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Both, DNA and RNA nucleoside modifications contribute to the complex multi-level regulation of gene expression. Modified bases in tRNAs modulate protein translation rates in a highly dynamic manner. Synonymous codons, which differ by the third nucleoside in the triplet but code for the same amino acid, may be utilized at different rates according to codon–anticodon affinity. Nucleoside modifications in the tRNA anticodon loop can favor the interaction with selected codons by stabilizing specific base pairs. Similarly, weakening of base pairing can discriminate against binding to near-cognate codons. mRNAs enriched in favored codons are translated in higher rates constituting a fine-tuning mechanism for protein synthesis. This so-called codon bias establishes a basic protein level, but sometimes it is necessary to further adjust the production rate of a particular protein to actual requirements, brought by, e.g., stages in circadian rhythms, cell cycle progression or exposure to stress. Such an adjustment is realized by the dynamic change of tRNA modifications resulting in the preferential translation of mRNAs coding for example for stress proteins to facilitate cell survival. Furthermore, tRNAs contribute in an entirely different way to another, less specific stress response consisting in modification-dependent tRNA cleavage that contributes to the general down-regulation of protein synthesis. In this review, we summarize control functions of nucleoside modifications in gene regulation with a focus on recent findings on protein synthesis control by tRNA base modifications.

Oxidatively damaged biomolecules impair cellular functions and contribute to the pathology of a variety of diseases. RNA is also attacked by reactive oxygen species, and oxidized RNA is increasingly recognized as an important contributor to neurodegenerative complications in humans. Recently, evidence has accumulated supporting the notion that tRNA is involved in cellular responses to various stress conditions. This review focuses on the intriguing consequences of oxidative modification of tRNA at the structural and functional level.

The results of our previous research indicated that some derivatives of benzofurans, particularly halogeno-derivatives, are selectively toxic towards human leukemia cells. Continuing our work with this group of compounds we here report new data on the synthesis as well as regarding the physico-chemical and biological characterization of fourteen new derivatives of benzofurans, including six brominated compounds. The structures of all new compounds were established by spectroscopic methods (1H- and, 13C-NMR, ESI MS), and elemental analyses. Their cytotoxicity was evaluated against K562 (leukemia), MOLT-4 (leukemia), HeLa (cervix carcinoma), and normal cells (HUVEC). Five compounds (1c, 1e, 2d, 3a, 3d) showed significant cytotoxic activity against all tested cell lines and selectivity for cancer cell lines. The SAR analysis (structure-activity relationship analysis) indicated that the presence of bromine introduced to a methyl or acetyl group that was attached to the benzofuran system increased their cytotoxicity both in normal and cancer cells.

Histidine triad nucleotide-binding protein 1 (HINT1) is the oldest and most widely distributed branch of the histidine triad superfamily of proteins. The HINT1 protein plays an important role in various biological processes and has been found in many species. Here we report the first nearly complete structure of the human HINT1 protein at 1.43 Å resolution obtained from a crystal of the P212121 orthorhombic space group. The final structure has an Rcryst = 22.4% (Rfree = 27.7%) and contains a fragment of the N-terminal part that was not determined in the previously deposited structures. In addition, selective binding of the L-malate ion was detected, which had not been observed previously.

Heteroduplexes composed of all-DNA and all-2′-OMe RNA strands do not occur in nature, but they have found application in the development of molecular beacons and could also be used as aptamers or elements of nucleic acid-based nanostructures that will contain such structural motifs. The crystallization experiments performed have shown that the introduction of overhangs at the ends of the duplex has a great influence on the success of crystallization, as well as on the DNA:2′-OMe-RNA heteroduplex crystal packing. The molecular and crystal structure of the DNA:2′-O-methyl-RNA heteroduplex in its overhanging and blunt-ended versions was determined at 100 K using synchrotron radiation with a resolution of 1.91 and 1.55 Å, respectively. The Zn-SAD method was used to resolve the original duplex structure when molecular replacement by many existing models of duplex structures failed. Both molecules analyzed adopted a conformation close to the A-RNA double helix. The presented structures provide the first insight into this type of heteroduplexes and allowed a comparative analysis with existing nucleic acid homo- and heteroduplex structures. The results of our research expand the knowledge of the structural properties of new heteroduplexes and may be useful for future applications, such as therapies using this class of compounds.

Epidermal growth factor receptor (EGFR) is one of the most promising molecular targets for anticancer therapy. We used boron clusters as a platform for generation of new materials. For this, functional DNA constructs conjugated with boron clusters (B-ASOs) were developed. These B-ASOs, built from 1,2-dicarba-closo-dodecaborane linked with two anti-EGFR antisense oligonucleotides (ASOs), form with their complementary congeners torus-like nanostructures, as previously shown by atomic force microscope (AFM) and transmission electron cryo-microscopy (cryo-TEM) imaging. In the present work, deepened studies were carried out on B-ASO’s properties. In solution, B-ASOs formed four dominant complexes as confirmed by non-denaturing polyacrylamide gel electrophoresis (PAGE). These complexes exhibited increased stability in cell lysate comparing to the non-modified ASO. Fluorescently labeled B-ASOs localized mostly in the cytoplasm and decreased EGFR expression by activating RNase H. Moreover, the B-ASO complexes altered the cancer cell phenotype, decreased cell migration rate, and arrested the cells in the S phase of cell cycle. The 1,2-dicarba-closo-dodecaborane-containing nanostructures did not activate NLRP3 inflammasome in human macrophages. In addition, as shown by inductively coupled plasma mass spectrometry (ICP MS), these nanostructures effectively penetrated the human squamous carcinoma cells (A431), showing their potential applicability as anticancer agents.

