Nucleic acid aptamers are artificial recognition elements with great potential in biotechnology. For their effective integration into nanodevices, rational strategies for optimizing aptamer affinity and regulating activity are essential. Artificial nucleotide analogs offer versatile tools for both fundamental and applied research in the aptamer field. Herein, the affinity of a thrombin-binding aptamer is increased tenfold through a single nucleotide modification with 7,8-dihydro-8-oxo-1, N6-ethenoadenine (oxo-εA) introduced at either the 4th or 13th position. Normally, thymines in these positions form a T:T base pair, which is broken during the thrombin binding. A double oxo-εA modification at both the 4th and 13th positions resulted in Ag+-dependence of aptamer activity. Ag+ ions formed a noncanonical oxo-εA:Ag+ 2:oxo-εA pair, mimicking the T:T pair found in the unmodified aptamer, which is a crucial part of the thrombin recognition interface. The intrinsic fluorescence of oxo-εA is used to investigate conformational rearrangements in the loops during G-quadruplex (GQ) folding. The rapid kinetics of aptamer folding are captured using a stopped-flow fluorimeter. The dissociation constants are estimated for the aptamer complexes with K+, Na+, and Ag+, revealing the tiny influence of the modifications on GQ folding.

Nucleic acid aptamers are artificial recognition elements with high potential in biotechnology. Approaches for aptamer affinity optimization and activity regulation are required for aptamer-based nanodevices. Here thrombin aptamer affinity was increased by a single nucleotide modification with 7,8-dihydro-8-oxo-1,N(SUP)6(/SUP)-ethenoadenine. Whereas a double modification makes the aptamer activity Ag(SUP)+(/SUP)-dependent. ### Competing Interest Statement The authors have declared no competing interest.

Non-canonical nucleic acid structures play significant roles in cellular processes through selective interactions with proteins. While both natural and artificial G-quadruplexes have been extensively studied, the functions of i-motifs remain less understood. This study investigates the artificial aptamer BV42, which binds strongly to influenza A virus hemagglutinin and unexpectedly retains its i-motif structure even at neutral pH. However, BV42 conformational heterogeneity hinders detailed structural analysis. Molecular dynamics simulations and chemical modifications of BV42 helped us to identify a potential binding site, allowing for aptamer redesign to eliminate the conformational diversity while retaining binding affinity. Nuclear magnetic resonance spectroscopy confirmed the i-motif/duplex junction with the three-cytosine loop nearby. This study highlights the unique structural features of the functional i-motif and its role in molecular recognition of the target.

Bacterial and viral RNA polymerases are promising targets for the development of new transcription inhibitors. One of the potential blockers of RNA synthesis is 7,8-dihydro-8-oxo-1,N6-ethenoadenine (oxo-εA), a synthetic compound that combines two adenine modifications: 8-oxoadenine and 1,N6-ethenoadenine. In this study, we synthesized oxo-εA triphosphate (oxo-εATP) and showed that it could be incorporated by the RNA-dependent RNA polymerase of SARS-CoV-2 into synthesized RNA opposite template residues A and G in the presence of Mn2+ ions. Escherichia coli RNA polymerase incorporated oxo-εATP opposite A residues in the template DNA strand. The presence of oxo-εA instead of adenine in the template DNA strand completely stopped transcription at the modified nucleotide. At the same time, oxo-εATP did not suppress RNA synthesis by both RNA polymerases in the presence of unmodified nucleotides. Therefore, the oxo-εA modification significantly disrupts nucleotide base pairing during RNA synthesis by RNA polymerases of different classes, and the corresponding nucleotide derivatives cannot be used as potential antiviral or antibacterial transcription inhibitors.

Non-canonical nucleic acid structures possess an ability to interact selectively with proteins, thereby exerting influence over various intracellular processes. Numerous studies indicate that genomic G-quadruplexes and imotifs are involved in the regulation of transcription. These structures are formed temporarily during the unwinding of the DNA double helix; and their direct determination is a rather difficult task. In addition, i-motif folding is pH-dependent, with most i-motifs having low stability at neutral pH. However, some genomic i-motifs with long cytosine repeats were shown to be stable at pH 7.3, suggesting their functionality within the nucleus. Here we studied pH-dependent behavior of a model i-motif with flanking sequences that forms a duplex motif. Kinetic studies on bimodular structures with cytosine residues replaced with an environment-sensitive fluorescent label reveal the stabilization of the i-motif structure near the i-motif-duplex junction. These results highlight the importance of the natural environment of i-motifs for the correct assessment of their stability.

