Cation-dependent transition between the quadruplex and Watson-Crick hairpin forms of d(CGCG3GCG).

The DNA oligonucleotide d(CGCG3GCG) can form either a Watson-Crick (WC) hairpin or a parallel-stranded quadruplex structure containing six G-quartet base pair assemblies. The exchange between these forms and single strands can be monitored using circular dichroism (CD). NMR results verified the assignment of specific CD bands to quadruplex and hairpin species, respectively. Cations stabilize the quadruplex in the order K+ greater than Ca2+ greater than Na+ greater than Mg2+ greater than Li+ and K+ greater than Rb+ greater than Cs+, indicating that K+ has an optimum ionic radius for complex formation and that ionic charge affects the extent of ion-induced stabilization. The quadruplex is stable in the presence of 40 mM K+ at micromolar DNA concentration and can be kinetically trapped as a metastable form when prepared at millimolar DNA concentration and then diluted into buffer containing 40 mM Na+. The concentration of K+ required to reverse the equilibrium from the hairpin to the quadruplex decreases sharply with increased DNA concentration. The quadruplex has an unusual pKa of ca. 6.8, indicating that C.C+ base pairs are probably forming. This system provides insights into some of the detailed structural characteristics of a ["G4-DNA".ion] complex and an experimental model for the recently proposed "sodium-potassium conformational switch" [Sen, D., & Gilbert, W. (1988) Nature 334, 364-366; Sen, D., & Gilbert, W. (1990) Nature 344, 410-414]. These results may help to explain the lack of cytidine residues in G-rich telomeric DNAs and suggest that methylation of GC-rich duplex DNAs in "GpC islands" may induce quadruplex formation within heterochromatin domains, resulting in reversible chromosomal condensation.     

Synthesis and characterization of DNA quadruplexes containing T-tetrads formed by bunch-oligonucleotides

The solid phase syntheses of the bunch oligonucleotides and based on the sequences of the natural oligodeoxynucleotides (ODNs) d(TG2TG2C) and d(CG2TG2T), respectively, attached to a non-nucleotidic tetrabranched linker, are reported. Bunch-ODNs and were shown to form more stable monomolecular parallel G-quadruplexes and when compared with their tetramolecular counterparts [d(TG2TG2C)]4 and [d(CG2TG2T)]4, respectively. The structure and stability of all the synthesized complexes have been investigated by circular dichroism (CD), CD thermal denaturation experiments, and 1H-NMR (nuclear magnetic resonance) experiments at variable temperatures. Particularly, the spectroscopic data confirmed that 1 adopts a T-tetrad containing parallel-stranded quadruplex structure as in the tetramolecular complex.     

Luminescence study of G-quadruplex formation in the presence of Tb3+ ion

The interactions of Tb3+ with the quadruplex-forming oligonucleotide bearing human telomeric repeat sequence d(G(3)T(2)AG(3)T(2)AG(3)T(2)AG(3)), (htel21), have been studied using luminescence spectroscopy and circular dichroism (CD). Enhanced luminescence of Tb3+, resulting from energy transfer from guanines, indicated encapsulation of Tb3+ ion in the central cavity of quadruplex core. The ability of lanthanide ions (Eu3+ and Tb3+) to mediate formation of quadruplex structure has been further evidenced by the fluorescence energy transfer measurements with the use of oligonucleotide probe labeled with fluorescein and rhodamine FRET partners, FAM-htel21-TAMRA. The CD spectra revealed that Tb3+/htel21 quadruplex possesses antiparallel strand orientation, similarly as sodium quadruplex. Tb3+ binding equilibria have been investigated in the absence and the presence of competing metal cations. At low Tb3+ concentration (8 microM) Tb3+/htel21 quadruplex stability is very high (5 x 10(6) M(-1)) and stoichiometry of 5-7 Tb3+ ions per one quadruplex molecule is observed. Luminescence and CD titration experiments suggested that the cavity of quadruplex accommodates two Tb3+ ions and the remaining Tb3+ ions bind probably to TTA loops of quadruplex. Higher concentration of Tb3+ (above 10 microM) results in the excessive binding of Tb3+ ions that finally destabilizes quadruplex, which undergoes transformation into differently organized assemblies. Such assemblies (probably possessing multiple positive charge) exhibit kinetic stability, which is manifested by a very slow kinetics of displacement of Tb3+ ion by competing cations (Li+, Na+, K+).     

