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PubMedDate not supplied

[Conformational polymorphism and extensibility of DNA quadruplexes formed from d(GT)n repeats].

We showed earlier that oligonucleotides 3'-d(GT)5-pO(CH2CH2O)3p-d(GT)5-3' form bimolecular quadruplexes with parallel orientation of their strands, which are held by guanine quartets alternating with unpaired thymines (GT quadruplex). This work deals with the conformational polymorphism and extensibility of G quadruplexes in complex with molecules of an intercalating agent ethidium bromide (EtBr). A cooperative mechanism of EtBr binding to the GT quadruplex was revealed. The binding constant K = (3.3 +/- 0.1) x 10(4) M-1, cooperativity coefficient omega = 2.5 +/- 0.2, and maximal amount of EtBr molecules intercalated in GT quadruplex (N = 8) were determined. It was proved experimentally by analysis of adsorption isotherms and theoretically by mathematical modeling that the GT quadruplex is capable of double extension, which is indicative of the high elasticity of this four-stranded helix. Two most stable conformations of GT quadruplexes with thymine residues intercalated and/or turned outside were found by mechanico-mathematical modeling. The equilibrium is shifted toward the conformation with the looped out thymine residues upon intercalation of EtBr molecules into the GT quadruplex.

Base Sequence↗
PubMedDate not supplied

Quadruplex structures in nucleic acids.

DNA oligonucleotides that have repetitive tracts of guanine bases can form G-quadruplex structures that display an amazing polymorphism. Structures of several new G-quadruplexes have been solved recently that greatly expand the known structural motifs observed in nucleic acid quadruplexes. Base triads, base hexads, and quartets that contain cytosine have recently been identified stacked over the familiar G-quartets. The current status of the diverse array of structural features in quadruplexes is described and used to provide insight into the polymorphism and folding pathways. This review also summarizes recent progress in the techniques used to probe the structures of G-quadruplexes and discusses the role of ion binding in quadruplex formation. Several of the quadruplex structures featured in this review can be accessed in the online version of this review as CHIME representations.

DNA↗
PubMedDate not supplied

Biological aspects of DNA/RNA quadruplexes.

Among the many unusual conformations of DNA and RNA, quadruplex structures, based on the guanine quartet, possess several unique properties. These properties, along with the general features of guanine quadruplexes, are described in the context of possible roles for these structures in biological systems. A variety of experimental observations supporting the notion that quadruplexes are important in vivo is presented, including proteins known to specifically bind to quadruplex structures, guanine-rich DNA, and RNA sequences endowed with the potential for forming quartet-based structures in telomeres and regulatory regions, such as gene promoters, quadruplexes as DNA aptamer folding motifs arising from in vitro selection experiments, and potential chemotherapeutic, quadruplex-forming oligonucleotides. Taken together, all of these observations argue cogently not only for the presence of quadruplexes in biological systems but also for their significance in terms of their roles in various biological processes.

Animals↗
PubMed2001

Dimeric DNA quadruplex containing major groove-aligned A-T-A-T and G-C-G-C tetrads stabilized by inter-subunit Watson-Crick A-T and G-C pairs.

We report on an NMR study of unlabeled and uniformly 13C,15N-labeled d(GAGCAGGT) sequence in 1 M NaCl solution, conditions under which it forms a head-to-head dimeric quadruplex containing sequentially stacked G-C-G-C, G-G-G-G and A-T-A-T tetrads. We have identified, for the first time, a slipped A-T-A-T tetrad alignment, involving recognition of Watson-Crick A-T pairs along the major groove edges of opposing adenine residues. Strikingly, both Watson-Crick G-C and A-T pairings within the direct G-C-G-C and slipped A-T-A-T tetrads, respectively, occur between rather than within hairpin subunits of the dimeric d(GAGCAGGT) quadruplex. The hairpin turns in the head-to-head dimeric quadruplex involve single adenine residues and adds to our knowledge of chain reversal involving edgewise loops in DNA quadruplexes. Our structural studies, together with those from other laboratories, definitively establish that DNA quadruplex formation is not restricted to G(n) repeat sequences, with their characteristic stacked uniform G-G-G-G tetrad architectures. Rather, the quadruplex fold is a more versatile and robust architecture, accessible to a range of mixed sequences, with the potential to facilitate G-C-G-C and A-T-A-T tetrad through major and minor groove alignment, in addition to G-G-G-G tetrad formation. The definitive experimental identification of such major groove-aligned mixed A-T-A-T and G-C-G-C tetrads within a quadruplex scaffold, has important implications for the potential alignment of duplex segments during homologous recombination.

