[Prime-Editing Methods and pegRNA Design Programs].

It has been 10 years since CRISPR/Cas technology was applied to edit the genomes of various organisms. Its ability to produce a double-strand break in a DNA region specified by the researcher started a revolution in bioengineering. Later, the Base Editing (BE) method was developed. BE is performed via the formation of single-strand breaks by the mutant form of Cas nuclease (nickase), fused with deaminases and other enzymes. It can be used to promote A ↔ G and C ↔ T transitions, and a C → G transversion. Just over 3 years ago, a new Prime Editing (PE) variant of CRISPR/Cas was invented. Unlike BE, in PE the nickase is fused with reverse transcriptase, capable of building a new DNA chain using the pegRNA template. The pegRNA consists of an elongated guide RNA with an extra sequence at the 3'-end. Prime editing makes it possible to insert the desired mutations into this extra sequence and to carry out any substitutions and indels of bases without the use of special donor DNA. To date, a number of PE variants have been proposed; they are briefly considered in this review with an emphasis on prime editing of plant genomes. Some attention is also paid to pegRNA design programs, as well as evaluation of the efficiency of the editing. Such a variety of PE techniques is due to the opportunities of high-precision introduction of desired changes with a rather low frequency of off-target mutations in the genomes of various organisms. The relatively low efficiency of prime editing inspires researchers to offer new approaches. There is hope that further development of the technology will improve PE enough to take its rightful place among the genome targeting methods that are suitable for any organisms, and will have a positive impact on the agricultural sector, industrial biotechnologies, and medicine.

[New polymorphic DNA marker to determine a person's sex from biological material]. / Novyi polimorfnyi DNK-marker dlya opredeleniya pola cheloveka po ego biologicheskomu materialu.

The objective of the study was to pre-evaluate the applicability of gender-specific nucleotide sequences in human neuroligin genes as alternative DNA markers of sex. A new polymorphic locus based on NLGNX and NLGNY genes was proposed to establish the sex attribute of human biomaterials. The significant difference in the location of these loci relative to the pseudoautosomal region (PAR), as well as the combination of different types of polymorphism on the one hand, and the possibility of using gender-specific primers «in one assay¼ on the other hand, warrants their use as an additional marker of human sex attribute, including utilization as part of systems for DNA registration in the population. The introduction of a new polymorphic locus based on the NLGNX and NLGNY genes will make it possible to reliably identify the sex attribute of biological material recovered from crime scenes.

Rolling Circle Amplification as a Universal Method for the Analysis of a Wide Range of Biological Targets.

Detection and quantification of biotargets are important analytical tasks, which are solved using a wide range of various methods. In recent years, methods based on the isothermal amplification of nucleic acids (NAs) have been extensively developed. Among them, a special place is occupied by rolling circle amplification (RCA), which is used not only for the detection of a specific NA but also for the analysis of other biomolecules, and is also a versatile platform for the development of highly sensitive methods and convenient diagnostic devices. The present review reveals a number of methodical aspects of RCA-mediated analysis; in particular, the data on its key molecular participants are presented, the methods for increasing the efficiency and productivity of RCA are described, and different variants of reporter systems are briefly characterized. Differences in the techniques of RCA-mediated analysis of biotargets of various types are shown. Some examples of using different RCA variants for the solution of specific diagnostic problems are given.

[Iatrogenic deficits of micronutrients].

Long-term use of certain drugs causes subclinical and clinically significant micronutrient deficiencies, which can affect the course of the disease, its prognosis, quality of life, and patient compliance with therapy. The aim of the study was to single out groups of drugs, which long-term use leads to micronutrient deficiency, and to determine vitamins, minerals and trace elements, which supply can be reduced as a result of pharmacotherapy, basing on the analysis of data published in the scientific literature. Material and methods. This review analyzes articles on medical sciences from MEDLINE and PubMed-NCBI bibliographic databases. Results. Combined oral contraceptives reduce woman's supply with B vitamins (B6, B12, B9), can cause hypomagnesemia, affect the calcium/magnesium blood ratio, reduce the amount of vitamin E circulating in blood. Proton pump inhibitors reduce the absorption of vitamin B12, calcium, magnesium, iron, zinc. Aspirin increases ascorbic acid metabolism. Loop diuretics increase urinary excretion of calcium, magnesium, thiamine, thiazide ones elevate zinc and vitamin B9 excretion. Loss of taste when taking captopril is associated with a decrease in zinc supply. The use of calcium channel blockers interfere with the absorption of folic acid by gingival fibroblasts. Conclusion. Given the growing prevalence of long-term drug use, it is necessary to be able to predict and prevent potential consequences of interactions with micronutrients. It is advisable, along with a varied and healthy diet, to provide patients with supplementation in order to prevent micronutrient deficiencies. Optimization of vitamin status of the population in terms of its significance for public health is comparable to drug therapy and is one of the technologies for reducing losses from chronic diseases.

