Energetics of the human Tel-22 quadruplex-telomestatin interaction: a molecular dynamics study

The formation and stabilization of telomeric quadruplexes has been shown to inhibit the activity of telomerase, thus establishing telomeric DNA quadruplex as an attractive target for cancer therapeutic intervention. In this context, telomestatin, a G-quadruplex-specific ligand known to bind and stabilize G-quadruplex, is of great interest. Knowledge of the three-dimensional structure of telomeric quadruplex and its complex with telomestatin in solution is a prerequisite for structure-based rational drug design. Here, we report the relative stabilities of human telomeric quadruplex (AG3[T2AG3]3) structures under K+ ion conditions and their binding interaction with telomestatin, as determined by molecular dynamics simulations followed by energy calculations. The energetics study shows that, in the presence of K+ ions, mixed hybrid-type Tel-22 quadruplex conformations are more stable than other conformations. The binding free energy for quadruplex-telomestatin interactions suggests that 1:2 binding is favored over 1:1 binding. To further substantiate our results, we also calculated the change in solvent-accessible surface area (DeltaSASA) and heat capacity (DeltaCp) associated with 1:1 and 1:2 binding modes. The extensive investigation performed for quadruplex-telomestatin interaction will assist in understanding the parameters influencing the quadruplex-ligand interaction and will serve as a platform for rational drug design.     

Role of locked nucleic acid modified complementary strand in quadruplex/Watson-Crick duplex equilibrium

In the human genome, the G-rich sequences that form quadruplexes are present along with their C-rich complementary strands; this suggests the existence of equilibrium between a quadruplex and a Watson-Crick duplex which allows the execution of their respective biological functions. We have investigated the sensitivity of this equilibrium to pharmacological agents by employing locked nucleic acid (LNA) modified complementary strands, and demonstrated successful invasion of the stable telomeric quadruplex d[(G(3)TTA)(3)G(3)]. Fluorescence, UV, ITC, and SPR studies were performed to understand the binding process involving the preformed quadruplex and LNA-modified complementary strands compared with that involving the unmodified complementary strand. Our data indicate that LNA modifications in the complementary strand shift the equilibrium toward the duplex state. These modifications confer increased thermodynamic stability to the duplex and increase the magnitude of relative free energy (DeltaDeltaG degrees) difference between duplex and quadruplex, thus favoring the predominance of duplex population over quadruplex. This superior ability of LNA-modified complementary strand can be exploited to pave an exploratory approach in which it hybridizes to a telomeric quadruplex and drives duplex formation, and inhibits the recognition of 3' G-rich overhang by RNA template of telomerase which guides telomere extension.     

Synthesis and preliminary evaluation of novel analogues of quindolines as potential stabilisers of telomeric G-quadruplex DNA

Telomeric DNA is a potential selective target for cancer therapy since the tumour-associated enzyme telomerase regulates telomere maintenance in most cancer cells. The 3′ single-stranded ends of telomeric DNA can be folded into quadruplex structures by appropriate small molecules. We describe the preparation of a new class of 2,7-disubstituted 10H-indolo[3,2-b]quinolines with enhanced selectivity for the stabilisation of quadruplex DNA compared to duplex DNA, and also the preparation of a key intermediate for the synthesis of trisubstituted quindolines.     

Structure of the parallel-stranded DNA quadruplex d(TTAGGGT)4 containing the human telomeric repeat: evidence for A-tetrad formation from NMR and molecular dynamics simulations

The structure of the intermolecular DNA quadruplex d(TTAGGGT)4, based on the human telomeric DNA sequence d(TTAGGG), has been determined in solution by NMR and restrained molecular dynamics simultations. The core GGG region forms a highly stable quadruplex with G-tetrads likely stabilised by K+ ions bound between tetrad plains. However, we have focused on the conformation of the adenines which differ considerably in base alignment, stability and dynamics from those in previously reported structures of d(AGGGT)4 and d(TAGGGT)4. We show unambiguously that the adenines of d(TTAGGGT)4 are involved in the formation of a relatively stable A-tetrad with well-defined glycosidic torsion angles (anti), hydrogen bonding network (adenine 6-NH2-adenine N1) defined by interbase NOEs, and base stacking interactions with the neighbouring G-tetrad. All of these structural features are apparent from NOE data involving both exchangeable and non-exchangeable protons. Thus, context-dependent effects appear to play some role in dictating preferred conformation, stability and dynamics. The structure of d(TTAGGGT)4 provides us with a model system for exploiting in the design of novel telomerase inhibitors that bind to and stabilise G-quadruplex structures.     