Base Pairing↗
PubMed2005

Stabilization of guanine quadruplex DNA by the binding of porphyrins with cationic side arms.

Many aromatic ligands, including tetra-(N-methyl-4-pyridyl)porphyrin (TMPyP4), have been reported to bind and stabilize quadruplex structure of telomeric DNA. We synthesized novel quadruplex-interacting porphyrins with cationic pyridinium and trimethylammonium arms at para- or meta-position of all phenyl groups of tetratolyl porphyrin. An antiparallel quadruplex structure was found to be stabilized more greatly by the meta-isomers than by the para-isomers and well-studied TMPyP4, as revealed by the increase in melting temperature of the quadruplex. One mole equivalent of the isomers was sufficient to stabilize the quadruplex. From the results of absorption, induced circular dichroism, and fluorescence resonance energy transfer spectroscopic methods, the unique site for the porphyrin binding is suggested to be the external guanine tetrad or groove of the quadruplex. The cationic side arms played a key role in the stabilization of the quadruplex structure.

Cations↗
PubMed2003

A comparative study on the interaction of acridine and synthetic bis-acridine with G-quadruplex structure.

DNA from the telomeres contains a stretch of simple tandemly repeated sequences in which clusters of G residues alternate with clusters of T/A sequences along one DNA strand. Model telomeric G-clusters form four-stranded structures in presence of Na(I), K(I) and NH(4)(I) ions. Electrophoretic and spectroscopic studies were made with the telomeric related sequences d(T6G16) or d(G4T2G4T2G4T2G4). It was noticed earlier that G-quadruplex may either be inter-molecular, or intra-molecular, or a mixture of both. CD spectral characteristics of various G-quadruplex DNA suggests that the CD maximum at 293 nm corresponds to that of an intra-molecular G-quadruplex structure or hairpin dimers. Fluorescence titration studies also show that acridine and the bis-acridine are interacting with G-quadruplex DNA and destabilize the K(I)-quadruplex structure more efficiently than the quadruplex formed by NH(4)(I) ion. Among the two drugs studied, acridine is more capable of breaking the G-quadruplex structure than bis-acridine. This result is further confirmed by the CD experiments.

Acridines↗
PubMed2002

Investigation of quadruplex oligonucleotide-drug interactions by electrospray ionization mass spectrometry.

Selectivity, binding stoichiometry, and mode of binding of Tel01, distamycin A, and diethylthiocarbocyanine iodide (DTC) to the parallel stranded G4-quadruplex [d(T2G5T)]4 were investigated by ESI-MS. The first drug/quadruplex complexes observed by ESI-MS are described. Tel01, distamycin A, and DTC all form complexes with quadruplex DNA, but only Tel01 is completely selective for quadruplex versus duplex oligonucleotide under the conditions employed. Previous solution determinations of the binding mode of Tel01 and distamycin A to quadruplex oligonucleotides indicate that Tel01 interacts through end-stacking with guanine tetrads of quadruplex DNA, while distamycin A interacts by binding to quadruplex grooves. When these two different drug/quadruplex complexes are subjected to collisionally activated dissociation in a mass spectrometer, the observed fragmentation patterns are distinct. Tel01/quadruplex complexes undergo facile loss of drug and dissociation to single-strand oligonucleotide ions, while distamycin/quadruplex complexes fragment into single-strand oligonucleotide ions in which the drug molecule is retained. Dissociation patterns for DTC/quadruplex complexes are similar to those of distamycin; therefore, it is concluded that DTC interacts with [d(T2G5T)]4 through groove-binding. These ESI-MS results are applicable to both the identification and characterization of G-quadruplex interactive agents and may also be useful in probing unusual DNA structures.

