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Impaired fibroblast growth factor receptor (FGFR) signaling is associated with many human conditions, including growth disorders, degenerative diseases, and cancer. Current FGFR therapeutics are based on chemical inhibitors of FGFR tyrosine kinase activity (TKIs). However, FGFR TKIs are limited in their target specificity as they generally inhibit all FGFRs and other receptor tyrosine kinases. In the search for specific inhibitors of human FGFR1, we identified VZ23, a DNA aptamer that binds to FGFR1b and FGFR1c with a KD of 55 nM and 162 nM, respectively, but not to the other FGFR variants (FGFR2b, FGFR2c, FGFR3b, FGFR3c, FGFR4). In cells, VZ23 inhibited the activation of downstream FGFR1 signaling and FGFR1-mediated regulation of cellular senescence, proliferation, and extracellular matrix homeostasis. Consistent with the specificity toward FGFR1 observed in vitro, VZ23 did not inhibit FGFR2-4 signaling in cells. We show that the VZ23 inhibits FGFR1 signaling in the presence of cognate fibroblast growth factor (FGF) ligands and its inhibitory activity is linked to its capacity to form unusual G-quadruplex structure. Our data suggest that targeting FGFR1 with DNA aptamers could be an effective alternative to TKIs for treating impaired FGFR1 signaling in human craniosynostoses.
- Publikační typ
- časopisecké články MeSH
A novel Gram-stain-negative, strictly aerobic, rod-shaped, light-yellow-pigmented, and chemo-organoheterotrophic bacterium, designated DF-77T, was isolated from dense mats of filamentous algae collected in March 2004 at Okinawa in Japan. The microorganism grew at 0-2.0% NaCl concentrations (w/v), pH 6.0-9.0, and 20-30 °C. The 16S rRNA gene sequence-based phylogenetic tree demonstrated that the strain DF-77T is a novel member of the family Flavobacteriaceae and was greatly related to Flagellimonas nanhaiensis SM1704T with sequence similarity of 95.5%. The main fatty acids were iso-C15:1 G, iso-C15:0, and iso-C17:0 3-OH, and the only isoprenoid quinone was menaquinone-6. The dominant polar lipids were phosphatidylethanolamine, two unidentified aminolipids, an unidentified phosphoaminolipid, and four unidentified lipids. The genome size of strain DF-77T was 3.60 Mbp with a DNA G + C content of 47.5%. The average nucleotide identity (ANI) value between the genomes of strain DF-77T and its closely related species was 69.8-70.7%. The digital DNA - DNA hybridization (dDDH) value of strain DF-77T with the strain of F. nanhaiensis SM1704T was 16.8%. The genome of the strain DF-77T revealed that it encoded several genes involved in bio-macromolecule degradation, indicating a high potential for producing industrially useful enzymes. Consequently, the strain is described as a new species in the genus Flagellimonas, for which the name Flagellimonas algarum sp. nov., is proposed with the type strain DF-77T (= KCTC 72791T = NBRC 114251T).
- MeSH
- DNA bakterií genetika chemie MeSH
- Flavobacteriaceae * klasifikace izolace a purifikace genetika MeSH
- fosfolipidy analýza MeSH
- fylogeneze MeSH
- genom bakteriální MeSH
- hybridizace nukleových kyselin MeSH
- mastné kyseliny analýza MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- techniky typizace bakterií MeSH
- vitamin K 2 analýza analogy a deriváty MeSH
- zastoupení bazí MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Japonsko MeSH
The activity of the light-oxygen-voltage/helix-turn-helix (LOV-HTH) photoreceptor EL222 is regulated through protein-protein and protein-DNA interactions, both triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV-visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using noncanonical amino acids. Kinetic experiments provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently bound N5 protonated, and noncovalently bound hydroquinone forms of FMN, respectively. Only subtle structural differences were observed between the monomeric forms of all three EL222 species (dark, lit1, and lit2). While the dark state is largely monomeric, both lit states undergo monomer-dimer exchange. Furthermore, molecular modeling revealed differential dynamics and interdomain separation times arising from the three FMN states (oxidized, adduct, and reduced). Unexpectedly, all three EL222 species can associate with DNA, but only upon blue-light irradiation, a high population of stable complexes is obtained. Overall, we propose a model of EL222 activation where photoinduced changes in the FMN moiety shift the population equilibrium toward an open conformation that favors self-association and DNA-binding.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- DNA * chemie metabolismus MeSH
- flavinmononukleotid * chemie metabolismus MeSH
- flaviny chemie metabolismus MeSH
- kinetika MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- oxidace-redukce * MeSH
- simulace molekulární dynamiky MeSH
- světlo * MeSH
- Thermosynechococcus metabolismus MeSH
- transkripční faktory metabolismus chemie MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
RNA secondary (2D) structure visualization is an essential tool for understanding RNA function. R2DT is a software package designed to visualize RNA 2D structures in consistent, recognizable, and reproducible layouts. The latest release, R2DT 2.0, introduces multiple significant features, including the ability to display position-specific information, such as single nucleotide polymorphisms or SHAPE reactivities. It also offers a new template-free mode allowing visualization of RNAs without pre-existing templates, alongside a constrained folding mode and support for animated visualizations. Users can interactively modify R2DT diagrams, either manually or using natural language prompts, to generate new templates or create publication-quality images. Additionally, R2DT features faster performance, an expanded template library, and a growing collection of compatible tools and utilities. Already integrated into multiple biological databases, R2DT has evolved into a comprehensive platform for RNA 2D visualization, accessible at https://r2dt.bio.