Solution NMR Structure of a Ligand/Hybrid‐2‐G‐Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding

Telomeric G‐quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G‐quadruplex that adopts the biologically relevant hybrid‐2 conformation in a ligand‐bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G‐quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G‐quadruplex. The ligand is sandwiched between one terminal G‐tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G‐quadruplex structure as observed for other G‐quadruplexes in different conformations, invalidating simple docking approaches to ligand–G‐quadruplex structure determination.     

Carbohydrate–DNA Interactions at G‐Quadruplexes: Folding and Stability Changes by Attaching Sugars at the 5′‐End

Quadruplex DNA structures are attracting an enormous interest in many areas of chemistry, ranging from chemical biology, supramolecular chemistry to nanoscience. We have prepared carbohydrate–DNA conjugates containing the oligonucleotide sequences of G‐quadruplexes (thrombin binding aptamer (TBA) and human telomere (TEL)), measured their thermal stability and studied their structure in solution by using NMR and molecular dynamics. The solution structure of a fucose–TBA conjugate shows stacking interactions between the carbohydrate and the DNA G‐tetrad in addition to hydrogen bonding and hydrophobic contacts. We have also shown that attaching carbohydrates at the 5′‐end of a quadruplex telomeric sequence can alter its folding topology. These results suggest the possibility of modulating the folding of the G‐quadruplex by linking carbohydrates and have clear implications in molecular recognition and the design of new G‐quadruplex ligands.     

Selective and Cooperative Ligand Binding to Antiparallel Human Telomeric DNA G‐Quadruplexes

The quest for ligands that specifically bind to particular G‐quadruplex nucleic acid structures is particularly important to conceive molecules with specific effects on gene expression or telomere maintenance, or conceive structure‐specific molecular probes. Using electrospray mass spectrometry in native conditions, we reveal a highly cooperative and selective 2:1 binding of Cu^II‐tolylterpyridine complexes to human telomeric G‐quadruplexes. Circular dichroism and comparisons of affinities for different sequences reveal a marked preference for antiparallel structures with diagonal loops and/or wide‐medium–narrow‐medium groove‐width order. The cooperativity is attributed to conformational changes in the polymorphic telomeric G‐quadruplex sequences, which convert preferably into an antiparallel three‐quartet topology upon binding of two ligands.     

Selective G-quadruplex DNA stabilizing agents based on bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine

Various biologically relevant G-quadruplex DNA structures offer a platform for therapeutic intervention for altering the gene expression or by halting the function of proteins associated with telomeres. One of the prominent strategies to explore the therapeutic potential of quadruplex DNA structures is by stabilizing them with small molecule ligands. Here we report the synthesis of bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine and their interaction with human telomeric DNA and promoter G-quadruplex forming DNAs. The interactions of ligands with quadruplex forming DNAs were studied by various biophysical, biochemical, and computational methods. Results indicated that bisquinolinium ligands bind tightly and selectively to quadruplex DNAs at low ligand concentration (～0.2-0.4 μM). Furthermore, thermal melting studies revealed that ligands imparted higher stabilization for quadruplex DNA (an increase in the T(m) of up to 21 °C for human telomeric G-quadruplex DNA and >25 °C for promoter G-quadruplex DNAs) than duplex DNA (ΔT(m) ≤ 1.6 °C). Molecular dynamics simulations revealed that the end-stacking binding mode was favored for ligands with low binding free energy. Taken together, the results indicate that the naphthyridine-based ligands with quinolinium and pyridinium side chains form a promising class of quadruplex DNA stabilizing agents having high selectivity for quadruplex DNA structures over duplex DNA structures.     