Binding Sites↗
PubMed2001

Cleavage of telomeric G-quadruplex DNA with perylene-EDTA*Fe(II).

Interest in the development of compounds that bind G-quadruplex DNA selectively has been sparked by the discovery that these compounds can inhibit the activity of telomerase. Compounds that bind to this unique structure, as well as compounds that recognize it and cleave it upon binding, have potential uses as anticancer agents and as probes for the presence of G-quadruplex structures in chromatin. Here we report on the G-quadruplex-specific cleavage properties of a perylene derivative, perylene-EDTA*Fe(II). This water-soluble complex cleaves G-quadruplex DNA selectively in the presence of dithiothreitol. We characterize the cleavage reaction with respect to perylene-EDTA*Fe(II) concentration, DNA structural type, cation type (Na+ vs K+), cation concentration, and pH. Visible spectroscopy confirms the selectivity of the perylene-EDTA*metal complex for G-quadruplex DNA. NMR titration of a parallel G-quadruplex with perylene-EDTA (without metal) indicates that the compound binds to G-quadruplex DNA by stacking externally on the 3' G-tetrad.

Binding Sites↗
PubMed2004

Parallel-stranded guanine quadruplex interactions with a copper cationic porphyrin.

G-quadruplexes are formed by association of DNA strands containing multiple contiguous guanines. The capability of drugs to induce formation of or stabilize G-quadruplexes is an active area of investigation. We report the interactions of CuTMpyP4, the Cu(2+) derivative of 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine, with the parallel-stranded G-quadruplexes formed by d(T(4)G(4)T(4)) (1) and d(T(4)G(8)T(4)) (3). Absorption titrations of CuTMpyP4 with (1)(4) or (3)(4) cause both bathochromicity and hypochromicity of the porphyrin Soret band, with larger changes observed for the longer oligonucleotide. An approximate binding constant for (1)(4) and CuTMpyP4 according to the Scatchard model is 5.6 x 10(6) M(-)(1) in terms of quadruplexes and according to the McGhee-von Hippel model is 1.3 x 10(6) M(-)(1) in terms of potential binding sites. An approximate binding constant for (3)(4) and CuTMpyP4 according to the Scatchard model is 5.2 x 10(7) M(-)(1) in terms of quadruplexes and in terms of the McGhee-von Hippel model is 2.4 x 10(6) M(-)(1) in terms of potential binding sites. The site size for CuTMpyP4 and (1)(4) is four using the McGhee-von Hippel model. We find a 2:1 binding stoichiometry for CuTMpyP4 and (1)(4) and a 3:1 binding stoichiometry for CuTMpyP4 and (3)(4) using the method of continuous variation analysis. Induced emission spectra of CuTMpyP4 with (1)(4) or (3)(4) indicate a mode of binding in which the ligand is protected from the solvent. Electron paramagnetic resonance spectra of CuTMpyP4 with added oligonucleotide show an increase in the Cu-N superhyperfine coupling constant as the length of the oligonucleotide increases. On the basis of these data, we propose that for both (1)(4) and (3)(4), CuTMpyP4 molecules externally stack at each end of the run of guanines, similar to other planar G-quadruplex ligands. For (3)(4), our data are consistent with intercalation of a CuTMpyP4 molecule into the G-quadruplex.

Circular Dichroism↗
PubMed1998

NMR-Based model of a telomerase-inhibiting compound bound to G-quadruplex DNA.

