In this work we explore the structure of a G-rich DNA aptamer termed AT11-L2 (TGGTGGTGGTTGTTGTTGGTGGTGGTGGT; derivative of AT11) by evaluating the formation and stability of G-quadruplex (G4) conformation under different experimental conditions such as KCl concentration, temperature, and upon binding with a variety of G4 ligands (360A, BRACO-19, PDS, PhenDC3, TMPyP4). We also determined whether nucleolin (NCL) can be a target of AT11-L2 G4. Firstly, we assessed by circular dichroism, UV and NMR spectroscopies the formation of G4 by AT11-L2. We observed that, for KCl concentrations of 65 mM or less, AT11-L2 adopts hybrid or multiple topologies. In contrast, a parallel topology predominates for buffer containing 100 mM of KCl. The Tm of AT11-L2 in 100 mM of KCl is 38.9 °C, proving the weak stability of this sequence. We also found that upon titration with two molar equivalents of 360A, BRACO-19 and PhenDC3, the G4 is strongly stabilized and its topology is maintained, while the addition of 3.5 molar equivalents of TMPyP4 promotes the disruption of G4. The KD values between AT11-L2 G4, ligands and NCL were obtained by fluorescence titrations and are in the range of µM for ligand complexes and nM when adding NCL. In silico studies suggest that four ligands bind to the AT11-L2 G4 structure by stacking interactions, while the RBD1,2 domains of NCL interact preferentially with the thymines of AT11-L2 G4. Finally, AT11-L2 G4 co-localized with NCL in NCL-positive tongue squamous cell carcinoma cell line.

• Klíčová slova
• G-quadruplex aptamer, aptamer–ligand interactions, biophysical techniques, ligands,
• MeSH
• aptamery nukleotidové * chemie   MeSH
• G-kvadruplexy * MeSH
• lidé MeSH
• ligandy MeSH
• nádory jazyka * MeSH
• spinocelulární karcinom * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aptamery nukleotidové * MeSH
• ligandy MeSH

Paraspeckles are RNA-protein structures within the nucleus of mammalian cells, capable of orchestrating various biochemical processes. An overexpression of the architectural component of paraspeckles, a long non-coding RNA called NEAT1 (Nuclear Enriched Abundant Transcript 1), has been linked to a variety of cancers and is often associated with poor patient prognosis. Thus, there is an accumulating interest in the role of paraspeckles in carcinogenesis, however there is a limited understanding of how NEAT1 expression is regulated. Here, we demonstrate that both nuclear G-quadruplex (G4) and paraspeckle formation are significantly increased in a human breast cancer cell line compared to non-tumorigenic breast cells. Moreover, we identified and characterized G4-forming sequences within the NEAT1 promoter and demonstrate stabilization of G4 DNA with a G4-stabilizing small molecule results in a significant alteration in both paraspeckle formation and NEAT1 expression. This G4-mediated alteration of NEAT1 at both the transcriptional and post-transcriptional levels was evident in U2OS osteosarcoma cells, MCF-7 breast adenocarcinoma and MDA-MB-231 triple negative breast cancer cells.

• Klíčová slova
• Breast cancer, G-quadruplex, G-quartet, G-tetraplex, G4 DNA, NEAT1, NEAT1_2, Paraspeckles,
• MeSH
• G-kvadruplexy * MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prsu genetika   metabolismus   MeSH
• nádory genetika   metabolismus   MeSH
• paraskvrny genetika   metabolismus   MeSH
• RNA dlouhá nekódující chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• NEAT1 long non-coding RNA, human MeSH Prohlížeč
• RNA dlouhá nekódující MeSH

Several small-molecule ligands specifically bind and stabilize G-quadruplex (G4) nucleic acid structures, which are considered to be promising therapeutic targets. G4s are polymorphic structures of varying stability, and their formation is dynamic. Here, we investigate the mechanisms of ligand binding to dynamically populated human telomere G4 DNA by using the bisquinolinium based ligand Phen-DC3 and a combination of single-molecule FRET microscopy, ensemble FRET and CD spectroscopies. Different cations are used to tune G4 polymorphism and folding dynamics. We find that ligand binding occurs to pre-folded G4 structures and that Phen-DC3 also induces G4 formation in unfolded single strands. Following ligand binding to dynamically populated G4s, the DNA undergoes pronounced conformational redistributions that do not involve direct ligand-induced G4 conformational interconversion. On the contrary, the redistribution is driven by ligand-induced G4 folding and trapping of dynamically populated short-lived conformation states. Thus, ligand-induced stabilization does not necessarily require the initial presence of stably folded G4s.

