G-quadruplexes are noncanonical nucleic acid structures formed by stacked guanosine tetrads. Despite their functional and structural diversity, a single consensus model is typically used to describe sequences with the potential to form G-quadruplex structures. We are interested in developing more specific sequence models for G-quadruplexes. In previous work, we functionally characterized each sequence in a 496-member library of variants of a monomeric reference G-quadruplex for the ability to bind GTP, promote a model peroxidase reaction, generate intrinsic fluorescence, and to form multimers. Here we used NMR to obtain a broad overview of the structural features of this library. After determining the 1H NMR spectrum of each of these 496 sequences, spectra were sorted into multiple classes, most of which could be rationalized based on mutational patterns in the primary sequence. A more detailed screen using representative sequences provided additional information about spectral classes, and confirmed that the classes determined based on analysis of 1H NMR spectra are correlated with functional categories identified in previous studies. These results provide new insights into the surprising structural diversity of this library. They also show how NMR can be used to identify classes of sequences with distinct mutational signatures and functions.

• Klíčová slova
• DNA, G-quadruplex, Multimeric structures, NMR,
• MeSH
• G-kvadruplexy * MeSH
• guanosintrifosfát chemie   metabolismus   MeSH
• magnetická rezonanční spektroskopie metody   MeSH
• mutace MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• guanosintrifosfát 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
• Názvy látek
• RNA * 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-quadruplexes are unusual DNA and RNA secondary structures ubiquitous in a variety of organisms including vertebrates, plants, viruses and bacteria. The folding topology and stability of intramolecular G-quadruplexes are determined to a large extent by their loops. Loop permutation is defined as swapping two or three of these regions so that intramolecular G-quadruplexes only differ in the sequential order of their loops. Over the past two decades, both length and base composition of loops have been studied extensively, but a systematic study on the effect of loop permutation has been missing. In the present work, 99 sequences from 21 groups with different loop permutations were tested. To our surprise, both conformation and thermal stability are greatly dependent on loop permutation. Loop permutation actually matters as much as loop length and base composition on G-quadruplex folding, with effects on Tm as high as 17°C. Sequences containing a longer central loop have a high propensity to adopt a stable non-parallel topology. Conversely, sequences containing a short central loop tend to form a parallel topology of lower stability. In addition, over half of interrogated sequences were found in the genomes of diverse organisms, implicating their potential regulatory roles in the genome or as therapeutic targets. This study illustrates the structural roles of loops in G-quadruplex folding and should help to establish rules to predict the folding pattern and stability of G-quadruplexes.

• MeSH
• algoritmy MeSH
• cirkulární dichroismus MeSH
• DNA chemie   MeSH
• G-kvadruplexy * MeSH
• konformace nukleové kyseliny * MeSH
• sekvence nukleotidů MeSH
• termodynamika * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH