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PubMed1998

Secondary structures in d(CGG) and d(CCG) repeat tracts.

Several studies have been made to elucidate the nature of secondary structures in the single strands of d(CGG).d(CCG) repeat tracts but with conflicting conclusions. Here, we review this work and attempt to come towards consensus. Some investigators find that the G-rich strand forms hairpins. Of these, some conclude that pairing is in the alignment d(GGC).d(GGC) with two Watson-Crick bonds and one G.G bond per duplex repeat, others conclude that the alignment is d(GCG).d(GCG) with two G.G bonds and one C.C bond per duplex repeat. Others find quadruplex formation and conclude that this is in the latter alignment with two G4-quartets per quadruplex repeat and C.C bonds. We investigate why these different results were obtained and conclude that quadruplexes are likely to form under physiological conditions. We argue that they are probably bonded in the alignment d(GGC).d(GGC) with one G4-quartet and two C.G.C.G. quartets per quadruplex repeat. The C-rich strand does not appear to form quadruplexes under physiological conditions but forms hairpins. Apparently, short hairpins adopt the alignment d(CCG).d(CCG) with mismatched cytosine residues stacked into the helix but with 15 or more repeat units, the dominant form is a distorted hairpin aligned as d(GCC).d(GCC) with unpaired cytosine residues possibly turned outwards and stacked in the minor groove.

Animals↗
PubMed2002

Site-specific probing of oxidative reactivity and telomerase function using 7,8-dihydro-8-oxoguanine in telomeric DNA.

Telomeres at the ends of human chromosomes contain the repeating sequence 5'-d[(TTAGGG)(n)]-3'. Oxidative damage of guanine in DNAs that contain telomeric and nontelomeric sequence generates 7,8-dihydro-8-oxoguanine (8OG) preferentially in the telomeric segment, because GGG sequences are more reactive in duplex DNA. We have developed a general strategy for probing site-specific oxidation reactivity in diverse biological structures through substitution of minimally modified building blocks that are more reactive than the parent residue, but preserve the parent structure. In this study, 8OG was substituted for guanine at G(8), G(9), G(14), or G(15) in the human telomeric oligonucleotide 5'-d[AGGGTTAG(8)G(9)GTT AG(14)G(15)GTTAGGGTGT]-3'. Replacement of G by 8OG in telomeric DNA can affect the formation of intramolecular G quadruplexes, depending on the position of substitution. When 8OG was incorporated in the 5'-position of a GGG triplet, G quadruplex formation was observed; however, substitution of 8OG in the middle of a GGG triplet produced multiple structures. A clear correspondence between structure and reactivity was observed when oligonucleotides containing 8OG in the 5'-position of a GGG triplet were prepared in the quadruplex or duplex forms and interrogated by mediated electrocatalytic oxidation with Os(bpy)(3)(2+) (bpy = 2,2'-bipyridine). The rate constant for one-electron oxidation of a single 8OG in the 5'-position of a GGG triplet was (6.2 +/- 1.7) x 10(4) M(-1) s(-1) in the G quadruplex form. The rate constant was 2-fold lower for the same telomeric sequence in the duplex form ((3.0 +/- 1.3) x 10(4) M(-1) s(-1)). The position of 8OG in the GGG triplet affects telomerase activity and synthesis of telomeric repeat products. Telomerase activity was decreased significantly when 8OG was substituted in the 5'-position of the GGG triplet, but not when 8OG was substituted in the middle of the triplet. Thus, biological oxidation of G to 8OG in telomeres has the potential to modulate telomerase activity. Further, small molecules that inhibit telomerase by stabilizing telomeric G quadruplexes may not be as effective under oxidative stress.

Binding Sites↗
PubMed2003

Trisubstituted acridine derivatives as potent and selective telomerase inhibitors.

The synthesis and evaluation for telomerase-inhibitory and quadruplex DNA binding properties of three related series of rationally designed trisubstituted acridine derivatives are described. These are substituted on the acridine ring at the 2,6,9; 2,7,9; and 3,6,9 positions. The ability of several of the most potent compounds to interact with and stabilize an intramolecular G-quadruplex DNA was evaluated by surface plasmon resonance methods, and affinities were found to correlate with potency in a telomerase assay. The interactions of a number of compounds with a parallel quadruplex DNA structure were simulated by molecular modeling methods. The calculated interaction energies were compared with telomerase activity and showed generally consistent correlations between quadruplex affinity and telomerase inhibition. These data support a model for the action of these compounds that involves the stabilization of intermediate quadruplex structures that inhibit the elongation of telomeric DNA by telomerase in tumor cells.