Boron cluster-modified therapeutic nucleic acids with improved properties are of interest in gene therapy and in cancer boron neutron capture therapy (BNCT). High metallacarborane-loaded antisense oligonucleotides (ASOs) targeting epidermal growth factor receptor (EGFR) were synthesized through post-synthetic Cu (I)-assisted “click” conjugation of alkyne-modified DNA-oligonucleotides with a boron cluster alkyl azide component. The obtained oligomers exhibited increased lipophilicity compared to their non-modified precursors, while their binding affinity to complementary DNA and RNA strands was slightly decreased. Multiple metallacarborane residues present in the oligonucleotide chain, each containing 18 B-H groups, enabled the use of IR spectroscopy as a convenient analytical method for these oligomers based on the diagnostic B-H signal at 2400–2650 cm−1. The silencing activity of boron cluster-modified ASOs used at higher concentrations was similar to that of unmodified oligonucleotides. The screened ASOs, when used in low concentrations (up to 50 μM), exhibited pro-oxidative properties by inducing ROS production and an increase in mitochondrial activities in HeLa cells. In contrast, when used at higher concentrations, the ASOs exhibited anti-oxidative properties by lowering ROS species levels. In the HeLa cells (tested in the MTT assay) treated (without lipofectamine) or transfected with the screened compounds, the mitochondrial activity remained equal to the control level or only slightly changed (±30%). These findings may be useful in the design of dual-action boron cluster-modified therapeutic nucleic acids with combined antisense and anti-oxidant properties.

One of the speculated reasons for the observed increase of type 1 diabetes incidence in children is weight gain causing accelerated disease development in predisposed individuals. This so-called accelerator hypothesis is, however, controversial. The aim of the study was to analyze whether in the ethnically homogeneous population of Lesser Poland an increase in the number of cases of diabetes among children was associated with younger age and higher BMI-SDS at the moment of diagnosis. Retrospective data analysis from medical records of all patients under the age of 14 (n=559; 50.6% male), with newly diagnosed type 1 diabetes in Lesser Poland between 1st of January 2006 and 31st of December 2017 (11 years). The incidence ratio ranged significantly (P<0.001) from the lowest in 2006 (11.2/100,000/year) to the highest in 2012 (21.9/100,000/year). The mean age of diagnosis was 8.2±3.5 years; there was no trend of decreasing age (P=0.43). The mean body mass index expressed as the standard deviation score was -0.4±1.2. Almost all children (99%) at the time of diagnosis presented BMI-SDS within the normal range, with only 2.7% cases of obesity at the moment of diagnosis. The results of the present study do not confirm that increase of type 1 incidence in paediatric population is associated with younger age of diagnosis and higher BMI-SDS. Therefore are insufficient to prove the .

The bacterial enzyme tRNA 2-selenouridine synthase (SelU) is responsible for the conversion of 5-substituted 2-thiouridine (R5S2U), present in the anticodon of some bacterial tRNAs, into 5-substituted 2-selenouridine (R5Se2U). We have already demonstrated using synthetic RNAs that transformation S2U→Se2U is a two-step process, in which the S2U-RNA is geranylated and the resulting geS2U-RNA is selenated. Currently, the question is how SelU recognizes its substrates and what the cellular pathway of R5S2U→R5Se2U conversion is in natural tRNA. In the study presented here, we characterized the SelU substrate requirements, identified SelU-associated tRNAs and their specific modifications in the wobble position. Finally, we explained the sequence of steps in the selenation of tRNA. The S2U position within the RNA chain, the flanking sequence of the modification, and the length of the RNA substrate, all have a key influence on the recognition by SelU. MST data on the affinity of SelU to individual RNAs confirmed the presumed process. SelU binds the R5S2U-tRNA and then catalyzes its geranylation to the R5geS2U-tRNA, which remains bound to the enzyme and is selenated in the next step of the transformation. Finally, the R5Se2U-tRNA leaves the enzyme and participates in the translation process. The enzyme does not directly catalyze the R5S2U-tRNA selenation and the R5geS2U-tRNA is the intermediate product in the linear sequence of reactions.

Antisense oligonucleotides conjugated with boron clusters (B-ASOs) have been described as potential gene expression inhibitors and carriers of boron for boron neutron capture therapy (BNCT), providing a dual-action therapeutic platform. In this study, we tested the nucleolytic stability of DNA oligonucleotides labeled with metallacarborane [(3,3’-iron-1,2,1’,2’-dicarbollide)(−1)]ate [Fe(C2B9H11)2] (FESAN) against snake venom phosphodiesterase (svPDE, 3’→5’-exonuclease). Contrary to the previously observed protective effect of carborane (C2B10H12) modifications, the B-ASOs containing a metallacarborane moiety at the 5’-end of the oligonucleotide chain were hydrolyzed faster than their parent nonmodified oligomers. Interestingly, an enhancement in the hydrolysis rate was also observed in the presence of free metallacarborane, and this reaction was dependent on the concentration of the metallacarborane. Microscale thermophoresis (MST) analysis confirmed the high affinity (Kd nM range) of the binding of the metallacarborane to the proteins of crude snake venom and the moderate affinity (Kd µM range) between the metallacarborane and the short single-stranded DNA. We hypothesize that the metallacarborane complex covalently bound to B-ASO holds DNA molecules close to the protein surface, facilitating enzymatic cleavage. The addition of metallacarborane alone to the ASO/svPDE reaction mixture provides the interface to attract freely floating DNA molecules. In both cases, the local DNA concentration around the enzymes increases, giving rise to faster hydrolysis. It was experimentally shown that an allosteric effect, possibly attributable to the observed boost in the 3’→5’-exonucleolytic activity of snake venom phosphodiesterase, is much less plausible.