[Botulinum therapy of spastic forms of cerebral palsy in various locomotor patterns]. / Botulinoterapiya spasticheskikh form detskogo tserebral'nogo paralicha pri razlichnykh lokomotornykh patternakh.

Spasticity in patients with cerebral palsy (CP) is the main impediment to normal locomotion. The function of the Central Pattern Generator (CPG), i.e. a group of neural chains in the spinal cord, stands at the core of any rhythmical movement. CPG can generate locomotion patterns without supraspinal control, which can have both positive and negative impact on the ability to move. Performing the motor tasks such as walking, running and swimming, creates the consistent rhythmical movement of legs and arms through interaction between CPGs of upper and lower extremities. This interaction can cause the activation of pathological movement patterns in lower extremities in response to upper limb spasticity. Thus, neural chains in the spinal cord become the generator of pathologically increased excitation which has developed as a result of a focal lesion in the CNS. All the statements described above show the importance of introducing the upper limb injections of bFotulinum toxin A in the protocol in order to develop normal locomotion. The PUL study approved the optimal level of efficacy and favourable safety profile of botulinum toxin A in children with CP and upper limb muscle spasticity.

[Design of Guide RNA for CRISPR/Cas Plant Genome Editing].

CRISPR/Cas technology of genome editing is a powerful tool for making targeted changes in the DNA of various organisms, including plants. The choice of the precise nucleotide sequence (protospacer) in the gene to be edited is important in the design of guide RNA, which can be carried out by specialized software. We review and compare all the known on-line and off-line resources for guide RNA design, with special attention paid to tools capable of searching for off-target edits sites in plant genomes. The use of Cas12a may be preferable to Cas9. Techniques allowing C→T and G→A base editing without DNA cleavage are discussed along with the basic requirements for the design of effective and highly specific guide RNAs. Ways for improving guide RNA design software are presented. We also discuss the lesser risks of off-target editing in plant genomes as opposed to animal genomes. Examples of edited plant genomes including those that do not lead to the creation of transgenic plants are reviewed.

[PCR-based evaluation of sequence specificity of DNA fragmentation by ultrasound].

Ultrasonic fragmentation, which is a simple and convenient method for the mechanical degradation of DNA, is widely used in modern genome studies as one of the sample preparation steps. It has been recently found that the DNA breaks occur more often in the regions containing 5'-CG-3' dinucleotides. We studied the influence of the 5'-CG-3' dinucleotides on the efficiency of the 28S rRNA gene amplification during PCR with sonicated DNA of Mantis religiosa. It was shown that the amplification rate depends on the template length and the number of 5'-CG-3' dinucleotides. Amplification of the DNA regions with a higher 5'-CG-3' density is less efficient because of their higher sensitivity to ultrasound. The amount of the amplified DNA templates is inversely proportional to the 5'-CG-3'number.

[The Effect of Cadmium on the Efficiency of Development of Legume-Rhizobium Symbiosis].

Screening of nodule bacteria (rhizobia) forming symbiotic relationships with legumes has been performed in order to isolate strains resistant to cadmium ions in a wide range of concentrations (6-132 mg/kg). The effect ofcadmium salts (6, 12, 24 mg/kg) on the legume-rhizobium symbiosis ofthe pea Pisum sativum L. with Rhizobium leguminosarum and of the fodder galega Galega orientalis Lam. with Rhizobium galegae has been studied under experimental laboratory conditions. No statistically significant differences have been revealed in the growth and biomass of plants with regard to the control in the range of concentrations given above. However, it was found that cadmium inhibited nodulation in P. sativum and stimulated it in G. orientalis.

[The Increase of Oligonucleotides--Gold Nanoparticles Conjugates Stability].

