ABSTRACT: G-quadruplexes are noncannonical four-stranded DNA or RNA structures formed by guanine-rich repeating sequences. Guanine nucleotides can hydrogen bond to form a planar tetrad structure. Such tetrads can stack to form quadruplexes of various molecularities with a variety of types of single-stranded loops joining the tetrads. High-resolution structures may be obtained by X-ray crystallography or NMR spectroscopy for quadruplexes formed by short (≈25 nt) sequences but these methods have yet to succeed in characterizing higher order quadruplex structures formed by longer sequences. An integrated computational and experimental approach was implemented in our laboratory to obtain structural models for higher order quadruplexes that might form in longer telomeric or promoter sequences. In our approach, atomic-level models are built using folding principles gleaned from available high-resolution structures and then optimized by molecular dynamics. The program HYDROPRO is then used to construct bead models of these structures to predict experimentally testable hydrodynamic properties. Models are validated by comparison of these properties with measured experimental values obtained by analytical ultracentrifugation or other biophysical tools. This chapter describes our approach and practical procedures.
Project description:The nuclease-hypersensitivity element III1 in the c-myc promoter is a good anticancer target since it largely controls transcriptional activation of the important c-myc oncogene. Recently, the guanine-rich strand of this element has been shown to form an equilibrium between G-quadruplex structures built from two different sets of G-stretches; two models of intramolecular fold-back antiparallel-stranded G-quadruplexes, called "basket" and "chair" forms, were proposed. Here, we show by NMR that two sequences containing these two sets of G-stretches form intramolecular propeller-type parallel-stranded G-quadruplexes in K(+)-containing solution. The two structures involve a core of three stacked G-tetrads formed by four parallel G-stretches with all anti guanines and three double-chain-reversal loops bridging three G-tetrad layers. The central loop contains two or six residues, while the two other loops contain only one residue.
Project description:G-quadruplexes (G4) are secondary structures formed by guanine-rich nucleic acid sequences and shown to exist in living cells where they participate in regulation of gene expression and chromosome maintenance. G-quadruplexes with solvent-exposed guanine tetrads show the tendency to associate together through cofacial stacking, which may be important for packaging of G4-forming sequences and allows for the design of higher-order G4 DNA structures. To understand the molecular driving forces for G4 association, here, we study the binding interaction between two parallel-stranded G-quadruplexes using all-atom molecular dynamics simulations. The predicted dimerization free energies show that direct binding through the 5'-G-tetrads is the most preferred of all possible end-to-end stacking orientations, consistently with all available experimental data. Decomposition of dimerization enthalpies in combination with simulations at varying ionic strength further indicate that the observed orientational preferences arise from a fine balance between the electrostatic repulsion of the sugar-phosphate backbones and favorable counterion binding at the dimeric interface. We also demonstrate how these molecular-scale findings can be used to devise means of controlling G4 dimerization equilibrium, e.g., by altering salt concentration and using G4-targeted ligands.
Project description:Unusual DNA/RNA structures of the C9orf72 repeat may participate in repeat expansions or pathogenesis of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded repeats are CpG methylated with unknown consequences. Typically, quadruplex structures form by G-rich but not complementary C-rich strands. Using CD, UV and electrophoresis, we characterized the structures formed by (GGGGCC)8 and (GGCCCC)8 strands with and without 5-methylcytosine (5mCpG) or 5-hydroxymethylcytosine (5hmCpG) methylation. All strands formed heterogenous mixtures of structures, with features of quadruplexes (at pH 7.5, in K(+), Na(+) or Li(+)), but no feature typical of i-motifs. C-rich strands formed quadruplexes, likely stabilized by G•C•G•C-tetrads and C•C•C•C-tetrads. Unlike G•G•G•G-tetrads, some G•C•G•C-tetrad conformations do not require the N7-Guanine position, hence C9orf72 quadruplexes still formed when N7-deazaGuanine replace all Guanines. 5mCpG and 5hmCpG increased and decreased the thermal stability of these structures. hnRNPK, through band-shift analysis, bound C-rich but not G-rich strands, with a binding preference of unmethylated > 5hmCpG > 5mCpG, where methylated DNA-protein complexes were retained in the wells, distinct from unmethylated complexes. Our findings suggest that for C-rich sequences interspersed with G-residues, one must consider quadruplex formation and that methylation of quadruplexes may affect epigenetic processes.
