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.     

Evidence from nitrogen-15 and solvent deuterium isotope effects on the chemical mechanism of adenosine deaminase

We have determined 15N isotope effects and solvent deuterium isotope effects for adenosine deaminase using both adenosine and the slow alternate substrate 7,8-dihydro-8-oxoadenosine. With adenosine, 15N isotope effects were 1.0040 in H2O and 1.0023 in D2O, and the solvent deuterium isotope effect was 0.77. With 7,8-dihydro-8-oxoadenosine, 15N isotope effects were 1.015 in H2O and 1.0131 in D2O, and the solvent deuterium isotope effect was 0.45. The inverse solvent deuterium isotope effect shows that the fractionation factor of a proton, which is originally less than 0.6, increases to near unity during formation of the tetrahedral intermediate from which ammonia is released. Proton inventories for 1/V and 1/(V/K) vs percent D2O are linear, indicating that a single proton has its fractionation factor altered during the reaction. We conclude that a sulfhydryl group on the enzyme donates its proton to oxygen or nitrogen during this step. pH profiles with 7,8-dihydro-8-oxoadenosine suggest that the pK of this sulfhydryl group is 8.45. The inhibition of adenosine deaminase by cadmium also shows a pK of approximately 9 from the pKi profile. Quantitative analysis of the isotope effects suggests an intrinsic 15N isotope effect for the release of ammonia from the tetrahedral intermediate of approximately 1.03 for both substrates; however, the partition ratio of this intermediate for release of ammonia as opposed to back-reaction is 14 times greater for adenosine (1.4) than for 7,8-dihydro-8-oxoadenosine (0.1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context

The telomeric DNA of vertebrates consists of d(TTAGGG)_n tandem repeats, which can form quadruplex DNA structures in vitro and likely in vivo. Despite the fact that the G-rich telomeric DNA is susceptible to oxidation, few biochemical studies of base excision repair in telomeric DNA and quadruplex structures have been done. Here, we show that telomeric DNA containing thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh), or spiroiminodihydantoin (Sp) can form quadruplex DNA structures in vitro. We have tested the base excision activities of five mammalian DNA glycosylases (NEIL1, NEIL2, mNeil3, NTH1, and OGG1) on these lesion-containing quadruplex substrates and found that only mNeil3 had excision activity on Tg in quadruplex DNA and that the glycosylase exhibited a strong preference for Tg in the telomeric sequence context. Although Sp and Gh in quadruplex DNA were good substrates for mNeil3 and NEIL1, none of the glycosylases had activity on quadruplex DNA containing 8-oxoG. In addition, NEIL1 but not mNeil3 showed enhanced glycosylase activity on Gh in the telomeric sequence context. These data suggest that one role for Neil3 and NEIL1 is to repair DNA base damages in telomeres in vivo and that Neil3 and Neil1 may function in quadruplex-mediated cellular events, such as gene regulation via removal of damaged bases from quadruplex DNA.     

Flexibility and structural conservation in a c-KIT G-quadruplex

A quadruplex sequence from the promoter region of the c-KIT gene forms a stable quadruplex, as characterized by crystallographic and NMR methods. Two new crystal structures are reported here, together with molecular dynamics simulation studies on these quadruplex crystal structures and an NMR structure. The new crystal structures, each in a distinct space group and lattice packing arrangement, together with the existing structures, demonstrate that the c-KIT quadruplex fold does not change with differing environments, suggesting that quadruplex topological dynamism is not a general phenomenon. The single and dinucleotide loops in these structures show a high degree of conformational flexibility within the three crystal forms and the NMR ensemble, with no evidence of clustering to particular conformers. This is in accord with the findings of high loop flexibility from the molecular dynamics studies. It is suggested that intramolecular quadruplexes can be grouped into two broad classes (i) those with at least one single-nucleotide loop, often showing singular topologies even though loops are highly flexible, and (ii) with all loops comprising at least two nucleotides, leading to topological dynamism. The loops can have more stable and less dynamic base-stacked secondary structures.     

Biophysical properties of quadruple helices of modified human telomeric DNA

Telomeric DNA of a variety of vertebrates including humans contains the tandem repeat d(TTAGGG)n. The guanine rich strand can fold into four-stranded G-quadruplex structures, which have recently become attractive for biomedical research. Indeed, the aptamers based on the quadruplex motif may prove useful as tools aimed at binding and inhibiting particular proteins, catalyzing various biochemical reactions, or even serving as pharmaceutically active agents. The incorporation of modified bases into oligonucleotides can have profound effects on their folding and may produce useful changes in physical and biological properties of the resulting DNA fragments. In this work, the adenines of the human telomeric repeat oligonucleotide d(TAGGGT) and d(AGGGT) were substituted by 2'-deoxy-8-(propyn-1-yl)adenosine (A-->APr) or by 8-bromodeoxyadenosine (A-->ABr). The biophysical properties of the resulting quadruplex structures were compared with the unmodified quadruplexes. NMR and CD spectra of the studied sequences were characteristic of parallel-stranded, tetramolecular quadruplexes. The analysis of the equilibrium melting curves reveals that the modifications stabilize the quadruplex structure. The results are useful when considering the design of novel aptameric nucleic acids with diverse molecular recognition capabilities that would not be present using native RNA/DNA sequences.     