A nanosecond molecular dynamics study of antiparallel d(G)7 quadruplex structures: effect of the coordinated cations

Nanosecond scale molecular dynamics simulations have been performed on antiparallel Greek key type d(G7) quadruplex structures with different coordinated ions, namely Na+ and K+ ion, water and Na+ counter ions, using the AMBER force field and Particle Mesh Ewald technique for electrostatic interactions. Antiparallel structures are stable during the simulation, with root mean square deviation values of approximately 1.5 A from the initial structures. Hydrogen bonding patterns within the G-tetrads depend on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate different cations. However, alternating syn-anti arrangement of bases along a chain as well as in a quartet is maintained through out the MD simulation. Coordinated Na+ ions, within the quadruplex cavity are quite mobile within the central channel and can even enter or exit from the quadruplex core, whereas coordinated K+ ions are quite immobile. MD studies at 400K indicate that K+ ion cannot come out from the quadruplex core without breaking the terminal G-tetrads. Smaller grooves in antiparallel structures are better binding sites for hydrated counter ions, while a string of hydrogen bonded water molecules are observed within both the small and large grooves. The hydration free energy for the K+ ion coordinated structure is more favourable than that for the Na+ ion coordinated antiparallel quadruplex structure.     

NMR structure refinement and dynamics of the K+-[d(G3T4G3)]2 quadruplex via particle mesh Ewald molecular dynamics simulations.

The solution structure and dynamical properties of the potassium-stabilized, hairpin dimer quadruplex formed by the oligonucleotide d(G3T4G3) have been elucidated by a combination of high-resolution NMR and molecular dynamics simulations. Refinement calculations were carried out both in vacuo, without internally coordinated K+ cations, and in explicit water, with internally coordinated K+ cations. In the latter case, the electrostatic interactions were calculated using the particle mesh Ewald (PME) method. The NMR restraints indicate that the K+ quadruplex has a folding arrangement similar to that formed by the same oligonucleotide in the presence of sodium, but with significant local differences. Unlike the Na+ quadruplex, the thymine loops found in K+ exhibit considerable flexibility, and appear to interconvert between two preferred conformations. Furthermore, the NMR evidence points toward K+-stabilized guanine quartets of slightly larger diameter relative to the Na+-stabilized structure. The characteristics of the quartet stem are greatly affected by the modeling technique employed: caged cations alter the size and symmetry of the quartets, and explicit water molecules form hydration spines within the grooves. These results provide insight into those factors that determine the overall stability of hairpin dimer quadruplexes and the effects of different cations in modulating the relative stability of the dimeric hairpin and linear, four-stranded, quadruplex forms.     

Characterization of an unusual folding pattern in a catalytically active guanine quadruplex structure

In the presence of certain metal ions, DNA and RNA can form guanine quadruplex structures, which have been proposed to play a functional role in a variety of biological processes. An 18-nucleotide DNA oligomer, PS2.M, d(GTG3TAG3CG3T2G2), was previously reported to bind hemin and the resulting complex exhibited peroxidase activity. It was proposed that PS2.M folds unimolecularly into an antiparallel quadruplex with unusual, single-base loops and terminal guanines positioned in adjacent quartets. Here we describe structural and stability properties of PS2.M alone in different buffers and metal ions, using gel electrophoresis, circular dichroism (CD), ultraviolet (UV)-visible spectroscopies, and one-dimensional 1H nuclear magnetic resonance (NMR). Native gel behavior of PS2.M in the presence of either Na+ or Pb2+ suggests the formation of unimolecular structures but, in the presence of K+, both unimolecular and multistranded structures are observed. In the presence of Pb2+ ions, PS2.M forms a unimolecular quadruplex containing three guanine quartets. CD titrations reveal that binding of Pb2+ ions to PS2.M is stoichiometric, and a single lead cation suffices to fully fold PS2.M. The PS2.M-Na+ system also forms a similar unimolecular quadruplex. In the presence of K+, the PS2.M-K+ system forms mixed species. With increasing time and PS2.M concentration, the contribution of unimolecular species decreases while that of multimolecular species increases, and this behavior is independent of buffer media. These results suggest that the catalytically active form, studied in the presence of K+, may be a parallel, multistranded quadruplex rather than an antiparallel, unimolecular quadruplex.     