For today the reagents based on oligonucleotides--gold nanoparticles conjugates and used for specific nucleic acids detection are actively being developed. Such molecular structures are stabilized through the bonds between thiol group in oligonucleotides and gold atoms in nanoparticle. The durability of oligonucleotides--gold nanoparticles binding affects directly on the stability of conjugates and on the possibility of further manipulations. In this paper, a method for the strengthening of oligonucleotides attachment on the gold nano-particles surface by means of anchor groups with dithiolane residues is proposed. A comparative study of the anchors molecular structure influence on the conjugates stability at conditions that typical for oligonucleotide probes was carried out.

[DNA amplification using PCR with abutting primers].

DNA analysis of ñîmplex biological objects (wastewater, soil, archaeological and forensic samples, etc.) is currently of great interest. DNA of these objects is characterized by low suitability for research due to the violation of its integrity and chemical structure; thus, the detection of specific nucleic acid fragments can be achieved by PCR with contiguous primers. In this paper, we present the results that clarify the specific characteristics of PCR with abutting primers. The 3'-ends of these primers are annealed at adjacent nucleotides of complementary chains of DNA target. It has been shown that the proximity of primers enables the formation of specific reaction products with a higher sensitivity and less reaction time. Using artificially damaged DNA and DNA from the soil we demonstrated that the abutting primers provide assured detection of specific DNA fragments. The results of this work may be taken into account in PCR with degraded (fragmented) DNA.

[The creation of transgenic tobacco plants expressing fragments of the ARGOS and NtEXPA4 genes in antisense orientation].

Transgenic tobacco plants expressing the fragments of the ARGOS and NtEXPA4 genes in antisense orientation have been created. Eleven lines of transgenic plants were investigated and five of them were characterized by a decrease in the sizes of the leaves and flowers as compared to control. Stalk sizes decreased when only the NtEXPA4 gene fragment was used. The organ size of the experimental plants decreased because of a reduction in the level of both cell division and cell expansion. Two lines of transgenic tobacco plants expressing the part of the ARGOS gene in antisense orientation were characterized by a reduction in the level of the NtEXPA1 and NtEXPA4 gene expression.

[Role of the expansin genes NtEXPA1 and NtEXPA4 in the regulation of cell extension during tobacco leaf growth].

The tobacco plant genes NtEXPA1 and NtEXPA4 encode the α-expansin proteins involved in the regulation of cell growth and extension. We examined the levels of expression of these genes in various plant organs and under the effect of exogenous phytohormones. The highest levels of NtEXPA1 expression were registered in the terminal bud and in the young growing leaves and flowers. NtEXPA1 expression ceased once the leaves stopped growing. The NtEXPA4 gene showed a similar expression profile, except for higher levels of mRNA in the leaves. In young leaves located near the terminal bud, high levels of NtEXPA1 and NtEXPA4 are induced by auxins. In the lower leaves, expansin expression is differentially regulated by brassinosteroids, which inhibit NtEXPA1 and upregulate NtEXPA4. We further showed that expression of the transgenic ARGOS-LIKE results in upregulation of NtEXPA1 and a reduction in the NtEXPA4 mRNA. In turn, overexpression of NtEXPA1 resulted in an increased size of the leaves and stems because of the larger size of the individual cells.

[Morphological analysis of transgenic tobacco plants expressing the PnEXPA3 gene of black poplar (Populus nigra)].

Transgenic tobacco plants overexpressing the PnEXPA3 gene of black poplar (Populus nigra), which encodes alpha-expansin, were obtained. The transgenic plants were characterized by increased size of epidermic and mesophyll cells of leaves. However, the size of leaves remained normal. Overexpression of the PnEXPA3 gene provided stimulatory effect only on the stem length. Other morphological traits of the transgenic plants remained unchanged.

[Effect of ectopic expression of NtEXPA5 gene on cell size and growth of organs of transgenic tobacco plants].

We obtained transgenic tobacco plants demonstrating overexpression of NtEXPA5 gene that encodes alpha-expansin of Nicotiana tabacum. The transgenic plants were characterized by increased size of leaves and stems. However, size of flowers remained almost unchanged. The increase of organ sizes was induced by cell stretching only. Moreover, the number of cell divisions was even decreased. The obtained data suggest tight interaction between cell stretching regulation and cell division, which together provide the basic mechanism aimed at the controlling of plant organ sizes.

[Ectopic expression of the PnANTL1 and PnANTL2 black poplar genes in transgenic tobacco plants].