人类端粒DNA的分子识别及其作用机制探讨

核酸中富含短的G-碱基重复的序列可以形成一种复杂的高级结构,称为G-四链体（G-quadruplex）.在基因组中,借助生物信息学发现这类富G序列广泛分布在基因的启动子区,特别是那些参与到复制中去的基因,例如癌基因.同时发现这类序列在mRNA的5′非翻译区（5′UTR）也广泛存在.这类序列在染色体末段端粒部位的存在及功能已得到充分阐明.已知端粒富含G-碱基序列,其3′末端以单链状态存在,这使得在一些小分子的选择性作用下端粒序列很容易形成G-四链体结构,进而破坏端粒结构,影响端粒酶活性.已知端粒酶在超过85%的肿瘤中过量表达,因此,端粒酶已经成为抗癌药物设计的特殊靶点,是目前本领域的研究热点之一.已发现系列配体通过有效抑制端粒酶而表现高的抗肿瘤活性.本文主要综述了近年来端粒G-四链体分子识别及其药物靶向的最新进展,并对其作用机理做了进一步的分析和探讨.     

Structural basis of telomeric RNA quadruplex--acridine ligand recognition

Human telomeric DNA is now known to be transcribed into noncoding RNA sequences, termed TERRA. These sequences, which are believed to play roles in the regulation of telomere function, can form higher-order quadruplex structures and may themselves be the target of therapeutic intervention. The crystal structure of a TERRA quadruplex-acridine small-molecule complex at a resolution of 2.60 ?, is reported here and contrasts remarkably with the structure of the analogous DNA quadruplex complex. The bimolecular RNA complex has a parallel-stranded topology with propeller-like UUA loops. These loops are held in particular conformations by multiple hydrogen bonds involving the O2' hydroxyl groups of the ribonucleotide sugars and play an active role in binding the acridine molecules to the RNA quadruplex. By contrast, the analogous DNA quadruplex complex has simpler 1:1 acridine binding, with no loop involvement. There are significant loop conformational changes in the RNA quadruplex compared to the native TERRA quadruplex (Collie, G. W.; Haider, S. M.; Neidle, S.; Parkinson, G. N. Nucleic Acids Res. 2010, 38, 5569 - 5580), which have implications for the future design of small molecules targeting TERRA quadruplexes, and RNA quadruplexes more generally.     

Cationic Pentaheteroaryls as Selective G‐Quadruplex Ligands by Solvent‐Free Microwave‐Assisted Synthesis

We report herein a solvent‐free and microwaved‐assisted synthesis of several water soluble acyclic pentaheteroaryls containing 1,2,4‐oxadiazole moieties ( 1 – 7 ). Their binding interactions with DNA quadruplex structures were thoroughly investigated by FRET melting, fluorescent intercalator displacement assay (G4‐FID) and CD spectroscopy. Among the G‐quadruplexes considered, attention was focused on telomeric repeats together with the proto‐oncogenic c‐kit sequences and the c‐myc oncogene promoter. Compound 1 , and to a lesser extent 2 and 5 , preferentially stabilise an antiparallel structure of the telomeric DNA motif, and exhibit an opposite binding behaviour to structurally related polyoxazole ( TOxaPy ), and do not bind duplex DNA. The efficiency and selectivity of the binding process was remarkably controlled by the structure of the solubilising moieties.     

Comparative binding of antitumor drugs to DNA containing the telomere repeat sequence