• Klíčová slova
• G-quadruplex, Phen-DC3, dynamics, ligand binding, smFRET,
• MeSH
• chinoliny chemie   metabolismus   MeSH
• G-kvadruplexy * MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• ligandy * MeSH
• rezonanční přenos fluorescenční energie MeSH
• simulace molekulární dynamiky MeSH
• telomery chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chinoliny MeSH
• ligandy * MeSH

The G-quadruplex (G4)-forming sequence within the AS1411 derivatives with alternative nucleobases and backbones can improve the chemical and biological properties of AS1411. Zn(II) phthalocyanine (ZnPc) derivatives have potential as high-affinity G4 ligands because they have similar size and shape to the G-quartets. The interactions of four Zn(II) phthalocyanines with the G4 AS1411 aptamer and its derivatives were determined by biophysical techniques, molecular docking and gel electrophoresis. Cell viability assay was carried out to evaluate the antiproliferative effects of Zn(II) phthalocyanines and complexes. CD experiments showed structural changes after addition of ZnPc 4, consistent with multiple binding modes and conformations shown by NMR and gel electrophoresis. CD melting confirmed that ZnPc 2 and ZnPc 4, both containing eight positive charges, are able to stabilize the AT11 G4 structure (ΔTm > 30 °C and 18.5 °C, respectively). Molecular docking studies of ZnPc 3 and ZnPc 4 suggested a preferential binding to the 3'- and 5'-end, respectively, of the AT11 G4. ZnPc 3 and its AT11 and AT11-L0 complexes revealed pronounced cytotoxic effect against cervical cancer cells and no cytotoxicity to normal human cells. Zn(II) phthalocyanines provide the basis for the development of effective therapeutic agents as G4 ligands.

• Klíčová slova
• Biophysical characterization, G-quadruplex aptamers, Phthalocyanines,
• MeSH
• antitumorózní látky chemie   farmakologie   MeSH
• aptamery nukleotidové chemie   farmakologie   MeSH
• buněčné linie MeSH
• G-kvadruplexy MeSH
• HeLa buňky MeSH
• indoly chemie   farmakologie   MeSH
• isoindoly MeSH
• lidé MeSH
• nádory farmakoterapie   MeSH
• oligodeoxyribonukleotidy chemie   farmakologie   MeSH
• organokovové sloučeniny chemie   farmakologie   MeSH
• simulace molekulového dockingu MeSH
• sloučeniny zinku MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AGRO 100 MeSH Prohlížeč
• antitumorózní látky MeSH
• aptamery nukleotidové MeSH
• indoly MeSH
• isoindoly MeSH
• oligodeoxyribonukleotidy MeSH
• organokovové sloučeniny MeSH
• sloučeniny zinku MeSH
• Zn(II)-phthalocyanine MeSH Prohlížeč

The clinical applicability of G-quadruplexes (G4s) as anticancer drugs is currently being evaluated. Several G4 ligands and aptamers are undergoing clinical trials following the notable examples of quarfloxin and AS1411, respectively. In this review, we summarize the latest achievements and breakthroughs in the use of G4 nucleic acids as both therapeutic tools ('friends', as healing anticancer drugs) and targets ('foes', within the harmful cancer cell), particularly using aptamers and quadruplex-targeted ligands, respectively. We explore the recent research on synthetic G4 ligands toward the discovery of anticancer therapeutics and their mechanism of action. Additionally, we highlight recent advances in chemical and structural biology that enable the design of specific G4 aptamers to be used as novel anticancer agents.