Project description:Guanine-rich DNA sequences can form G-quadruplexes stabilized by stacked G-G-G-G tetrads in monovalent cation-containing solution. The length and number of individual G-tracts and the length and sequence context of linker residues define the diverse topologies adopted by G-quadruplexes. The review highlights recent solution NMR-based G-quadruplex structures formed by the four-repeat human telomere in K(+) solution and the guanine-rich strands of c-myc, c-kit and variant bcl-2 oncogenic promoters, as well as a bimolecular G-quadruplex that targets HIV-1 integrase. Such structure determinations have helped to identify unanticipated scaffolds such as interlocked G-quadruplexes, as well as novel topologies represented by double-chain-reversal and V-shaped loops, triads, mixed tetrads, adenine-mediated pentads and hexads and snap-back G-tetrad alignments. The review also highlights the recent identification of guanine-rich sequences positioned adjacent to translation start sites in 5'-untranslated regions (5'-UTRs) of RNA oncogenic sequences. The activity of the enzyme telomerase, which maintains telomere length, can be negatively regulated through G-quadruplex formation at telomeric ends. The review evaluates progress related to ongoing efforts to identify small molecule drugs that bind and stabilize distinct G-quadruplex scaffolds associated with telomeric and oncogenic sequences, and outlines progress towards identifying recognition principles based on several X-ray-based structures of ligand-G-quadruplex complexes.
Project description:DNA G-rich sequences are able to form four-stranded structures organized in stacked guanine tetrads. These structures, called G-quadruplexes, were found to have an important role in the regulation of oncogenes expression and became, for such a reason, appealing targets for anticancer drugs. Aiming at finding selective G-quadruplex binders, we have designed, synthesized and characterized a new water soluble Salen-like Schiff base ligand and its Ni(II) and Cu(II) metal complexes. UV-Vis, circular dichroism and FRET measurements indicated that the nickel complex can stabilize oncogene promoter G-quadruplexes with high selectivity, presenting no interactions with duplex DNA at all. The same compound exhibited dose-dependent cytotoxic activity in MCF-7 breast cancer cells when combined with lipofectamine as lipophilic carrier.
Project description:Guanine-rich DNA repeat sequences located at the terminal ends of chromosomal DNA can fold in a sequence-dependent manner into G-quadruplex structures, notably the terminal 150-200 nucleotides at the 3' end, which occur as a single-stranded DNA overhang. The crystal structures of quadruplexes with two and four human telomeric repeats show an all-parallel-stranded topology that is readily capable of forming extended stacks of such quadruplex structures, with external TTA loops positioned to potentially interact with other macromolecules. This study reports on possible arrangements for these quadruplex dimers and tetramers, which can be formed from 8 or 16 telomeric DNA repeats, and on a methodology for modeling their interactions with small molecules. A series of computational methods including molecular dynamics, free energy calculations, and principal components analysis have been used to characterize the properties of these higher-order G-quadruplex dimers and tetramers with parallel-stranded topology. The results confirm the stability of the central G-tetrads, the individual quadruplexes, and the resulting multimers. Principal components analysis has been carried out to highlight the dominant motions in these G-quadruplex dimer and multimer structures. The TTA loop is the most flexible part of the model and the overall multimer quadruplex becoming more stable with the addition of further G-tetrads. The addition of a ligand to the model confirms the hypothesis that flat planar chromophores stabilize G-quadruplex structures by making them less flexible.
