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-quadruplexes are noncanonical nucleic acid structures formed by stacked guanine tetrads. They are capable of a range of functions and thought to play widespread biological roles. This diversity raises an important question: what determines the biochemical specificity of G-quadruplex structures? The answer is particularly important from the perspective of biological regulation because genomes can contain hundreds of thousands of G-quadruplexes with a range of functions. Here we analyze the specificity of each sequence in a 496-member library of variants of a reference G-quadruplex with respect to five functions. Our analysis shows that the sequence requirements of G-quadruplexes with these functions are different from one another, with some mutations altering biochemical specificity by orders of magnitude. Mutations in tetrads have larger effects than mutations in loops, and changes in specificity are correlated with changes in multimeric state. To complement our biochemical data we determined the solution structure of a monomeric G-quadruplex from the library. The stacked and accessible tetrads rationalize why monomers tend to promote a model peroxidase reaction and generate fluorescence. Our experiments support a model in which the sequence requirements of G-quadruplexes with different functions are overlapping but distinct. This has implications for biological regulation, bioinformatics, and drug design.

• MeSH
• DNA chemie   MeSH
• fluorescence MeSH
• G-kvadruplexy * MeSH
• guanosintrifosfát chemie   MeSH
• molekulární modely MeSH
• mutace MeSH
• peroxidasy chemie   MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• guanosintrifosfát MeSH
• peroxidasy MeSH

The formation of a G-quadruplex motif in the promoter region of the c-MYC protooncogene prevents its expression. Accordingly, G-quadruplex stabilization by a suitable ligand may be a viable approach for anticancer therapy. In our study, we used the 4-(4-methylpiperazin-1-yl)aniline molecule, previously identified as a fragment library screen hit, as a template for the SAR-guided design of a new small library of clickable fragments and subjected them to click reactions, including kinetic target-guided synthesis in the presence of a G-quadruplex forming oligonucleotide Pu24. We tested the clickable fragments and products of click reactions for their G-quadruplex stabilizing activity and determined their mode of binding to the c-MYC G-quadruplex by NMR spectroscopy. The enhanced stabilizing potency of click products in biology assays (FRET, Polymerase extension assay) matched the increased yields of in situ click reactions. In conclusion, we identified the newly synthesized click products of bis-amino derivatives of 4-(4-methylpiperazin-1-yl)aniline as potent stabilizers of c-MYC G-quadruplex, and their further evolution may lead to the development of an efficient tool for cancer treatment.

• Klíčová slova
• G-quadruplex, NMR, anticancer therapy, click chemistry, secondary structures,
• MeSH
• aniliny chemická syntéza   chemie   farmakologie   MeSH
• click chemie MeSH
• G-kvadruplexy účinky léků   MeSH
• geny myc genetika   MeSH
• kinetika MeSH
• ligandy MeSH
• simulace molekulární dynamiky MeSH
• techniky syntetické chemie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aniline MeSH Prohlížeč
• aniliny MeSH
• ligandy MeSH

Bone remodeling is a fine-tuned process principally regulated by a cascade triggered by interaction of receptor activator of NF-κB (RANK) and RANK ligand (RANKL). Excessive activity of the RANKL gene leads to increased bone resorption and can influence the incidence of osteoporosis. Although much has been learned about the intracellular signals activated by RANKL/RANK complex, significantly less is known about the molecular mechanisms of regulation of RANKL expression. Here, we report on the structure of an unprecedented DNA G-quadruplex, well-known secondary structure-mediated gene expression regulator, formed by a G-rich sequence found in the regulatory region of a RANKL gene. Solution-state NMR structural study reveals the formation of a three-layered parallel-type G-quadruplex characterized by an unique features, including a G-A bulge. Although a guanine within a G-tract occupies syn glycosidic conformation, bulge-forming residues arrange in a pseudo-loop conformation to facilitate partial 5/6-ring stacking, typical of G-quadruplex structures with parallel G-tracts orientation. Such distinctive structural features protruding from the core of the structure can represent a novel platform for design of highly specific ligands with anti-osteoporotic function. Additionally, our study suggests that the expression of RANKL gene may be regulated by putative folding of its G-rich region into non-B-DNA structure(s).

• Klíčová slova
• G-quadruplex, NMR spectroscopy, osteoporosis, structure,
• MeSH
• adenin chemie   MeSH
• G-kvadruplexy MeSH
• guanin chemie   MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• ligand RANK genetika   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• osteoporóza genetika   MeSH
• protein RANK genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenin MeSH
• guanin MeSH
• ligand RANK MeSH
• protein RANK MeSH
• TNFRSF11A protein, human MeSH Prohlížeč
• TNFSF11 protein, human MeSH Prohlížeč