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

Guanine quadruplex (GQ) is a noncanonical nucleic acid structure formed by guanine-rich DNA and RNA sequences. Folding of GQs is a complex process, where several aspects remain elusive, despite being important for understanding structure formation and biological functions of GQs. Pulling experiments are a common tool for acquiring insights into the folding landscape of GQs. Herein, we applied a computational pulling strategy─steered molecular dynamics (SMD) simulations─in combination with standard molecular dynamics (MD) simulations to explore the unfolding landscapes of tetrameric parallel GQs. We identified anisotropic properties of elastic conformational changes, unfolding transitions, and GQ mechanical stabilities. Using a special set of structural parameters, we found that the vertical component of pulling force (perpendicular to the average G-quartet plane) plays a significant role in disrupting GQ structures and weakening their mechanical stabilities. We demonstrated that the magnitude of the vertical force component depends on the pulling anchor positions and the number of G-quartets. Typical unfolding transitions for tetrameric parallel GQs involve base unzipping, opening of the G-stem, strand slippage, and rotation to cross-like structures. The unzipping was detected as the first and dominant unfolding event, and it usually started at the 3'-end. Furthermore, results from both SMD and standard MD simulations indicate that partial spiral conformations serve as a transient ensemble during the (un)folding of GQs.

• MeSH
• biomechanika MeSH
• DNA chemie   MeSH
• G-kvadruplexy * MeSH
• mechanické jevy MeSH
• simulace molekulární dynamiky * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH

BACKGROUND: Canonical telomeres (telomerase-synthetised) are readily forming G-quadruplexes (G4) on the G-rich strand. However, there are examples of non-canonical telomeres among eukaryotes where telomeric tandem repeats are invaded by specific retrotransposons. Drosophila melanogaster represents an extreme example with telomeres composed solely by three retrotransposons-Het-A, TAHRE and TART (HTT). Even though non-canonical telomeres often show strand biased G-distribution, the evidence for the G4-forming potential is limited. RESULTS: Using circular dichroism spectroscopy and UV absorption melting assay we have verified in vitro G4-formation in the HTT elements of D. melanogaster. Namely 3 in Het-A, 8 in TART and 2 in TAHRE. All the G4s are asymmetrically distributed as in canonical telomeres. Bioinformatic analysis showed that asymmetric distribution of potential quadruplex sequences (PQS) is common in telomeric retrotransposons in other Drosophila species. Most of the PQS are located in the gag gene where PQS density correlates with higher DNA sequence conservation and codon selection favoring G4-forming potential. The importance of G4s in non-canonical telomeres is further supported by analysis of telomere-associated retrotransposons from various eukaryotic species including green algae, Diplomonadida, fungi, insects and vertebrates. Virtually all analyzed telomere-associated retrotransposons contained PQS, frequently with asymmetric strand distribution. Comparison with non-telomeric elements showed independent selection of PQS-rich elements from four distinct LINE clades. CONCLUSION: Our findings of strand-biased G4-forming motifs in telomere-associated retrotransposons from various eukaryotic species support the G4-formation as one of the prerequisites for the recruitment of specific retrotransposons to chromosome ends and call for further experimental studies.

• Klíčová slova
• Drosophila, G-quadruplex, Het-A, Retrotransposon, TAHRE, TART, Telomere,
• Publikační typ
• časopisecké články 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

Parasitic helminths infecting humans are highly prevalent infecting ∼2 billion people worldwide, causing inflammatory responses, malnutrition and anemia that are the primary cause of morbidity. In addition, helminth infections of cattle have a significant economic impact on livestock production, milk yield and fertility. The etiological agents of helminth infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms). G-quadruplexes (G4) are unusual nucleic acid structures formed by G-rich sequences that can be recognized by specific G4 ligands. Here we used the G4Hunter Web Tool to identify and compare potential G4 sequences (PQS) in the nuclear and mitochondrial genomes of various helminths to identify G4 ligand targets. PQS are nonrandomly distributed in these genomes and often located in the proximity of genes. Unexpectedly, a Nematode, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This species can tolerate high-stability G4 structures, which are not counter selected at all, in stark contrast to most other species. We experimentally confirmed G4 formation for sequences found in four different parasitic helminths. Small molecules able to selectively recognize G4 were found to bind to Schistosoma mansoni G4 motifs. Two of these ligands demonstrated potent activity both against larval and adult stages of this parasite.