Chemical ligation of oligodeoxynucleotides by X-irradiation and its application to regulation of G-quadruplex formation

We demonstrated radiolytic ligation of oligodeoxynucleotides (ODNs) possessing disulfide bond and its application to regulation of DNA quadruplex formation. G-rich hexamer ODNs had poor ability to form quadruplex, while X-irradiation of the ODNs induced interstrand exchange reaction at disulfide bond to form ligated 12 mer ODNs, leading to the ready formation of quadruplex due to the entropic effect. Since complexation of the ligated ODNs with hemin in the presence of K⁺ showed strong soret band absorption and also catalyzed the H₂O₂-mediated oxidation of luminol, it appears that the quadruplex formed from ligated ODNs showed a function similar to native DNA quadruplex.     

Effects of the introduction of inversion of polarity sites in the quadruplex forming oligonucleotide TGGGT

Insight into the influence of inversion of polarity sites on the structural features of quadruplex structures is presented. The NMR and CD studies concern modified oligodeoxynucleotides (ODNs) based on the quadruplex forming sequence TGGGT. The presence of inversion of polarity sites not only does not compromise the formation of quadruplexes, but in some cases it increases the thermal stability of modified complexes compared with that of the unmodified one.     

Effect of coordinated ions on structure and flexiblity of parallel G-quandruplexes: a molecular dynamics study

Single tract guanine residues can associate to form stable parallel quadruplex structures in the presence of certain cations. Nanosecond scale molecular dynamics simulations have been performed on fully solvated fibre model of parallel d(G7) quadruplex structures with Na+ or K+ ions coordinated in the cavity formed by the 06 atoms of the guanine bases. The AMBER 4.1 force field and Particle Mesh Ewald technique for electrostatic interactions have been used in all simulations. These quadruplex structures are stable during the simulation, with the middle four base tetrads showing root mean square deviation values between 0.5 to 0.8 A from the initial structure as well the high resolution crystal structure. Even in the absence of any coordinated ion in the initial structure, the G-quadruplex structure remains intact throughout the simulation. During the 1.1 ns MD simulation, one Na+ counter ion from the solvent as well as several water molecules enter the central cavity to occupy the empty coordination sites within the parallel quadruplex and help stabilize the structure. Hydrogen bonding pattern depends on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate cations of different sizes. In the absence of any coordinated ion, due to strong mutual repulsion, 06 atoms within G-tetrad are forced farther apart from each other, which leads to a considerably different hydrogen bonding scheme within the G-tetrads and very favourable interaction energy between the guanine bases constituting a G-tetrad. However, a coordinated ion between G-tetrads provides extra stacking energy for the G-tetrads and makes the quadruplex structure more rigid. Na+ ions, within the quadruplex cavity, are more mobile than coordinated K+ ions. A number of hydrogen bonded water molecules are observed within the grooves of all quadruplex structures.     

A comparison of the properties and the solution structure for RNA and DNA quadruplexes which contain two GGAGG sequences joined with a tetranucleotide linker

We have determined solution structure of r(GGAGGUUUUGGAGG) (R14) by NMR; the RNA 14-mer forms an intra-strand parallel quadruplex with a G-tetrad and a hexad, in which a G-tetrad core is augmented by association of two A residues. The quadruplex further forms a dimer through stacking interaction between the hexads. In order to obtain insight into the difference between RNA and DNA quadruplexes, we synthesized the corresponding DNA 14-mer, d(GGAGGTTTTGGAGG) (D14), and examined its properties and structure by CD, gel electrophoresis, and NMR. K+ ions increased the thermal stability of both R14 and D14 structures. The binding affinity of K+ ions to R14 was much higher than that to D14. The CD and gel electrophoretic studies suggest that D14 forms a quadruplex entirely different from that of R14 in the presence of K+ ions; two molecules of D14 form a quadruplex with both antiparallel and parallel strand alignments and with diagonal loops at both ends of the stacked G-tetrads. The NMR study also gave results that are consistent with such structure: alternate glycosidic conformation, 5'G(syn)-G(anti)3', and characteristic chemical shift data observed for many quadruplexes containing diagonal TTTT loops.     

