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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 hardto- 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 (VHHFc) 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 LD50) of BoNT/A. During the COVID-19 pandemic, mRNAvaccines 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 LD50 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. [ABSTRACT FROM AUTHOR]

A benzothiazole-substituted derivative (X) of 1,3-diaza-2-oxophenoxazine was evaluated with respect to its ability to engage in Ag(I)-mediated homo base pair formation in two different DNA duplexes. The metal binding was determined by a combination of temperature-dependent UV spectroscopy, CD spectroscopy, and fluorescence spectroscopy, indicating the incorporation of two Ag(I) ions to generate a dinuclear X–Ag(I)2–X base pair. Interestingly, a luminescence increase was observed upon metal binding. Theoretical luminescence spectra were calculated using time-dependent density functional theory (TDDFT) for all possible Ag(I)-mediated X : X base pair geometries to identify the species responsible for the increase in luminescence. The study shows that even bulky non-planar artificial nucleobases can be applied to form stabilizing metal-mediated base pairs. [ABSTRACT FROM AUTHOR]

Protein-based therapeutics is an emerging technology to combat various pathologies, including lung diseases. Particle-mediated delivery promotes its accumulation and increases therapeutic efficacy, minimizing side effects. Barnase (Bn) is a highly stable bacterial protein with ribonuclease (RNase) activity that affects cell proliferation. Encapsulation of Bn in particulate forms like liposomes or inorganic nanoparticles leads to its prolonged circulation in blood, but effective accumulation in targeted sites and subsequent internalization are still limited. In this work, we focus on the development of an effective delivery system with ribonuclease activity and specific accumulation in lung tissues. We fabricate multilayer Bn-loaded vaterite nanoparticles conjugated with targeting vector–aptamer specific to the epithelial cell adhesion molecule (EpCAM). Although two Bn layers in the particles do not result in an increase in loading efficiency, they do contribute significantly to ribonuclease activity (44.9 nM/min for (DS/Bn)2/DS vs 14.8 nM/min for DS/Bn/Ds) and enzyme stability (up to 90% of Bn activity for 7 days). We show that the conjugation of particles with EpCAM aptamer drastically increases particle internalization in human lung adenocarcinoma cells with a significant cytotoxic effect. In vivo biodistribution analysis reveals that intratracheal instillation of EpCAM-modified (DS/Bn)2/DS particles causes better accumulation of particles in lung tissues as identified by fluorescence imaging tomography and confocal microscopy. Thus, the developed targeted bilayer vaterite nanoparticles can be a promising protein-based delivery system for the treatment of lung diseases. [ABSTRACT FROM AUTHOR]

Autologous macrophage transfer is an emerging platform for cell therapy. It is anticipated that conventional macrophage reprogramming based on ex vivo polarization using cytokines and ligands of TLRs may enhance the therapeutic effect. We describe an alternative approach based on small interfering RNA (siRNA) knockdown of selected molecular cues of macrophage polarization, namely EGR2, IRF3, IRF5, and TLR4 in Raw264.7 monocyte/macrophage cell line and mouse-bone-marrow-derived macrophages (BMDMs). The impact of IRF5 knockdown was most pronounced, curtailing the expression of other inflammatory mediators such as IL-6 and NOS2, especially in M1-polarized macrophages. Contrary to IRF5, EGR2 knockdown potentiated M1-associated markers while altogether abolishing M2 marker expression, which is indicative of the principal role of EGR2 in the maintenance of alternative phenotypes. IRF3 knockdown suppressed M1 polarization but upregulated Arg 1, a canonical marker of alternative polarization in M1 macrophages. As anticipated, the knockdown of TLR4 also attenuated the M1 phenotype but, akin to IRF3, significantly induced Arginase 1 in M0 and M1, driving the phenotype towards M2. This study validates RNAi as a viable option for the alteration and maintenance of macrophage phenotypes. [ABSTRACT FROM AUTHOR]

Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible to investigate structural features by fixing complexes at different stages of DNA–protein recognition. The most common cross-linking methods are based on reactive groups that interact with native or engineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferential fixation of early-stage complexes or even non-selective cross-linking. We synthesised a set of DNA reagents carrying an acrylamide group attached to the C5 atom of a 2′-deoxyuridine moiety via various linkers and studied cross-linking with MutS as a model protein. MutS scans DNA for mismatches and damaged nucleobases and can form multiple non-specific complexes with DNA that may cause non-selective cross-linking. By varying the length of the linker between DNA and the acrylamide group and by changing the distance between the reactive nucleotide and a mismatch in the duplex, we showed that cross-linking occurs only if the distance between the acrylamide group and cysteine is optimal within the DNA–protein complex. Thus, acrylamide-modified DNA duplexes are excellent tools for studying DNA–protein interactions because of high selectivity of cysteine trapping. [ABSTRACT FROM AUTHOR]

The present study focuses on the immobilization of the bacterial ribonuclease barnase (Bn) into submicron porous calcium carbonate (CaCO3) particles. For encapsulation, we apply adsorption, freezing-induced loading and co-precipitation methods and study the effects of adsorption time, enzyme concentration and anionic polyelectrolytes on the encapsulation efficiency of Bn. We show that the use of negatively charged dextran sulfate (DS) and ribonucleic acid from yeast (RNA) increases the loading capacity (LC) of the enzyme on CaCO3 particles by about 3-fold as compared to the particles with Bn itself. The ribonuclease (RNase) activity of encapsulated enzyme depends on the LC of the particles and transformation of metastable vaterite to stable calcite, as studied by the assessment of enzyme activities in particles. [ABSTRACT FROM AUTHOR]

The lack of high throughput screening (HTS) techniques for small molecules that stabilize DNA iMs limits their development as perspective drug candidates. Here we showed that fluorescence monitoring for probing the effects of ligands on the iM stability using the FAM–BHQ1 pair provides incorrect results due to additional dye-related interactions. We developed an alternative system with fluorescent phenoxazine pseudonucleotides in loops that do not alter iM unfolding. At the same time, the fluorescence of phenoxazine residues is sensitive to iM unfolding that enables accurate evaluation of ligand-induced changes of iM stability. Our results provide the basis for new approaches for HTS of iM ligands. [ABSTRACT FROM AUTHOR]

Purpose: Liver cancers are among the deadliest malignancies due to a limited efficacy of early diagnostics, the lack of appropriate biomarkers and insufficient discrimination of different types of tumors by classic and molecular methods. In this study, we searched for novel long non-coding RNA (lncRNA) as well as validated several known candidates suitable as probable biomarkers for primary liver tumors of various etiology. Methods: We described a novel lncRNA HELIS (aka "HEalthy LIver Specific") and estimated its expression by RT-qPCR in 82 paired tissue samples from patients with hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), combined HCC-CCA, pediatric hepatoblastoma (HBL) and non-malignant hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH). Additionally, we examined expression of cancer-associated lncRNAs HULC, MALAT1, UCA1, CYTOR, LINC01093 and H19, which were previously studied mainly in HCC. Results: We demonstrated that down-regulation of HELIS strongly correlates with carcinogenesis; whereas in tumors with non-hepatocyte origin (HBL, CCA) or in a number of poorly differentiated HCC, this lncRNA is not expressed. We showed that recently discovered LINC01093 is dramatically down-regulated in all malignant liver cancers; while in benign tumors LINC01093 expression is just twice decreased in comparison to adjacent samples. Conclusion: Our study revealed that among all measured biomarkers only down-regulated HELIS and LINC01093, up-regulated CYTOR and dysregulated HULC are perspective for differential diagnostics of liver cancers; whereas others demonstrated discordant results and cannot be considered as potential universal biomarkers for this purpose. [ABSTRACT FROM AUTHOR]