The molecular beacon (MB) strategy finds diverse applications in bioimaging, biosensing and disorders diagnostics. Classic MBs are stem-loop oligonucleotides modified with a reporter and a quencher at the 5'- and 3'- ends. Hybridization with a specific target leads to spatial separation of the quencher and the reporter with subsequent changes in spectral signal, for example, an increase in reporter fluorescence emission. However, hydrophobic interactions between a conventional bulky quencher and the reporter might influence hybridization properties and limit stem length in MB design. In this work, we studied the synthetic nucleobase analog tCoAzo mimicking cytosine, capable of quenching reporter (FAM) fluorescence in folded MBs while minimally affecting their tharmal stability compared to MBs with classic FAM/BHQ1 labels. In addition, we conducted a preliminary assessment of the applicability of FAM/tCoAzo-modified MBs for SERS-based techniques using short single-stranded and long double-stranded DNA fragments from Fusarium avenaceum elongation factor 1a DNA as matrices. SERS intensity was decreased in the presence of matrix DNA compared to the scrambled sequence; and the MB modified with three tCoAzo residues provided a concentration-dependent signal. The results indicate that tCoAzo is a promising tool for fine tuning MB properties for fluorescent- and SERS-based diagnostic applications.

The oncoprotein Gankyrin/PSMD10 is upregulated in various cancers, including glioblastoma multiforme (GBM), the most difficult tumor to treat. The heterogeneity of this type of tumor requires a multi-targeted approach to overcome radio- and chemoresistance. Here we aim to investigate the effects of Gankyrin inhibition on glioma cell proliferation and its impact on sensitivity to chemotherapy both in vitro and in vivo. We have employed the RNA interference approach as a valuable tool to downregulate the expression of target genes. Small interfering RNA (siRNA) were formulated into lipid nanoparticles (LNP) to facilitate delivery and overcome challenges, including RNase activity. We demonstrate that knockdown of Gankyrin promotes cell growth arrest of murine (GL261, CT-2A) and human (U87, GBM23, GBM24) glioma cells and induces the generation of reactive oxygen species and apoptosis. Importantly, Gankyrin inhibition enhances the therapeutic effect of temozolomide in murine glioma cell lines and decreases the IC50 of doxorubicin and cisplatin for U87 and GL261 cells. The therapeutic potential of Gankyrin inhibition was shown in patient-derived glioblastoma stem cells, which are responsible for tumor initiation and resistance to therapy. We showed that downregulation of Gankyrin led to decreased cell proliferation in vitro and improved survival in xenograft glioma model mice. Our results suggest that harnessing siRNA against Gankyrin may serve as a novel therapeutic strategy for glioblastoma therapy in a multi-drug approach and address challenges in current treatment by sensitizing glioma cells to chemotherapy. ### Competing Interest Statement The authors have declared no competing interest.

Phosphorothioate (PS) group is a key component of a majority of FDA approved oligonucleotide drugs that increase stability to nucleases whilst maintaining interactions with many proteins, including RNase H in the case of antisense oligonucleotides (ASOs). At the same time, uniform PS modification increases nonspecific protein binding that can trigger toxicity and pro-inflammatory effects, so discovery and characterization of alternative phosphate mimics for RNA therapeutics is an actual task. Here we evaluated the effects of the introduction of several N-alkane sulfonyl phosphoramidate groups such as mesyl (methanesulfonyl) or busyl (1-butanesulfonyl) phosphoramidates into gapmer ASOs on the efficiency and pattern of RNase H cleavage, cellular uptake in vitro, and intracellular localization. Using Malat1 lncRNA as a target, we have identified patterns of mesyl or busyl modifications in the ASOs for optimal knockdown in vitro. Combination of the PSMA ligand-mediated delivery with optimized mesyl and busyl ASOs resulted in the efficient target depletion in the prostate cancer cells. Our study demonstrated that other N-alkanesulfonyl phosphoramidate groups apart from a known mesyl phosphoramidate can serve as an essential component of mixed backbone gapmer ASOs to reduce drawbacks of uniformly PS-modified gapmers, and deserve further investigation in RNA therapeutics.