Four putative orthologs of the AINTEGUMENTA gene were found in the poplar (Populus trichocarpa) genome. Two of them, which we called PnANTL1 and PnANTL2, were isolated from black poplar (Populus nigra L.) and transgenic tobacco plants were generated on their basis. Tobacco plants that were transgenic for the PnANTL1 gene were characterized by increased leaf length, smaller flower size, and different defects in the development of the corolla and stamens. Tobacco plants that were transgenic for the PnANTL2 gene were characterized by an increased length of leaves and larger flowers. The increase in the leaf size in all transgenic plants was determined by stimulation of cell expansion; the number of cells was even reduced in the case of the PnANTL1 gene. Ectopic expression of the PnANTL1 and PnANTL2 genes promoted an increase in the level of mRNA of some tobacco expansins. The data we obtained demonstrate the involvement of transcription factors of the AP2 subfamily in the regulation of cell expansion.

[Bioengineering of symbiotic systems: creation of new associative symbiosis with the use of lectins on the example of tobacco and colza].

"Barbate roots" in tobacco and colza transgenic on lectin gene were obtained with the use of a wild strain of Agrobacterium rhizogenes 15834 transformed with pCAMBIA1305.1 plasmid containing the full-size lectin gene (psl) from the Pisum sativum. Influence of expression oflectin gene on colonization oftransgenic roots with symbiont of pea (Rhizobium leguminosarum) was investigated. The number of adhered bacteria onto the roots transformed with lectin gene was 14-fold and 37-fold higher in comparison with the control; this confirms the interaction of R. leguminosarum with pea lectin at the surface of the transformed roots of tobacco and colza. The developed experimental approach, based on the simulation of recognition processes and early symbiotic interactions with lectins of pea plants, may, in perspective, be used for obtaining stable associations of economically valuable, nonsymbiotrophic plant species with rhizobia.

[Obtaining of fluorescent-labeled nodule bacteria strains of wild legumes for their detection in vive and in vitro].

A series of expression vectors containing genes of fluorescent proteins TurboGFP and TurboRFP under the phage T5 constitutive promoter regulation, intended for lifetime marking of nodule bacteria is created: a series of vectors based on a broad-host-range replicon BBRI, for marking strains with an expression of reporter gene from a transformed plasmid and a series of vectors based on a plasmid pRL765gfp for marking strains by introduction genes of fluorescent proteins in a bacterial chromosome. It was shown that transformation is the most preferable method of constructions transfer in nodule bacteria cells, as in the presence of mob locus in the vectors necessary for conjugation, exists the possibility of occasional plasmid mobilization and its transition from marked strain cells in other soil bacteria. With application of the created vector constructions we obtained fluorescent tagged strains of Rhizobium sp., Mesorhizobium sp., Ensifer (Sinorhizobium) sp., Bradyrhizobium sp., Phyllobacterium sp., Agrobacterium sp. Also their suitability for experiments in vivo and in vitro is shown.

[New approaches to the real-time detection of nucleotide mismatches by means of chimeric hybridization probes].

New approaches to the detection of impaired nucleotides based on the allele specific ligation of a "C probe" followed by rolling circle amplification have been developed. The detection of amplification products was realized by using enzymatic and deoxyribozyme digestion of fluorescently-labeled DNA-RNA-DNA chimeric oligonucleotide structures in cycling probe technology (CPT) in real-time mode.

[Polymorphism of the prion protein PRNP gene and risk of multiple sclerosis development in ethnic Russians from Bashkortostan].

M129V polymorphism of prion protein gene PRNP has been studied in patients with multiple sclerosis (MS) and healthy ethnic Russians from Bashkortostan using allele-specific PCR. The genotype frequency distribution of the examined polymorphism in Russians from Bashkortostan was similar to that in European populations. MM, MV, and VV genotype frequencies in control group and in the MS patients were 50.24%, 42.58%, 7.18% and 43.33%, 45.83%, 10.84%, respectively. It was shown that in the group of MS patients with onset of the disease at the age of 21 and older, the frequency of the VV genotype was higher than in the control group (14.3% versus 6.18%, respectively, P = 0.041). We suggest that the VV genotype is associated with higher risk factor of MS development in the patients aged 21 years and older.

[Immunogenicity of N-protein of Puumala hantavirus for noninbred mice in the intramuscular administration of its gene DNA].

The outbreaks of hantavirus infections in some regions of the Russian Federation in some years involve considerable material and social losses. In this connection, the designing of the most effective types of vaccines is an urgent task. The authors have created plasmid constructions containing the gene of hemorrhagic fever with renal syndrome virus, with whose Intramuscular injection there is a specific immune response and plasmid DNA is detectable in the adjacent tissues within a month after injection.