Human telomeric G-quadruplex formed from duplex under near physiological conditions: Spectroscopic evidence and kinetics

The structural interconversion between the G-quadruplex and duplex in vivo is an important subject. In the present study, we used human telomeric DNA duplex composed of GGG(TTAGGG)₃/CCC(TAACCC)₃ as a model system to investigate its properties under near physiological conditions by spectroscopic methods. Circular dichroism and fluorescence spectra demonstrated that G-quadruplex structure can be formed from duplex at near physiological pH (pH 7.4), salt concentration (150 mM K⁺), and temperature (37 °C) in the presence of molecular crowding agent PEG (400 g/l), whereas the G-quadruplex structure cannot be formed at 25 °C in buffer containing 150 mM K⁺ in the presence of PEG. It is found that the formation rate of G-quadruplex structure depends on the temperature and the concentrations of both PEG and K⁺. This work suggests that human telomeric G-quadruplex structure may be potentially formed from Watson–Crick duplex in vivo.     

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.     

Hydration regulates the thermodynamic stability of DNA structures under molecular crowding conditions

Although water is an integral part of DNA structures, the effects of water molecules on various DNA structures which are formed by not only Watson-Crick but also Hoogsteen base pairs are still unclear. Here, we studied quantitatively the effects of molecular crowding on the thermodynamics of a parallel G-quadruplex formation of [d(TG 4)2]4 with Hoogsteen base pairs. It was demonstrated that molecular crowding conditions stabilized the parallel G-quadruplex. Moreover, the plot of stability of the parallel G-quadruplex structure versus water activity suggested that water molecules were released through the G-quadruplex formation. The stabilization of the DNA structures consisting of Hoogsteen base pairs under cell-like conditions may lead to a structural polymorphism of various DNA sequences regulated by water molecules.     

Identification of nonplanar small molecule for G-quadruplex grooves: molecular docking and molecular dynamic study

DNA G-quadruplex is an attractive drug target for anticancer therapy. Most G-quadruplex ligands have little selectivity, due to π-stacking interaction with common G-tetrads surface. Thanks to the varieties of G-quadruplex grooves, the groove-binding ligand is expected to create high selectivity. Therefore, developing novel molecular geometries that target G-quadruplex groove has been paid growing attention. In this work, steroid FG, a special nonplanar and nonaromatic small molecule, interacting with different conformations of G-quadruplexes has been studied by molecular docking and molecular dynamics simulations. The results showed the selectivity of the hydrophobic group of steroid FG for the wide groove of antiparallel G-quadruplex. The methyl groups on the tetracyclic ring of steroid represent the specific binding ability for the small hydrophobic cavity formed by reversed stacking of G-tetrads in antiparallel G-quadruplex groove. This work provides new insight for developing new classes of G-quadruplex groove-binding ligands.     

Spectroscopic,biological, and molecular modeling studies on the interactions of [Fe(III)-meloxicam] with G-quadruplex DNA and investigation of its release from bovine serum albumin (BSA) nanoparticles

The guanine-rich sequence, specifically in DNA, telomeric DNA, is a potential target of anticancer drugs. In this work, a mononuclear Fe(III) complex containing two meloxicam ligands was synthesized as a G-quadruplex stabilizer. The interaction between the Fe(III) complex and G-quadruplex with sequence of 5′-G₃(T₂AG₃)₃-3′ (HTG21) was investigated using spectroscopic methods, molecular modeling, and polymerase chain reaction (PCR) assays. The spectroscopic methods of UV–vis, fluorescence, and circular dichroism showed that the metal complex can effectively induce and stabilize G-quadruplex structure in the G-rich 21-mer sequence. Also, the binding constant between the Fe(III) complex and G-quadruplex was measured by these methods and it was found to be 4.53(±0.30)?×?10⁵ M^?1). The PCR stop assay indicated that the Fe(III) complex inhibits DNA amplification. The cell viability assay showed that the complex has significant antitumor activities against Hela cells. According to the UV–vis results, the interaction of the Fe(III) complex with duplex DNA is an order of magnitude lower than G-quadruplex. Furthermore, the release of the complex incorporated in bovine serum albumin nanoparticles was also investigated in physiological conditions. The release of the complex followed a bi-phasic release pattern with high and low releasing rates at the first and second phases, respectively. Also, in order to obtain the binding mode of the Fe(III) complex with G-quadruplex, molecular modeling was performed. The molecular docking results showed that the Fe(III) complex was docked to the end-stacked of the G-quadruplex with a π–π interaction, created between the meloxicam ligand and the guanine bases of the G-quadruplex.     