Project description:G-quadruplexes are non-canonical structures of nucleic acids, in which guanine bases form planar G-tetrads (G·G·G·G) that stack on each other in the core of the structure. G-quadruplexes generally contain multiple times of four (4n) guanines in the core. Here, we study the structure of G-quadruplexes with only (4n - 1) guanines in the core. The solution structure of a DNA sequence containing 11 guanines showed the formation of a parallel G-quadruplex involving two G-tetrads and one G-triad with a vacant site. Molecular dynamics simulation established the formation of a stable G-triad·water complex, where water molecules mimic the position of the missing guanine in the vacant site. The concept of forming G-quadruplexes with missing guanines in the core broadens the current definition of G-quadruplex-forming sequences. The potential ability of such structures to bind different metabolites, including guanine, guanosine and GTP, in the vacant site, could have biological implications in regulatory functions. Formation of this unique binding pocket in the G-triad could be used as a specific target in drug design.
Project description:Isoguanine (2-oxo-6-amino-guanine), a natural but non-standard base, exhibits unique self-association properties compared to its isomer, guanine, and results in formation of different higher order DNA structures. In this work, the higher order structures formed by oligonucleotides containing guanine repeats or isoguanine repeats after annealing in solutions containing various cations are evaluated by electrospray ionization mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy. The guanine-containing strand (G9) consistently formed quadruplexes upon annealing, whereas the isoguanine strand (Ig9) formed both pentaplexes and quadruplexes depending on the annealing cation. Quadruplex formation with G9 showed some dependence on the identity of the cation present during annealing with high relative quadruplex formation detected with six of ten cations. Analogous annealing experiments with Ig9 resulted in complex formation with all ten cations, and the majority of the resulting complexes were pentaplexes. CD results indicated most of the original complexes survived the desalting process necessary for ESI-MS analysis. In addition, several complexes, especially the pentaplexes, were found to be capable of cation exchange with ammonium ions. Ab initio calculations were conducted for isoguanine tetrads and pentads coordinated with all ten cations to predict the most energetically stable structures of the complexes in the gas phase. The observed preference of forming quadruplexes versus pentaplexes as a function of the coordinated cation can be interpreted by the calculated reaction energies of both the tetrads and pentads in combination with the distortion energies of tetrads.
Project description:G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential quadruplex sequences have been identified in G-rich eukaryotic telomeres, and more recently in non-telomeric genomic DNA, e.g. in nuclease-hypersensitive promoter regions. The natural role and biological validation of these structures is starting to be explored, and there is particular interest in them as targets for therapeutic intervention. This survey focuses on the folding and structural features on quadruplexes formed from telomeric and non-telomeric DNA sequences, and examines fundamental aspects of topology and the emerging relationships with sequence. Emphasis is placed on information from the high-resolution methods of X-ray crystallography and NMR, and their scope and current limitations are discussed. Such information, together with biological insights, will be important for the discovery of drugs targeting quadruplexes from particular genes.
Project description:G-quadruplexes constitute a class of nucleic acid structures defined by stacked guanine tetrads (or G-tetrads) with guanine bases from neighboring tetrads stacking with one another within the G-tetrad core. Individual G-quadruplexes can also stack with one another at their G-tetrad interface leading to higher-order structures as observed in telomeric repeat-containing DNA and RNA. In this study, we investigate how guanine base stacking influences the stability of G-quadruplexes and their stacked higher-order structures. A structural survey of the Protein Data Bank is conducted to characterize experimentally observed guanine base stacking geometries within the core of G-quadruplexes and at the interface between stacked G-quadruplex structures. We couple this survey with a systematic computational examination of stacked G-tetrad energy landscapes using quantum mechanical computations. Energy calculations of stacked G-tetrads reveal large energy differences of up to 12 kcal/mol between experimentally observed geometries at the interface of stacked G-quadruplexes. Energy landscapes are also computed using an AMBER molecular mechanics description of stacking energy and are shown to agree quite well with quantum mechanical calculated landscapes. Molecular dynamics simulations provide a structural explanation for the experimentally observed preference of parallel G-quadruplexes to stack in a 5'-5' manner based on different accessible tetrad stacking modes at the stacking interfaces of 5'-5' and 3'-3' stacked G-quadruplexes.