Distinctive features in the structure and dynamics of the DNA repeat sequence GGCGGG

G-rich DNA has been known to form a variety of folded and multistranded structures, with even single base modifications causing important structural changes. But, very little is known about the dynamic characteristics of the structures, which may play crucial roles in facilitating the structural transitions. In this background, we report here NMR investigations on the structure and dynamics of a DNA repeat sequence GGCGGG in aqueous solution containing Na+ ions at neutral pH. The chosen sequence d-TGGCGGGT forms a parallel quadruplex with a C-tetrad in the middle, formed by symmetrical pairing of four Cs in a plane via NH2-O2 H-bonds. 13C relaxation measurements at natural abundance for C' sugar carbons provided valuable insight into the sequence specific dynamism of G and C-tetrads in the quadruplex. The C4 tetrad seems to introduce high conformational dynamism at milli- to micro-second time scale in the quadruplex. Concomitantly, there is a decrease in the pico-second time scale dynamics. Interestingly, these effects are seen more prominently at the G-tetrads on the 3' end of C-tetrad than on its 5' end. These observations would have important implications for the roles the tetrads may play in many biological functions.     

Small-Molecule Selectively Recognizes Human Telomeric G-Quadruplex DNA and Regulates Its Conformational Switch

Structural complexity is an inherent feature of the human telomeric sequence, and it presents a major challenge for developing ligands of pharmaceutical interest. Recent studies have pointed out that the induction of a quadruplex or change of a quadruplex conformation on binding may be the most powerful method to exert the desired biological effect. In this study, we demonstrate a quadruplex ligand that binds selectively to different forms of the human telomeric G-quadruplex structure and regulates its conformational switch. The results show that not only can oxazine750 selectively induce parallel quadruplex formation from a random coil telomeric oligonucleotide in the absence of added cations, it also can easily surpass the energy barrier between two structures and change the G-quadruplex conformation in Na⁺ or K⁺ solution. The combination of its unique properties, including the size and shape of the G-quadruplex and the small molecule, is proposed as the predominant force for regulating the special structural formation and transitions. These results may stimulate the design of new quadruplex binders that would be capable of discriminating different G-quadruplex structures as well as controlling biological phenomena, functional molecules, and nanomaterials.     

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.     

A further contribution to the extreme variability of quadruplex structures from oligodeoxyribonucleotides containing inversion of polarity sites in the G-tract

Structural insight into DNA quadruplex structures formed by oligodeoxyribonucleotides 3'TG5'-5'GGGT3' (QS55) and 5'TG3'-3'GGGT5' (QS33) is presented. NMR analysis reveals that QS33 forms a parallel-like four-fold symmetric quadruplex, while QS55 possesses a two-fold symmetry and is characterized by a tetrameric antiparallel quadruplex embedded between two parallel tracts. The results reported here describe unprecedented quadruplex complexes provided by peculiar structural features never reported to date. These structures might inspire the design of new aptameric nucleic acids characterized by novel structural motifs hardly realizable with unmodified DNA/RNA.     

Binding properties of human telomeric quadruplex multimers: a new route for drug design

Human telomeric G-quadruplex structures are known to be promising targets for an anticancer therapy. In the past decade, several research groups have been focused on the design of new ligands trying to optimize the interactions between these small molecules and the G-quadruplex motif. In most of these studies, the target structures were the single quadruplex units formed by short human DNA telomeric sequences (typically 21-26 nt). However, the 3′-terminal single-stranded human telomeric DNA is actually 100-200 bases long and can form higher-order structures by clustering several consecutive quadruplex units (multimers). Despite the increasing number of structural information on longer DNA telomeric sequences, very few data are available on the binding properties of these sequences compared with the shorter DNA telomeric sequences.In this paper we use a combination of spectroscopic (CD, UV and fluorescence) and calorimetric techniques (ITC) to compare the binding properties of the (TTAGGG)₈TT structure formed by two adjacent quadruplex units with the binding properties of the (AG₃TT)₄ single quadruplex structure. The three side-chained triazatruxene derivative azatrux and TMPyP4 cationic porphyrin were used as quadruplex ligands. We found that, depending on the drug, the number of binding sites per quadruplex unit available in the multimer structure was smaller or greater than the one expected on the basis of the results obtained from individual quadruplex binding studies. This work suggests that the quadruplex units along a multimer structure do not behave as completely independent. The presence of adjacent quadruplexes results in a diverse binding ability not predictable from single quadruplex binding studies. The existence of quadruplex-quadruplex interfaces in the full length telomeric overhang may provide an advantageous factor in drug design to enhance both affinity and selectivity for DNA telomeric quadruplexes.     