Tetrasubstituted naphthalene diimide ligands with selectivity for telomeric G-quadruplexes and cancer cells

A series of tetrasubstituted naphthalene diimide compounds with N-methylpiperazine end groups has been synthesized and evaluated as G-quadruplex ligands. They have high affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA. CD studies show that they induce formation of a parallel G-quadruplex topology. They inhibit the binding of hPOT1 and topoisomerase IIIα to telomeric DNA and inhibit telomerase activity in MCF7 cells. The compounds have potent activity in a panel of cancer cell lines, with typical IC(50) values of ～0.1 μM, and up to 100-fold lower toxicity in a normal human fibroblast cell line.     

Natural isoflavones regulate the quadruplex–duplex competition in human telomeric DNA

Effects of natural isoflavones on the structural competition of human telomeric G-quadruplex d[AG₃(T₂AG₃)₃] and its related Watson–Crick duplex d[AG₃(T₂AG₃)₃-(C₃TA₂)₃C₃T] are investigated by using circular dichroism (CD), ESI-MS, fluorescence quenching measurement, CD stopped-flow kinetic experiment, UV spectroscopy and molecular modeling methods. It is intriguing to find out that isoflavones can stabilize the G-quadruplex structure but destabilize its corresponding Watson–Crick duplex and this discriminated interaction is intensified by molecular crowding environments. Kinetic experiments indicate that the dissociation rate of quadruplex (k_{obs290 nm}) is decreased by 40.3% at the daidzin/DNA molar ratio of 1.0 in K⁺, whereas in Na⁺ the observed rate constant is reduced by about 12.0%. Furthermore, glycosidic daidzin significantly induces a structural transition of the polymorphic G-quadruplex into the antiparallel conformation in K⁺. This is the first report on the recognition of isoflavones with conformational polymorphism of G-quadruplex, which suggests that natural isoflavone constituents potentially exhibit distinct regulation on the structural competition of quadruplex versus duplex in human telomeric DNA.     

Molecular crowding regulates the structural switch of the DNA G-quadruplex

Almost all biochemical reactions in vitro have been investigated through numerous experiments conducted in dilute solutions containing low concentrations of solutes. However, biomacromolecules such as nucleic acids, proteins, and polysaccharides are designed to function and/or form their native structures in a living cell containing high concentrations of biomacromolecules, substrates, cofactors, salts, and so on. In the present study, we have demonstrated quantitatively the effect of molecular crowding on structures and stabilities of the G-quadruplex of d(G(4)T(4)G(4)). Molecular crowding with poly(ethylene glycol) (PEG) induced a structural transition from the antiparallel to the parallel G-quadruplex of d(G(4)T(4)G(4)), while molecular crowding with polycations did not alter the structure of the antiparallel G-quadruplex. The binding constants of putrescine, one of the polycations, for d(G(4)T(4)G(4)) in the absence and presence of Na(+) are calculated to be 277 and 2.5 M(-)(1), respectively. This indicates that the polycations coordinate to d(G(4)T(4)G(4)) with electrostatic interactions. The thermodynamic parameters of the antiparallel G-quadruplex formation under the crowding and noncrowding conditions induced by putrescine were also estimated. The stability of the antiparallel G-quadruplex decreased (-DeltaG degrees (25) decreased from 28 to 22 kcal mol(-)(1)) with molecular crowding by putrescine. Also, enthalpy and entropy changes in the structural formation under crowding and noncrowding conditions clearly showed that destabilization was entropy-driven. These quantitative parameters indicated that both the volume excluded by PEG and chemical interactions such as electrostatic interaction with solute polycations are critical for determining how molecular crowding affects the structure and stability of highly ordered DNA structures.     