• Klíčová slova
• G-quadruplex, anticancer strategy, aptamers, cell regulation, drug discovery, ligands,
• MeSH
• antitumorózní látky farmakologie   terapeutické užití   MeSH
• aptamery nukleotidové farmakologie   terapeutické užití   MeSH
• G-kvadruplexy účinky léků   MeSH
• lidé MeSH
• ligandy MeSH
• nádory farmakoterapie   MeSH
• nukleové kyseliny farmakologie   MeSH
• oligodeoxyribonukleotidy farmakologie   terapeutické užití   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• AGRO 100 MeSH Prohlížeč
• antitumorózní látky MeSH
• aptamery nukleotidové MeSH
• ligandy MeSH
• nukleové kyseliny MeSH
• oligodeoxyribonukleotidy MeSH

Retroviruses are among the most extensively studied viral families, both historically and in contemporary research. They are primarily investigated in the fields of viral oncogenesis, reverse transcription mechanisms, and other infection-specific aspects. These include the integration of endogenous retroviruses (ERVs) into host genomes, a process widely utilized in genetic engineering, and the ongoing search for HIV/AIDS treatment. G-quadruplexes (G4) have emerged as potential therapeutic targets in antiviral therapy and have been identified in important regulatory regions of viral genomes. In this study, we examine the presence of potential G-quadruplex-forming sequences (PQS) across all currently available unique retroviral genomes. Given that these retroviral genomes typically consist of single-stranded RNA (ssRNA) molecules, we also investigated whether the localization of PQSs is strand-dependent. This is particularly relevant since antisense transcripts have been detected in HIV, and ERV integration into the host genome involves reverse transcription from genomic positive strand ssRNA to double-stranded DNA (dsDNA), implicating both strands in this process. We show that in most mammalian retroviruses, including human retroviruses, PQSs are significantly more prevalent on the negative (antisense) strand, with some notable exceptions such as HIV-1. In sharp contrast, avian retroviruses exhibit a higher prevalence of PQSs on the positive (sense) strand.

• Klíčová slova
• Bioinformatics, G-quadruplex, G4Hunter, Persistent infection, Retroviral genome,
• MeSH
• endogenní retroviry genetika   MeSH
• G-kvadruplexy * MeSH
• genom virový * MeSH
• lidé MeSH
• Retroviridae * genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Recent advancements have finally delivered a complete human genome assembly, including the elusive Y chromosome. This accomplishment closes a significant knowledge gap. Prior efforts were hampered by challenges in sequencing repetitive DNA structures such as direct and inverted repeats. We used the G4Hunter algorithm to analyze the presence of G-quadruplex forming sequences (G4s) within the current human reference genome (GRCh38) and the new telomere-to-telomere (T2T) Y chromosome assemblies. This analysis served a dual purpose: identifying the location of potential G4s within the genomes and exploring their association with functionally annotated sequences. Compared to GRCh38, the T2T assembly exhibited a significantly higher prevalence of G-quadruplex forming sequences. Notably, these repeats were abundantly located around precursor RNA, exons, genes, and within protein binding sites. This remarkable co-occurrence of G4-forming sequences with these critical regulatory regions suggests their role in fundamental DNA regulation processes. Our findings indicate that the current human reference genome significantly underestimated the number of G4s, potentially overlooking their functional importance.

• Klíčová slova
• Chromosome Y, G-quadruplex, Gapless assembly, Genome analysis,
• MeSH
• G-kvadruplexy * MeSH
• genom lidský * MeSH
• lidé MeSH
• lidský chromozom Y * genetika   MeSH
• telomery genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

A high level of nucleolin (NCL) expression is often associated with a poor prognosis of patients with lung cancer (LC), suggesting that NCL can be used as a possible biomarker. NCL has been shown to display a marked preference for the binding to G-quadruplexes (G4). Here, we investigate the formation of an RNA quadruplex structure in a sequence found in the human precursor pre-MIR150 with the potential to recognize NCL. Circular dichroism (CD) spectra of pre-MIR150 G4-forming sequence (designated by rG4) indicate the formation of a parallel quadruplex structure in KCl or when complexed with the well-known G4 ligand PhenDC3. The thermal stability of rG4 is very high, and further increases in the presence of PhenDC3. The binding affinities of rG4 to PhenDC3 and NCL RBD1,2 are similar with KD values in the nanomolar range. PAGE results suggest the formation of a ternary quadruplex-ligand-protein complex (rG4-PhenDC3-NCL RBD1,2), indicative that PhenDC3 does not prevent the binding of rG4 to NCL RBD1,2. Finally, rG4 can recognize NCL-positive cells and, when fluorescently labeled, can be used as a probe for this protein. ELISA experiments indicate altered NCL expression patterns in liquid biopsies of LC patients in a non-invasive manner, potentially helping the diagnosis, prognosis, and patient response to treatment. Hence, labeled rG4 could be used as a detection probe of LC in liquid biopsies.