The effect of tautomeric constant on the specificity of nucleotide incorporation during DNA replication: support for the rare tautomer hypothesis of substitution mutagenesis

The nucleoside analogue dP (6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one) displays ambivalent hydrogen bonding characteristics whereby the imino tautomer of P can base-pair with adenine and its amino tautomer can base-pair with guanine. Fixed imino and amino tautomers of 6-methyl-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one (N-methyl P) have been synthesised and their structures obtained by X-ray crystallography. The tautomeric constant of N-methyl P has been calculated from pK(a) values of the fixed tautomers and the kinetic parameters for the incorporation of its 5'-triphosphate (dPTP) by exonuclease-free Klenow fragment of DNA polymerase I have been determined. A strong correlation between the tautomeric constant and the incorporation specificity of dPTP is found. These results lend support to the proposal that the minor tautomeric forms of the natural bases may play an important role in substitution mutagenesis during DNA replication. Furthermore, they imply that DNA polymerases impose specific steric requirements on the base-pair during nucleotide incorporation.     

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

The paramagnetic metal ion Mn2+ has been used to probe the electrostatic potentials of a DNA quadruplex that has two quartets with an overall fold of the chair type. A quadruplex with a basket type structure has also been examined. The binding of the paramagnetic ion manganese to these quadruplex DNAs has been investigated by solution state electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopies. The EPR results indicate that the DNA aptamer, d(GGTTGGTGTGGTTGG), binds two manganese ions and that the binding constants for each of these sites is approximately 10(5) M-1. The NMR results indicate that the binding sites of the manganese are in the narrow grooves of this quadruplex DNA. The binding sites of the DNA quadruplex formed by dimers of d(GGGGTTTTGGGG) which forms a basket structure are also in the narrow groove. These results indicate that the close approach of phosphates in the narrow minor grooves of the quadruplex structures provide strong binding sites for the manganese ions and that EPR and NMR monitoring of manganese binding can be used to distinguish between the different types of quadruplex structures.     

Arrangements of human telomere DNA quadruplex in physiologically relevant K+ solutions

The arrangement of the human telomeric quadruplex in physiologically relevant conditions has not yet been unambiguously determined. Our spectroscopic results suggest that the core quadruplex sequence G₃(TTAG₃)₃ forms an antiparallel quadruplex of the same basket type in solution containing either K⁺ or Na⁺ ions. Analogous sequences extended by flanking nucleotides form a mixture of the antiparallel and hybrid (3 + 1) quadruplexes in K⁺-containing solutions. We, however, show that long telomeric DNA behaves in the same way as the basic G₃(TTAG₃)₃ motif. Both G₃(TTAG₃)₃ and long telomeric DNA are also able to adopt the (3 + 1) quadruplex structure: Molecular crowding conditions, simulated here by ethanol, induced a slow transition of the K⁺-stabilized quadruplex into the hybrid quadruplex structure and then into a parallel quadruplex arrangement at increased temperatures. Most importantly, we demonstrate that the same transitions can be induced even in aqueous, K⁺-containing solution by increasing the DNA concentration. This is why distinct quadruplex structures were detected for AG₃(TTAG₃)₃ by X-ray, nuclear magnetic resonance and circular dichrosim spectroscopy: Depending on DNA concentration, the human telomeric DNA can adopt the antiparallel quadruplex, the (3 + 1) structure, or the parallel quadruplex in physiologically relevant concentrations of K⁺ ions.     

Biophysical and biological properties of quadruplex oligodeoxyribonucleotides

Single-stranded guanosine-rich oligodeoxyribonucleotides (GROs) have a propensity to form quadruplex structures that are stabilized by G-quartets. In addition to intense speculation about the role of G-quartet formation in vivo, there is considerable interest in the therapeutic potential of quadruplex oligonucleotides as aptamers or non-antisense antiproliferative agents. We previously have described several GROs that inhibit proliferation and induce apoptosis in cancer cell lines. The activity of these GROs was related to their ability to bind to a specific cellular protein (GRO-binding protein, which has been tentatively identified as nucleolin). In this report, we describe the physical properties and biological activity of a group of 12 quadruplex oligonucleotides whose structures have been characterized previously. This group includes the thrombin-binding aptamer, an anti-HIV oligonucleotide, and several quadruplexes derived from telomere sequences. Thermal denaturation and circular dichroism (CD) spectropolarimetry were utilized to investigate the stability, reversibility and ion dependence of G-quartet formation. The ability of each oligonucleotide to inhibit the proliferation of cancer cells and to compete for binding to the GRO-binding protein was also examined. Our results confirm that G-quartet formation is essential for biological activity of GROs and show that, in some cases, quadruplex structures formed in the presence of potassium ions are significantly more active than those formed in the presence of sodium ions. However, not all quadruplex structures exhibit antiproliferative effects, and the most accurate factor in predicting biological activity was the ability to bind to the GRO-binding protein. Our data also indicate that the CD spectra of quadruplex oligonucleotides may be more complex than previously thought.     

