Unusual monomolecular DNA quadruplex structures using bunch-oligonucleotides

The chemical synthesis of several G-rich bunch-oligonucleotides and the structural characterization of the corresponding monomolecular G-quadruplexes (I-IV) have been reported. The synthetic method allow the achievement of monomolecular DNA quadruplex structures having unusual and predeterminable oligodeoxyribonucleotide (ODN) strand orientation.     

Effects of a 8-oxoadenosine incorporation on quadruplex structures: thermal stabilities and structural studies

The effects of incorporation of 8-oxoadenosine in two different truncations of human telomeric sequence forming quadruplex structures are reported. In order to characterise their structures, a combination of NMR and UV spectroscopy and computational techniques were used. Both oligonucleotides have been found to form fourfold symmetric quadruplex structures. As a tautomeric equilibrium between keto and enol forms of 8-oxoadenosine may establish in solution and intrinsic stabilities effects, such as internal H-bonds, for example, may determine the predominance of some particular tautomer, molecular modelling studies were performed on quadruplex structures containing both the tautomeric forms. Both molecules resulted to be thermally less stable than the natural.     

Interaction of an acridine dimer with DNA quadruplex structures.

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 an intramolecular G-quadruplex structure in vitro, which has been shown to inhibit telomerase activity. The C-rich sequence can also adopt a quadruplex (intercalated) structure (i-DNA). Two acridine derivatives were shown to increase the melting temperature of the G- quadruplex and the C-quadruplex at 1 microM dye concentration. The increase in Tm value of the G-quadruplex was associated with telomerase inhibition in vitro. The most active compound, "BisA", showed an IC(50) value of 0.75 microM in a standard TRAP assay.     

8-methyl-2'-deoxyguanosine incorporation into parallel DNA quadruplex structures

This paper concerns the Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) structural studies of the quadruple helix arrangements adopted by three tailored oligodeoxyribonucleotide analogues, namely d(TG^MeGGT), d(TGG^MeGT) and d(TGGG^MeT), where dG^Me represents a 8-methyl-2′-deoxyguanosine residue. The results of this study clearly demonstrate that the effects of the incorporation of dG^Me instead of a dG residue are strongly dependant upon the positioning of a single base replacement along the sequence. As such, d(TG^MeGGT), d(TGG^MeGT) have been found to form 4-fold symmetric quadruplexes with all strands parallel and equivalent to each other, each more stable than their natural counterpart. NMR experiments clearly indicate that [d(TG^MeGGT)]₄ possesses a G^Me-tetrad with all dG^Me residues in a syn-glycosidic conformation while an anti-arrangement is apparent for the four dG^Me of [d(TGG^MeGT)]₄. As the two complexes show a quite different CD behaviour, a possible relationship between the presence of residues adopting syn-glycosidic conformations and CD profiles is briefly discussed. As far as d(TGGG^MeT) is concerned, NMR data indicate that at 25°C it exists primarily as a single-strand conformation in equilibrium with minor amounts of a quadruplex structure.     

FANCJ promotes DNA synthesis through G‐quadruplex structures

Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ^−/− cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes.     

Predictive modelling of topology and loop variations in dimeric DNA quadruplex structures

We have used a combination of simulated annealing (SA), molecular dynamics (MD) and locally enhanced sampling (LES) methods in order to predict the favourable topologies and loop conformations of dimeric DNA quadruplexes with T2 or T3 loops. This follows on from our previous MD simulation studies on the influence of loop lengths on the topology of intramolecular quadruplex structures [P. Hazel et al. (2004) J. Am. Chem. Soc., 126, 16405–16415], which provided results consistent with biophysical data. The recent crystal structures of d(G4T3G4)2 and d(G4BrUT2G4) (P. Hazel et al. (2006) J. Am. Chem. Soc., in press) and the NMR-determined topology of d(TG4T2G4T)2 [A.T. Phan et al. (2004) J. Mol. Biol., 338, 93–102] have been used in the present study for comparison with simulation results. These together with MM-PBSA free-energy calculations indicate that lateral T3 loops are favoured over diagonal loops, in accordance with the experimental structures; however, distinct loop conformations have been predicted to be favoured compared to those found experimentally. Several lateral and diagonal loop conformations have been found to be similar in energy. The simulations suggest an explanation for the distinct patterns of observed dimer topology for sequences with T3 and T2 loops, which depend on the loop lengths, rather than only on G-quartet stability.     