Acridines↗
PubMed1991

Self-association of telomeric short oligodeoxyribonucleotides containing a dG cluster.

Oligonucleotides containing a dG cluster, d(TmGnTm), are models of single-stranded parts of telomeric deoxyribonucleic acid and substitutes for poly(dG). Electrophoretic and spectroscopic analyses of the oligomers indicate that the oligomers can form two alternative structures, single- and quadruple-stranded helices, in solution at room temperature. The transformation of the single-strandef form into the quadruple-stranded form or vice versa is undetectable in 0.1 M NaCl at 4.3 x 10(-5) M strand concentration at room temperature. However, at a 50-fold higher strand concentration, the single-stranded oligomer is gradually converted into the quadruplex. An increase in ionic strength stabilizes the single-stranded structure, so it seems to inhibit the formation of the quadruplex. The quadruplex, [d(TTGGGGTT)]4, is resistant to denaturation in 7 M urea, in which the Watson-Crick type d(TTGGGGTT).d(AACCCCAA) duplex dissociates. An increase in the number of T residues facilitates the dissociation of the quadruplex by heating. Thus the number of T residues surrounding the dG cluster might control the rigidity of the quadruplex structure.

Animals↗
PubMed2002

The cationic porphyrin TMPyP4 down-regulates c-MYC and human telomerase reverse transcriptase expression and inhibits tumor growth in vivo.

Cationic porphyrins are being studied as possible anticancer agents because of their ability to bind to and stabilize DNA guanine quadruplexes (G-quadruplexes). We have shown previously that the cationic porphyrin TMPyP4 is able to bind to and stabilize G-quadruplexes in human telomere sequences, resulting in inhibition of telomerase activity. To better understand the mechanism of action behind telomerase inhibition by TMPyP4, we performed a cDNA microarray analysis on cells treated with TMPyP4 and TMPyP2, a positional isomer of TMPyP4 that has low affinity for G-quadruplexes. Analysis of time course data from the microarray experiments revealed that TMPyP4 and TMPyP2 treatment altered the expression of several gene clusters. We found that c-MYC, an oncogene nearly ubiquitous in human tumors that bears the potential in its promoter to form a G-quadruplex, was among the genes specifically down-regulated by TMPyP4, but not by TMPyP2. The hTERT gene, which encodes the catalytic subunit of telomerase, is transcriptionally regulated by c-MYC, and we have found that TMPyP4 also causes a decrease in human telomerase reverse transcriptase transcripts, suggesting two possible mechanisms for the effect of TMPyP4 on telomerase activity. We also show that TMPyP4, but not TMPyP2, is able to prolong survival and decrease tumor growth rates in two xenograft tumor models. We believe that, because of the actions of TMPyP4 in decreasing both c-MYC protein levels and telomerase activity, as well as its anticancer effects in vivo, it is a worthwhile agent to pursue and develop further.

Angiogenesis Inhibitors↗
PubMed2005

205Tl NMR methods for the characterization of monovalent cation binding to nucleic acids.

Monovalent cations play an important role in many biological functions. The guanine rich sequence, d(G4T4G4), requires monovalent cations for formation of the G-quadruplex, d(G4T4G4)2. This requirement can be satisfied by thallium (Tl+), a potassium (K+) surrogate. To verify that the structure of d(G4T4G4)2 in the presence of Tl+ is similar to the K+-form of the G-quadruplex, the solution structure of the Tl+-form of d(G4T4G4)2 was determined. The 10 lowest energy structures have an all atom RMSD of 0.76 +/- 0.16 A. Comparison of this structure to the identical G-quadruplex formed in the presence of K+ validates the isomorphous nature of Tl+ and K+. Using a 1H-205Tl spin-echo difference experiment we show that, in the Tl+-form of d(G4T4G4)2, small scalar couplings (<1 Hz) exist between 205Tl and protons in the G-quadruplex. These data comprise the first 1H-205Tl scalar couplings observed in a biological system and have the potential to provide important constraints for structure determination. These experiments can be applied to any system in which the substituted Tl+ cations are in slow exchange with the bulk ions in solution.

Cations, Monovalent↗
PubMed2004

Interaction of telomestatin with the telomeric single-strand overhang.

