DNA quadruplexes Dotaz Zobrazit nápovědu
For mimicking macromolecular crowding of DNA quadruplexes, various crowding agents have been used, typically PEG, with quadruplexes of micromolar strand concentrations. Thermal and thermodynamic stabilities of these quadruplexes increased with the concentration of the agents, the rise depended on the crowder used. A different phenomenon was observed, and is presented in this article, when the crowder was the quadruplex itself. With DNA strand concentrations ranging from 3 µM to 9 mM, the thermostability did not change up to ∼2 mM, above which it increased, indicating that the unfolding quadruplex units were not monomolecular above ∼2 mM. The results are explained by self-association of the G-quadruplexes above this concentration. The ΔG(°) 37 values, evaluated only below 2 mM, did not become more negative, as with the non-DNA crowders, instead, slightly increased. Folding topology changed from antiparallel to hybrid above 2 mM, and then to parallel quadruplexes at high, 6-9 mM strand concentrations. In this range, the concentration of the DNA phosphate anions approached the concentration of the K(+) counterions used. Volume exclusion is assumed to promote the topological changes of quadruplexes toward the parallel, and the decreased screening of anions could affect their stability.
- MeSH
- cirkulární dichroismus MeSH
- denzitometrie MeSH
- DNA chemie MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- entropie MeSH
- G-kvadruplexy * MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- spektrofotometrie ultrafialová MeSH
- telomery chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA guanine quadruplexes are all based on stacks of guanine tetrads, but they can be of many types differing by mutual strand orientation, topology, position and structure of loops, and the number of DNA molecules constituting their structure. Here we have studied a series of nine DNA fragments (G(3)Xn)(3)G(3), where X = A, C or T, and n = 1, 2 or 3, to find how the particular bases and their numbers enable folding of the molecule into quadruplex and what type of quadruplex is formed. We show that any single base between G(3) blocks gives rise to only four-molecular parallel-stranded quadruplexes in water solutions. In contrast to previous models, even two Ts in potential loops lead to tetramolecular parallel quadruplexes and only three consecutive Ts lead to an intramolecular quadruplex, which is antiparallel. Adenines make the DNA less prone to quadruplex formation. (G(3)A(2))(3)G(3) folds into an intramolecular antiparallel quadruplex. The same is true with (G(3)A(3))(3)G(3) but only in KCl. In NaCl or LiCl, (G(3)A(3))(3)G(3) prefers to generate homoduplexes. Cytosine still more interferes with the quadruplex, which only is generated by (G(3)C)(3)G(3), whereas (G(3)C(2))(3)G(3) and (G(3)C(3))(3)G(3) generate hairpins and/or homoduplexes. Ethanol is a more potent DNA guanine quadruplex inducer than are ions in water solutions. It promotes intramolecular folding and parallel orientation of quadruplex strands, which rather corresponds to quadruplex structures observed in crystals. c) 2007 Wiley Periodicals, Inc.
G -kvadruplexy (G4) se za půl století studia staly z přírodní hříčky jednou z nejstudovanějších struktur nukleových kyselin. Lokalizace G4 do funkčních oblastí DNA a RNA poukazuje na množství buněčných procesů, které je možné regulovat formováním G4. Důkazy o jejich existenci in vivo, stejně jako možnost jejich stabilizace prostřednictvím nízkomolekulárních látek – ligandů G4, přináší nové terapeutické možnosti zejména v oblasti onkologie, kde narušení funkce telomer, replikace DNA, či exprese onkogenů vede k inhibici růstu nádoro-vých buněk.
Within the last 50 years, G -quadruplexes (G4) have become one of the most studied structures of nucleic acids. Localization of the G4 into the functional regions of the DNA and RNA indicates a number of cellular processes that can be regulated by G4 formation. Evi-dence of their existence in vivo as well as the possibility of their stabilization by low molecular weight G4 ligands brings new treatment opportunities, particularly in the field of oncology, where telomere disruption, inhibition of DNA replication, or oncogenic expression may affect tumor cell growth.
- MeSH
- DNA genetika MeSH
- G-kvadruplexy * účinky léků MeSH
- lidé MeSH
- ligandy MeSH
- nádory farmakoterapie genetika MeSH
- onkogeny účinky léků MeSH
- protinádorové látky MeSH
- regulace genové exprese u nádorů MeSH
- replikace DNA genetika MeSH
- RNA metabolismus MeSH
- rostliny MeSH
- telomerasa antagonisté a inhibitory metabolismus MeSH
- telomery mikrobiologie MeSH
- vyvíjení léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
Some metallo-supramolecular helical assemblies with size, shape, charge and amphipathic architectures similar to short cationic α-helical peptides have been shown to target and stabilise DNA G-quadruplexes (G4s) in vitro and downregulate the expression of G4-regulated genes in human cells. To expand the library of metallohelical structures that can act as efficient DNA G4 binders and downregulate genes containing G4-forming sequences in their promoter regions, we investigated the interaction of the two enantiomeric pairs of asymmetric Fe(II) triplex metallohelices with a series of five different DNA G4s formed by the human telomeric sequence (hTelo) and in the promoter regions of c-MYC, c-KIT, and k-RAS oncogenes. The metallohelices display preferential binding to G4s over duplex DNA in all investigated G4-forming sequences and induced arrest of DNA polymerase on template strands containing G4-forming sequences. Moreover, the investigated metallohelices suppressed the expression of c-MYC and k-RAS genes at mRNA and protein levels in HCT116 human cancer cells, as revealed by RT-qPCR analysis and western blotting.
We studied the effect of antitumor cisplatin and inefficient transplatin on the structure and stability of G quadruplexes formed by the model human telomere sequence 5'-GGG(TTAGGG)(3)-3' using circular dichroism, UV-monitored thermal denaturation, and gel electrophoresis. In addition, to investigate whether there is a connection between the ability of cisplatin or transplatin to affect telomerase activity and stability of G quadruplexes, we also used a modified telomere repeat amplification protocol assay that uses an oligonucleotide substrate for telomerase elongation susceptible to forming a G quadruplex. The results indicate that cisplatin is more efficient than transplatin in disturbing the quadruplex structure, thereby precluding telomeric sequences from forming quadruplexes. On the other hand, the results of this work also demonstrate that in absence of free platinum complex, DNA adducts of antitumor cisplatin inhibit telomerase catalysis, so the mechanism underlying this inhibition does not involve formation of the G quadruplexes which are not elongated by telomerase.
- MeSH
- adukty DNA genetika metabolismus účinky léků MeSH
- biokatalýza MeSH
- cirkulární dichroismus MeSH
- cisplatina farmakologie chemie MeSH
- denaturace nukleových kyselin MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- G-kvadruplexy účinky léků MeSH
- lidé MeSH
- protinádorové látky farmakologie chemie MeSH
- sekvence nukleotidů MeSH
- spektrofotometrie ultrafialová MeSH
- techniky amplifikace nukleových kyselin MeSH
- telomerasa antagonisté a inhibitory metabolismus MeSH
- telomery genetika chemie MeSH
- tranzitní teplota MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Circular dichroism (CD) is remarkably sensitive to the conformational states of nucleic acids; therefore, CD spectroscopy has been used to study most features of DNA and RNA structures. Quadruplexes are among the significant noncanonical nucleic acids architectures that have received special attentions recently. This article presents examples on the contribution of CD spectroscopy to our knowledge of quadruplex structures and their polymorphism. The examples were selected to demonstrate the potential of this simple method in the quadruplex field. As CD spectroscopy detects only the global feature of a macromolecule, it should preferably be used in combination with other techniques. On the other hand, CD spectroscopy, often as a pioneering approach, can reveal the formation of particular structural arrangements, to search for the conditions stabilizing the structures, to follow the transitions between various structural states, to explore kinetics of their appearance, to determine thermodynamic parameters and also detect formation of higher order structures. This article aims to show that CD spectroscopy is an important complementary technique to NMR spectroscopy and X-ray diffraction in quadruplex studies.
- MeSH
- cirkulární dichroismus metody MeSH
- difrakce rentgenového záření MeSH
- DNA chemie MeSH
- G-kvadruplexy * MeSH
- guanin chemie MeSH
- kinetika MeSH
- konformace nukleové kyseliny * MeSH
- oligonukleotidy chemie MeSH
- termodynamika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Guanine-rich sequences of DNA are able to create tetrastranded structures known as G-quadruplexes; they are formed by the stacking of planar G-quartets composed of four guanines paired by Hoogsteen hydrogen bonding. G-quadruplexes act as ligands for metal ions and aptamers for various molecules. Interestingly, the G-quadruplexes form a complex with anionic porphyrin hemin and exhibit peroxidase-like activity. This review focuses on overview of sensing techniques based on G-quadruplex complexes with anionic porphyrins for detection of various analytes, including metal ions such as K+, Ca2+, Ag+, Hg2+, Cu2+, Pb2+, Sr2+, organic molecules, nucleic acids, and proteins. Principles of G-quadruplex-based detection methods involve DNA conformational change caused by the presence of analyte which leads to a decrease or an increase in peroxidase activity, fluorescence, or electrochemical signal of the used probe. The advantages of various detection techniques are also discussed.
