Recently, the 1H-detected in-cell NMR spectroscopy has emerged as a unique tool allowing the characterization of interactions between nucleic acid-based targets and drug-like molecules in living human cells. Here, we assess the application potential of 1H and 19F-detected in-cell NMR spectroscopy to profile drugs/ligands targeting DNA G-quadruplexes, arguably the most studied class of anti-cancer drugs targeting nucleic acids. We show that the extension of the original in-cell NMR approach is not straightforward. The severe signal broadening and overlap of 1H in-cell NMR spectra of polymorphic G-quadruplexes and their complexes complicate their quantitative interpretation. Nevertheless, the 1H in-cell NMR can be used to identify drugs that, despite strong interaction in vitro, lose their ability to bind G-quadruplexes in the native environment. The in-cell NMR approach is adjusted to a recently developed 3,5-bis(trifluoromethyl)phenyl probe to monitor the intracellular interaction with ligands using 19F-detected in-cell NMR. The probe allows dissecting polymorphic mixture in terms of number and relative populations of individual G-quadruplex species, including ligand-bound and unbound forms in vitro and in cellulo. Despite the probe's discussed limitations, the 19F-detected in-cell NMR appears to be a promising strategy to profile G-quadruplex-ligand interactions in the complex environment of living cells.

• Klíčová slova
• BRACO19, Bcl2, G-quadruplex, KRAS, NMM, PhenDC3, drug, in-cell NMR, ligand, telomeric DNA,
• MeSH
• DNA chemie   účinky léků   MeSH
• G-kvadruplexy účinky léků   MeSH
• konformace nukleové kyseliny účinky léků   MeSH
• léčivé přípravky chemie   MeSH
• lidé MeSH
• ligandy MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární modely MeSH
• protony MeSH
• vazebná místa účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH
• léčivé přípravky MeSH
• ligandy MeSH
• protony 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

Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.

• Klíčová slova
• EBNA1, Epstein-Barr virus (EBV), G-quadruplexes, PhenDC3, protein-mRNA interactions, pyridostatin, structure-activity relationship,
• MeSH
• aminochinoliny chemie   MeSH
• biotest metody   MeSH
• fosfoproteiny metabolismus   MeSH
• G-kvadruplexy * MeSH
• kyseliny pikolinové chemie   MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• nádorové buněčné linie MeSH
• nukleolin MeSH
• proteiny vázající RNA metabolismus   MeSH
• virus Epsteinův-Barrové - jaderné antigeny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminochinoliny MeSH
• EBV-encoded nuclear antigen 1 MeSH Prohlížeč
• fosfoproteiny MeSH
• kyseliny pikolinové MeSH
• messenger RNA MeSH
• proteiny vázající RNA MeSH
• pyridostatin MeSH Prohlížeč
• virus Epsteinův-Barrové - jaderné antigeny MeSH

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

G-rich aptamers such as AS1411 are small oligonucleotides that present several benefits comparatively to monoclonal antibodies, since they are easier to manufacture and store, have small size and do not stimulate an immune response. We analyzed AT11-B1, a modified sequence of AT11 (itself a modified version of AS1411), in which one thymine was removed from the bulge region. We studied G-quadruplex (G4) formation/stabilization using PhenDC3, PDS, BRACO-19, TMPyP4 and 360A ligands by different biophysical techniques, namely circular dichroism (CD), Förster resonance energy transfer (FRET-melting) and nuclear magnetic resonance (NMR). The CD spectra showed that AT11-B1 adopts a predominant G4 of parallel topology when the buffer contains KCl or when ligands are added. PhenDC3 induced a ΔTm of 30 °C or more of the G4 structure as shown by CD- and FRET-melting experiments. The ligands demonstrate high affinity for AT11-B1 G4 and the NMR studies revealed that the AT11-B1 G4 involves four G-tetrad layers. The in silico studies suggest that all ligands bind AT11-B1 G4, namely, by stacking interactions, with the possible exception of PDS that may bind to the loop/groove interface. In addition, molecular dynamics simulations revealed that nucleolin (NCL) interacts with the AT11-B1 G4 structure through the RNA binding domain (RBD) 2 and the 12-residue linker between RBD1,2. Moreover, AT11-B1 G4 was internalized into a NCL-positive tongue squamous cell carcinoma cell line. In a nutshell, this study may help the identification of the ligands scaffolds to bind and stabilize AT11-B1, improving the targeting towards NCL that is overexpressed in cancer cells.

• 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

Herein, we report, for the first time, the screening of several ligands in terms of their ability to bind and stabilize G-quadruplexes (G4) found in seven human Papillomavirus (HPV) genomes. Using a variety of biophysical assays, HPV G-quadruplexes were shown to possess a high degree of structural polymorphism upon ligand binding, which may have an impact on transcription, replication, and viral protein production. A sequence found in high-risk HPV16 genotype folds into multiple non-canonical DNA structures; it was converted into a major G4 conformation upon interaction with a well-characterized highly selective G4 ligand, PhenDC3, which may have an impact on the viral infection. Likewise, HPV57 and 58, which fold into multiple G4 structures, were found to form single stable complexes in the presence of two other G4 ligands, C8 and pyridostatin, respectively. In addition, one of the selected compounds, the acridine derivative C8, demonstrated a significant antiviral effect in HPV18-infected organotypic raft cultures. Altogether, these results indicate that targeting HPV G4s may be an alternative route for the development of novel antiviral therapies.

• Klíčová slova
• G-quadruplex, antiviral, human papillomavirus, ligands, organotypic rafts,
• MeSH
• aminochinoliny farmakologie   MeSH
• cílená molekulární terapie MeSH
• DNA vazebné proteiny genetika   MeSH
• G-kvadruplexy účinky léků   MeSH
• genom virový účinky léků   genetika   MeSH
• genotyp MeSH
• komplement C8 genetika   farmakologie   MeSH
• konformace nukleové kyseliny účinky léků   MeSH
• kyseliny pikolinové farmakologie   MeSH
• lidé MeSH
• lidský papilomavirus 16 účinky léků   genetika   patogenita   ultrastruktura   MeSH
• lidský papilomavirus 18 účinky léků   genetika   ultrastruktura   MeSH
• ligandy MeSH
• virové nemoci farmakoterapie   genetika   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminochinoliny MeSH
• DNA vazebné proteiny MeSH
• komplement C8 MeSH
• kyseliny pikolinové MeSH
• ligandy MeSH
• pyridostatin MeSH Prohlížeč