The extremities of chromosomes end in a G-rich single-stranded overhang that has been implicated in the onset of the replicative senescence. The repeated sequence forming a G-overhang is able to adopt a peculiar four-stranded DNA structure in vitro called a G-quadruplex, which is a poor substrate for telomerase. Small molecule ligands that selectively stabilize the telomeric G-quadruplex induce telomere shortening and a delayed growth arrest. Here we show that the G-quadruplex ligand telomestatin has a dramatic effect on the conformation of intracellular G-overhangs. Competition experiments indicate that telomestatin strongly binds in vitro and in vivo to the telomeric overhang and impairs its single-stranded conformation. Long-term treatment of cells with telomestatin greatly reduces the G-overhang size, as evidenced by specific hybridization or telomeric oligonucleotide ligation assay experiments, with a concomitant delayed loss of cell viability. In vivo protection experiments using dimethyl sulfate also indicate that telomestatin treatment alters the dimethyl sulfate effect on G-overhangs, a result compatible with the formation of a local quadruplex structure at telomeric overhang. Altogether these experiments strongly support the hypothesis that the telomeric G-overhang is an intracellular target for the action of telomestatin.

Base Sequence↗
PubMed2003

Ethidium probing of the parallel double- and four-stranded structures formed by the telomeric DNA sequences dG(GT)4G and d(GT)5.

Oligonucleotides 3'-d(GT)(5)-(CH(2)CH(2)O)(3)-d(GT)(5)-3' (parGT), containing GT repeats present in the telomeric DNA from Saccharomyces cerevisiae, had been demonstrated to form bimolecular structure, GT-quadruplex (qGT) [O. F. Borisova et al. FEBS Letters 306, 140-142 (1992)]. Four d(GT)(5) strands of the GT-quadruplex are parallel and form five G-quartets while thymines are bulged out. The four GT repeats when flanked by guanines, 3'-dG(TG)(4)G-(CH(2)CH(2)O)(3)-dG(GT)(4)G-3' (hp-GT), had been shown to form a novel parallel-stranded (ps) double helix with G.G and T.T base pairs (hp-GT ps-DNA) [A. K. Shchyolkina et al. J. Biomol. Struct. Dyn. 18, 493-503 (2001)]. In the present study the intercalator ethidium bromide (Et) was used for probing the two structures. The mode of Et binding and its effect on thermostability of qGT and hp-GT were compared. The quantum yield (q) and the fluorescence lifetime (tau) of Et:qGT (q = 0.15 +/- 0.01 and tau = 24 +/- 1 ns) and Et:hp-GT (q = 0.10 +/- 0.01 and tau = 16.5 +/- 1 ns) indicative of intercalation mode of Et binding were determined. Et binding to qGT was found to be cooperative with corresponding coefficient omega = 3.9 +/- 0.1 and the binding constant Kappa = (6.4 +/- 0.1).10(4) M(-1). The maximum number of Et molecules intercalating into GT-quadruplex is as high as twice the number of innerspaces between G-quartets (eight in our case). The data conform to the model of Et association with GT-quadruplex suggested earlier [O. F. Borisova et al. Mol. Biol. (Russ) 35, 732-739 (2001)]. The anticooperative type of Et binding was observed in case of hp-GT ps-DNA, with the maximum number of bound Et molecules, N = 4 / 5, and the association constant Kappa = (1.5 +/- 0.1).10(5) M(-1). Thermodynamic parameters of formation of Et:qGT and EtBr:hp-GT complexes were calculated from UV thermal denaturation profiles.

Animals↗
PubMed2005

Affinity and selectivity of G4 ligands measured by FRET.

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analyzed the stabilization and selectivity of two well-known quadruplex ligands (telomestatin and a cationic porphyrin) towards the human telomeric G-quadruplex species, with FRET. Both molecules strongly stabilize the G-quadruplex, but telomestatin appears much more selective, as shown by competition experiments with double-stranded DNA.

DNA↗
PubMed2005

Modulation of highly ordered structures of human telomeric sequence by cationic copolymers.

Human telomeric DNA, d(GGGTTA)n, tends to adopt complex intramolecular G-quadruplex structure in solution. The sequence folds into G-quadruplex with the antiparallel orientation in the presence of sodium ions, while antiparallel and parallel conformations were coexisted in solution containing relatively high concentration of potassium ions. Here, we describe that cationic comb-type copolymer, poly(l-lysine)-graft-dextran (PLL-g-Dex), triggers selective folding of d[GGG(TTAGGG)3] into G-quadruplex with the parallel orientation. The parallel G-quadruplex formed in the presence of the copolymer has the melting temperature > 90 degrees C, including strong stabilization effect of the copolymer.

Base Sequence↗
PubMed2006

The new models of the human telomere DNA in K+ solution revealed by NMR analysis assisted by the incorporation of 8-bromoguanines.