Research progress of RNA quadruplex

RNA/DNA sequences rich in guanine (G) can form a 4-strand structure, G-quadruplex, which has been extensively researched and observed in mammalian, fungi, and plants, with in vivo existence in eukaryotic cells. Compared with DNA quadruplex, the potential existence of RNA quadruplex appears to be generally rare; however, it is believed by some researchers to be more inevitable in vivo and speculated to play an important role where it exists. Recently, researches concerning the function of G-quadruplexes in RNAs commence, making much progress. However, there is no available review particularly focusing on RNA quadruplex till now as we know. Therefore, we decide to give a review to comprehensively summarize research progress on it. This review highlights the diverse topologies for RNA quadruplex structure and its effect factors; outlines the current knowledge of RNA quadruplex's physiological functions in biological systems, especially in gene expression; and presents the prospects of RNA quadruplex.     

Effect of 1-methyladenine on thermodynamic stabilities of double-helical DNA structures

1-Methyladenine (m1A) alters T·A Watson-Crick to T·m1A Hoogsteen base pair. Owing to its conversion to N6-methyladenine (m6A) at higher temperatures, thermodynamic studies of m1A-containing DNAs using conventional melting methods are subject to the influence of m6A species. In this study, we applied nuclear magnetic resonance spectroscopy to determine the base pairing modes and effect of m1A on thermodynamic stability of double-helical DNA. The observed base pairing modes account for the destabilizing trend which follows the order T·m1A ∼ G·m1A < A·m1A < C·m1A, providing insights into the m1A flipping process and enhancing our understandings of the mutagenicity of m1A.     

PNA-DNA chimeras forming quadruplex structures

1H-NMR, CD, and UV spectroscopy have been used to investigate the structure of PNA/DNA chimeras forming quadruplex structures. In particular, we synthesized 5'TGGG3'-t (1) and 5'TGG3'-gt (2), where lower and upper case letters indicate PNA and DNA residues, respectively. CD spectrum and all NMR data of (1) are typical of quadruplexes involving four parallel strands. UV melting profile of (1) indicates that its thermal stability is quite similar to that observed for the reference structure [d(TGGGT)]4. 1H-NMR spectrum for 5'TGG3'-gt (2) shows that this oligonucleotide is not able to fold into a single, well-defined species.     

Polymorphism of human telomeric quadruplex structures

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). Compounds that can stabilize the intramolecular DNA G-quadruplexes formed in the human telomeric sequence have been shown to inhibit the activity of telomerase and telomere maintenance, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. Knowledge of intramolecular human telomeric G-quadruplex structure(s) formed under physiological conditions is important for structure-based rational drug design and thus has been the subject of intense investigation. This review will give an overview of recent progress on the intramolecular human telomeric G-quadruplex structures formed in K(+) solution. It will also give insight into the structure polymorphism of human telomeric sequences and its implications for drug targeting.     

Dual sensing of hairpin and quadruplex DNA structures using multicolored peptide nucleic acid fluorescent probes

Synthesis of water-soluble 5-mer peptide nucleic acids (PNAs) functionalized at their 5'- and 3'-ends with two original precursors of pentamethine cyanine dye synthesis is reported. The successful use of these PNA probes for sensing DNA hairpin structures in vitro was also demonstrated where specific hairpin formation was associated with the appearance of a characteristic fluorescence signal at 660 nm. A comparative study between three different strategies where PNAs were targeting either the stem or the loop of the hairpin was carried out. Best sensitivity was obtained using PNA sequences complementary to the loop sequence and directing both functional moieties toward the base of loop. Unprecedented proof-of-concept for the simultaneous sensing of hairpin and quadruplex DNAs with a nonoverlapping two-color system (C3 and C5) is also demonstrated.     