Summary: Translation efficiency contributes several orders of magnitude difference in the overall yield of exogenous gene expression in bacteria. In diverse bacteria, the translation initiation site, whose sequence is the primary determinant of the translation performance, is comprised of the start codon and the Shine–Dalgarno box located upstream. Here, we have examined how the sequence of a spacer between these main components of the translation initiation site contributes to the yield of synthesized protein. We have created a library of reporter constructs with the randomized spacer region, performed fluorescently activated cell sorting and applied next‐generation sequencing analysis (the FlowSeq protocol). As a result, we have identified sequence motifs for the spacer region between the Shine–Dalgarno box and AUG start codon that may modulate the translation efficiency in a 100‐fold range. [ABSTRACT FROM AUTHOR]

1,3-Diaza-2-oxophenoxazine (X) has been introduced as a ligand in silver(I)-mediated base pairing in a parallel DNA duplex. This fluorescent cytosine analog is capable of forming stabilizing X–Ag(I)–X and X–Ag(I)–C base pairs in DNA duplexes, as confirmed by temperature-dependent UV spectroscopy and luminescence spectroscopy. DFT calculations of the silver(I)-mediated base pairs suggest the presence of a synergistic hydrogen bond. Molecular dynamics (MD) simulations of entire DNA duplexes nicely underline the geometrical flexibility of these base pairs, with the synergistic hydrogen bond facing either the major or the minor groove. Upon silver(I) binding to the X:X or X:C base pairs, the luminescence emission maximum experiences a red shift from 448 to 460 nm upon excitation at 370 nm. Importantly, the luminescence of the 1,3-diaza-2-oxophenoxazine ligand is not quenched significantly upon binding a silver(I) ion. In fact, the luminescence intensity even increases upon formation of a X–Ag(I)–C base pair, which is expected to be beneficial for the development of biosensors. As a consequence, the silver(I)-mediated phenoxazinone base pairs represent the first strongly fluorescent metal-mediated base pairs. [ABSTRACT FROM AUTHOR]

Nicking endonucleases (NEases) selectively cleave single DNA strands in double-stranded DNAs at a specific site. They are widely used in bioanalytical applications and in genome editing; however, the peculiarities of DNA–protein interactions for most of them are still poorly studied. Previously, it has been shown that the large subunit of heterodimeric restriction endonuclease BspD6I (Nt.BstD6I) acts as a NEase. Here we present a study of interaction of restriction endonuclease BspD6I with modified DNA containing single non-nucleotide insertion with an azobenzene moiety in the enzyme cleavage sites or in positions of sugar-phosphate backbone nearby. According to these data, we designed a number of effective stimulus-responsive oligonucleotide inhibitors bearing azobenzene or triethylene glycol residues. These modified oligonucleotides modulated the functional activity of Nt.BspD6I after cooling or heating. We were able to block the cleavage of T7 phage DNA by this enzyme in the presence of such inhibitors at 20–25°C, whereas the Nt.BspD6I ability to hydrolyze DNA was completely restored after heating to 45°C. The observed effects can serve as a basis for the development of a platform for regulation of NEase activity in vitro or in vivo by external signals. [ABSTRACT FROM AUTHOR]

Molecular beacons (MBs) are valuable tools in molecular biology, clinical diagnostics and analytical chemistry. Here we describe a novel approach for the design of MBs with nucleotide or non-nucleotide linkers between the stem and loop regions. Such modified MBs have significantly improved specificity and performance for single nucleotide polymorphism (SNP) detection. These advantages are especially distinct, when compared to the classic MBs, in the case of possible interactions between the stem and loop regions. We demonstrated the applicability of such modified MBs for the discrimination of common Factor V, NOS3 and ADRB2 SNPs in model plasmids and in clinical samples. The developed approach could be applicable not only to fluorescently labeled MBs, but also to other biosensors based on nucleic acids with stem–loop structures. [ABSTRACT FROM AUTHOR]