• Klíčová slova
• Liquid biopsies, Lung Cancer, Molecular recognition, Nucleolin, pre-MIR150 G-quadruplex forming sequence,
• MeSH
• aminokyselinové motivy fyziologie   MeSH
• dospělí MeSH
• fosfoproteiny biosyntéza   genetika   MeSH
• G-kvadruplexy * MeSH
• genový targeting metody   MeSH
• kultivované buňky MeSH
• leukocyty mononukleární metabolismus   MeSH
• lidé MeSH
• nádory plic genetika   metabolismus   terapie   MeSH
• nukleolin MeSH
• proteiny vázající RNA biosyntéza   genetika   MeSH
• regulace genové exprese u nádorů MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoproteiny MeSH
• proteiny vázající RNA MeSH

G-quadruplexes are four-stranded nucleic acid structures occurring in the genomes of all living organisms and viruses. It is increasingly evident that these structures play important molecular roles; generally, by modulating gene expression and overall genome integrity. For a long period, G-quadruplexes have been studied specifically in the context of human promoters, telomeres, and associated diseases (cancers, neurological disorders). Several of the proteins for binding G-quadruplexes are known, providing promising targets for influencing G-quadruplex-related processes in organisms. Nonetheless, in plants, only a small number of G-quadruplex binding proteins have been described to date. Thus, we aimed to bioinformatically inspect the available protein sequences to find the best protein candidates with the potential to bind G-quadruplexes. Two similar glycine and arginine-rich G-quadruplex-binding motifs were described in humans. The first is the so-called "RGG motif"-RRGDGRRRGGGGRGQGGRGRGGGFKG, and the second (which has been recently described) is known as the "NIQI motif"-RGRGRGRGGGSGGSGGRGRG. Using this general knowledge, we searched for plant proteins containing the above mentioned motifs, using two independent approaches (BLASTp and FIMO scanning), and revealed many proteins containing the G4-binding motif(s). Our research also revealed the core proteins involved in G4 folding and resolving in green plants, algae, and the key plant model organism, Arabidopsis thaliana. The discovered protein candidates were annotated using STRINGdb and sorted by their molecular and physiological roles in simple schemes. Our results point to the significant role of G4-binding proteins in the regulation of gene expression in plants.

• Klíčová slova
• G-quadruplex folding, G-quadruplex resolving, G-quadruplex-binding proteins, NIQI, RGG motif, regulation of gene expression,
• Publikační typ
• časopisecké články MeSH

G-quadruplex (G4) structures are non-canonical DNA/RNA secondary structures able to form within guanine rich nucleic acids sequences. They are present in several regions of the human genome including gene promoters, untranslated sequences, and telomeres. Due to their biological relevance G4 structures are considered important drug targets, in particular for anticancer therapies, leading to the development of G4 stabilizing small molecules. Telomeric regions have received special attention in this field since they can fold into several distinct intramolecular G-quadruplexes topologies. Herein, we report the synthesis of 2,9-disubstituted-1,10-phenanthroline derivatives and their ability to stabilize different intramolecular telomeric G4 sequences. We evaluated ligand-induced stabilization, selectivity and specificity of ligands using Förster Resonance Energy Transfer (FRET) melting experiments and circular dichroism (CD). In addition, we assessed the cytotoxicity of ligands against two cancer cell lines (A549 and H1299) and one healthy cell line (NHDF).

• Klíčová slova
• 2,9-disubstituted-1,10-phenanthroline derivatives, Biophysical evaluation, G-quadruplex structures, In vitro studies,
• MeSH
• cirkulární dichroismus MeSH
• DNA chemie   MeSH
• fenantroliny MeSH
• G-kvadruplexy * MeSH
• guanin MeSH
• lidé MeSH
• ligandy MeSH
• RNA MeSH
• telomery MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,10-phenanthroline MeSH Prohlížeč
• DNA MeSH
• fenantroliny MeSH
• guanin MeSH
• ligandy MeSH
• RNA MeSH