Hepatic fibrosis remains a significant clinical challenge due to ineffective treatments. 4-methylumbelliferone (4MU), a hyaluronic acid (HA) synthesis inhibitor, has proven safe in phase one clinical trials. In this study, we aimed to ameliorate liver fibrosis by inhibiting HA synthesis. We compared two groups of mice with CCl4-induced fibrosis, treated with 4-methylumbelliferone (4MU) and hyaluronan synthase 2 (HAS2) targeting siRNA (siHAS2). The administration of 4MU and siHAS2 significantly reduced collagen and HA deposition, as well as biochemical markers of hepatic damage induced by repeated CCl4 injections. The transcriptomic analysis revealed converging pathways associated with downstream HA signalling. 4MU- and siHAS2-treated fibrotic livers shared 405 upregulated and 628 downregulated genes. These genes were associated with xenobiotic and cholesterol metabolism, mitosis, endoplasmic reticulum stress, RNA processing, and myeloid cell migration. The functional annotation of differentially expressed genes (DEGs) in siHAS2-treated mice revealed attenuation of extracellular matrix-associated pathways. In comparison, in the 4MU-treated group, DEGs were related to lipid and bile metabolism pathways and cell cycle. These findings confirm that HAS2 is an important pharmacological target for suppressing hepatic fibrosis using siRNA.

Abstract Background Prostate adenocarcinoma (PRAD) is the second leading cause of cancer-related deaths in men. High variability in DNA methylation and a high rate of large genomic rearrangements are often observed in PRAD. Results To investigate the reasons for such high variance, we integrated DNA methylation, RNA-seq, and copy number alterations datasets from The Cancer Genome Atlas (TCGA), focusing on PRAD, and employed weighted gene co-expression network analysis (WGCNA). Our results show that only single cluster of co-expressed genes is associated with genomic and epigenomic instability. Within this cluster, TP63 and TRIM29 are key transcription regulators and are downregulated in PRAD. We discovered that TP63 regulates the level of enhancer methylation in prostate basal epithelial cells. TRIM29 forms a complex with TP63 and together regulates the expression of genes specific to the prostate basal epithelium. In addition, TRIM29 binds DNA repair proteins and prevents the formation of the TMPRSS2:ERG gene fusion typically observed in PRAD. Conclusion Our study demonstrates that TRIM29 and TP63 are important regulators in maintaining the identity of the basal epithelium under physiological conditions. Furthermore, we uncover the role of TRIM29 in PRAD development.

Non-canonical nucleic acid structures have been shown the capacity to selectively interact with proteins, thereby exerting influence over various intracellular processes. Among these structures, both natural and artificial G-quadruplexes have been extensively studied in relation to their structure-activity relationships. In contrast, the role of i-motifs remains incompletely evaluated. In this study the artificial aptamer BV42 possessing a high affinity for hemagglutinin of influenza A virus was proven to contain the i-motif structure even at neutral pH. However, conformational heterogeneity of BV42 poses challenges for in-depth structural investigations. Using molecular dynamics simulations and introducing chemical modifications for molecular probing, a putative binding site in the aptamer was suggested. These findings have enabled us to redesign the aptamer, eliminating the conformational diversity associated with the i-motif while preserving its binding affinity. Subsequent validation through NMR spectroscopy confirms the presence of the i-motif/duplex junction with the 3-cytosine loop located near the junction. This study elucidates a distinctive instance of an unusual nucleic acid architecture involved in molecular recognition, thereby shedding light on the structural peculiarities of functional i-motifs.

