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The liquid−liquid phase separation phase separation phenomenon has been described for some biopolymers and has been studied in sufficient detail for a few proteins with intrinsically disordered regions. One example of such proteins is the nucleocapsid (N) protein of the severe acute respiratory syndrome coronavirus 2. In this review, we have analyzed available data on N-protein separation in the presence of viral RNA. Particular attention has been paid to transient contacts within the condensates and the N-protein/RNA fragments that form these contacts. The modern concepts of the role of the condensates in the SARS-CoV-2 life cycle and their influence on the host-protective machinery have been summarized. Finally, comments on the possibility of regulating the viral condensates using synthetic or natural small molecules (phase separation modulators), which can provide a new option in the design of antiviral agents, are given. [ABSTRACT FROM AUTHOR]

Entomopathogenic bacteria of the genus Photorhabdus secrete protease S (PrtS), which is considered a virulence factor. We found that in the Photorhabdus genomes, immediately after the prtS genes, there are genes that encode small hypothetical proteins homologous to emfourin, a recently discovered protein inhibitor of metalloproteases. The gene of emfourin-like inhibitor from Photorhabdus laumondii subsp. laumondii TT01 was cloned and expressed in Escherichia coli cells. The recombinant protein, named photorin (Phin), was purified by metal-chelate affinity and gel permeation chromatography and characterized. It has been established that Phin is a monomer and inhibits activity of protealysin and thermolysin, which, similar to PrtS, belong to the M4 peptidase family. Inhibition constants were 1.0 ± 0.3 and 10 ± 2 µM, respectively. It was also demonstrated that Phin is able to suppress proteolytic activity of P. laumondii culture fluid (half-maximal inhibition concentration 3.9 ± 0.3 nM). Polyclonal antibodies to Phin were obtained, and it was shown by immunoblotting that P. laumondii cells produce Phin. Thus, the prtS genes in entomopathogenic bacteria of the genus Photorhabdus are colocalized with the genes of emfourin-like inhibitors, which probably regulate activity of the enzyme during infection. Strict regulation of the activity of proteolytic enzymes is essential for functioning of all living systems. At the same time, the principles of regulation of protease activity by protein inhibitors remain poorly understood. Bacterial protease-inhibitor pairs, such as the PrtS and Phin pair, are promising models for in vivo studies of these principles. Bacteria of the genus Photorhabdus have a complex life cycle with multiple hosts, being both nematode symbionts and powerful insect pathogens. This provides a unique opportunity to use the PrtS and Phin pair as a model for studying the principles of protease activity regulation by proteinaceous inhibitors in the context of bacterial interactions with different types of hosts. [ABSTRACT FROM AUTHOR]

In this work, a comparative analysis of the conditions of transglycosylation reactions catalyzed by E. coli nucleoside phosphorylases was carried out, and the optimal conditions for the formation of various nucleosides were determined. Under the optimized conditions of transglycosylation reaction, fluorine-containing derivatives of N6-benzyl-2'-deoxyadenosine, potential inhibitors of replication of enteroviruses in a cell, were obtained starting from the corresponding ribonucleosides. [ABSTRACT FROM AUTHOR]

A cytosine-rich sequence from the human genome that is presumably involved in the regulation of SHANK1 gene expression through the formation of an intercalated motif (iM) has been investigated. A modification that imitates base excision in the iM loops was shown to significantly accelerate the conformational transitions induced by small pH changes. These results supplement the understanding of the relation between DNA repair and the formation of noncanonical secondary structures. Moreover, they open up new possibilities in the development of oligonucleotide pH sensors with a fast response. [ABSTRACT FROM AUTHOR]