Circular dichroism of quadruplex DNAs: applications to structure, cation effects and ligand binding

Circular dichroism, CD, spectra can be used to gain information about quadruplex structures of DNAs as well as the effects of sequence, cations, chemical modification and ligand binding on quadruplex structure. There is not yet a validated approach to calculate a CD spectrum from a quadruplex structure nor is their one to go from a CD spectrum to a structure. However, it is possible to empirically correlate CD spectra features with quadruplex structural type in many cases. In this article four case studies are presented to indicate the strengths and limitations of CD in investigations of the properties of quadruplex structures formed by telomere repeat sequences. The case studies include determination of the quadruplex structural type present as a function of potassium concentration, the effect of sequence on the equilibrium between quadruplex structural types as a function of potassium concentration, the effect of ligand binding on quadruplex structure and the effect of 5' phosphorylation on quadruplex structural type.     

Relative stability of G‐quadruplex structures: Interactions between the human Bcl2 promoter region and derivatives of carbazole and diphenylamine

The bcl2 promoter region forms a G‐quadruplex structure, which is a crucial target for anticancer drug development. In this study, we provide theoretical predictions of the stability of different G‐quadruplex folds of the 23‐mer bcl2 promoter region and G‐quadruplex ligand. We take into account the whole G‐quadruplex structure, including bound‐cations and solvent effects, in order to compute the ligand binding free energy using molecular dynamics simulation. Two series of the carbazole and diphenylamine derivatives are used to screen for the most potent drug in terms of stabilization. The energy analysis identifies the predominant energy components affecting the stability of the various different G‐quadruplex folds. The energy associated with the stability of the G‐quadruplex‐K⁺ structures obtained displays good correlation with experimental T_m measurements. We found that loop orientation has an intrinsic influence on G‐quadruplex stability and that the basket structure is the most stable. Furthermore, parallel loops are the most effective drug binding site. Our studies also demonstrate that rigidity and planarity are the key structural elements of a drug that stabilizes the G‐quadruplex structure. BMVC‐4 is the most potential G‐quadruplex ligand. This approach demonstrates significant promise and should benefit drug design. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1038–1050, 2014.     

Oligonucleotide aggregates bind to the macrophage scavenger receptor.

We have prepared a model receptor containing a Lys cluster (320-340) in the collagen-like domain of the bovine macrophage scavenger receptor, and have shown that it has a similar binding specificity to the native scavenger receptor. The native scavenger receptor is reported to bind the quadruplex structure of nucleotides. In this study, we analyzed the model receptor binding of nucleotides with various structures, random, parallel or antiparallel quadruplex and aggregate forms. This was carried out by direct binding assays using labeled oligonucleotides or surface plasmon resonance, and by an inhibition assay using Chinese hamster ovary (CHO) cells expressing the scavenger receptor. The results showed that the nucleotides forming the quadruplex structure did not exhibit any binding. Only the aggregate forms of the nucleotide could bind to the model receptor. They also inhibited the degradation of acetylated low density lipoprotein by CHO cells expressing the native scavenger receptor, whereas nucleotides that did not bind to the model receptor had no effect on cellular acetylated low density lipoprotein degradation. Our results suggest that the quadruplex structure is not essential but may be required for the formation of the nucleotide aggregates, which can interact with the scavenger receptor.     

G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates

Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes. We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117-38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs. We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures. The apparent K(d) values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 +/- 6 and 77 +/- 6 Pm, respectively, as measured by gel mobility shift assay. In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution. Down-regulation of G4R1/RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells. This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/RHAU in targeting in vivo RNA quadruplex structures.     

Mix and measure fluorescence screening for selective quadruplex binders

The human genome contains thousands of regions, including that of the telomere, that have the potential to form quadruplex structures. Many of these regions are potential targets for therapeutic intervention. There are many different folding patterns for quadruplex DNAs and the loops exhibit much more variation than do the quartets. The successful targeting of a particular quadruplex structure requires distinguishing that structure from all of the other quadruplex structures that may be present. A mix and measure fluorescent screening method has been developed, that utilizes multiple reporter molecules that bind to different features of quadruplex DNA. The reporter molecules are used in combination with DNAs that have a variety of quadruplex structures. The screening is based on observing the increase or decrease in the fluorescence of the reporter molecules. The selectivity of a set of test molecules has been determined by this approach.