Complicated behavior of G-quadruplexes and evaluating G-quadruplexes' ligands in various systems mimicking cellular circumstance

Environments surrounding G-rich sequences remarkably affect the conformations of these structures. A proper evaluation system mimicking the crowded environment in a cell with macromolecules should be developed to perform structural and functional studies on G-quadruplexes. In this study, the topology and stability of a G-quadruplex formed by human telomeric repeat sequences were investigated in a macromolecule-crowded environment created by polyethylene glycol 200 (PEG200), tumor cell extract, and Xenopus laevis egg extract. The interactions between small molecules and telomeric G-quadruplexes were also evaluated in the different systems. The results suggested that the actual behavior of G-quadruplex structures in cells extract is quite different from that in the PEG crowding system, and proteins or other factors in extracts might play a very important role in G-quadruplex structures.     

Conformational change of a G-quadruplex under molecular crowding conditions

Abstract

This study examined the influence of the molecular crowding condition induced by polyethylene glycol (PEG) on the G-quadruplex structure of the thrombin-binding aptamer sequence, 5′-GGGTTGGGTGTGGGTTGGG (G3), in a solution containing a sufficient concentration of mono cations (K⁺ and Na⁺). Although the G3 sequence preferably formed the antiparallel type G-quadruplex structure in a Na⁺ solution, conversion to the parallel type occurred when PEG was added. The antiparallel type was maintained at low PEG concentrations. When the PEG concentration reached 30%, the antiparallel type and parallel type coexist. At PEG concentrations above 40%, the G-quadruplex structure adopted the parallel type completely. In the presence of K⁺ ions, G3 showed a parallel conformation and remained as a parallel conformation with increasing PEG concentration. The dissociation temperature increased with increasing PEG concentration in all cases, suggesting that the G-quadruplex conformation is more stable under molecular crowding conditions.

Communicated by Ramaswamy H. Sarma     

Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). The formation and stabilization of DNA G-quadruplexes in the human telomeric sequence have been shown to inhibit the activity of telomerase, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. However, knowledge of the intact human telomeric G-quadruplex structure(s) formed under physiological conditions is a prerequisite for structure-based rational drug design. Here we report the folding structure of the human telomeric sequence in K⁺ solution determined by NMR. Our results demonstrate a novel, unprecedented intramolecular G-quadruplex folding topology with hybrid-type mixed parallel/antiparallel G-strands. This telomeric G-quadruplex structure contains three G-tetrads with mixed G-arrangements, which are connected consecutively with a double-chain-reversal side loop and two lateral loops, each consisting of three nucleotides TTA. This intramolecular hybrid-type telomeric G-quadruplex structure formed in K⁺ solution is distinct from those reported on the 22 nt Tel22 in Na⁺ solution and in crystalline state in the presence of K⁺, and appears to be the predominant conformation for the extended 26 nt telomeric sequence Tel26 in the presence of K⁺, regardless of the presence or absence of Na⁺. Furthermore, the addition of K⁺ readily converts the Na⁺-form conformation to the K⁺-form hybrid-type G-quadruplex. Our results explain all the reported experimental data on the human telomeric G-quadruplexes formed in the presence of K⁺, and provide important insights for understanding the polymorphism and interconversion of various G-quadruplex structures formed within the human telomeric sequence, as well as the effects of sequence and cations. This hybrid-type G-quadruplex topology suggests a straightforward pathway for the secondary structure formation with effective packing within the extended human telomeric DNA. The hybrid-type telomeric G-quadruplex is most likely to be of pharmacological relevance, and the distinct folding topology of this G-quadruplex suggests that it can be specifically targeted by G-quadruplex interactive small molecule drugs.     