Three evolutionary conserved (G-rich) sites of Alu repeats (PQS2, PQS3, and PQS4) could form in vitro stable inter- and intramolecular G-quadruplexes (GQs). Structures and topologies of these GQs were elucidated using spectral methods. The study of self-association of G-rich Alu fragments performed using a FRET-based method revealed dimeric GQ formation from two distally located sites (PQS2), (PQS3) or PQS2−PQS3 within one extended single stranded DNA. Using DOSY NMR, AFM microscopy and differential CD spectroscopy it has been demonstrated that oligomer PQS4 (folded into a parallel intramolecular GQ) forms stacks of quadruplexes stabilized by stacking interactions of external G-tetrads. Comparative analysis of the properties of various GQs suggests involvement of two universal general mechanisms of GQ-dependent genomic rearrangements: (i) formation of dimeric GQs from fragments of different molecules; (ii) formation of GQ-GQ-stacks from pre-folded intramolecular parallel GQs from different strands. Thus, association of G-rich Alu motifs with sensitivity to double-strand breaks and rearrangements may be attributed not to structural features of G-rich Alu fragments, but also to their high abundance. [ABSTRACT FROM AUTHOR]

We report results of the first systematic study of conformational polymorphism of G-rich DNA fragments of Alu-repeats. Alu retrotransposons are primate-specific short interspersed elements. Using the Alu sequence of the prooncogen bcl2 intron and the consensus AluS sequence as representative examples, we have determined characteristic Alu sites that are capable of adopting G-quadruplex (GQ) conformations (i.e., potential quadruplex sites-PQSAlu), and demonstrated by bioinformatics methods that these sites are Alu-specific in the human genome. Genomic frequencies of PQS Alu were assessed (~1/10000 bp). These sites were found to be characteristic of young (active) Alu families (Alu-Y). A recombinant DNA sequence bearing the Alu element of the human bcl2 gene (304 bp) and its PQS-mutant (Alu-PQS) were constructed. The formation of noncanonical structures in Alu dsDNA and their absence in the case of Alu-PQS have been shown using DMS-footprinting and atomic force microscopy (AFM). Expression vectors bearing wild-type and mutant Alu insertions in the promoter regions of the reporter gene have been prepared, and their regulatory effects have been compared during transfection of НЕК293 and HeLa cells. We suggest that the dynamic study of the spatial organization of Alu repeats may provide insight into the mechanisms of genomic rearrangements responsible for the development of many oncological and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Since potential therapeutic significance had been discovered for quadruplex secondary structures of nucleic acids, many compounds stabilizing these targets were identified. The progress was due to understanding the structural features of G-quadruplexes. Quadruplex ligands selectively suppress the growth of tumor cells by indirectly attenuating telomerase activity or inhibiting oncogene expression. A therapeutic effect demonstrated in vivo supports the idea that G-quadruplexes are promising for the development of targeted anticancer drugs. The review highlights the significance of G-quadruplexes as therapeutic targets and focuses on the biochemical properties of low-molecular-weight quadruplex ligands. [ABSTRACT FROM AUTHOR]

Supramolecular complexes of biopolymers based on magnetic nanoparticles play an important role in creation of biosensors, implementation of theragnostic and gene therapeutic methods and biosafety evaluation. We investigated the impact of DNA interactions with nanoparticles of nickel (nNi) on the integrity and functionality of DNA. Data obtained by mass spectrometry, electrophoresis, TEM and AFM microscopy techniques, bacterial transformation, and real-time PCR provide evidence that ssDNA and plasmid DNA (pDNA) efficiently form complexes with nNi. AFM data suggest that the complexes are necklace-type structures, in which nanoparticles are randomly distributed along the DNA chains, rather than highly entangled clot-type structures. After desorption, observed DNA characteristics in bioanalytical and biological systems remain unchanged. Only supercoiled pDNA was nicked, but remained, as well as a plasmid-nNi complex, active in expression vector assays. These results are very important for creation of new methods of DNA immobilization and controlled manipulation. [ABSTRACT FROM AUTHOR]

