Characterization of structure and stability of long telomeric DNA G-quadruplexes

In the current study, we used a combination of gel electrophoresis, circular dichroism, and UV melting analysis to investigate the structure and stability of G-quadruplexes formed by long telomeric DNAs from Oxytricha and human, where the length of the repeat (n)=4 to 12. We found that the Oxytricha telomeric DNAs, which have the sequence (TTTTGGGG)n, folded into intramolecular and intermolecular G-quadruplexes depending on the ionic conditions, whereas human telomeric DNAs, which have the sequence (TTAGGG)n, formed only intramolecular G-quadruplexes in all the tested conditions. We further estimated the thermodynamic parameters of the intramolecular G-quadruplex. We found that thermodynamic stabilities of G-quadruplex structures of long telomeric DNAs (n=5 to 12) are mostly independent of sequence length, although telomeric DNAs are more stable when n=4 than when n>or=5. Most importantly, when n is a multiple of four, the change in enthalpy and entropy for G-quadruplex formation increased gradually, demonstrating that the individual G-quadruplex units are composed of four repeats and that the individual units do not interact. Therefore, we propose that the G-quadruplexes formed by long telomeric DNAs (n>or=8) are bead-on-a-string structures in which the G-quadruplex units are connected by one TTTT (Oxytricha) or TTA (human) linker. These results should be useful for understanding the structure and function of telomeres and for developing improved therapeutic agents targeting telomeric DNAs.     

Structure of human telomeric DNA in crowded solution

G-quadruplex structures formed by DNA at the human telomeres are attractive anticancer targets. Human telomeric sequences can adopt a diverse range of intramolecular G-quadruplex conformations: a parallel-stranded conformation was observed in the crystalline state, while at least four other forms were seen in K(+) solution, raising the question of which conformation is favored in crowded cellular environment. Here, we report the first NMR structure of a human telomeric G-quadruplex in crowded solution. We show that four different G-quadruplex conformations are converted to a propeller-type parallel-stranded G-quadruplex in K(+)-containing crowded solution due to water depletion. This study also reveals the formation of a new higher-order G-quadruplex structure under molecular crowding conditions. Our molecular dynamics simulations of solvent distribution provide insights at molecular level on the formation of parallel-stranded G-quadruplex in environment depleted of water. These results regarding human telomeric DNA can be extended to oncogenic promoters and other genomic G-rich sequences.     

Telomestatin and diseleno sapphyrin bind selectively to two different forms of the human telomeric G-quadruplex structure

The human telomeric sequence d[T(2)AG(3)](4) has been demonstrated to form different types of G-quadruplex structures, depending upon the incubation conditions. For example, in sodium (Na(+)), a basket-type G-quadruplex structure is formed. In this investigation, using circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and a polymerase stop assay, we have examined how the addition of different G-quadruplex-binding ligands affects the conformation of the telomeric G-quadruplex found in solution. The results show that while telomestatin binds preferentially to the basket-type G-quadruplex structure with a 2:1 stoichiometry, 5,10,15,20-[tetra-(N-methyl-3-pyridyl)]-26-28-diselena sapphyrin chloride (Se2SAP) binds to a different form with a 1:1 stoichiometry in potassium (K(+)). CD studies suggest that Se2SAP binds to a hybrid G-quadruplex that has strong parallel and antiparallel characteristics, suggestive of a structure containing both propeller and lateral, or edgewise, loops. Telomestatin is unique in that it can induce the formation of the basket-type G-quadruplex from a random coil human telomeric oligonucleotide, even in the absence of added monovalent cations such as K(+) or Na(+). In contrast, in the presence of K(+), Se2SAP was found to convert the preformed basket G-quadruplex to the hybrid structure. The significance of these results is that the presence of different ligands can determine the type of telomeric G-quadruplex structures formed in solution. Thus, the biochemical and biological consequences of binding of ligands to G-quadruplex structures found in telomeres and promoter regions of certain important oncogenes go beyond mere stabilization of these structures.     

An intramolecular G-quadruplex structure is required for binding of telomeric repeat-containing RNA to the telomeric protein TRF2

Telomeric repeat-containing RNA (TERRA) is important for telomere regulation, but the structural basis for how TERRA localizes to chromosome ends is unknown. Here we report on studies exploring whether the TERRA G-quadruplex structure is critical for binding to telomeres. We demonstrate that the telomeric protein TRF2 binds TERRA via interactions that necessitate the formation of a G-quadruplex structure rather than the TERRA sequence per se. We also show that TRF2 simultaneously binds TERRA and telomeric duplex or G-quadruplex DNA. These observations suggest that the TERRA G-quadruplex is a key feature of telomere organization.     

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.     

电喷雾质谱法研究天然产物小分子识别人类端粒G-四链体及复合物的热稳定性

利用电喷雾质谱(ESI-MS)研究了12种天然产物小分子与人类端粒G-四链体结构的非共价相互作用和识别功能, 比较了不同小分子与人类端粒G-四链体的结合强弱, 发现了一种新的识别小分子——防己诺林碱对人类端粒G-四链体有很好的结合. 通过质谱升温实验比较了小分子结合对G-四链体热稳定性的影响, 防己诺林碱的结合使G-四链体的离子的解离温度(T1/2)上升到200 ℃. 利用分子模拟对G-四链体DNA与小分子结合的模式以及稳定性进行了探讨, 给出了防己诺林碱可能的结合位点和结合模式, Autodock计算出来的结合能约为-31.5 kJ·mol-1. 同原来的平面型分子不同, 防己诺林碱是一类新型结构的分子, 为设计合成新型G-四链体识别分子提供了新的结构模型.     