The structure of human telomeric DNA has been controversial: the solution structure in the presence of Na(+) has been reported to be antiparallel basket-type quadruplex by NMR, while the crystal structure in the presence of K(+) has been reported to be parallel propeller-type quadruplex. The solution structure in the presence of K(+) has drawn intense interest, as the intracellular K(+) concentration is higher than that of Na(+), but the structure is still open to address. Recently Sugiyama et al. has suggested that the DNA exists as a mixture of mixed-parallel/antiparallel quadruplex and antiparallel chair-type quadruplex on the basis of the combination of a series of 8-bromoguanine mutations and CD analysis. Here, we have started NMR analysis of the DNAs with the mutations to evaluate the proposed model. So far, NMR analysis is qualitatively consistent with the proposal. The structure determination is in progress to evaluate the model at atomic resolution.

DNA↗
PubMed2005

Characterization of the monovalent ion position and hydrogen-bond network in guanine quartets by DFT calculations of NMR parameters.

Conformational stability of G-quartets found in telomeric DNA quadruplex structures requires the coordination of monovalent ions. Here, an extensive Hartree-Fock and density functional theory analysis of the energetically favored position of Li+, Na+, and K+ ions is presented. The calculations show that at quartet-quartet distances observed in DNA quadruplex structures (3.3 A), the Li+ and Na+ ions favor positions of 0.55 and 0.95 A outside the plane of the G-quartet, respectively. The larger K+ ion prefers a central position between successive G-quartets. The energy barrier separating the minima in the quartet-ion-quartet model are much smaller for the Li+ and Na+ ions compared with the K+ ion; this suggests that K+ ions will not move as freely through the central channel of the DNA quadruplex. Spin-spin coupling constants and isotropic chemical shifts in G-quartets extracted from crystal structures of K+- and Na+-coordinated DNA quadruplexes were calculated with B3LYP/6-311G(d). The results show that the sizes of the trans-hydrogen-bond couplings are influenced primarily by the hydrogen bond geometry and only slightly by the presence of the ion. The calculations show that the R(N2N7) distance of the N2-H2...N7 hydrogen bond is characterized by strong correlations to both the chemical shifts of the donor group atoms and the (h2)J(N2N7) couplings. In contrast, weaker correlations between the (h3)J(N1C6') couplings and single geometric factors related to the N1-H1...O6=C6 hydrogen bond are observed. As such, deriving geometric information on the hydrogen bond through the use of trans-hydrogen-bond couplings and chemical shifts is more complex for the N1-H1...O6=C6 hydrogen bond than for the N2-H2...N7 moiety. The computed trans-hydrogen-bond couplings are shown to correlate with the experimentally determined couplings. However, the experimental values do not show such strong geometric dependencies.

Guanine↗
PubMed2001

Modelling studies on neurodegenerative disease-causing triplet repeat sequences d(GGC/GCC)n and d(CAG/CTG)n.

Model building and molecular mechanics studies have been carried out to examine the potential structures for d(GGC/GCC)5 and d(CAG/CTG)5 that might relate to their biological function and association with triplet repeat expansion diseases. Model building studies suggested that hairpin and quadruplex structures could be formed with these repeat sequences. Molecular mechanics studies have demonstrated that the hairpin and hairpin dimer structures of triplet repeat sequences formed by looping out of the two strands are as favourable as the corresponding B-DNA type hetero duplex structures. Further, at high salt condition, Greek key type quadruplex structures are energetically comparable with hairpin dimer and B-DNA type duplex structures. All tetrads in the quadruplex structures are well stacked and provide favourable stacking energy values. Interestingly, in the energy minimized hairpin dimer and Greek key type quadruplex structures, all the bases even in the non-G tetrads are cyclically hydrogen bonded, even though the A, C and T-tetrads were not hydrogen bonded in the starting structures.

Humans↗
PubMed2006

[Telomeres and telomerase, new targets for anticancer chemotherapy].

Telomeres are composed of single-strand DNA rich in guanine which can adopt particular structures such as T-loop or G-quadruples, a four-strand DAN structure formed by guanine repeats. Telomeric single-strand DNA is the substrate of telomerase, an enzyme necessary for telomeric replication which is suppressed in most cancer cells and which participates in tumor genesis. The formation of a telomeric G-quadruplex blocks telomerase activity and offers an original strategy for new anti-cancer agents. Using an original approach combining rational screening and synthesis, several series of compounds have been identified which specifically bind to the telomeric quadruplex. These derivatives, called "G-quadruplex DNA ligands", are able to block telomeric replication in cancer cells and provoke replicative senescence and/or apoptosis after a few cell cycles. Our team is working on characterizing the cellular and molecular mechanisms of action of these ligands. Using mutant cell models resistant to these ligands or expressing a protein cuff covering the telomere in tumor lines, we have demonstrated that the telomere is the principal intracellular target of action of these compounds and the implicit existence of the G-quadruplex structure. In collaboration with academic and industrial partners, optimization of these ligands to develop pharmacologically active products should enable in vivo validation of a new therapeutic concept.

