Institute of Chemical Biology and Fundamental Medicine

Abstract DNA polymerase β (Polβ) is a central player of base excision repair (BER), performing gap-filling synthesis on damaged DNA. While nucleosome core particles (NCPs) are known to impede activity of BER enzymes, the regulation of this process in linker DNA adjacent to nucleosomes remains unclear. Here we demonstrate an unexpected stimulation of Polβ-catalyzed gap-filling and strand-displacement synthesis in linker DNA by the adjacent NCP. Notably, the nucleosomal context reinforces the regulatory modulation of Polβ activity by PARP1/PARP2 and FEN1. While linker histone H1 restricts strand-displacement synthesis at the nucleosome entry/exit site, PARP1 and PARP2 modulate Polβ function through competitive binding to DNA gaps or nicks and via poly(ADP-ribosyl)ation (PARylation). At the same time, PARPs binding differentially regulates BER sub-pathway choice, and PARylation alleviates H1-mediated inhibition. These findings reveal a multi-layered regulatory system wherein the nucleosome acts as a dynamic platform coordinating Polβ activity and its interplay with chromatin-associated factors, influencing the balance between short- and long-patch BER. The research advances understanding of chromatin-mediated control of BER DNA repair synthesis and the functional specialization of PARP1 and PARP2 in maintaining genome stability. Highlights The nucleosome core particle acts not only as a barrier but also as a stimulator of Polβ-mediated DNA repair synthesis in adjacent linker DNA. The nucleosome acts as an allosteric platform that enhances the regulatory functions of chromatin-associated factors (PARP1, PARP2, H1) in the linker DNA repair synthesis. PARP1 suppresses overall Polβ synthesis, while PARP2 specifically inhibits strand displacement, thereby gating the choice between short- and long-patch BER pathways.

Argonautes are an evolutionarily conserved family of proteins that use guide oligonucleotides for specific recognition of nucleic acid targets. While eukaryotic Argonautes share a conserved structure and act in RNA interference, prokaryotic Argonautes (pAgos) display remarkable structural and functional variations, including various guide and target specificities. Most studied catalytically active pAgos use 5′-phosphorylated DNA guides to recognize and cleave DNA targets. Here, we describe a new group of pAgos from mesophilic bacteria that use RNA guides to cleave DNA targets and are active at physiological temperatures. In contrast to most characterized pAgo nucleases, these proteins can utilize guides of varying lengths containing either a 5′-phosphate or a 5′-hydroxyl group with similar efficiencies. Measurements of the kinetics of target DNA binding show that the annealing of the guide–target duplex in pAgo occurs in an ordered way and that pAgo increases the rate of guide–target interaction in the seed region. We show that the analyzed pAgos can produce alternative DNA products depending on the structure of guide RNA and the cleavage conditions. The results demonstrate that RNA-guided pAgo nucleases are more widespread than previously anticipated and that these pAgos have a relaxed specificity for target DNA cleavage.

Abstract Chemically modified nucleic acids have become a powerful platform for basic research and applied technologies. Universal nucleobases are used in PCR,sequencing, and the design of nanodevices and aptamers. Fluorescent universal nucleobases have an even wider range of applications, including the development of nucleic acid-based sensors, switches, and relay logic gates. However, few such nucleobases have been proposed to date, and most of them have suboptimal optical properties. Here, we propose an adenine-based molecular rotor, 7,8-dihydro-8-oxo-6-(3-methylbenzo[d]thiazol-2(3H)-ylidene)adenine ( oxo-Ade BZT ), as a new, remarkably bright and potent fluorescent universal nucleobase. Its brightness in both oligodeoxyribonucleotides (ODNs) and DNA duplexes (4200 - 10000 M -1 × cm -1 ) originates from a high molar extinction coefficient (averaged ε 368 37000 M -1 × cm -1 ), provided by the appended 3-methylbenzo[d]thiazolyl moiety, and a relatively high quantum yield (0.11 – 0.27). Melting temperature variations observed upon the incorporation of oxo-Ade BZT opposite native nucleobases in a duplex context did not exceed 10%. The basis of these universal hybridizing properties was unveiled using computational methods. According to molecular dynamics simulations, oxo-Ade BZT pushes the opposite nucleobase out of the DNA double helix and forms multiple hydrophobic contacts with the flanking base pairs. At the same time, the rotational mobility of the bonds between the oxo-Ade BZT -constituting heterobicycles decreases, and oxo-Ade BZT adopts a planar conformation in both ODNs and their duplexes, resulting in the light-up effect. These properties make oxo-Ade BZT a promising molecular tool for analytical, biophysical and biochemical studies.