The single-stranded (TTAGGG)n tail of human telomeric DNA is known to form stable G-quadruplex structures. Optimal telomerase activity requires the nonfolded single-stranded form of the primer, and stabilization of the G-quadruplex form is known to interfere with telomerase binding. We have identified 3,4,9, 10-perylenetetracarboxylic diimide-based ligands as potent inhibitors of human telomerase by using a primer extension assay that does not use PCR-based amplification of the telomerase primer extension products. A set of NMR titrations of the ligand into solutions of G-quadruplexes using various oligonucleotides related to human telomeric DNA showed strong and specific binding of the ligand to the G-quadruplex. The exchange rate between bound and free DNA forms is slow on the NMR time scale and allows the unequivocal determination of the binding site and mode of binding. In the case of the 5'-TTAGGG sequence, the ligand-DNA complex consists of two quadruplexes oriented in a tail-to-tail manner with the ligand sandwiched between terminal G4 planes. Longer telomeric sequences, such as TTAGGGTT, TTAGGGTTA, and TAGGGTTA, form 1:1 ligand-quadruplex complexes with the ligand bound at the GT step by a threading intercalation mode. On the basis of 2D NOESY data, a model of the latter complex has been derived that is consistent with the available experimental data. The determination of the solution structure of this telomerase inhibitor bound to telomeric quadruplex DNA should help in the design of new anticancer agents with a unique and novel mechanism of action.

Anthracenes↗
PubMed2001

Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay.

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. Intramolecular folding of an oligonucleotide with four repeats of the human telomeric sequence into a G-quadruplex structure led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5' and 3' ends of the oligonucleotide, respectively. The melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the fluorescence emission of the donor. A series of compounds (pentacyclic crescent-shaped dibenzophenanthroline derivatives) was shown to increase the melting temperature of the G-quadruplex by 2-20 degrees C at 1 microM dye concentration. This increase in T(m) value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC(50) value of 28 nM in a standard telomerase repeat amplification protocol assay. Fluorescence energy transfer can thus be used to reveal the formation of four-stranded DNA structures, and its stabilization by quadruplex-binding agents, in an effort to discover new potent telomerase inhibitors.

DNA↗
PubMed1995

Single strand targeted triplex-formation. Destabilization of guanine quadruplex structures by foldback triplex-forming oligonucleotides.

Oligonucleotides that can hybridize to single-stranded complementary polypurine nucleic acid targets by Watson-Crick base pairing as well as by Hoogsteen base pairing, referred to here as foldback triplex-forming oligonucleotides (FTFOs), have been designed. These oligonucleotides hybridize with target nucleic acid sequences with greater affinity than antisense oligonucleotides, which hybridize to the target sequence only by Watson-Crick hydrogen bonding [Kandimalla, E. R. and Agrawal, S. Gene(1994) 149, 115-121 and references cited therein]. FTFOs have been studied for their ability to destabilize quadruplexes formation by RNA or DNA target sequences. The influence of various DNA/RNA compositions of FTFOs on their ability to destabilize RNA and DNA quadruplexes has been examined. The ability of the FTFOs to destabilize quadruplex structures is related to the structurally and thermodynamically stable foldback triplex formed between the FTFO and its target sequence. Antisense oligonucleotides (DNA or RNA) that can form only a Watson-Crick double helix with the target sequence are unable to destabilize quadruplex structures of RNA and DNA target sequences and are therefore limited in their repertoire of target sequences. The quadruplex destabilization ability of FTFOs is dependent on the nature of the cation present in solution. The RNA quadruplex destabilization ability of FTFOs is -20% higher in the presence of sodium ion than potassium ion. The use of FTFOs, which can destabilize quadruplex structure, opens up new areas for development of oligonucleotide-based therapeutics, specifically, targeting guanine-rich sequences that exist at the ends of pro- and eukaryotic chromosomes and dimerization regions of retroviral RNA.

Base Sequence↗
PubMed1997

The contribution of thymine-thymine interactions to the stability of folded dimeric quadruplexes.