Animals↗
PubMed2002

Telomeres and telomerases as drug targets.

Recent advances in telomerase inhibition have been achieved by using antisense oligonucleotides and ribozymes to target the telomerase mRNA or the telomerase RNA template. Also, small molecules are potent catalytic inhibitors of telomerase. However, therapeutic regimes incorporating these agents will be challenging to implement in the clinic because of their delayed effectiveness. Drugs that directly bind to the telomeres and stabilize secondary DNA structures such as G-quadruplexes are also potent inhibitors of telomerase and disrupt telomere structure. These G-quadruplex-interactive drugs could feasibly be used in synergy with more conventional cytotoxic agents to bring about more immediate responses in cancer cells that are less dependent upon telomere length. Recently, an emerging possible novel use of G-quadruplex-interactive drugs employs their ability to target G-quadruplexes in promoter regions of genes (such as c-MYC), which then serves to repress the production of the human telomerase reverse transcriptase protein.

Animals↗
PubMed1998

Human telomerase inhibition by regioisomeric disubstituted amidoanthracene-9,10-diones.

Telomerase is an attractive target for the design of new anticancer drugs. We have previously described a series of 1,4- and 2, 6-difunctionalized amidoanthracene-9,10-diones that inhibit human telomerase via stabilization of telomeric G-quadruplex structures. The present study details the preparation of three further, distinct series of regioisomeric difunctionalized amidoanthracene-9,10-diones substituted at the 1,5-, 1,8-, and 2,7-positions, respectively. Their in vitro cytotoxicity and Taq DNA polymerase and human telomerase inhibition properties are reported and compared with those of their 1,4- and 2,6-isomers. Potent telomerase inhibition (telIC50 values 1.3-17.3 microM) is exhibited within each isomeric series. In addition, biophysical and molecular modeling studies have been conducted to examine binding to the target G-quadruplex structure formed by the folding of telomeric DNA. These studies indicate that the isomeric diamidoanthracene-9,10-diones bind to the human telomeric G-quadruplex structure with a stoichiometry of 1:1. Plausible G-quadruplex-ligand complexes have been identified for each isomeric family, with three distinct modes of intercalative binding being proposed. The exact mode of intercalative binding is dictated by the positional placement of substituent side chains. Furthermore, in contrast to previous studies directed toward triplex DNA, it is evident that stringent control over positional attachment of substituents is not a necessity for effective telomerase inhibition.

Anthracenes↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed2003

Development and assessment of a quantitative reverse transcription-PCR assay for simultaneous measurement of four amplicons.

BACKGROUND: High-throughput and forward-deployable biological dosimetry capabilities are required for tactical and medical decisions after radiologic events. We previously reported a quantitative reverse transcription (QRT)-PCR assay for human radiation-responsive gene targets using a whole-blood ex vivo irradiation model, but we needed a multitarget assay on a smaller, less costly, real-time PCR detection system. METHODS: We developed a quadruplex QRT-PCR assay in a 96-well, closed-plate format suitable for use with RNA extracted from whole blood. Four cDNA targets were simultaneously amplified in a sealed tube by hybridization to exonuclease probes, each conjugated to distinct fluorogenic reporters. A novel primer-limited 18S rRNA reference target was validated from serial dilutions of human total RNA. To test assay precision, we incorporated a positive-control cDNA mimic into duplex and quadruplex PCR reactions. The master mixture was supplemented with more enzyme, MgCl(2), and deoxyribonucleotides. Simultaneous detection of four targets was evaluated in comparison with respective duplex QRT-PCR assays. RESULTS: The simultaneous detection of three radiation-responsive genes by quadruplex QRT-PCR was quantitative, with gene expression changes similar to those observed with optimized duplex and triplex QRT-PCR assays. The 18S rRNA and GADD45 calibration curves (threshold cycle vs log(10) cDNA) were linear and reproducible and showed optimal PCR efficiencies as indicated by slopes statistically equivalent to the theoretical value of -3.322. CONCLUSIONS: This is the first study of a quadruplex QRT-PCR assay. Our approach has diagnostic utility in the detection of biomarkers, biological and toxicologic agents, and genes of inherited diseases and cancer.

DNA Repair↗