Objective: Rolling circle amplification (RCA) is a sensitive and specific method of isothermal amplification of nucleic acids. RCA from the genomic DNA is complicated by the fact that the template target is present in the form of a long double-stranded DNA molecule that is difficult to access for ligase. One of the ways to prepare DNA for RCA analysis is the pre-amplification of genomic DNA by PCR, which leads to the formation of shorter products (amplicons) from which the ligation of the detecting oligonucleotide “padlock probe” is performed. The disadvantage of this method is the pre-amplification with thermal cycling (multiple melting of duplexes), which requires specialized equipment. Methods: A combined HDA-RCA approach is proposed to increase the sensitivity of RCA analysis in the study of full-genomic double-stranded DNA samples. Results and Discussion: It has been demonstrated that the use of pre-amplification increases the sensitivity of RCA in genomic DNA analysis. The PCR protocols are well reproducible, however, pre-amplification by PCR requires thermal cycling. With the HDA, it is possible to carry out pre-amplification in an isothermal mode, facilitating the unification of all stages into one, that is, the creation of a “one-pot” system for genomic DNA analysis using the RCA method. Conclusions: The possibility of using the proposed combined HDA-RCA method for detecting genome DNA in a sample is demonstrated. It has been demonstrated that pre-amplification using HDA increases the sensitivity of the RCA analysis, opening up the possibility of developing test systems for point-of-care diagnostics and in a single test tube without transferring material (“one-pot”).

In PCR with Taq polymerase on the Staphylococcus aureus genomic DNA template, the substrate properties of eight fluorescently labeled deoxyuridine and deoxycytidine triphosphates (Cy5-dUTP and Cy5-dCTP), which are dU-dC pairs with similar cyanine substituents, were compared during simultaneous introduction of such pairs in PCR. The different Cy5-dUTP and Cy5-dCTP pairs each had substituents with different linker lengths between the nitrogenous base and the fluorophore and between the quaternary ammonium group and the second heterocycle of the Cy5 fluorophore. The amplification efficiency, as well as the yield of the product, and the density of label incorporation were determined. It was found that, with the simultaneous introduction of Cy5-modified dU and dC into the reaction at equimolar concentrations, the inhibitory effect was not directly proportional to the concentration, in contrast to that with separate (individual) introduction of fluorescently labeled dNTPs. This allows one to use the simultaneous introduction of Cy5-modified dU and dC into PCR to increase sensitivity in methods based on the detection of a fluorescent signal, for example, in DNA-microarray technology.

The kinetics of amplification and the features of individual and simultaneous incorporation of modified deoxynucleoside triphosphates in DNA during rolling circle amplification (RCA) have been studied. This study was carried out for six pairs of Sy5-labeled triphosphates of deoxyuridine (dU) and deoxycytidine (dC) previously synthesized with similar fluorescent substituents inside the pair. The effect of the linker length between the fluorophore and the pyrimidine base on the incorporation density was determined: nucleotides with a linker length of six carbon atoms are embedded in a growing DNA chain better than with three carbon atoms. It was found that the combined introduction of triphosphates into the reaction in an equivalent total concentration does not enhance the inhibitory effect, which gives grounds for a more detailed study of the simultaneous use of labeled dU and dC.