Extraction of information on the buildup and consumption of reactive intermediates from quadruplex DNA assembly time courses

A method was developed to detect the time course of the overall presence of intermediate species during K+-induced DNA quadruplex assembly from single-stranded d(TG4) oligonucleotides in experiments in which only the combined circular dichroisms (CD) of all species present could be measured directly. The presence of intermediate species is determined unambiguously but quantitative estimates can be made only to the extent that the CD characteristics of all intermediates are known. The method consists of (i) obtaining CD spectra of known concentrations of initial and final species to determine their molar ellipticity coefficients, (ii) carrying out CD measurements of the kinetics of quadruplex assembly reactions at two different wavelengths, chosen to give optimal differentiation between the initial and final species, and (iii) using the results of (ii) to detect discrepancies between the rates of consumption of single strands and the generation of quadruplex to infer the presence of intermediate species. The analysis was facilitated by the validation and use of biphasic exponential expressions obtained from the SAS nonlinear curve fitting procedure NLIN in place of the raw CD data. The general method is described, then applied to data from [d(TG4)4.(K+)3] quadruplex assembly experiments.     

Efficient procedure of preparation and properties of long uniform G4-DNA nanowires

Here we describe a novel and efficient procedure for preparation of long uniform G4-DNA wires. The procedure includes (i) enzymatic synthesis of double-stranded DNA molecules consisting of long (up to 10,000 bases), continuous G strands and chains of complementary (dC)20-oligonucleotides, poly(dG)-n(dC)20; (ii) size exclusion HPLC separation of the G strands from the (dC)20 oligonucleotides in 0.1M NaOH; and (iii) folding of the purified G strands into G4-DNA structures by lowering the pH to 7.0. We show by atomic force microscopy (AFM) that the preparation procedure yielded G4-DNA wires with a uniform morphology and a narrow length distribution. The correlation between the total amount of nucleotides in the G strands and the contour length of the G4-DNA molecules estimated by AFM suggests monomolecular folding of the G strands into quadruplex structures. The folding takes place either in the presence or in the absence of stabilizing ions (K+ or Na+). The addition of these cations leads to a dramatic change in the circular dichroism spectrum of the G4-DNA.     

The insertion of two 8-methyl-2'-deoxyguanosine residues in tetramolecular quadruplex structures: trying to orientate the strands

In this article, we report a structural study, based on NMR and CD spectroscopies, and molecular modelling of all possible d(TG(3)T) and d(TG(4)T) analogues containing two 8-methyl-2'-deoxyguanosine residues (M). Particularly, the potential ability of these modified residues to orientate the strands and then to affect the folding topology of tetramolecular quadruplex structures has been investigated. Oligodeoxynucleotides (ODNs) TMMGT (T12) and TMMGGT (F12) form parallel tetramolecular quadruplexes, characterized by an all-syn M-tetrad at the 5'-side stacked to all-anti M- and G-tetrads. ODNs TMGMT (T13) and TMGGMT (F14) form parallel tetramolecular quadruplexes, in which an all-anti G core is sandwiched between two all-syn M-tetrads at the 5'- and the 3'-side. Notably, the quadruplex formed by T13 corresponds to an unprecedented structure in which the syn residues exceed in number the anti ones. Conversely, ODN TGMGMT (F24) adopts a parallel arrangement in which all-anti G-tetrads alternate with all-syn M-tetrads. Most importantly, all data strongly suggest that ODN TMGMGT (F13) forms an unprecedented anti-parallel tetramolecular quadruplex in which G and M residues adopt anti and syn glycosidic conformations, respectively. This article opens up new understandings and perspectives about the intricate relationship between the quadruplex strands orientation and the glycosidic conformation of the residues.     

Quadruplex to Watson-Crick duplex transition of the thrombin binding aptamer: a fluorescence resonance energy transfer study

Thermodynamic parameters of closing up of guanine-rich thrombin binding element, upon binding to K(+) and Na(+) ions to form quadruplexes and opening up of these quadruplexes upon binding to its complementary strand, were investigated. For this purpose, 15mer deoxynucleotide, d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), labeled with 5'-fluorescein and 3'-tetramethylrhodamine was taken and fluorescence resonance energy transfer was monitored as a function of either metal ions or complementary strand concentrations. Equilibrium association constant obtained from FRET studies demonstrates that K(+) ions bind with higher affinity than the Na(+) ions. The enthalpy changes, DeltaH, obtained from temperature dependence of equilibrium association constant studies revealed that formation of quadruplex upon binding of metal ions is primarily enthalpy driven. Binding studies of complementary strand to the quadruplex suggest that opening of a quadruplex in NaCl buffer in presence of the complementary strand is enthalpic as well as entropic driven and can occur easily, whereas opening of the same quadruplex in KCl buffer suffers from enthalpic barrier. Comparison of overall thermodynamic parameters along with kinetics studies indicates that, although quadruplexes cannot efficiently compete with duplex formation at physiological pH, they delay the association of two strands.     