Bacteriophages, traditionally viewed solely as antibacterial agents, are increasingly being studied for their immunomodulatory properties. In this study, we demonstrate that PM16 phage therapy not only effectively controls subcutaneous Proteus mirabilis infection in mice but also induces long-term specific humoral immunity against subsequent reinfection. This immunomodulatory effect was dose-dependent. In vitro, PM16 directly activates macrophages, leading to increased production of proinflammatory cytokines (tumor necrosis factor-α and interleukin-1β) and inducible nitric oxide synthase, and enhances macrophage bactericidal activity against P. mirabilis. We assume that the enhancement of the adaptive immune response is mediated not by the phage acting as a classical antigenic adjuvant but by its ability to prime innate immune cells, specifically macrophages. This priming leads to more efficient bacterial clearance, antigen presentation, and the formation of protective immunological memory.

Protein-based materials such as human serum albumin (HSA) have demonstrated significant potential for the development of novel wound management materials. For the first time, the formation of HSA-based hydrogels was proposed using a combination of thermal- and ethanol-induced approaches. The combination of phosphate-buffered saline (PBS) and limited (up to 20% v/v) ethanol content offers a promising strategy for fabricating human serum albumin-based hydrogels with tunable properties. The hydrogel formation was studied using in situ dynamic light scattering (DLS) for qualitative and semi-quantitative analysis of the patterns of protein hydrogel formation through thermally induced gelation. The rheological properties of human serum albumin-based hydrogels were investigated. Hydrogels synthesized via thermally induced gelation using a denaturing agent exhibit a dynamic viscosity ranging from 100 to 10,000 mPa·s. The biocompatibility, biodegradability, and structural stability of human serum albumin-based hydrogels were comprehensively evaluated in physiologically relevant media. These human serum albumin-based hydrogels represent a promising platform for developing topical therapeutic agents for wound management and tissue engineering applications. This study investigated the kinetics of tetracycline release from human serum albumin-based hydrogels in PBS and fetal bovine serum (FBS). All tested formulations of HSA-based hydrogels loaded with tetracycline (1 mg/mL) demonstrated antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Corynebacterium striatum strains.

Aeromonas veronii is a mesophilic bacterium that is widely distributed in terrestrial and aquatic environments and is able to cause disease in various species of fish and other animals. In humans, pathogenic A. veronii causes mainly foodborne infections due to increased consumption of chilled seafood. In this study, the novel lytic Aeromonas veronii phage AerV_348 was isolated and characterized. The phage and its bacterial host, A. veronii CEMTC 9558, were isolated from Lake Pestroe, North Kazakhstan. AerV_348 is a dsDNA virus with a genome size of 43,779 bp. The phage is lytic and has a very narrow host range, reproducing only in its host strain. The latent time was determined to be 50 minutes, and the burst size was 55 plaque-forming units per cell. The AerV_348 genome content corresponds to a lytic phage with myovirus morphology that does not contain genetic signs of being a temperate phage. Comparative analysis revealed that AerV_348 belongs to a large group of unclassified tailed phages that infect aquatic bacteria such as Vibrio, Shewanella, and Oceanospirillum. All of these phages have similar genome sizes and structures and share a conserved cluster of genes that encode structural proteins. However, these phages demonstrate high variability in a cluster of genes responsible for DNA metabolism and translation. Currently, the classification of this phage group is not clear, and new phage genomic data may help to separate this group as a new taxonomic unit within the class Caudoviricetes.

