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Numerous studies have shown that oxidative modifications of guanine (7,8-dihydro-8-oxoguanine, 8-oxoG) can affect cellular functions. 7,8-Dihydro-8-oxoadenine (8-oxoA) is another abundant paradigmatic ambiguous nucleobase but findings reported on the mutagenicity of 8-oxoA in bacterial and eukaryotic cells are incomplete and contradictory. Although several genotoxic studies have demonstrated the mutagenic potential of 8-oxoA in eukaryotic cells, very little biochemical and bioinformatics data about the mechanism of 8-oxoA-induced mutagenesis are available. In this review, we discuss dual coding properties of 8-oxoA, summarize historical and recent genotoxicity and biochemical studies, and address the main protective cellular mechanisms of response to 8-oxoA. We also discuss the available structural data for 8-oxoA bypass by different DNA polymerases as well as the mechanisms of 8-oxoA recognition by DNA repair enzymes. [ABSTRACT FROM AUTHOR]

Rose Bengal (RB) is an anionic xanthene dye with multiple useful biological features, including photosensitization properties. RB was studied extensively as a photosensitizer, mostly for antibacterial and antitumor photodynamic therapy (PDT). The application of RB to virus inactivation is rather understudied, and no RB derivatives have been developed as antivirals. In this work, we used a synthetic approach based on a successful design of photosensitizing antivirals to produce RB derivatives for virus photoinactivation. A series of n-alkyl-substituted RB derivatives was synthesized and evaluated as antiviral photosensitizers. The compounds exhibited similar 1O2 generation rate and efficiency, but drastically different activities against SARS-CoV-2, CHIKV, and HIV; with comparable cytotoxicity for different cell lines. Submicromolar-to-subnanomolar activities and high selectivity indices were detected for compounds with C4-6 alkyl (SARS-CoV-2) and C6-8 alkyl (CHIKV) chains. Spectrophotometric assessment demonstrates low aqueous solubility for C8-10 congeners and a significant aggregation tendency for the C12 derivative, possibly influencing its antiviral efficacy. Initial evaluation of the synthesized compounds makes them promising for further study as viral inactivators for vaccine preparations. [ABSTRACT FROM AUTHOR]

Advances in peptide and protein therapeutics increased the need for rapid and cost-effective polypeptide prototyping. While in vitro translation systems are well suited for fast and multiplexed polypeptide prototyping, they suffer from misfolding, aggregation and disulfide-bond scrambling of the translated products. Here we propose that efficient folding of in vitro produced disulfide-rich peptides and proteins can be achieved if performed in an aggregation-free and thermodynamically controlled folding environment. To this end, we modify an E. coli-based in vitro translation system to allow co-translational capture of translated products by affinity matrix. This process reduces protein aggregation and enables productive oxidative folding and recycling of misfolded states under thermodynamic control. In this study we show that the developed approach is likely to be generally applicable for prototyping of a wide variety of disulfide-constrained peptides, macrocyclic peptides with non-native bonds and antibody fragments in amounts sufficient for interaction analysis and biological activity assessment. Generic approach for rapid prototyping is essential for the progress of synthetic biology. Here the authors modify the cell-free translation system to control protein aggregation and folding and validate the approach by using single conditions for prototyping of various disulfide-constrained polypeptides. [ABSTRACT FROM AUTHOR]

Perylene-based compounds are attracting significant attention due to their high broad-spectrum antiviral activity against enveloped viruses. Despite unambiguous results of in vitro studies and high selectivity index, the poor water solubility of these compounds prevented in vivo evaluation of their antiviral properties. In this work, we synthesized a series of compounds with a perylene pharmacophore bearing positively charged substituents to improve the aqueous solubility of this unique type of antivirals. Three types of charged groups were introduced: (1) quaternary morpholinium salts (3a–b); (2) a 2′-O-l-valinyl-uridine hydrochloride residue (8), and (3) a 3-methylbenzothiazolium cation (10). The synthesized compounds were evaluated based both on antiviral properties in vitro (CHIKV, SARS-CoV-2, and IAV) and on solubility in aqueous media. Compound 10 has the greatest aqueous solubility, making it preferable for pre-evaluation by intragastrical administration in a mouse model of lethal influenza pneumonia. The results indicate that the introduction of a positively charged group is a viable strategy for the design of drug candidates with a perylene scaffold for in vivo studies. [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]

Advances in peptide and protein therapeutics increased the need for rapid and cost-effective polypeptide prototyping. While in vitro translation systems are well suited for fast and multiplexed polypeptide prototyping, they suffer from misfolding, aggregation and disulfide-bond scrambling of the translated products. Here we propose that efficient folding of in vitro produced disulfide-rich peptides and proteins can be achieved if performed in an aggregation-free and thermodynamically controlled folding environment. To this end, we modify an E. coli-based in vitro translation system to allow co-translational capture of translated products by affinity matrix. This process reduces protein aggregation and enables productive oxidative folding and recycling of misfolded states under thermodynamic control. In this study we show that the developed approach is likely to be generally applicable for prototyping of a wide variety of disulfide-constrained peptides, macrocyclic peptides with non-native bonds and antibody fragments in amounts sufficient for interaction analysis and biological activity assessment. Generic approach for rapid prototyping is essential for the progress of synthetic biology. Here the authors modify the cell-free translation system to control protein aggregation and folding and validate the approach by using single conditions for prototyping of various disulfide-constrained polypeptides. [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]

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]

Background: The initial stage of the biosynthesis of steroid hormones in animals occurs in the mitochondria of steroidogenic tissues, where cytochrome P450SCC (CYP11A1) encoded by the CYP11A1 gene catalyzes the conversion of cholesterol into pregnenolone – the general precursor of all the steroid hormones, starting with progesterone. This stage is missing in plants where mitochondrial cytochromes P450 (the mito CYP clan) have not been found. Generating transgenic plants with a mitochondrial type P450 from animals would offer an interesting option to verify whether plant mitochondria could serve as another site of P450 monooxygenase reaction for the steroid hormones biosynthesis. Results: For a more detailed comparison of steroidogenic systems of Plantae and Animalia, we have created and studied transgenic tobacco and tomato plants efficiently expressing mammalian CYP11A1 cDNA. The detailed phenotypic characterization of plants obtained has shown that through four generations studied, the transgenic tobacco plants have reduced a period of vegetative development (early flowering and maturation of bolls), enlarged biomass and increased productivity (quantity and quality of seeds) as compared to the only empty-vector containing or wild type plants. Moreover, the CYP11A1 transgenic plants show resistance to such fungal pathogen as Botrytis cinerea. Similar valuable phenotypes (the accelerated course of ontogenesis and/or stress resistance) are also visible in two clearly distinct transgenic tomato lines expressing CYP11A1 cDNA: one line (No. 4) has an accelerated rate of vegetative development, while the other (No. 7) has enhanced immunity to abiotic and biotic stresses. The progesterone level in transgenic tobacco and tomato leaves is 3-5 times higher than in the control plants of the wild type. Conclusions: For the first time, we could show the compatibility in vivo of even themost specific components of the systems of biosynthesis of steroid hormones in Plantae and Animalia. The hypothesis is proposed and substantiated that the formation of the above-noted special phenotypes of transgenic plants expressing mammalian CYP11A1 cDNA is due to the increased biosynthesis of progesterone that can be considered as a very ancient bioregulator of plant cells and the first real hormone common to plants and animals. [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]

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]