The substrate properties of six pairs of fluorescently labeled deoxyuridine and deoxycytidine triphosphates (Cy5-dUTPs and Cy5-dCTPs) in PCR with Taq polymerase were compared. In each pair, the modified dU and dC contained identical fluorescently labeled Cy5 substituents; for different pairs, the substituent structures differed in the length of the linker between the nitrogenous base and the fluorophore, the length of the linker between the quaternary ammonium group and the second heterocycle of the fluorophore, as well as the structure of the fluorophore itself. DNA fragments of Staphylococcus aureus (AT-rich template) and Mycobacterium tuberculosis (GC-rich) were used as templates. With both templates, deoxycytidine derivatives showed slightly higher amplification efficiency (E). The influence of the fluorophore structure and the GC-composition of the template on the kinetics of the reaction was insignificant. At the same time, a high incorporation efficiency was observed on the AT-rich template for uridine derivatives, and on the GC-rich template for cytidine derivatives (and in both cases, for substituents with a longer linker length). Nevertheless, the specific incorporation density, which takes into account the number of similar nucleotides in the DNA chain, was in all cases higher for dU derivatives. It was found that in pairs with similar fluorophore modifications, uridine derivatives, compared with cytidine, are characterized by a higher incorporation density, regardless of the composition of the template, but at the same time they have a greater inhibitory effect. The results obtained will increase the sensitivity of fluorescence analysis using the immobilized phase (microarray analysis).

Objective: To develop a multifactorial, highly sensitive nucleic acid analysis method on biological microarrays within an environmentally sealed system. This approach eliminates the need for transferring biological material between vessels and avoids adding components during the assay, thereby reducing contamination risk and enabling process automation. Methods: Polyethylene terephthalate (PET) film substrates for biochip fabrication were treated with corona discharge. A thin layer of photoactive polyvinyl acetate was deposited on the surface by spin coating. A matrix of cells composed of brush polymers was obtained through photoinitiated radical polymerization of monomers “from the surface” using photolithographic patterning under UV irradiation through a photomask. Reactive carboxyl groups on polymer chains were activated, and primers with C6-amino modification at the 5′-end were immobilized within biochip cells. PCR with extension of immobilized primers using Cy5-fluorescently labeled nucleotides was performed within the biochip cells. Results and Discussion: A method for nucleic acid analysis by solid-phase PCR with immobilized primer extension in a closed film biochip during thermocycling was successfully developed. Detection was achieved through endpoint digital fluorescence microscopy monitoring Cy5-fluorescently labeled nucleotide incorporation. The developed “film biochip” consists of PET film with an internal chamber, brush polymer cells containing immobilized primers, and channels for solution supply and removal. The biochip features low heat capacity and high thermal conductivity of thin-film components, thermocycling capability, and result detection by digital fluorescence microscopy through the lid and washing solution layer without biochip disassembly in an environmentally isolated lab-on-a-chip system. The method′s performance and functional suitability were demonstrated by analyzing samples containing DNA from pathogenic bacteria Staphylococcus aureus and Legionella pneumophila. Conclusions: Film biochips made of commercially available PET film with brush polymer cells show promise for further development and application in solid-phase PCR for multiplex nucleic acid analysis, lab-on-a-chip microanalysis technologies, and clinical laboratory applications.

A matrix of hydrogel cells containing polymer “brushes” with active groups was obtained by photolithography on polymer substrates. Covalent immobilization of reverse primers specific to Staphylococcus aureus or Legionella pneumophila into the corresponding cells was performed, followed by amplification in a sealed chamber of the assembled biochips.

A method has been developed for manufacturing biological microchips on an aluminum substrate with hydrophilic cells from brush copolymers with the formation of a matrix of cells using photolithography. The surface of aluminum substrates was previously coated with a thin, durable, moderately hydrophobic layer of cross-linked polymer to prevent contact with the aluminum surface of the components used in the analysis of nucleic acids. Aluminum biochip substrates have high thermal conductivity and low heat capacity, which is important for the development of methods for multiplex PCR analysis on a chip. Oligonucleotide probes were covalently immobilized in the cells of the biochip. The preservation of the hybridization activity of the immobilized DNA probes was demonstrated in a hybridization analysis with a synthetic DNA target representing a section of the sequence of the seventh exon of the human ABO gene. The methods developed can be used in the development of a technology for parallel multiple rapid microanalysis of nucleic acids “lab on a chip” for the detection of human somatic and infectious diseases.