Lipid-mediated delivery of active pharmaceutical ingredients (API) opened new possibilities in advanced therapies. By encapsulating an API into a lipid nanocarrier (LNC), one can safely deliver APIs not soluble in water, those with otherwise strong adverse effects, or very fragile ones such as nucleic acids. However, for the rational design of LNCs, a detailed understanding of the composition-structure-function relationships is missing. This review presents currently available computational methods for LNC investigation, screening, and design. The state-of-the-art physics-based approaches are described, with the focus on molecular dynamics simulations in all-atom and coarse-grained resolution. Their strengths and weaknesses are discussed, highlighting the aspects necessary for obtaining reliable results in the simulations. Furthermore, a machine learning, i.e., data-based learning, approach to the design of lipid-mediated API delivery is introduced. The data produced by the experimental and theoretical approaches provide valuable insights. Processing these data can help optimize the design of LNCs for better performance. In the final section of this Review, state-of-the-art of computer simulations of LNCs are reviewed, specifically addressing the compatibility of experimental and computational insights.
- MeSH
- léčivé přípravky chemie MeSH
- lékové transportní systémy metody MeSH
- lidé MeSH
- lipidy * chemie MeSH
- nanočástice chemie MeSH
- nosiče léků chemie MeSH
- počítačová simulace MeSH
- simulace molekulární dynamiky * MeSH
- strojové učení MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The RNA chaperone Hfq plays crucial roles in bacterial gene expression and is a major facilitator of small regulatory RNA (sRNA) action. The toroidal architecture of the Hfq hexamer presents three well-characterized surfaces that allow it to bind sRNAs to stabilize them and engage target transcripts. Hfq-interacting sRNAs are categorized into two classes based on the surfaces they use to bind Hfq. By characterizing a systematic alanine mutant library of Hfq to identify amino acid residues that impact survival of Escherichia coli experiencing nitrogen (N) starvation, we corroborated the important role of the three RNA-binding surfaces for Hfq function. We uncovered two, previously uncharacterized, conserved residues, V22 and G34, in the hydrophobic core of Hfq, to have a profound impact on Hfq's RNA-binding activity in vivo. Transcriptome-scale analysis revealed that V22A and G34A Hfq mutants cause widespread destabilization of both sRNA classes, to the same extent as seen in bacteria devoid of Hfq. However, the alanine substitutions at these residues resulted in only modest alteration in stability and structure of Hfq. We propose that V22 and G34 have impact on Hfq function, especially critical under cellular conditions when there is an increased demand for Hfq, such as N starvation.
- MeSH
- bakteriální RNA * metabolismus genetika chemie MeSH
- dusík metabolismus MeSH
- Escherichia coli * genetika metabolismus MeSH
- hydrofobní a hydrofilní interakce * MeSH
- konzervovaná sekvence MeSH
- malá nekódující RNA * metabolismus genetika chemie MeSH
- mutace MeSH
- protein hostitelského faktoru 1 * metabolismus genetika chemie MeSH
- proteiny z Escherichia coli * metabolismus genetika chemie MeSH
- regulace genové exprese u bakterií MeSH
- stabilita RNA * genetika MeSH
- stanovení celkové genové exprese MeSH
- transkriptom genetika MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
Protein synthesis (translation) consumes a substantial proportion of cellular resources, prompting specialized mechanisms to reduce translation under adverse conditions. Ribosome inactivation often involves ribosome-interacting proteins. In both bacteria and eukaryotes, various ribosome-interacting proteins facilitate ribosome dimerization or hibernation, and/or prevent ribosomal subunits from associating, enabling the organisms to adapt to stress. Despite extensive studies on bacteria and eukaryotes, understanding factor-mediated ribosome dimerization or anti-association in archaea remains elusive. Here, we present cryo-electron microscopy structures of an archaeal 30S dimer complexed with an archaeal ribosome dimerization factor (designated aRDF), from Pyrococcus furiosus, resolved at a resolution of 3.2 Å. The complex features two 30S subunits stabilized by aRDF homodimers in a unique head-to-body architecture, which differs from the disome architecture observed during hibernation in bacteria and eukaryotes. aRDF interacts directly with eS32 ribosomal protein, which is essential for subunit association. The binding mode of aRDF elucidates its anti-association properties, which prevent the assembly of archaeal 70S ribosomes.