Relative stabilities of RNA/DNA hybrids: effect of RNA chain length in competitive hybrization

Escherichia coli DNA and fragmented rRNA were used as a model system to study the effect of RNA fragment size in hybridization-competition experiments. Though no difference in hybridization rates was observed, the relative stabilities of the RNA/DNA hybrids were found to be largely affected by the fragment size of the RNA molecule. Intact rRNA was shown to replace shorter homologous rRNA sequences in their hybrids, the rate of the displacement being dependent on the molecular size of the RNA fragments. Hybridization-competition experiments between molecules of different lengths are expected to be complicated by the displacement reaction. The synthesis of tRNA^Tyr-like sequences transcribed in vitro on φ80psu₃⁺ bacteriophage DNA was measured by hybridization competition assays. Indirect competition with labelled E. coli tRNA^Tyr hybridization revealed that the in vitro-synthesized RNA contained significant amounts of tRNA^Tyr; these sequences could not, however, be detected by the direct competition method in which labelled in vitro-synthesized RNA competes with E. coli tRNA^Tyr for hybridization to φ80psu₃⁺ DNA. These contradictory results can be traced to the differences in size of the competing molecules in the hybridization-competition reaction. Indeed, in vitro-transcribed tRNA^Tyr-like sequences, longer than mature tRNA, were found to displace efficiently E. coli tRNA^Tyr from its hybrids with φ80psu₃⁺ DNA. These findings explain why such sequences could not be detected by direct competition with E. coli tRNA^Tyr.     

Cross-links of quadruplex structures from human telomeric DNA by dinuclear platinum complexes show the flexibility of both structures

The folding of AG(3)(T(2)AG(3))(3) was investigated in the presence of Na(+) or K(+) ions, by using the dinuclear platinum complexes [{trans-PtCl(NH(3))(2)}(2)H(2)N(CH(2))(n)NH(2)]Cl(2) (n = 2 or 6). AG(3)(T(2)AG(3))(3) has been previously found to adopt two different quadruplex structures: the antiparallel one in a solution containing Na(+) and the parallel one in a K(+)-containing crystal. The two structures are strikingly distinct and are not expected to form the same platinum cross-links. Therefore, characterization of the cross-links formed with platinum complexes in solution allowed the predominant conformation(s) to be identified. The bases coordinating the platinum atoms were identified by chemical and 3'-exonuclease digestions. The observed cross-links showed that the parallel structure exists in solution whatever the cation and confirmed the existence of the antiparallel structure in the presence of both cations as previously reported from cross-linking experiments of AG(3)(T(2)AG(3))(3) by mononuclear platinum complexes. Furthermore, the major platinum cross-links were unexpectedly formed between two guanines belonging to the same G-quartet. Their formation was rationalized using molecular dynamics simulations in implicit solvent of the two quadruplex structures. It was shown that they were flexible, allowing some guanines to leave reversibly the top G-quartet and thus rendering their N(7) atom accessible to platinum complexes. Our results also suggest that the human telomere sequence could be a target for such platinum complexes.     

Human telomere quadruplex: refolding and selection of individual conformers via RNA/DNA chimeric editing

The conformation of the guanine quadruplex formed by the human telomere (HT) repeat in solutions containing physiological concentrations of K+ ions has been a topic of intensive investigation during the past several years. Of particular interest are the directionality of the overall folding pattern, i.e., parallel, antiparallel, or a combination of these two modes, and the alternation, if any, of the glycosidic bond conformation between syn and anti. An additional issue involves resolving mixtures of conformations when more than one species is present. We approach these questions using selective substitution of riboguanosine, rG, for deoxyriboguanosine, dG. Using a combination of circular dichroism, gel electrophoresis, equilibrium ultracentrifugation, and imino proton NMR, we are able to show that these modifications can yield sequences which fold into parallel or antiparallel conformations consisting of one or two strands. We also demonstrate that chimeric editing of the HT sequence permits isolating one of two conformational isomers existing in solution in the presence of KCl. The ability to engineer and control quadruplex folding motifs illustrated here with HT may prove useful more generally for a variety of quadruplex-forming sequences.