• MeSH
• cizopasní červi genetika   MeSH
• G-kvadruplexy * MeSH
• genom MeSH
• hlístice * genetika   MeSH
• lidé MeSH
• ligandy MeSH
• paraziti genetika   MeSH
• ploštěnci * genetika   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ligandy MeSH

Genomic sequences susceptible to form G-quadruplexes (G4s) are always flanked by other nucleotides, but G4 formation in vitro is generally studied with short synthetic DNA or RNA oligonucleotides, for which bases adjacent to the G4 core are often omitted. Herein, we systematically studied the effects of flanking nucleotides on structural polymorphism of 371 different oligodeoxynucleotides that adopt intramolecular G4 structures. We found out that the addition of nucleotides favors the formation of a parallel fold, defined as the 'flanking effect' in this work. This 'flanking effect' was more pronounced when nucleotides were added at the 5'-end, and depended on loop arrangement. NMR experiments and molecular dynamics simulations revealed that flanking sequences at the 5'-end abolish a strong syn-specific hydrogen bond commonly found in non-parallel conformations, thus favoring a parallel topology. These analyses pave a new way for more accurate prediction of DNA G4 folding in a physiological context.

• MeSH
• cirkulární dichroismus MeSH
• DNA genetika   ultrastruktura   MeSH
• G-kvadruplexy * MeSH
• konformace nukleové kyseliny MeSH
• nukleotidy chemie   genetika   MeSH
• oligonukleotidy chemie   genetika   MeSH
• polymorfismus genetický genetika   MeSH
• RNA genetika   ultrastruktura   MeSH
• simulace molekulární dynamiky MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• nukleotidy MeSH
• oligonukleotidy MeSH
• RNA MeSH

Recently, we reported an inhibitory effect of guanine substitutions on the conformational switch from antiparallel to parallel quadruplexes (G4) induced by dehydrating agents. As a possible cause, we proposed a difference in the sensitivity of parallel and antiparallel quadruplexes to the guanine substitutions in the resulting thermodynamic stability. Reports on the influence of guanine substitutions on the biophysical properties of intramolecular parallel quadruplexes are rare. Moreover, such reports are often complicated by the multimerisation tendencies of parallel quadruplexes. To address this incomplete knowledge, we employed circular dichroism spectroscopy (CD), both as stopped-flow-assisted fast kinetics measurements and end-point measurements, accompanied by thermodynamic analyses, based on UV absorption melting profiles, and electrophoretic methods. We showed that parallel quadruplexes are significantly more sensitive towards guanine substitutions than antiparallel ones. Furthermore, guanine-substituted variants, which in principle might correspond to native genomic sequences, distinctly differ in their biophysical properties, indicating that the four guanines in each tetrad of parallel quadruplexes are not equal. In addition, we were able to distinguish by CD an intramolecular G4 from intermolecular ones resulting from multimerisation mediated by terminal tetrad association, but not from intermolecular G4s formed due to inter-strand Hoogsteen hydrogen bond formation. In conclusion, our study indicates significant variability in parallel quadruplex structures, otherwise disregarded without detailed experimental analysis.

• Klíčová slova
• DNA secondary structure, circular dichroism, multimerisation, parallel guanine quadruplex, stopped-flow,
• MeSH
• cirkulární dichroismus MeSH
• DNA chemie   genetika   MeSH
• G-kvadruplexy MeSH
• guanin chemie   MeSH
• konformace nukleové kyseliny MeSH
• molekulární modely MeSH
• substituce aminokyselin * MeSH
• termodynamika MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH
• guanin MeSH

G-quadruplexes (G4) are non-canonical DNA and/or RNA secondary structures formed in guanine-rich regions. Given their over-representation in specific regions in the genome such as promoters and telomeres, they are likely to play important roles in key processes such as transcription, replication or RNA maturation. Putative G4-forming sequences (G4FS) have been reported in humans, yeast, bacteria, viruses and many organisms. Here we present the first mapping of G-quadruplex sequences in Dictyostelium discoideum, the social amoeba. 'Dicty' is an ameboid protozoan with a small (34 Mb) and extremely AT rich genome (78%). As a consequence, very few G4-prone motifs are expected. An in silico analysis of the Dictyostelium genome with the G4Hunter software detected 249-1055 G4-prone motifs, depending on G4Hunter chosen threshold. Interestingly, despite an even lower GC content (as compared to the whole Dicty genome), the density of G4 motifs in Dictyostelium promoters and introns is significantly higher than in the rest of the genome. Fourteen selected sequences located in important genes were characterized by a combination of biophysical and biochemical techniques. Our data show that these sequences form highly stable G4 structures under physiological conditions. Five Dictyostelium genes containing G4-prone motifs in their promoters were studied for the effect of a new G4-binding porphyrin derivative on their expression. Our results demonstrated that the new ligand significantly decreased their expression. Overall, our results constitute the first step to adopt Dictyostelium discoideum as a 'G4-poor' model for studies on G-quadruplexes.

• MeSH
• Dictyostelium genetika   MeSH
• G-kvadruplexy * MeSH
• genom genetika   MeSH
• konformace nukleové kyseliny MeSH
• počítačová simulace MeSH
• porfyriny genetika   MeSH
• promotorové oblasti (genetika) * MeSH
• telomery genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• porfyriny 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