1,721,004 research outputs found
G-quadruplexes formation within the promoter of TEAD4 oncogene and their interaction with Vimentin
Full text linkG-quadruplexes (G4s) are nucleic acid secondary structures detected within human chromosomes, that cluster at gene promoters and enhancers. This suggests that G4s may play specific roles in the regulation of gene expression. Within a distinct subgroup of G-rich domains, the formation of two or more adjacent G4 units (G4-repeats) is feasible. Recently it was shown that Vimentin, a protein highly expressed within mesenchymal cells, selectively recognizes these arrangements. Putative G4-repeats have been searched within the human gene proximal promoters by the bioinformatics tool QPARSE and they resulted to be enriched at genes related to epithelial-to-mesenchymal transition (EMT). This suggested that Vimentin binding at these sites might be relevant for the maintenance of the mesenchymal phenotype. Among all the identified sequences, in the present study we selected the one located within the promoter of the TEAD4 oncogene. TEAD4 codifies for a transcriptional enhancer factor, TEAD4, that actively promotes EMT, supporting, cell proliferation and migration. Moreover, in colorectal cancer cells TEAD4 directly enhances the expression of Vimentin. Thus, the possible interaction of Vimentin with TEAD4 promoter could highlight a positive feedback loop between these two factors, associated to important tumor metastasis related events. Here, we exploited spectroscopic and electrophoretic measurements under different conditions to address the folding behavior of the selected sequence. This allowed us to validate the folding of TEAD4 promoter into a G4-repeat able to interact with Vimentin
Polymorphic and Higher-Order G-Quadruplexes as Possible Transcription Regulators: Novel Perspectives for Future Anticancer Therapeutic Applications
Full text linkIn the past two decades, significant efforts have been put into designing small molecules to target selected genomic sites where DNA conformational rearrangements control gene expression. G-rich sequences at oncogene promoters are considered good points of intervention since, under specific environmental conditions, they can fold into non-canonical tetrahelical structures known as G-quadruplexes. However, emerging evidence points to a frequent lack of correlation between small molecule targeting of G-quadruplexes at gene promoters and the expression of the associated protein, which hampers pharmaceutical applications. The wide genomic localization of G-quadruplexes along with their highly polymorphic behavior may account for this scenario, suggesting the need for more focused drug design strategies. Here, we will summarize the G4 structural features that can be considered to fulfill this goal. In particular, by comparing a telomeric sequence with the well-characterized G-rich domain of the KIT promoter, we will address how multiple secondary structures might cooperate to control genome architecture at a higher level. If this holds true, the link between drug–DNA complex formation and the associated cellular effects will need to be revisited
Nucleoside analogs for management of respiratory virus infections: mechanism of action and clinical efficacy
No full textThe COVID-19 pandemic has accelerated the development of nucleoside analogs to treat respiratory virus infections, with remdesivir being the first compound to receive worldwide authorization and three other nucleoside analogs (i.e. favipiravir, molnupiravir, and bemnifosbuvir) in the pipeline. Here, we summarize the current knowledge concerning their clinical efficacy in suppressing the virus and reducing the need for hospitalization or respiratory support. We also mention trials of favipiravir and lumicitabine, for influenza and respiratory syncytial virus, respectively. Besides, we outline how nucleoside analogs interact with the polymerases of respiratory viruses, to cause lethal virus mutagenesis or disturbance of viral RNA synthesis. In this way, we aim to convey the key findings on this rapidly evolving class of respiratory virus medication
Synthesis, antiviral activity, and computational study of β-D-xylofuranosyl nucleoside phosphonates
Full text linkMolecular dynamics (MD) simulations provided insights into the favorable interactions between xylose nucleosides bearing a phosphonate moiety at their 3'-position and specific residues at the active site of the archetypal RNA-dependent RNA-polymerase (RdRp) of Enterovirus 71. Therefore, a series of xylosyl nucleoside phosphonates with adenine, uracil, cytosine, guanosine, and hypoxanthine as nucleobases were synthesized through multistep sequences starting from a single common precursor. Following antiviral activity evaluation, the adenine containing analogue was found to possess good antiviral activity against RNA viruses displaying an EC50 of 12 and 16 μM against measles virus (MeV) and enterovirus-68 (EV-68), respectively, whereas lacking cytotoxicity.sponsorship: KUL|C14/19/102status: Publishe
Modulation of the tetrameric I-motif folding of C-rich Tetrahymena telomeric sequences by hexitol nucleic acid (HNA) modifications