Oligonucleotide analogues containing one or a few glycine, L-, and D-alanine residues instead of phosphodiester internucleotide linkages were synthesized (C3′-NH-C(O)-CH(X)-NH-C(O)-C4′, where X = H, ( S)-CH3, and ( R)-CH3. The stability of the duplexes of modified oligonucleotides with their wild-type complements was studied. The incorporation of glycine and L-alanine residues into internucleotide linkages was shown to noticeably decrease the stability of modified duplexes as compared to that of native ones (Δ Tm∼−2°C per modification), whereas analogues containing D-alanine linkers form duplexes with increased stability (Δ Tm∼+2°C per modification). [ABSTRACT FROM AUTHOR]

A dinucleotide containing a C3′-NH-C(O)-CH2-C5′ amide internucleotide bond was synthesized by the interaction of 3′-deoxy-3′-amino-5′- O-( tert-butyldimethylsilyl)thymidine with 3′- O-benzyl-2′- O-tert-butyldimethylsilyl-5′-deoxy-5′-carboxymethylribosylthymine, which was obtained from 2′- O-acetyl-3′- O-benzyl-5′-deoxy-5′-ethoxycarbonylmethylribosylthymine through the methanolysis of the acetyl group followed by silylation of liberated hydroxyl and ester saponification. After standard manipulation with protecting groups, the dinucleotide was converted into 3′- O(2-cyanoethyl- N,N-diisopropylphosphoramidite), which was used for the synthesis of modified oligonucleotides on an automated synthesizer. The melting curves of the duplexes formed by modified and complementary natural oligonucleotides were registered, and the melting temperatures and thermodynamic parameters of the duplex formation were calculated. The introduction of a single modified bond into the oligonucleotide led to an insignificant decrease in the melting temperature of these duplexes as compared to unmodified ones. [ABSTRACT FROM AUTHOR]

Abstract  A method for the synthesis of 5′-deoxy-5′-ethoxycarbonylmethyl nucleosides has been developed. 3-O-benzyloxymethyl-1,2-O-isopropylidene-α-D-allofuranose was oxidized by sodium periodate to form a 5′-aldo derivative, which was converted by the reaction with triethylphosphonoacetate in the presence of sodium hydride into a 5-deoxy-5-ethoxycarbonylmethylene derivative. The hydration of the unsaturated compound gave 5-deoxy-5-ethoxycarbonylmethyl-1,2-O-isopropylidene-α-D-ribofuranose. After the benzylation of 3-hydroxyl, the removal of the isopropylidene group by heating with acetic acid, and the subsequent acetylation, 1,2-di-O-acetyl-3-O-benzyl-5-deoxy-5-ethoxycarbonylmethyl-D-ribofuranose was obtained, which reacted with persilylated nucleic acid bases to form 5′-deoxy-5′-ethoxycarbonylmethyl nucleosides. [ABSTRACT FROM AUTHOR]

Abstract  A dinucleoside bearing an amide internucleotide C3′-CH2-C(O)-NH-C5′ bond was synthesized by the interaction of 3′-deoxy-3′-carboxylmethylribothymidine-2′,3′-lactone obtained by hydrolysis of 2′-O-acetyl-5′-O-benzoyl-3′-deoxy-3′-ethoxycarboxylmethylribothymidine with 5′-deoxy-5′-amino-3′-O-(tert-butyldimethylsilyl)thymidine. After standard manipulations with protective groups, the dinucleoside was converted into 3′-O-(2-cyanoethyl-N,N′-diisopropylphosphoroamidite), which was used for the synthesis of modified oligonucleotides on an automatic synthesizer. Duplex melting curves formed by modified and complementary natural oligonucleotides were measured and the melting temperatures and thermodynamic parameters of duplex formation were calculated. The introduction of one modified bond into oligonucleotides caused only an insignificant decrease in the duplex melting temperatures compared with the nonmodified ones. [ABSTRACT FROM AUTHOR]