Objective: Polyethylene terephthalate (PET) is thermally stable, biocompatible, transparent in visible and near-infrared light. The study of grafting conditions and the distribution of reactive amino groups on the PET surface without affecting the polymer array makes it possible to change the surface properties in a directed manner. Methods: A method for obtaining active amino groups on the surface of polyethylene terephthalate (PET) substrate by reaction with ethylenediamine was developed. A method for quantitative estimation of the concentration and distribution of chemically accessible amino groups on the surface of PET substrate using cyanine dye Cy5 and digital fluorescence microscopy was developed. Results and Discussion: The PET surface during chemical modification remains without visible damage up to the concentration of amino groups 8 pmol/cm2, while surface degradation is observed at higher concentrations. Chemically available amino groups capable of covalently binding to Cy5 dye are distributed unevenly, which is probably due to the presence of amorphous and crystalline areas on the surface of PET substrates. Amino groups can be used for further chemical modification of the PET surface, grafting of various functional groups, and covalent binding to biomolecules, which opens up prospects for the wide use of inexpensive PET as functional substrates in biochips, biosensors, lab-on-a-chip devices, and other biotechnological applications.

Objective: The introduction of fluorescently labeled nucleotides in the process of DNA amplification in molecular genetic analysis is a very attractive alternative to post-labeling or the use of labeled primers in the reaction (RCA). Methods: To study the substrate efficiency, the kinetic index (amplification efficiency) was studied, which allowed us to evaluate the inhibitory effect of modified substrates; as well as the yield of the isothermal amplification reaction product and the density of embedding the label into the growing DNA chain. Results and Discussion: Two pairs of Cy5-labeled dU and dC triphosphates with similar electroneutral fluorophore structures, differing in the length of the hydrocarbon linker between the fluorophore and the nitrogenous base were synthesized. A comparative analysis of their substrate behavior in the rolling circle amplification (RCA) reaction using Bst 3.0 DNA polymerase was carried out. It was found that nucleotides with a long linker between the fluorophore and the pyrimidine base are more efficiently incorporated into the growing DNA chain, while the nucleotides with a short linker cause lesser inhibition of RCA. In each of the pairs consisting of dU and dC with similar fluorophores and linkers, the fluorescently labeled uridine derivatives demonstrated a higher incorporation density. It was revealed that, under simultaneous incorporation of the labeled dU and dC, the inhibitory effects do not sum up. This gives grounds for a more careful study of different Cy5-dC variants with a view to increasing the sensitivity of the analysis under simultaneous introduction of the labeled dU and dC. Conclusions: The work presents the results of comparative study of the substrate properties of fluorescently labeled deoxynucleoside triphosphates in RCA. The influence of the chemical nature of the nucleotide (dU or dC) and the structure of the fluorophore on the substrate properties of modified deoxynucleoside triphosphates has been studied. Deoxyuridines with a long linker between the fluorophore and the nitrogenous base were found to be the most effective substrates. In addition, the possibility of simultaneous use of labeled dU and dC in the process of isothermal amplification has been shown, which is potentially capable of increasing the sensitivity of the analysis method.

Objectives. To study the substrate properties of Cy5-labeled deoxynucleoside triphosphates of various natures (dU and dC) in the process of incorporation in the DNA chain during recombinase polymerase amplification (RPA).Methods. The work used the real-time RPA method. The method of horizontal electrophoresis was used to control the quality of the amplification products obtained.Results. The influence of the fluorophore structure and linker lengths on the substrate properties for deoxynucleoside triphosphates Cy5-dUTP and Cy5-dCTP was studied. The following values of the substrate efficiency parameters were determined: amplification efficiency (kinetic indicator), normalized product yield, and embedding coefficient.Conclusions. Modified deoxynucleoside triphosphates (dNTP) with long linkers between the fluorophore and the nitrogenous base, as well as between the quaternary ammonium group and the second heterocycle of the fluorophore, showed greater substrate efficiency than fluorescently labeled dNTP with short linkers. The modified dU in each pair demonstrated greater substrate efficiency compared to the modified dC.