Full text linkI-motifs are non-canonical DNA structures consisting of two parallel strands held together by hemiprotonated cytosine-cytosine thorn base pairs, which intercalate to form a ordered column of stacked base pairs. This unique structure covers potential relevance in various fields, including gene regulation and biotechnological applications. A unique structural feature of I-motifs (iM), is the presence of sugar-sugar interactions through their extremely narrow minor grooves. Consistently, oligonucleotides containing pentose derivatives such as ribose, 20-deoxyribose, arabinose, and 20-deoxy-20-fluoroarabinose highlighted a very different attitude to fold into iM. On the other hand, there is significant attention focused on exploring sugar-modifications that can increase nucleic acids resistance to nuclease degradation, a crucial requirement for therapeutic applications. An interesting example, not addressed in the iM field yet, is represented by hexitol nucleic acid (HNA), a metabolically stable six-membered ring analogue compatible with A-like double helix formation. Herein, we selected two DNA C-rich Tetrahymena telomeric sequences whose tetrameric iMs were already resolved by NMR and we investigated the iM folding of related HNA and RNA oligonucleotides by circular dichroism, differential scanning calorimetry and NMR. The comparison of their behaviours vs the DNA counterparts provided interesting insights into the influence of the sugar on iM folding. In particular, ribose and hexitol prevented iM formation. However, by clustering the hexitol-containing residues at the 30-end, it was possible to modulate the distribution of the different topological species described for the DNA iMs. These data open new avenues for the exploitation of sugar modifications for Imotif characterization and applications. (c) 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
CRISPR-Cas9 recognition of enzymatically synthesized base-modified nucleic acids
Full text linkAn enzymatic method has been successfully established enabling the generation of partially base-modified RNA (previously named RZA) constructs, in which all G residues were replaced by isomorphic fluorescent thienoguanosine (thG) analogs, as well as fully modified RZA featuring thG, 5-bromocytosine, 7-deazaadenine and 5-chlorouracil. The transcriptional efficiency of emissive fully modified RZA was found to benefit from the use of various T7 RNA polymerase variants. Moreover, dthG could be incorporated into PCR products by Taq DNA polymerase together with the other three base-modified nucleotides. Notably, the obtained RNA products containing thG as well as thG together with 5-bromocytosine could function as effectively as natural sgRNAs in an in vitro CRISPR-Cas9 cleavage assay. N1-Methylpseudouridine was also demonstrated to be a faithful non-canonical substitute of uridine to direct Cas9 nuclease cleavage when incorporated in sgRNA. The Cas9 inactivation by 7-deazapurines indicated the importance of the 7-nitrogen atom of purines in both sgRNA and PAM site for achieving efficient Cas9 cleavage. Additional aspects of this study are discussed in relation to the significance of sgRNA-protein and PAM--protein interactions that were not highlighted by the Cas9-sgRNA-DNA complex crystal structure. These findings could expand the impact and therapeutic value of CRISPR-Cas9 and other RNA-based technologies.sponsorship: China Scholarship Council|G085321N], Fonds Wetenschappelijk Onderzoek|12Q8619N, Fonds Wetenschappelijk Onderzoek|1509920N, China Scholarship Council|201707040069 to H.Y., European Union’s Horizon 2020 research and innovation program|965135];status: Publishe
Reshaping an Acyclic Nucleoside Phosphonate into a Selective Anti-hepatitis B Virus Compound
No full textMinor structural modifications of acyclic nucleoside phosphonates can dramatically affect their antiviral properties. This work discloses a shift in the selectivity spectrum of 3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) nucleotides from herpesviruses toward hepatitis B virus (HBV) induced by their acyclic chain 2-substitution with a nonpolar group. Two series of racemic (R,S)-2-methyl-3-hydroxy-2-(phosphonomethoxy)propyl (MHPMP) and (R,S)-2-ethynyl-3-hydroxy-2-(phosphonomethoxy)propyl (EHPMP) nucleotides were initially synthesized. Among these, guanine-containing derivatives exhibited significant anti-HBV activities in the submicromolar range. Enantioenriched MHPMPG and EHPMPG analogues were subsequently obtained by Sharpless asymmetric epoxidation. The (S)-enantiomers possessed an 8- to 26-fold higher potency than the relative (R)-forms. A further comparison of the EC90 values indicated that (S)-EHPMPG inhibited HBV replication more effectively than its 2-methyl analogue. A phosphonodiamidate prodrug of (S)-EHPMPG was thus prepared and found to exert a remarkably high anti-HBV activity (EC50 = 9.27 nM) with excellent selectivity (SI50 > 10,787), proving to be a promising candidate for anti-HBV drug development.sponsorship: China Scholarship Council|201606220077status: Published onlin
Synthesis and evaluation of purine C-nucleoside analogues as versatile tools for therapeutic applications