DNA-intercalated motifs (iMs) are facile scaffolds for the design of various pH-responsive nanomachines, including biocompatible pH sensors. First, DNA pH sensors relied on complex intermolecular scaffolds. Here, we used a simple unimolecular dual-labeled iM scaffold and minimized it by replacing the redundant loop nucleosides with abasic or alkyl linkers. These modifications improved the thermal stability of the iM and increased the rates of its pH-induced conformational transitions. The best effects were obtained upon the replacement of all three native loops with short and flexible linkers, such as the propyl one. The resulting sensor showed a pH transition value equal to 6.9 ± 0.1 and responded rapidly to minor acidification (tau1/2 <1 s for 7.2 → 6.6 pH jump). We demonstrated the applicability of this sensor for pH measurements in the nuclei of human lung adenocarcinoma cells (pH = 7.4 ± 0.2) and immortalized embryonic kidney cells (pH = 7.0 ± 0.2). The sensor stained diffusely the nucleoplasm and piled up in interchromatin granules. These findings highlight the prospects of iMs in the studies of normal and pathological pH-dependent processes in the nucleus, including the formation of biomolecular condensates.

Progress in RNA metabolism and function studies relies largely on molecular imaging systems, including those comprising a fluorogenic dye and an aptamer-based fluorescence-activating tag. G4 aptamers of the Mango family, typically combined with a duplex/hairpin scaffold, activate the fluorescence of a green light-emitting dye TO1-biotin and hold great promise for intracellular RNA tracking. Here, we report a new Mango-based imaging platform. Its key advantages are the tunability of spectral properties and applicability for visualization of small RNA molecules that require minimal tag size. The former advantage is due to an expanded (green-to-red-emitting) palette of TO1-inspired fluorogenic dyes, and the truncated duplex scaffold ensures the latter. To illustrate the applicability of the improved platform, we tagged Mycobacterium tuberculosis sncRNA with the shortened aptamer-scaffold tag. Then, we visualized it in bacteria and bacteria-infected macrophages using the new red light-emitting Mango-activated dye.

Reactive oxygen species (ROS) are highly reactive products of the cell metabolism derived from oxygen molecules, and their abundant level is observed in many diseases, particularly tumors, such as hepatocellular carcinoma (HCC). In vivo imaging of ROS is a necessary tool in preclinical research to evaluate the efficacy of drugs with antioxidant activity and for diagnosis and monitoring of diseases. However, most known sensors cannot be used for in vivo experiments due to low stability in the blood and rapid elimination from the body. In this work, we focused on the development of an effective delivery system of fluorescent probes for intravital ROS visualization using the HCC model. We have synthesized various lipid nanoparticles (LNPs) loaded with ROS-inducible hydrocyanine pro-fluorescent dye or plasmid DNA (pDNA) with genetically encoded protein sensors of hydrogen peroxide (HyPer7). LNP with an average diameter of 110 ± 12 nm, characterized by increased stability and pDNA loading efficiency (64 ± 7%), demonstrated preferable accumulation in the liver compared to 170 nm LNPs. We evaluated cytotoxicity and demonstrated the efficacy of hydrocyanine-5 and HyPer7 formulated in LNP for ROS visualization in mouse hepatocytes (AML12 cells) and in the mouse xenograft model of HCC. Our results demonstrate that obtained LNP could be a valuable tool in preclinical research for visualization ROS in liver diseases.

We propose catalytically synthesized nanozymes based on Prussian Blue (PB) and azidomethyl-substituted poly (3,4-ethylenedioxythiophene) (azidomethyl-PEDOT) as novel electrocatalytic labels for DNA/RNA sensors. Catalytic approach allowed to synthesize highly redox and electrocatalytically active Prussian Blue nanoparticles functionalized with azide groups that enable 'click' conjugation with alkyne-modified oligonucleotides. Both competitive and sandwich-type schemes were realized. As the sensor response the direct (mediator-free) electrocatalytic current of H2O2 reduction can be measured, which is proportional to the concentration of the hybridized labeled sequences. The current of H2O2 electrocatalytic reduction is only 3-8 times increased in the presence of the freely diffusing mediator catechol, which indicates high efficiency of direct electrocatalysis with the elaborated labels. Electrocatalytic amplification of the signal allows robust detection of (63-70)-base target sequences with concentrations below 0.2 nM in blood serum within an hour. We believe, the use of advanced Prussian Blue based electrocatalytic labels sets new avenues for point-of-care DNA/RNA sensing.