- MeSH
- archeální proteiny * chemie metabolismus ultrastruktura MeSH
- dimerizace MeSH
- elektronová kryomikroskopie * MeSH
- malé podjednotky ribozomu archebakteriální chemie metabolismus MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- Pyrococcus furiosus * metabolismus MeSH
- ribozomální proteiny * chemie metabolismus MeSH
- ribozomy metabolismus ultrastruktura chemie MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
DNA double-strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death or initiate oncogenesis. The activity of RNA polymerase II (RNAPII) at the break site is required for efficient DSB repair. However, the regulatory mechanisms governing the transcription cycle at DSBs are not well understood. Here, we show that Integrator complex subunit 6 (INTS6) associates with the heterotrimeric sensor of ssDNA (SOSS1) complex (comprising INTS3, INIP and hSSB1) to form the tetrameric SOSS1 complex. INTS6 binds to DNA:RNA hybrids and promotes Protein Phosphatase 2A (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-associated RNA transcripts (DARTs) and the stabilization of DNA:RNA hybrids at DSB sites. INTS6 interacts with and promotes the recruitment of senataxin (SETX) to DSBs, facilitating the resolution of DNA:RNA hybrids/R-loops. Our results underscore the significance of the tetrameric SOSS1 complex in the autoregulation of DNA:RNA hybrids and efficient DNA repair.
- MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA-helikasy metabolismus genetika MeSH
- DNA * metabolismus chemie MeSH
- dvouřetězcové zlomy DNA * MeSH
- fosforylace MeSH
- homeostáza genetika MeSH
- lidé MeSH
- oprava DNA * MeSH
- proteinfosfatasa 2 metabolismus genetika MeSH
- R-smyčka MeSH
- RNA-helikasy metabolismus genetika MeSH
- RNA-polymerasa II * metabolismus MeSH
- RNA * metabolismus genetika chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
G-quadruplexes (G4s) formed within RNA are emerging as promising targets for therapeutic intervention in cancer, neurodegenerative disorders and infectious diseases. Sequences containing a succession of short GG blocks, or uneven G-tract lengths unable to form three-tetrad G4s (GG motifs), are overwhelmingly more frequent than canonical motifs involving multiple GGG blocks. We recently showed that DNA is not able to form stable two-tetrad intramolecular parallel G4s. Whether RNA GG motifs can form intramolecular G4s under physiological conditions and play regulatory roles remains a burning question. In this study, we performed a systematic analysis and experimental evaluation of a number of biologically important RNA regions involving RNA GG motifs. We show that most of these motifs do not form stable intramolecular G4s but need to dimerize to form stable G4 structures. The strong tendency of RNA GG motif G4s to associate may participate in RNA-based aggregation under conditions of cellular stress.
- MeSH
- dimerizace MeSH
- G-kvadruplexy * MeSH
- genetická transkripce MeSH
- lidé MeSH
- nukleotidové motivy * MeSH
- RNA * chemie metabolismus genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mechanismus účinku většiny léčiv je založen na jejich interakci s molekulovými cíli v organismu, tj. biologickými makromolekulami, jako jsou proteiny nebo nukleové kyseliny. Mezi faktory ovlivňující sílu navázání molekuly léčiva na jeho biologický cíl patří celkový počet interakcí, jejich charakter a z něj vyplývající energie vazby. Hodnota energie vazby je zásadním parametrem pro odhad síly interakce. Základní typy těchto intermolekulárních interakcí jsou v přehledovém článku definovány, schematicky znázorněny a doplněny údaji o energii vazby. Dále jsou uvedeny další aspekty navazování léčiv na molekulové cíle, např. solvatace molekul ve vodném pro středí nebo vzdálenost interagujících chemických funkčních skupin. Znalost struktur molekulárních cílů i díky úspěchu současných modelů nám umožňuje tyto interakce využívat pro návrh nových léčiv.
The mechanism of action of most drugs is based on their interaction with molecular targets in the organism, i.e., biological macromolecules such as proteins or nucleic acids. Factors influencing the strength of binding of a drug molecule to its biological target include the total number of interactions, their character, and the resulting binding energy. The value of binding energy is an essential parameter for estimating the strength of the interaction. The basic types of these intermolecular interactions are defined, schematically illustrated, and supported with data on binding energy in this review article. Other aspects of drug binding to molecular targets are also presented, e.g., the solvation of molecules in aqueous environment or the distance of interacting chemical functional groups. Knowledge of the structures of molecular targets and the progress of current models allows us to use these interactions to design new drugs.