Abstract  An improved method for the synthesis of 3-deoxy-3-carboxymethyl nucleosides was suggested. Oxidation of 5-O-benzoyl-1,2-O-isopropylidene-α-D-xylofuranose resulted in the 3-keto derivative, which was treated with triethylphosphonoacetate in the presence of sodium hydride to obtain the 3-deoxy-3-ethoxycarbonylmethylene derivative. Hydrogenation of the unsaturated compound proceeded strictly stereospecifically and gave the product with the ribo-configuration. Acetolysis of the resulting compound with AcOH-Ac2O-CH3SO3H led to 1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-3-ethoxycarbonylmethyl-D-ribofuranose, whose interaction with persilylated nucleic bases gave 3-deoxy-3-ethoxycarbonylmethylnucleosides in a total yield of 42–49% from the starting compound. [ABSTRACT FROM AUTHOR]

Silicateins play a key role in biosynthesis of spicules in marine sponges; they are also capable to catalyze formation of amorphous silica in vitro. Silicateins are highly homologous to cathepsins L - a family of cysteine proteases. Molecular mechanisms of silicatein activity remain controversial. Here site-directed mutagenesis was used to clarify significance of selected residues in silica polymerization. A number of mutations were introduced into two sponge proteins - silicatein A1 and cathepsin L from Latrunculia oparinae, as well as into human cathepsin L. First direction was alanine scanning of the proposed catalytic residues. Also, reciprocal mutations were introduced at selected positions that differ between cathepsins L and silicateins. Surprisingly, all the wild type and mutant proteins were capable to catalyze amorphous silica formation with a water-soluble silica precursor tetra(glycerol)orthosilicate. Some mutants possessed several-fold enhanced silica-forming activity and can potentially be useful for nanomaterial synthesis applications. Our findings contradict to the previously suggested mechanisms of silicatein action via a catalytic triad analogous to that in cathepsins L. Instead, a surface-templated biosilification by silicateins and related proteins can be proposed. [ABSTRACT FROM AUTHOR]

The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing the mNeonGreen fluorescent part in NTnC with EYFP, we engineered an NTnC-like indicator, referred to as YTnC, that had a positive and substantially improved calcium response and faster kinetics. YTnC had a 3-fold higher calcium response and 13.6-fold lower brightness than NTnC in vitro. According to stopped-flow experiments performed in vitro, YTnC had 4-fold faster calcium-dissociation kinetics than NTnC. In HeLa cells, YTnC exhibited a 3.3-fold lower brightness and 4.9-fold increased response to calcium transients than NTnC. The spontaneous activity of neuronal cultures induced a 3.6-fold larger ΔF/F response of YTnC than previously shown for NTnC. On patched neurons, YTnC had a 2.6-fold lower ΔF/F than GCaMP6s. YTnC successfully visualized calcium transients in neurons in the cortex of anesthetized mice and the hippocampus of awake mice using single- and two-photon microscopy. Moreover, YTnC outperformed GCaMP6s in the mitochondria and endoplasmic reticulum of cultured HeLa and neuronal cells. [ABSTRACT FROM AUTHOR]

In this study we describe new modified analogs of the thrombin binding aptamer (TBA) containing 5-nitroindole residues. It has been shown that all modified TBAs form an anti-parallel G-quadruplex structure and retain the ability to inhibit thrombin. The most advanced TBA variant (TBA-N8) has a substantially increased clotting time and two-fold lower IC50 value compared to the unmodified prototype. Molecular modelling studies suggest that the improved anticoagulant properties of TBA-N8 result from changes in the binding mode of the analog. A modified central loop in TBA-N8 is presumed to participate in the binding of the target protein. Studies of FAM labelled TBA and TBA-N8 showed an improved binding affinity of the modified aptamer and provided evidence of a direct interaction between the modified central loop and thrombin. Our findings have implications for the design of new aptamers with improved binding affinities. [ABSTRACT FROM AUTHOR]