTHOR]

The expression of human telomerase catalytic subunit in HL-60 and HT-1080 malignant transformed cells and telomerized fibroblasts was studied by quantitative PCR. It was found that the number of transcripts of human telomerase catalytic subunit per cell in telomerized fibroblasts could be hundreds of times higher than in HL-60 and HT-1080 cells. Telomerized fibroblast cultures are suggested as experimental systems for selection of basal compounds for creation of anticancer drug prototypes, the molecular target of which is human telomerase catalytic subunit. The effects of human telomerase catalytic subunit expression on the fibroblast proteome are analyzed. [ABSTRACT FROM AUTHOR]