No full textreservedGli oligonucleotidi sono emersi come validi strumenti diagnostici e terapeutici, come dimostra il numero crescente di farmaci a base di acidi nucleici approvati negli ultimi anni. Esempi chiave con un notevole potenziale per la cura e la prevenzione di un'ampia gamma di patologie, come malattie neurodegenerative e infettive, includono gli oligonucleotidi antisenso (ASOs), i piccoli RNA interferenti (siRNAs), gli aptameri e i vaccini a base di mRNA. Tuttavia, la loro efficacia terapeutica è spesso ostacolata da limiti quali l'instabilità chimica ed enzimatica, lo scarso assorbimento cellulare e l'inefficiente consegna mirata a organi e tessuti. Per affrontare queste criticità sono state pertanto studiate diverse strategie di modifica chimica che coinvolgono la struttura portante, gli zuccheri o le basi azotate, oltre alla formazione di coniugati. Come parte di un più ampio sforzo di ricerca volto a indagare l'impatto delle modifiche strutturali del nucleo eterociclico così come del legame glicosidico sull'affinità di legame e sulla specificità dei corrispondenti oligonucleotidi modificati per varie applicazioni biomediche e biotecnologiche, questo progetto di tesi cerca di identificare candidati nucleosidici opportunamente modificati che possano servire agli scopi sopra menzionati. Il progetto è stato avviato con la valutazione di diversi sintoni eterociclici e la selezione finale di due 4-aza-7,9-dideazapurine come partner agliconici. Queste nucleobasi sono state poi associate a una frazione di zucchero ribosio tramite un legame C-C, con l'obiettivo di aumentare la resistenza alla scissione enzimatica e idrolitica. Inoltre, la geometria alterata del legame C-C, rispetto al legame C-N-glicosidico presente nei nucleosidi naturali, può determinare modelli di legame idrogeno modificati, influenzando la stabilità e le proprietà di interazione con gli enzimi e/o le sequenze di acidi nucleici target.
Poiché gli analoghi dei C-nucleosidi hanno dimostrato promettenti proprietà antivirali, come il remdesivir, farmaco antivirale ad ampio spettro approvato dall'FDA, gli analoghi sintetizzati saranno valutati anche per le loro proprietà antivirali contro diversi virus a RNA. Infine, i C-nucleosidi ottenuti saranno sfruttati come elementi costitutivi per sintetizzare sequenze oligonucleotidiche parzialmente e completamente modificate, previa conversione nei rispettivi fosforamiditi. La capacità degli oligonucleotidi C-modificati di ibridarsi con l'RNA naturale fornirà nuove conoscenze utili per future applicazioni nei campi della tecnologia antisenso e dell'interferenza dell'RNA.Oligonucleotides have emerged as powerful diagnostic and therapeutic tools, as demonstrated by the increasing number of approved nucleic acid drugs in recent years. Key examples with considerable potential for curing and preventing a wide range of conditions, such as neurodegenerative and infectious diseases, include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), aptamers, and mRNA vaccines. However, their therapeutic efficacy is often hindered by shortcomings such as chemical and enzymatic instability, poor cellular uptake, and inefficient targeted delivery to organs and tissues. Therefore, a variety of chemical modification strategies at the backbone, sugar, or nucleobase moieties, along with conjugate formation have been explored to tackle these issues. As part of a larger research endeavour aimed at investigating the impact of structural modifications at the heterocyclic nucleobase core as well as glycosidic bond on the binding affinity and specificity of the correspondingly modified oligonucleotides for various biomedical and biotechnological applications, this thesis project seeks to identify suitably modified nucleoside candidates that could serve the above purposes. The project commenced with the evaluation of diverse heterocyclic synthons and ultimate selection of two 4-aza-7,9-dideazapurines as aglycone partners. These nucleobases were then connected to a ribose sugar moiety via a C-C bond, aiming to enhance resistance to enzymatic and hydrolytic cleavage. In addition, the altered geometry of C-C bond, as opposed to the C-N-glycosidic bond found in natural nucleosides, can result in modified hydrogen bonding patterns, influencing the stability and interaction properties with target enzymes and/or nucleic acid sequences.
Since C-nucleoside analogues showed promise as potential antivirals, such as the FDA-approved broad-spectrum antiviral drug remdesivir, the synthesised analogues will also be evaluated for their antiviral properties against diverse RNA viruses. Eventually, the obtained C-nucleosides will be exploited as building blocks for synthesising partially and fully modified oligonucleotide sequences previous conversion to the respective phosphoramidites. The ability of C-modified oligonucleotides to hybridise with natural RNA will provide useful new knowledge for future applications in the fields of antisense and RNA interference technology
Alkene and alkyne metathesis reactions using ruthenium initiators
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