A template-assisted assembly approach to a C24 fullerene-like double-stranded DNA polyhedral shell is proposed. The assembly employed a supramolecular oligonucleotide dendrimer as a 3D template that was obtained via the hybridization of siRNA strands and a single-stranded DNA oligonucleotide joined to three- or four-way branched junctions. A four-way branched oligonucleotide building block (a starlet) was designed for the assembly of the shell composed of three identical self-complementary DNA single strands and a single RNA strand for hybridization to the DNA oligonucleotides of the template. To prevent premature auto-hybridization of the self-complementary oligonucleotides in the starlet, a photolabile protecting group was introduced via the N3-substituted thymidine phosphoramidite. Cleavable linkers such as a disulfide linkage, RNase A sensitive triribonucleotides, and di- and trideoxynucleotides were incorporated into the starlet and template at specific points to guide the post-assembly disconnection of the shell from the template, and enzymatic disassembly of the template and the shell in biological media. At the same time, siRNA strands were modified with 2′-OMe ribonucleotides and phosphorothioate groups in certain positions to stabilize toward enzymatic digestion. We report herein a solid-phase synthesis of branched oligodeoxy and oligoribonucleotide building blocks for the DNA/RNA dendritic template and the branched DNA starlet for a template-assisted construction of a C24 fullerene-like DNA shell after initial molecular modeling, followed by the assembly of the shell around the DNA-coated RNA dendritic template, and visualization of the resulting nanostructure by transmission electron microscopy.

To overcome immune tolerance to cancer, the immune system needs to be exposed to a multi-target action intervention. Here, we investigated the activating effect of CpG oligodeoxynucleotides (ODNs), mesyl phosphoramidate CpG ODNs, anti-OX40 antibodies, and OX40 RNA aptamers on major populations of immunocompetent cells ex vivo. Comparative analysis of the antitumor effects of in situ vaccination with CpG ODNs and anti-OX40 antibodies, as well as several other combinations, such as mesyl phosphoramidate CpG ODNs and OX40 RNA aptamers, was conducted. Antibodies against programmed death 1 (PD1) checkpoint inhibitors or their corresponding PD1 DNA aptamers were also added to vaccination regimens for analytical purposes. Four scenarios were considered: a weakly immunogenic Krebs-2 carcinoma grafted in CBA mice; a moderately immunogenic Lewis carcinoma grafted in C57Black/6 mice; and an immunogenic A20 B cell lymphoma or an Ehrlich carcinoma grafted in BALB/c mice. Adding anti-PD1 antibodies (CpG+αOX40+αPD1) to in situ vaccinations boosts the antitumor effect. When to be used instead of antibodies, aptamers also possess antitumor activity, although this effect was less pronounced. The strongest effect across all the tumors was observed in highly immunogenic A20 B cell lymphoma and Ehrlich carcinoma.

Single-domain antibodies (sdAbs, VHHs, or nanobodies) are a promising tool for the treatment of both infectious and somatic diseases. Their small size greatly simplifies any genetic engineering manipulations. Such antibodies have the ability to bind hard-to-reach antigenic epitopes through long parts of the variable chains, the third complementarity-determining regions (CDR3s). VHH fusion with the canonical immunoglobulin Fc fragment allows the Fc-fusion single-domain antibodies (VHH-Fc) to significantly increase their neutralizing activity and serum half-life. Previously we have developed and characterized VHH-Fc specific to botulinum neurotoxin A (BoNT/A), that showed a 1000-fold higher protective activity than monomeric form when challenged with five times the lethal dose (5 LD 50 ) of BoNT/A. During the COVID-19 pandemic, mRNA vaccines based on lipid nanoparticles (LNP) as a delivery system have become an important translational technology that has significantly accelerated the clinical introduction of mRNA platforms. We have developed an mRNA platform that provides long-term expression after both intramuscular and intravenous application. The platform has been extensively characterized using firefly luciferase (Fluc) as a reporter. An intramuscular administration of LNP-mRNA encoding VHH-Fc antibody made it possible to achieve its rapid expression in mice and resulted in 100% protection when challenged with up to 100 LD 50 of BoNT/A. The presented approach for the delivery of sdAbs using mRNA technology greatly simplifies drug development for antibody therapy and can be used for emergency prophylaxis.