A diagnostic system based on recombinase polymerase amplification (RPA) has been developed to identify six bacterial pathogens of human pneumonia. Species-specific primers have been designed and optimized to conduct a multiplex reaction in one common volume. Labeled primers were used for reliable discrimination of amplification products that are similar in size. Identification of the pathogen was carried out by visual analysis of an electrophoregram. The analytical sensitivity of the developed multiplex RPA was 10(2)-10(3) copies of DNA. The specificity of the system was determined by the absence of cross-amplification of the studied DNA samples of pneumonia pathogens for each pair of primers, as well as for the DNA of Mycobacterium tuberculosis H37(rv), and amounted to 100%. The execution time of the analysis is less than an 1 h, including the electrophoretic reaction control. The test system can be used in specialized clinical laboratories for rapid analysis of samples from patients with suspected pneumonia.

Fluorescently labeled nucleotides that contain a zwitterionic indodicarbocyanine dye have been synthesized. The dye has been attached at the C5 position of the pyrimidine base through a trans-alkene spacer. The substrate efficiency of the labeled nucleotides has been tested in recombinase polymerase amplification (RPA). As a result of RPA, full-sized target products of the ebpS gene fragment of the causative agent of bacterial pneumonia ( Staphylococcus aureus ) have been formed with a high density of fluorescent label.

“Polymer brushes” with functional epoxy groups on an aluminum substrate of a biochip were obtained by UV-initiated radical polymerization. The conditions of immobilization of fluorescently labeled oligonucleotide probes with various chaotropic agents were selected on the epoxy groups of the biochip.

— The appearance of mutations in the genes encoding the surface proteins of the new type of coronavirus SARS-CoV-2, when it circulates in the host population, makes it difficult to use monoclonal antibodies for its species identification. In such cases, the choice of conservative genetic targets allows identification by molecular biological methods. In this work, previously developed primers specific to the E- gene fragment were tested to detect a new type of coronavirus on six isolates belonging to different genetic variants (the original Wuhan strain, delta and omicron). The choice of a conservative site of the E gene encoding the small transmembrane protein E as a target for reverse transcription with subsequent amplification (RT-PCR) made it possible to detect coronavirus regardless of its subtypes characterized by antigenic heterogeneity in N- and S-proteins. The possibility of species-level identification of the COVID-19 pathogen circulating in Russia is shown, both in the total reaction volume (in a single test tube) and on biological microarrays.

A method for the photolithographic fabrication of biochips with hydrogel cells made of brush acrylic acid–acrylamide copolymers fixed by one end on the surface of a polymer substrate has been developed. Hydrogel cells with reactive carboxyl groups were used for covalent immobilization of oligonucleotide probes. The efficiency of the method was demonstrated by the hybridization analysis of DNA targets of different lengths, matching a segment of exon 7 of the human ABO gene.

A method has been developed for the immobilization of short DNA sequences in agarose cells fixed on the surface of a polymer substrate, with their subsequent partial cleavage from agarose when heated while protecting functional properties. The effectiveness of the method was demonstrated on oligonucleotides, which, after cleavage from agarose, retained the ability to participate as primers in the PCR amplification of the sequence site 7 of the exon of the human ABO gene.

The substrate properties of nitrogen-base modified derivatives of purine and pyrimidine deoxynucleoside triphosphates during their simultaneous pairwise insertion into the growing DNA strand have been studied. Modified nucleotides were introduced using real-time PCR and the primer extension reaction; in one reaction, derivatives with both different and similar functional substituents were used. Genomic bacterial DNA, specially constructed synthetic DNA fragments, and SELEX libraries were used as templates. The reactions were performed using DNA polymerases with no 3'–5' correcting exonuclease activity: Taq, Vent (exo-), DeepVent (exo-), and KOD XL. It was shown that the substrate efficiency is affected by both the size of the substituent group and the chemical nature of deoxynucleoside triphosphate. The effectiveness varies significantly depending on the polymerase used. The most effective of the studied substrates are pyrimidine deoxynucleoside triphosphates in combination with Vent (exo-) DNA polymerase. DNAs modified by pairs of dissimilar nucleotides (dU + dC, dU + dA, dC + dA) with similar and different functional substituents were obtained.