Background: Seasonal influenza remains a significant public health problem, and the constant antigenic drift of viruses requires regular vaccine updates. mRNA vaccines offer a promising platform for the development of new, effective influenza vaccines. Administration of the naked mRNA vaccine using a needle-free jet injection system further enhances its safety, reduces cost, and eliminates the need for lipid nanoparticles, which are traditionally used for mRNA delivery. Lyophilization of naked mRNA allows for long-term storage at +4 °C. Methods: We designed and produced an mRNA vaccine against seasonal influenza, designated mRNA-Vector-Flu, encoding the hemagglutinin (HA) of the A/Wisconsin/67/2022(H1N1)pdm09, A/Darwin/9/2021(H3N2), and B/Austria/1359417/2021 strains. The vaccine was lyophilized and stored for 1 month in a refrigerator (+4 °C). A comparative immunogenicity study was conducted between synthesized immediately before use prepared and lyophilized naked mRNA-Vector-Flu. The preparations were administered to BALB/c mice using a jet needleless injection twice, 3 weeks apart. Immunogenicity was assessed on day 35 of the study. Results: A comparative immunogenicity study of naked mRNA-Vector-Flu demonstrated that both the synthesized immediately before use prepared formulation and the lyophilized form, stored at +4 °C for a month, induced similar levels of virus-specific antibodies and generated a pronounced T-cell immune response. Conclusions: Delivery of the naked mRNA vaccine using a needle-free jet injection ensures a high-level immune response, which improves its safety, reduces its cost, and eliminates the need for lipid nanoparticles traditionally used for mRNA delivery. At the same time, lyophilization of the naked mRNA vaccine preserves its biological activity and ensures its storage for at least a month at +4 °C temperatures. Our results demonstrate that our proposed approach can be considered a promising direction for the development and improvement of the mRNA vaccine platform.

Salmonellosis remains one of the leading causes of bacterial gastrointestinal infections in humans and animals. Molecular diagnostics has dramatically reshaped the diagnostic landscape for Salmonella infection; however, it remains time- and resource-intensive. Isothermal DNA amplification, for example loop isothermal amplification (LAMP), performed at a constant temperature, is the basis for the development of rapid diagnostic tests that can be adapted to the point-of-care (PoC) formats and implemented in resource-limited settings or remote from centralized laboratories. The aim of this study was to develop and validate a novel LAMP-based method for detecting Salmonella enterica in human stool samples, wherein amplification results are monitored using a loop primer labeled with a fluorophore and an internal quencher. The proposed method achieves a limit of detection (LoD95) of 250 copies per reaction, with a sensitivity of 86.84% (95% CI: 71.91–95.59%) and specificity of 96.49% (95% CI: 87.89–99.57%) relative to qPCR, and demonstrates increased robustness against DNA amplification inhibitors present in fecal samples. Incorporation of distinct fluorophores into loop primers for FLP-LAMP targeting different genes could potentially enable multiplexing and simultaneous detection of multiple pathogens, thereby expanding the diagnostic utility of isothermal amplification.

The aim of the present study was to investigate the effect of a synthetic microbial consortium (SMC) containing five functionally different bacterial strains (Rahnella aquatilis, Rothia endophytica, Stenotrophomonas indicatrix, Burkholderia contaminans, Lelliotia amnigena) on the growth and development of three agricultural crops (wheat, buckwheat, and rapeseed) on two soil types (chernozem and gray forest soil) under field conditions. The experiment was conducted from June to September 2024 under extreme field conditions, with temperatures reaching 43.8 °C. This study evaluates SMC efficacy under severe abiotic stress, reflecting increasingly common climate extremes. Metagenomic data analysis showed that the introduced strains did not establish stable populations in the soil, possibly due to heat-induced bacterial mortality, though other factors including competition with indigenous microflora and lack of protective formulations may have also contributed. No statistically significant effects on plant morphometric parameters were observed. The extreme temperature and water stress conditions appear to have been the dominant limiting factors, overriding any potential benefits from microbial inoculation, as evidenced by the lack of response to mineral fertilizer application as well. Crop-specific effects were revealed: when cultivating rapeseed on chernozem, a significant increase in available phosphorus content was noted (from 278 ± 45 to 638 ± 92 mg/kg with SMC application, p < 0.001).