Telomeres are the ends of the linear chromosomes of eukaryotes and consist of tandem GT-rich repeats in telomere sequence i.e. 500-3000 repeats of 5'-TTAGGG-3' in human somatic cells, which are shortened gradually with age. The G-rich overhang of telomere sequence can adopt different intramolecular fold-backs and tetra-stranded DNA structures, in vitro, which inhibit telomerase activity. In this report, DNA binding agents to telomere sequence were studied novel therapeutic possibility to destabilize telomeric DNA sequences. Oligonucleotides containing the guanine repeats in human telomere sequence were synthesized and used for screening potential antitumor drugs. Telomeric DNA sequence was characterized using spectral measurements and CD spectroscopy. CD spectrum indicated that the double-stranded telomeric DNA is in a right-handed conformation. Polyacrylamide gel electrophoresis was performed for binding behaviors of antitumor compounds with telomeric DNA sequence. Drugs interacted with DNA sequence caused changes in the electrophoretic mobility and band intensity of the gels. Depending on the binding mode of the anticancer drugs, telomeric DNA sequence was differently recognized and the efficiency of cleavage of DNA varies in the bleomycin-treated samples under different conditions. DNA cleavage occurred at about 1% by the increments of 1 micromM bleomycin-Fe(III). These results imply that the stability of human telomere sequence is important in conjunction with the cancer treatment and aging process.     

Selective recognition of G-qQuadruplex telomeric DNA by a bis(quinacridine) macrocycle

The interaction of G-quadruplex DNA with the macrocyclic compound BOQ1, which possesses two dibenzophenanthroline (quinacridine) subunits, has been investigated by a variety of methods. The oligonucleotide 5'-A(GGGT(2)A)(3)G(3), which mimics the human telomeric repeat sequence and forms an intramolecular quadruplex, was used as one model system. Equilibrium binding constants measured by biosensor surface plasmon resonance (SPR) methods indicate a high affinity of the macrocycle for the quadruplex conformation (K > 1 x 10(7) M(-)(1)) with two equivalent binding sites. The affinity of BOQ1 for DNA duplexes is at least 1 order of magnitude lower. In addition, the macrocycle is more selective than the monomeric control compound (MOQ2), which is not able to discriminate between the two DNA structures (K(duplex) approximately K(quadruplex) approximately 10(6) M(-)(1)). Strong binding of BOQ1 to G4 DNA sequences was confirmed by fluorometric titrations with a tetraplex-forming oligonucleotide. Competition dialysis experiments with a panel of different DNA structures, from single strands to quadruplexes, clearly established the quadruplex binding specificity of BOQ1. Fluorescence resonance energy transfer (FRET) T(m) experiments with a doubly labeled oligonucleotide also revealed a strong stabilization of the G4 conformation in the presence of BOQ1 (DeltaT(m) = +28 degrees C). This DeltaT(m) value is one of the highest values measured for a G-quadruplex ligand and is significantly higher than observed for the monomer control compounds (DeltaT(m) = +10-12 degrees C). Gel mobility shift assays indicated that the macrocycle efficiently induces the formation of G-tetraplexes. Strong inhibition of telomerase was observed in the submicromolar range (IC(50) = 0.13 microM). These results indicate that macrocycles represent an exciting new development opportunity for targeting DNA quadruplexes.     

Formation of a PNA2-DNA2 hybrid quadruplex

DNA guanine (G) quadruplexes are stabilized by an interesting variation of the hydrogen-bonding schemes encountered in nucleic acid duplexes and triplexes. In an attempt to use this mode of molecular recognition, we target a dimeric G-quadruplex formed by the Oxytricha nova telomeric sequence d(G(4)T(4)G(4)) with a peptide nucleic acid (PNA) probe having a homologous rather than complementary sequence. UV-vis and CD spectroscopy reveal that a stable hybrid possessing G-quartets is formed between the PNA and DNA. The four-stranded character of the hybrid and the relative orientation of the strands is determined by fluorescence resonance energy transfer (FRET) experiments. FRET results indicate that (i) the two PNA strands are parallel to each other, (ii) the two DNA strands are parallel to each other, and (iii) the 5'-termini of the DNA strands align with the N-termini of the PNA strands. The resulting PNA(2)-DNA(2) quadruplex shows a preference of Na(+) over Li(+) and displays thermodynamic behavior consistent with alternating PNA and DNA strands in the hybrid. The formation of this novel supramolecular structure demonstrates a new high-affinity DNA recognition mechanism and expands the scope of molecular recognition by PNA.     