The loop of four thymines in the sodium form of the dimeric folded quadruplex [d(G3T4G3)]2 assumes a well-defined structure in which hydrogen bonding between the thymine bases appears to contribute to the stability and final conformation of the quadruplex. We have investigated the importance of the loop interactions by systematically replacing each thymine in the loop with a cytosine. The quadruplexes formed by d(G3CT3G3), d(G3TCT2G3), d(G3T2CTG3) and d(G3T3CG3) in the presence of 150 mM Na+ were studied by gel mobility, circular dichroism and 1H NMR spectroscopy. The major species formed by d(G3CT3G3), d(G3TCT2G3) and d(G3T3CG3) at 1 mM strand concentration at neutral pH is a dimeric folded quadruplex. d(G3T2CTG3) has anomalous behaviour and associates into a greater percentage of linear four-stranded quadruplex than the other three oligonucleotides at neutral pH and at the same concentration. The linear four-stranded quadruplex has a greater tendency to oligomerize to larger ill-defined structures, as demonstrated by broad 1H NMR resonances. At pH 4, when the cytosine is protonated, there is a greater tendency for each of the oligonucleotides to form some four-stranded linear quadruplex, except for d(G3T2CTG3), which has the reverse tendency. The experimental results are discussed in terms of hydrogen bonding within the thymine loop.

Circular Dichroism↗
PubMed2006

Resolution of a structural competition involving dimeric G-quadruplex and its C-rich complementary strand.

The resolution of the dimeric intermolecular G-quadruplex/duplex competition of the telomeric DNA sequence 5'-TAG GGT TAG GGT-3' and of its complementary 5' ACC CTA ACC CTA-3' is reported. To achieve this goal, melting experiments of both sequences and of the mixtures of these sequences were monitored by molecular absorption, molecular fluorescence and circular dichroism spectroscopies. Molecular fluorescence measurements were carried out using molecular beacons technology, in which the 5'-TAG GGT TAG GGT-3' sequence was labelled with a fluorophore and a quencher at the ends of the strand. Mathematical analysis of experimental spectroscopic data was performed by means of multivariate curve resolution, allowing the calculation of concentration profiles and pure spectra of all resolved structures (dimeric antiparallel and parallel G-quadruplexes, Watson-Crick duplex and single strands) present in solution. Our results show that parallel G-quadruplex is more stable than antiparallel G-quadruplex. When the complementary C-rich strand is present, a mixture of both G-quadruplex structures and Watson-Crick duplex is observed, the duplex being the major species. In addition to melting temperatures, equilibrium constants for the parallel/antiparallel G-quadruplex equilibrium and for the G-quadruplex/duplex equilibrium were determined from the concentration profiles.

Circular Dichroism↗
PubMed2006

Kinetics of double-chain reversals bridging contiguous quartets in tetramolecular quadruplexes.

Repetitive 5'GGXGG DNA segments abound in, or near, regulatory regions of the genome and may form unusual structures called G-quadruplexes. Using NMR spectroscopy, we demonstrate that a family of 5'GCGGXGGY sequences adopts a folding topology containing double-chain reversals. The topology is composed of two bistranded quadruplex monomeric units linked by formation of G:C:G:C tetrads. We provide a complete thermodynamic and kinetic analysis of 13 different sequences using absorbance spectroscopy and DSC, and compare their kinetics with a canonical tetrameric parallel-stranded quadruplex formed by TG4T. We demonstrate large differences (up to 10(5)-fold) in the association constants of these quadruplexes depending on primary sequence; the fastest samples exhibiting association rate equal or higher than the canonical TG4T quadruplex. In contrast, all sequences studied here unfold at a lower temperature than this quadruplex. Some sequences have thermodynamic stability comparable to the canonical TG4T tetramolecular quadruplex, but with faster association and dissociation. Sequence effects on the dissociation processes are discussed in light of structural data.

Base Sequence↗
PubMed2005

Quadruplex-forming oligonucleotides as tools in anticancer therapy and aptamers design: energetic aspects.