Pharmacophore-based discovery of triaryl-substituted imidazole as new telomeric G-quadruplex ligand

Discovery of potent and selective ligands for telomeric G-quadruplex DNA is a challenging work. Through a combination approach of pharmacophore model construction, model validation, database virtual screening, chemical synthesis and interaction evaluation, we discovered and confirmed triaryl-substituted imidazole TSIZ01 to be a new telomeric G-quadruplex ligand with potent binding and stabilizing activity to G-quadruplex DNA, as well as a 8.7-fold selectivity towards telomeric G-quadruplex DNA over duplex DNA.     

Structure of the Hybrid-2 type intramolecular human telomeric G-quadruplex in K+ solution: insights into structure polymorphism of the human telomeric sequence

Formation of the G-quadruplex in the human telomeric sequence can inhibit the activity of telomerase, thus the intramolecular telomeric G-quadruplexes have been considered as an attractive anticancer target. Information of intramolecular telomeric G-quadruplex structures formed under physiological conditions is important for structure-based drug design. Here, we report the first structure of the major intramolecular G-quadruplex formed in a native, non-modified human telomeric sequence in K⁺ solution. This is a hybrid-type mixed parallel/antiparallel-G-stranded G-quadruplex, one end of which is covered by a novel T:A:T triple capping structure. This structure (Hybrid-2) and the previously reported Hybrid-1 structure differ in their loop arrangements, strand orientations and capping structures. The distinct capping structures appear to be crucial for the favored formation of the specific hybrid-type intramolecular telomeric G-quadruplexes, and may provide specific binding sites for drug targeting. Our study also shows that while the hybrid-type G-quadruplexes appear to be the major conformations in K⁺ solution, human telomeric sequences are always in equilibrium between Hybrid-1 and Hybrid-2 structures, which is largely determined by the 3′-flanking sequence. Furthermore, both hybrid-type G-quadruplexes suggest a straightforward means for multimer formation with effective packing in the human telomeric sequence and provide important implications for drug targeting of G-quadruplexes in human telomeres.     

The parallel G-quadruplex structure of vertebrate telomeric repeat sequences is not the preferred folding topology under physiological conditions

G-quadruplex topologies of telomeric repeat sequences from vertebrates were investigated in the presence of molecular crowding (MC) mimetics, namely polyethylene glycol 200 (PEG), Ficoll 70 as well as Xenopus laevis egg extract by CD and NMR spectroscopy and native PAGE. Here, we show that the conformational behavior of the telomeric repeats in X. laevis egg extract or in Ficoll is notably different from that observed in the presence of PEG. While the behavior of the telomeric repeat in X. laevis egg extract or in Ficoll resembles results obtained under dilute conditions, PEG promotes the formation of high-order parallel topologies. Our data suggest that PEG should not be used as a MC mimetic.     

Spectroscopic, molecular modeling, and NMR-spectroscopic investigation of the binding mode of the natural alkaloids berberine and sanguinarine to human telomeric G-quadruplex DNA

G-quadruplex structures can be formed at the single-stranded overhang of telomeric DNA, and ligands able to stabilize this structure have recently been identified as potential anticancer drugs. Among the potential G-quadruplex binders, we have studied the binding ability of berberine and sanguinarine, two members of the alkaloid family, an important class of natural products long known for medicinal purpose. Our spectroscopic (CD, NMR, and fluorescence) studies and molecular modeling approaches revealed binding modes at ligand-complex stoichiometries >1:1 and ligand self-association induced by DNA for the interactions of the natural alkaloids berberine and sanguinarine with the human telomeric G-quadruplex DNA.     