- MeSH
- biosenzitivní techniky * MeSH
- delece genu MeSH
- DNA katalytická chemie MeSH
- DNA chemie MeSH
- G-kvadruplexy * MeSH
- ionty analýza chemie MeSH
- kovy analýza chemie MeSH
- lidé MeSH
- nádorový supresorový protein p53 chemie genetika MeSH
- nanočástice chemie MeSH
- nukleové kyseliny analýza chemie MeSH
- organické látky analýza chemie MeSH
- proteiny analýza chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
G-quadruplexes can multimerize under certain conditions, but the sequence requirements of such structures are not well understood. In this study, we investigated the ability of all possible variants of the central tetrad in a monomeric, parallel-strand G-quadruplex to form higher-order structures. Although most of these 256 variants existed primarily as monomers under the conditions of our screen, ∼10% formed dimers or tetramers. These structures could form in a wide range of monovalent and divalent metal ions, and folding was highly cooperative in both KCl and MgCl2. As was previously shown for G-quadruplexes that bind GTP and promote peroxidase reactions, G-quadruplexes that form dimers and tetramers have distinct sequence requirements. Some mutants could also form heteromultimers, and a second screen was performed to characterize the sequence requirements of these structures. Taken together, these experiments provide new insights into the sequence requirements and structures of both homomultimeric and heteromultimeric G-quadruplexes.
G-Quadruplexes are four-stranded nucleic acid structures typically stabilized by GGGG tetrads. These structures are intrinsically fluorescent, which expands the known scope of nucleic acid function and raises the possibility that they could eventually be used as signaling components in label-free sensors constructed from DNA or RNA. In this study, we systematically investigated the effects of mutations in tetrads, loops, and overhanging nucleotides on the fluorescence intensity and maximum emission wavelength of >500 sequence variants of a reference DNA G-quadruplex. Some of these mutations modestly increased the fluorescence intensity of this G-quadruplex, while others shifted its maximum emission wavelength. Mutations that increased the fluorescence intensity were distinct from those that increased the maximum emission wavelength, suggesting a trade-off between these two biochemical properties. The fluorescence intensity and maximum emission wavelength were also correlated with multimeric state: the most fluorescent G-quadruplexes were monomers, while those with the highest maximum emission wavelengths typically formed dimeric structures. Oligonucleotides containing multiple G-quadruplexes were in some cases more fluorescent than those containing a single G-quadruplex, although this depended on the length and sequence of the spacer linking the G-quadruplexes. These experiments provide new insights into the properties of fluorescent G-quadruplexes and should aid in the development of improved label-free nucleic acid sensors.
Guanine quadruplexes (G4s) are non-canonical nucleic acids structures common in important genomic regions. Parallel-stranded G4 folds are the most abundant, but their folding mechanism is not fully understood. Recent research highlighted that G4 DNA molecules fold via kinetic partitioning mechanism dominated by competition amongst diverse long-living G4 folds. The role of other intermediate species such as parallel G-triplexes and G-hairpins in the folding process has been a matter of debate. Here, we use standard and enhanced-sampling molecular dynamics simulations (total length of ∼0.9 ms) to study these potential folding intermediates. We suggest that parallel G-triplex per se is rather an unstable species that is in local equilibrium with a broad ensemble of triplex-like structures. The equilibrium is shifted to well-structured G-triplex by stacked aromatic ligand and to a lesser extent by flanking duplexes or nucleotides. Next, we study propeller loop formation in GGGAGGGAGGG, GGGAGGG and GGGTTAGGG sequences. We identify multiple folding pathways from different unfolded and misfolded structures leading towards an ensemble of intermediates called cross-like structures (cross-hairpins), thus providing atomistic level of description of the single-molecule folding events. In summary, the parallel G-triplex is a possible, but not mandatory short-living (transitory) intermediate in the folding of parallel-stranded G4.
- MeSH
- DNA chemie genetika metabolismus MeSH
- G-kvadruplexy * MeSH
- guanin chemie metabolismus MeSH
- jednovláknová DNA chemie genetika metabolismus MeSH
- kinetika MeSH
- konformace nukleové kyseliny * MeSH
- lidé MeSH
- sekvence nukleotidů MeSH
- simulace molekulární dynamiky * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