An alkyl azide derivative of 1-phenylethynylpyrene (PEPy) dye was prepared and used in the functionalization of oligonucleotides via click chemistry. Spectral and photo-physical properties of the PEPy-modified oligonucleotides as a single strand, and in perfect or mismatched duplexes, have been studied. A series of PEPy–Dabcyl fluorogenic TaqMan probes were synthesized and tested in qPCR. PEPy proved to be a superior substitute for AMCA as a short wavelength fluorescent dye for qPCR probes. PEPy probes were shown to significantly reduce Cq (a fractional PCR cycle used for quantification) vs. AMCA labeled probes, thus improving on the reliability of detection. Moreover, a larger increase of fluorescence during amplification was observed in the case of PEPy probes that makes this dye very suitable for an end-point PCR technique. This study broadens the panel of fluorescent dyes suitable for the use in probes for quantitative real-time PCR. [ABSTRACT FROM AUTHOR]

Modified oligonucleotides bearing 2′-reactive groups or 2′-conjugated molecules have found wide application as structural tools in molecular biology. Of principal interest has been the use of 2′-reactive oligonucleotides for cross-linking with biomolecules and of 2′-conjugated oligonucleotides in hybridization assays. In this review we compare a range of electrophilic, nucleophilic and photoreactive groups for cross-linking and conjugation studies. IUBMB Life, 56: 209-214, 2004 [ABSTRACT FROM AUTHOR]

Simple Summary: Apoptosis is one of the best-known types of programmed cell death. This process is regulated by a number of genes and proteins, among which the Bcl-2 protein family plays a key role. This family includes anti- and proapoptotic proteins. Cancer cell resistance to apoptosis is commonly associated with overexpression of the antiapoptotic members of Bcl-2 family proteins, in particular, Bcl-2, Bcl-xL, and Mcl-1. Subsequently, these proteins represent perspective targets for anticancer therapy. Here, using an inhibitory approach, we found that Bak and Bcl-xL regulate sensitivity of cancer cells to Mcl-1 inhibition. BH3 mimetics represent a promising tool in cancer treatment. Recently, the drugs targeting the Mcl-1 protein progressed into clinical trials, and numerous studies are focused on the investigation of their activity in various preclinical models. We investigated two BH3 mimetics to Mcl-1, A1210477 and S63845, and found their different efficacies in on-target doses, despite the fact that both agents interacted with the target. Thus, S63845 induced apoptosis more effectively through a Bak-dependent mechanism. There was an increase in the level of Bcl-xL protein in cells with acquired resistance to Mcl-1 inhibition. Cell lines sensitive to S63845 demonstrated low expression of Bcl-xL. Tumor tissues from patients with lung adenocarcinoma were characterized by decreased Bcl-xL and increased Bak levels of both mRNA and proteins. Concomitant inhibition of Bcl-xL and Mcl-1 demonstrated dramatic cytotoxicity in six of seven studied cell lines. We proposed that co-targeting Bcl-xL and Mcl-1 might lead to a release of Bak, which cannot be neutralized by other anti-apoptotic proteins. Surprisingly, in Bak-knockout cells, inhibition of Mcl-1 and Bcl-xL still resulted in pronounced cell death, arguing against a sole role of Bak in the studied phenomenon. We demonstrate that Bak and Bcl-xL are co-factors for, respectively, sensitivity and resistance to Mcl-1 inhibition. [ABSTRACT FROM AUTHOR]

Green fluorescent protein (GFP) chromophore and its congeners draw significant attention mostly for bioimaging purposes. In this work we probed these compounds as antiviral agents. We have chosen LTR-III DNA G4, the major G-quadruplex (G4) present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), as the target for primary screening and designing antiviral drug candidates. The stabilization of this G4 was previously shown to suppress viral gene expression and replication. FRET-based high-throughput screening (HTS) of 449 GFP chromophore-like compounds revealed a number of hits, sharing some general structural features. Structure-activity relationships (SAR) for the most effective stabilizers allowed us to establish structural fragments, important for G4 binding. Synthetic compounds, developed on the basis of SAR analysis, exhibited high LTR-III G4 stabilization level. NMR spectroscopy and molecular modeling revealed the possible formation of LTR-III G4-ligand complex with one of the lead selective derivative ZS260.1 positioned within the cavity, thus supporting the LTR-III G4 attractiveness for drug targeting. Selected compounds showed moderate activity against HIV-I (EC50 1.78–7.7 μM) in vitro, but the activity was accompanied by pronounced cytotoxicity. [ABSTRACT FROM AUTHOR]