Recent investigations on the G-quadruplex motif propose a new strategy for the making of antitumour drugs. Quadruplex-drug complexes have been suggested to inhibit telomerase activity; further, aptamers based on the quadruplex motif have been proved useful as tools aimed at binding and inhibiting particular proteins, thus serving as pharmaceutically active agents. However, the design of new aptamers is difficult because many factors affecting their activity and stability have not still been clarified. The knowledge of the energetics of quadruplex formation is a crucial point in view of their potential therapeutic utilization both as targets as well as therapeutic agents. In this review the energetic aspects of both quadruplex assembly and quadruplex-ligand interactions are discussed together with a summary of recent studies on physico-chemical properties in solution of quadruplex structures obtained from synthetic aptamers, including PNA-DNA chimeras.

Animals↗
PubMed1996

Determination of the number and location of the manganese binding sites of DNA quadruplexes in solution by EPR and NMR in the presence and absence of thrombin.

The interaction of a DNA quadruplex with thrombin has been studied by first determining the sites of manganese binding to the quadruplex in the absence of thrombin. This has been followed by determining if the interactions with thrombin displace the bound manganese. A different DNA quadruplex has also been studied as a control. The refined solution structures of two DNA quadruplexes have been used to predict the electrostatic potentials of these DNAs. The calculated electrostatic potentials have been used to predict the locations of the binding sites of the paramagnetic ion manganese to these DNAs. The enhanced relaxation of DNA protons due to the binding of the paramagnetic metal ion Mn2+ has been used to experimentally determine the locations of the binding sites. The NMR results and the predictions based on the electrostatic potentials both place the binding sites of the manganese in the narrow grooves of these quadruplex DNAs. The predicted locations are spatially close to those experimentally observed, and the predicted and experimental locations also have similar electrostatic potential energy. These results have allowed a validation of the predictions of electrostatic potentials from structure. The 15mer quadruplex has two strong Mn2+ binding sites with one in each narrow groove. Both Mn2+ are released when the 15mer is complexed with thrombin, indicating that both narrow grooves are involved in the 15mer-thrombin interactions. The dimer quadruplex has a different structural motif than the 15mer and the presence of thrombin does not appreciably affect its interactions with Mn2+.

Base Sequence↗
PubMed1998

Solution structure of a Na cation stabilized DNA quadruplex containing G.G.G.G and G.C.G.C tetrads formed by G-G-G-C repeats observed in adeno-associated viral DNA.

We have applied NMR and molecular dynamics computations including intensity based refinement to define the structure of the d(G-G-G-C-T4-G-G-G-C) dodecanucleotide in 100 mM NaCl solution. The G-G-G-C sequence is of interest since it has been found as tandem repeats in the DNA sequence of human chromosome 19. The same G-G-G-C sequence is also seen as islands in adeno-associated virus, a human parvovirus, which is unique amongst eukaryotic DNA viruses in its ability to integrate site-specifically into a defined region of human chromosome 19. The d(G-G-G-C-T4-G-G-G-C) sequence forms a quadruplex in Na cation containing solution through head-to-tail dimerization of two symmetry-related stem-hairpin loops with adjacent strands antiparallel to each other around the quadruplex. The connecting T4 loops are of the lateral type, resulting in a quadruplex structure containing two internal G.G.G.G tetrads flanked by G.C.G.C tetrads. The G(anti).G(syn).G(anti).G(syn) tetrads are formed through dimerization associated hydrogen bonding alignments of a pair of Hoogsteen G(anti).G(syn) mismatch pairs, while the G(anti).C(anti).G(anti).C(anti) tetrads are formed through dimerization associated bifurcated hydrogen bonding alignments involving the major groove edges of a pair of Watson-Crick G.C base-pairs. The quadruplex contains two distinct narrow and two symmetric wide grooves with extensive stacking between adjacent tetrad planes. The structure of the quadruplex contains internal cavities that can potentially accommodate Na cations positioned between adjacent tetrad planes. Three such Na cations have been modeled into the structure of the d(G-G-G-C-T4-G-G-G-C) quadruplex. Finally, we speculate on the potential role of quadruplex formation involving G.G.G.G and G.C.G.C tetrads during the integration of the adeno-associated parvovirus into its target on human chromosome 19, both of which involve stretches of G-G-G-C sequence elements.

Chromosomes, Human, Pair 19↗