Interaction between human telomere and a carbazole derivative: a molecular dynamics simulation of a quadruplex stabilizer and telomerase inhibitor

The mechanism of inhibition of telomerase by drugs is a key factor in an understanding of guanine-quadruplex complex stabilization during human cancer. This study describes a simulated annealing docking and molecular dynamics simulation to investigate a synthesized potent inhibitor, 3,6-bis(1-methyl-4-vinylpyridinium iodine) carbazole (BMVC), which stabilizes the quadruplex structure of the human telomeric DNA sequence d[AG3(T(2)AG(3))3] and inhibits telomerase activity. The compound was predicted to selectively interact with the quadruplex structure. During our simulation, the binding affinities were calculated and used to predict the best drug-binding sites as well as enhanced selectivity compared with other compounds. Our studies suggest that the simulation results quite coincide with the experimental results. In addition, molecular modeling shows that a 2:1 binding model involving the external binding of BMVC to both ends of the G-quartet of d[AG(3)(T(2)AG)3))3] is the most stable binding mode and this agrees with the absorbance titration results that show two binding sites. Of particular interest is that one pyridinium ring and carbazole moiety of the BMVC can stack well at the end of G-quartet. This implies that BMVC is a good human quadruplex stabilizer and also a good telomerase inhibitor.     

Dinuclear Ruthenium(II) Complexes That Induce and Stabilise G‐Quadruplex DNA

A series of dinuclear ruthenium(II) complexes were synthesised, and the complexes were determined to be new highly selective compounds for binding to telomeric G‐quadruplex DNA. The interactions of these complexes with telomeric G‐quadruplex DNA were studied by using circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET) melting assays, isothermal titration calorimetry (ITC) and molecular modelling. The results showed that the complexes 1 , 2 and 4 induced and stabilised the formation of antiparallel G‐quadruplexes of telomeric DNA in the absence of salt or in the presence of 100 mM K⁺‐containing buffer. Furthermore, complexes 1 and 2 strongly bind to and effectively stabilise the telomeric G‐quadruplex structure and have significant selectivity for G‐quadruplex over duplex DNA. In comparison, complex 3 had a much lesser effect on the G‐quadruplex, suggesting that possession of a suitably sized plane for good π–π stacking with the G‐quadruplets is essential for the interaction of the dinuclear ruthenium(II) complexes with the G‐quadruplex. Moreover, telomerase inhibition by the four complexes and their cellular effects were studied, and complex 1 was determined to be the most promising inhibitor of both telomerase and HeLa cell proliferation.     

吲哚并喹啉衍生物与G-四链体相互作用的分子模拟研究

G-四链体是富含鸟嘌呤碱基的DNA序列通过氢键相互作用形成的四链螺旋结构. 通过小分子化合物诱导与稳定端粒G-四链体从而抑制端粒酶活性是一种新的抗癌策略. 为了研究一系列吲哚并喹啉衍生物与端粒G-四链体的相互作用, 探究其相互作用模式, 从而为实现基于G-四链体结构的药物合理设计提供依据, 使用分子对接的方法构建了吲哚并喹啉衍生物与G-四链体复合物结构, 在此基础上进行分子动力学模拟, 并使用线性相互作用能(LIE)方法计算了化合物与G-四链体的结合自由能. 结果表明: 化合物与G-四链体的主要相互作用方式由氢键、静电与π-π堆积作用构成, 侧链末端基团类型和侧链的长短是影响相互作用强弱的重要因素. 通过LIE方法计算的结合自由能与实验结果基本吻合, 相关度达到r²＝0.79. 并且, 基于预测的结合模式, 总结了拥有更高活性的新型吲哚并喹啉衍生物应具有的几个结构特征.     