The coupling of alternative splicing with the nonsense-mediated decay (NMD) pathway maintains quality control of the transcriptome in eukaryotes by eliminating transcripts with premature termination codons (PTC) and fine-tunes gene expression. Long noncoding RNA (lncRNA) can regulate multiple cellular processes, including alternative splicing. Previously, murine Morrbid (myeloid RNA repressor of Bcl2l11 induced death) lncRNA was described as a locus-specific controller of the lifespan of short-living myeloid cells via transcription regulation of the apoptosis-related Bcl2l11 protein. Here, we report that murine Morrbid lncRNA in hepatocytes participates in the regulation of proto-oncogene NRAS (neuroblastoma RAS viral oncogene homolog) splicing, including the formation of the isoform with PTC. We observed a significant increase of the NRAS isoform with PTC in hepatocytes with depleted Morrbid lncRNA. We demonstrated that the NRAS isoform with PTC is degraded via the NMD pathway. This transcript is presented almost only in the nucleus and has a half-life ~four times lower than other NRAS transcripts. Additionally, in UPF1 knockdown hepatocytes (the key NMD factor), we observed a significant increase of the NRAS isoform with PTC. By a modified capture hybridization (CHART) analysis of the protein targets, we uncovered interactions of Morrbid lncRNA with the SFPQ (splicing factor proline and glutamine rich)-NONO (non-POU domain-containing octamer-binding protein) splicing complex. Finally, we propose the regulation mechanism of NRAS splicing in murine hepatocytes by alternative splicing coupled with the NMD pathway with the input of Morrbid lncRNA. [ABSTRACT FROM AUTHOR]

Recognition of danger signals by a cell initiates a powerful cascade of events generally leading to inflammation. Inflammatory caspases and several other proteases become activated and subsequently cleave their target proinflammatory mediators. The irreversible nature of this process implies that the newly generated proinflammatory fragments need to be sequestered, inhibited, or degraded in order to cancel the proinflammatory program or prevent chronic inflammation. The Arg/N-degron pathway is a ubiquitin-dependent proteolytic pathway that specifically degrades protein fragments bearing N-degrons, or destabilizing residues, which are recognized by the E3 ligases of the pathway. Here, we report that the Arg/N-degron pathway selectively degrades a number of proinflammatory fragments, including some activated inflammatory caspases, contributing in tuning inflammatory processes. Partial ablation of the Arg/N-degron pathway greatly increases IL-1β secretion, indicating the importance of this ubiquitous pathway in the initiation and resolution of inflammation. Thus, we propose a model wherein the Arg/N-degron pathway participates in the control of inflammation in two ways: in the generation of inflammatory signals by the degradation of inhibitory anti-inflammatory domains and as an "off switch" for inflammatory responses through the selective degradation of proinflammatory fragments. [ABSTRACT FROM AUTHOR]

Aptasensors became popular instruments in bioanalytical chemistry and molecular biology. To increase specificity, perspective signaling elements in aptasensors can be separated into a G-quadruplex (G4) part and a free fluorescent dye that lights up upon binding to the G4 part. However, current systems are limited by relatively low enhancement of fluorescence upon dye binding. Here, we added duplex modules to G4 structures, which supposedly cause the formation of a dye-binding cavity between two modules. Screening of multiple synthetic GFP chromophore analogues and variation of the duplex module resulted in the selection of dyes that light up after complex formation with two-module structures and their RNA analogues by up to 20 times compared to parent G4s. We demonstrated that the short duplex part in TBA25 is preferable for fluorescence light up in comparison to parent TBA15 molecule as well as TBA31 and TBA63 stabilized by longer duplexes. Duplex part of TBA25 may be partially unfolded and has reduced rigidity, which might facilitate optimal dye positioning in the joint between G4 and the duplex. We demonstrated dye enhancement after binding to modified TBA, LTR-III, and Tel23a G4 structures and propose that such architecture of short duplex-G4 signaling elements will enforce the development of improved aptasensors. [ABSTRACT FROM AUTHOR]