The substrate properties of nitrogen-base modified derivatives of purine and pyrimidine deoxynucleoside triphosphates during their simultaneous pairwise insertion into the growing DNA strand have been studied. Modified nucleotides were introduced using real-time PCR and the primer extension reaction; in one reaction, derivatives with both different and similar functional substituents were used. Genomic bacterial DNA, specially constructed synthetic DNA fragments, and SELEX libraries were used as templates. The reactions were performed using DNA polymerases with no 3'-5' correcting exonuclease activity: Taq, Vent (exo-), DeepVent (exo-), and KOD XL. It was shown that the substrate efficiency is affected by both the size of the substituent group and the chemical nature of deoxynucleoside triphosphate. The effectiveness varies significantly depending on the polymerase used. The most effective of the studied substrates are pyrimidine deoxynucleoside triphosphates in combination with Vent (exo-) DNA polymerase. DNAs modified by pairs of dissimilar nucleotides (dU + dC, dU + dA, dC + dA) with similar and different functional substituents were obtained.

A new method for evaluating the substrate efficiency of deoxynucleoside triphosphates containing functional groups for the selection of modified aptamers (mod-SELEX) is proposed. The method involves conducting three consecutive rounds of PCR with a combinatorial library and a modified dNTP candidate for mod-SELEX. The conclusion about the applicability of a specific dNTP derivative is made by the nature of the change in the amplification curve during the three rounds of PCR in real time and does not require SELEX rounds. If the library degenerates during amplification (becomes less representative), it means that the specific modification of dNTP cannot be used with the selected polymerase and the other selected library amplification conditions, since it leads to competitive amplification. When the nature of the signal accumulation curve does not change, it is concluded that the modified triphosphate does not affect the distribution of oligonucleotides with different sequences in the library, that is, it does not lead to a change in its composition from the point of view of the applied detection method. It is these derivatives that can be applied with the selected conditions for the selection of aptamers. The method is applicable for quick assessment of the substrate suitability of modifications introduced into deoxynucleoside triphosphates for mod-SELEX and will be useful in the selection of aptamers for clinical diagnostics, medicine and scientific research.

Маркировании ДНК пpоизводными 5′-тpифоcфатов 2′-дезокcиуpидина, содержащими цианиновые красители типа Сy5 и Cy7 в качестве флуорофора. Были выбраны два Cy5-dUTP и два Cy7-dUTP, способные эффективно встраиваться в растущую цепь ДНК в ходе ПЦР. Продукты ПЦР, флуоресцентно-меченные разными красителями, гибридизовали с матрицей олигонуклеотидных зондов, иммобилизованных в гидрогелевых ячейках биологического микрочипа. Результаты гибридизации регистрировали с помощью цифровой люминесцентной микроскопии в красном (для Cy5-dUTP) и ближнем инфракрасном (для Cy7-dUTP) диапазонах. Методом последовательных разведений определена минимальная исходная концентрация ДНК, при которой возможно определение генотипа в результате специфического взаимодействия флуоресцентно-меченного ПЦР-продукта и олигонуклеотидных зондов на биочипе. Показано, что флуоресцентно-меченные Cy7-dUTP обеспечивают более высокую чувствительность гибридизационного анализа по сравнению с Cy5-dUTP. Исследована чувствительность гибридизационного анализа с использованием биологического микрочипа при маркировании ДНК пpоизводными 5′-тpифоcфатов 2′-дезокcиуpидина, содержащими цианиновые красители типа Сy5 и Cy7 в качестве флуорофора...