Effect of loop orientation on quadruplex-TMPyP4 interaction

G-quadruplexes are believed to be potential targets for therapeutic intervention and this has resulted in designing of various quadruplex interacting ligands. Moreover, reports about existence of quadruplex forming sequences across the genome have propelled greater interest in understanding their interaction with small molecules. An intramolecular quadruplex sequence can adopt different conformations, owing to different orientation of loops in the structure. The differences in the loop orientation can affect their molecular recognition. Herein, we have studied the interaction of 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H, 23H-porphine (TMPyP4), a well-known G quadruplex binding ligand with three DNA quadruplexes differing in loop orientations. Results obtained from UV, ITC, and SPR studies have coherently revealed that the TMPyP4 molecule shows preferential binding to parallel G-quadruplex ( c-myc and c-kit) over its antiparallel counterpart (human telomeric). The binding affinity for parallel quadruplex was (10(7)) 1 order of magnitude higher than that for antiparallel DNA quadruplex (10 ). The study shows two binding modes, stronger binding (10(7)) of TMPyP4 involving end stacking and a weaker external binding (10 ), while TMPyP4 shows only one binding mode with duplex with a binding affinity of the order of 10(6). Overall, the study emphasizes that differences in the loop orientation give rise to different conformations of quadruplex, which in turn govern its binding to small molecules, and thereby play a pivotal role in molecular recognition.     

Discrimination of G‐Quadruplexes from Duplex and Single‐Stranded DNAs with Fluorescence and Energy‐Transfer Fluorescence Spectra of Crystal Violet

G‐rich nucleic acid sequences with the potential to form G‐quadruplex structures are common in biologically important regions. Most of these sequences are present with their complementary strands, so the development of a sensitive biosensor to distinguish G‐quadruplex and duplex structures and to determine the competitive ability of quadruplex to duplex structures has received a great deal of attention. In this work, the interactions between two triphenylmethane dyes (malachite green (MG) and crystal violet (CV)) and G‐quadruplex, duplex, or single‐stranded DNAs were studied by fluorescence spectroscopy and energy‐transfer fluorescence spectroscopy. Good discrimination between quadruplexes and duplex or single‐stranded DNAs can be achieved by using the fluorescence spectrum of CV or the energy‐transfer fluorescence spectra of CV and MG. In addition, by using energy‐transfer fluorescence titrations of CV with G‐quadruplexes, the binding‐stoichiometry ratios of CV to G‐quadruplexes can be determined. By using the fluorescence titrations of G‐quadruplex–CV complexes with C‐rich complementary strands, the fraction of G‐rich oligonucleotide that engages in G‐quadruplex structures in the presence of the complementary sequence can be measured. This study may provide a simple method for discrimination between quadruplexes and duplex or single‐stranded DNAs and for measuring G‐quadruplex percentages in the presence of the complementary C‐rich sequences.     

Formation of Human Telomeric G-quadruplex Structures Induced by the Quaternary Benzophenanthridine Alkaloids:Sanguinarine,Nitidine,and Chelerythrine

The ligands which can facilitate the formation and stabilize G‐quadruplex structures have attracted enormous attention due to their potential ability of inhibiting the telomerase activity and halting tumor cell proliferation. It is noteworthy that the abilities of the quaternary benzophenanthridine alkaloids (QBAs), the very important G‐quadruplex binders, in inducing the formation of human telomeric DNA G‐quadruplex structures, have not been reported. Herein, the interaction between single‐strand human telomeric DNA and three QBAs: Sanguinarine (San), Nitidine (Nit) and Chelerythrine (Che), has been investigated. Although these molecules are very similar in structure, they exhibit significantly different abilities in inducing oligonucleotide d(TTAGGG)₄ (HT4) to specific G‐quadruplex structures. Our experimental results indicated that the best ligand San could convert HT4 into antiparallel G‐quadruplex structure completely, followed by Nit, which could transform to mixed‐type or hybrid G‐quadruplex structure partially, whereas Che could only transform to antiparallel G‐quadruplex structure in small quantities. The relative QBAs' inducing abilities as indicated by the CD data are in the order of San>Nit>Che. Further investigation revealed that the G‐quadruplex structures from HT4 induced by QBAs are of intramolecular motif. And only sequences with certain length could be induced by QBAs because of their positive charges which could not attract short chain DNA molecules to close to each other and form intermolecular G‐quadruplex. In addition, the factors that affect the interaction between HT4 and QBAs were discussed. It is proposed that the thickness of the molecular frame and the steric hindrance are the primary reasons why the subtle differences in QBAs' structure lead to their remarkable differences in inducing the formation of the G‐quadruplex structures.