Possibility of an antiparallel (tetramer) quadruplex exhibited by the double repeat of the human telomere

Under physiological concentrations of Na+ and K+, human telomeric DNA can self-associate into G-quadruplexes. On the basis of circular dichroism, gel electrophoresis, gel filtration, and UV-melting experiments, we report here that the double repeat of human telomere (d-TTAGGGTTAGGG; HUM2) forms parallel as well as antiparallel quadruplexes in the presence of K+, whereas Na+ facilitates only the antiparallel form. Here, the gel techniques and CD studies have proved to be complementary in detecting the molecularity and pattern of strand orientation. By correlating the gel and CD experiments, the antiparallel G-quadruplex was identified as a tetrameric species, whereas the parallel G-quadruplex was found to be dimeric. Both structural species were separated through gel filtration, which when run on native polyacrylamide gel electrphoresis (PAGE), confirmed their molecularity. UV-melting profiles also confirm the presence of two biphasic and one monophasic structural species in the presence of K+ and Na+, respectively. Though our observation is consistent with the recent NMR report (Phan, A. T., and Patel, D. J. (2003) J. Am. Chem. Soc. 125, 15021-15027), it seems to differ in terms of the molecularity of the antiparallel quadruplex. A model is proposed for an antiparallel tetrameric quadruplex, showing the possibility of Watson-Crick hydrogen bonds between intervening bases on antiparallel strands. This article expands the known structural motifs of DNA quadruplexes. To the best of our knowledge, four-stranded antiparallel quadruplexes have not been characterized to date. On the basis of the model, we hypothesize a possible mechanism for telomere-telomere association involving their G-overhangs, during certain stages of the cell cycle. The knowledge of peculiar geometries of the G-quadruplexes may also have implications for its specific recognition by ligands.     

Effects of catecholamines injected into the septal area of the rat brain on natriuresis, kaliuresis and diuresis.

Urinary output of Na+ and K+, and volume of urine have been studied in conscious, unrestrained, water-loaded male rats following the intraseptal injection of catecholamines. Natriuresis and kaliuresis increased after injecting noradrenaline (NA), the intensity being dose related. The dose-response curve suggests that a monomolecular interacting takes place between NA and pharmacological receptors present in the septal area. No change was observed in diuresis. Systematic mapping of the septal area yielded about the same results for all sites except a zone located in the lateral nucleus that was more sensitive. An alpha blocker (dibenamine), injected intraseptally before NA, showed an inhibitory effect while a beta blocker (propranolol) yielded a potentiation effect. These same effects of the blocking agents were observed when adrenaline was used instead of NA. Lidocaine, which inhibits the re-uptake of NA, showed an enhancement of the natriuretic and kaliuretic effect of NA, and the same effect was observed when the enzymatic destruction of NA was prevented by nialamide, an inhibitor of monoaminoxidase. Dopamine showed a natriuretic effect, but no effect was observed on K+ and urine output. Serotonin had no action on natriuresis, kaliuresis and diuresis.     

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.     

8-oxoguanine in a quadruplex of the human telomere DNA sequence

8-Oxoguanine is a ubiquitous oxidative base lesion. We report here on the effect of this lesion on the structure and stability of quadruplexes formed by the human telomeric DNA sequence 5'-dG(3)(TTAG(3))(3) in NaCl and KCl. CD, PAGE and absorption-based thermodynamic stability data showed that replacement of any of the tetrad-forming guanines by 8-oxoguanine did not hinder the formation of monomolecular, antiparallel quadruplexes in NaCl. The modified quadruplexes were, however, destabilized in both salts, the extent of this depending on the position of the lesion. These results and the results of previous studies on guanine-to-adenine exchanges and guanine abasic lesions in the same quadruplex show a noticeable trend: it is not the type of the lesion but the position of the modification that determines the effect on the conformation and stability of the quadruplex. The type of lesion only governs the extent of changes, such as of destabilization. Most sensitive sites were found in the middle tetrad of the three-tetrad quadruplex, and the smallest alterations were observed if guanines of the terminal tetrad with the diagonal TTA loop were substituted, although even these substitutions brought about unfavorable enthalpic changes. Interestingly, the majority of these base-modified quadruplexes did not adopt the rearranged folding induced in the unmodified dG(3)(TTAG(3))(3) by potassium ions, an observation that could imply biological relevance of the results.