The first derivative of a function of circular dichroism spectra: biophysical study of human telomeric G-quadruplex

Depending on conditions and base modifications, telomeric repeats can form many topological structures; parallel, antiparallel and hybrid forms. The influence of salts and some specific ligands on conformational changes has already been established. In this study, we analyze the human telomeric repeats 5′-GGG(TTAGGG)₃-3′ because this sequence forms topologically different structures under various conditions which have been well described by many authors. CD results are compared with electrophoretic and UV absorption spectroscopy results obtained under corresponding conditions in the presence of different ratios of sodium and potassium ions and polyethylene glycol (PEG). We confirmed that the most stable G-quadruplexes could only form under crowding conditions with PEG-200 and K⁺ ion, but the molecularity is increased. Other monovalent ions without the presence of K⁺ are unable to form the parallel quadruplex conformer and no change of stoichiometry is observed, even when PEG-200 is present. The first derivative of a function applied to CD spectra seems to be a powerful tool for spectra evaluation of any G-quadruplex, and could be more unambiguous than a direct analysis of original spectra.     

Dual mode of binding of anti cancer drug epirubicin to G-quadruplex [d-(TTAGGGT)]4 containing human telomeric DNA sequence induces thermal stabilization

Epirubicin exerts its anti cancer action by blocking DNA/RNA synthesis and inhibition of topoisomerase-II enzyme. Recent reports on its influence on telomere maintenance, suggest interaction with G-quadruplex DNA leading to multiple strategies of action. The binding of epirubicin with parallel stranded inter molecular G-quadruplex DNA [d-(TTAGGGT)]₄ comprising human telomeric DNA sequence TTAGGG was investigated by absorption, fluorescence, circular dichroism and nuclear magnetic resonance spectroscopy. The epirubicin binds as monomer to G-quadruplex DNA with affinity, K_b1 = 3.8 × 10⁶ M⁻¹ and K_b2 = 2.7 × 10⁶ M⁻¹, at two independent sites externally. The specific interactions induce thermal stabilization of DNA by 13.2–26.3 °C, which is likely to come in the way of telomere association with telomerase enzyme and contribute to epirubicin-induced apoptosis in cancer cell lines. The findings pave the way for drug designing in view of the possibility of altering substituent groups on anthracyclines to enhance efficacy using alternate mechanism of its interaction with G4 DNA, causing interference in telomere maintenance pathway by inducing telomere dysfunction.     

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.     

BRACO19 analog dimers with improved inhibition of telomerase and hPot 1

Human chromosomes terminate with telomeres, which contain double-stranded G-rich, repetitive DNA followed by a single-stranded overhang of the G-rich sequence. Single-stranded oligonucleotides containing G-rich telomeric repeats have been observed in vitro to fold into a variety of G-quadruplex topologies depending on the solution conditions. G-quadruplex structures are notable in part because G-quadruplex ligands inhibit both the enzyme telomerase and other telomere-binding proteins. Because telomerase is required for growth by the majority of cancers, G-quadruplex-stabilizing ligands have become an attractive platform for anticancer drug discovery. Here, we present the preparation and biochemical activities of a novel series of 3,6-disubstituted acridine dimers modeled after the known G-quadruplex ligand BRACO19. These BRACO19 Analog Dimer (BAD) ligands were shown to bind to human telomeric DNA and promote the formation of intramolecular G-quadruplexes in the absence of monovalent cations. As expected, the BAD ligands bound to telomeric DNA with a 1:1 stoichiometry, whereas the parent compound BRACO19, a monomer, bound with a 2:1 stoichiometry. The BAD ligands exhibited potent inhibition of human telomerase with IC₅₀ values similar to or lower than those of BRACO19. Furthermore, the BAD ligands displayed greater potency in the inhibition of hPot1 and increased selectivity for G-quadruplex DNA when compared to BRACO19. Collectively, these experiments support the hypothesis that there is an increased potency and selectivity to be gained in the design of G-quadruplex-stabilizing agents that incorporate multiple interactions.