(3 + 1) Assembly of three human telomeric repeats into an asymmetric dimeric G-quadruplex

We present an NMR study on the structure of a DNA fragment of the human telomere containing three guanine-tracts, d(GGGTTAGGGTTAGGGT). This sequence forms in Na(+) solution a unique asymmetric dimeric quadruplex, in which the G-tetrad core involves all three G-tracts of one strand and only the last 3'-end G-tract of the other strand. We show that a three-repeat human telomeric sequence can also associate with a single-repeat human telomeric sequence into a structure with the same topology that we name (3 + 1) quadruplex assembly. In this G-quadruplex assembly, there are one syn.syn.syn.anti and two anti.anti.anti.syn G-tetrads, two edgewise loops, three G-tracts oriented in one direction and the fourth oriented in the opposite direction. We discuss the possible implications of the new folding topology for understanding the structure of telomeric DNA, including t-loop formation, and for targeting G-quadruplexes in the telomeres.     

Structural basis for telomeric G-quadruplex targeting by naphthalene diimide ligands

The folding of the single-stranded 3' end of the human telomere into G-quadruplex arrangements inhibits the overhang from hybridizing with the RNA template of telomerase and halts telomere maintenance in cancer cells. The ability to thermally stabilize human telomeric DNA as a four-stranded G-quadruplex structure by developing selective small molecule compounds is a therapeutic path to regulating telomerase activity and thereby selectively inhibit cancer cell growth. The development of compounds with the necessary selectivity and affinity to target parallel-stranded G-quadruplex structures has proved particularly challenging to date, relying heavily upon limited structural data. We report here on a structure-based approach to the design of quadruplex-binding ligands to enhance affinity and selectivity for human telomeric DNA. Crystal structures have been determined of complexes between a 22-mer intramolecular human telomeric quadruplex and two potent tetra-substituted naphthalene diimide compounds, functionalized with positively charged N-methyl-piperazine side-chains. These compounds promote parallel-stranded quadruplex topology, binding exclusively to the 3' surface of each quadruplex. There are significant differences between the complexes in terms of ligand mobility and in the interactions with quadruplex grooves. One of the two ligands is markedly less mobile in the crystal complex and is more quadruplex-stabilizing, forming multiple electrostatic/hydrogen bond contacts with quadruplex phosphate groups. The data presented here provides a structural rationale for the biophysical (effects on quadruplex thermal stabilization) and biological data (inhibition of proliferation in cancer cell lines and evidence of in vivo antitumor activity) on compounds in this series and, thus, for the concept of telomere targeting with DNA quadruplex-binding small molecules.     

Structure of the human telomere in K+ solution: an intramolecular (3 + 1) G-quadruplex scaffold

We present the intramolecular G-quadruplex structure of human telomeric DNA in physiologically relevant K(+) solution. This G-quadruplex, whose (3 + 1) topology differs from folds reported previously in Na(+) solution and in a K(+)-containing crystal, involves the following: one anti.syn.syn.syn and two syn.anti.anti.anti G-tetrads; one double-chain reversal and two edgewise loops; three G-tracts oriented in one direction and the fourth in the opposite direction. The topological characteristics of this (3 + 1) G-quadruplex scaffold should provide a unique platform for structure-based anticancer drug design targeted to human telomeric DNA.     

Specific recognition of human telomeric G-quadruplex DNA with small molecules and the conformational analysis by ESI mass spectrometry and circular dichroism spectropolarimetry

Electrospray ionization mass spectrometry (ESI-MS) was utilized to investigate the binding affinity and stoichiometry of small molecules with human telomeric G-quadruplex DNA. The binding-affinity order obtained for the (AGGGTT)(4) quadruplex was: Tel01>ImImImbetaDp>PyPyPygammaImImImbetaDp. The specific binding of Tel01 and PyPyPygammaImImImbetaDp in one system consisting of human telomeric G-quadruplex and duplex DNA was observed directly for the first time. This revealed that PyPyPygammaImImImbetaDp has a binding specificity for the duplex DNA, whereas Tel01 selectively recognizes the G-quadruplex DNA. Moreover, both ESI-MS and circular dichroism (CD) spectra indicated that Tel01 favored the formation and stabilization of the antiparallel G-quadruplex, and a structural transition of the (AGGGTT)(4) sequence from a coexistence of parallel and antiparallel G-quadruplexes to a parallel G-quadruplex induced by annealing.     

Affinity of the anthracycline antitumor drugs Doxorubicin and Sabarubicin for human telomeric G-quadruplex structures

Combining various techniques in solution we proved that Doxorubicin, also called Adriamycin, and Sabarubicin, also known as MEN 10755, bind to the human telomeric sequence, 5'-d[GGG(TTAGGG)(3)]-3' (21-mer), assuming a G-quadruplex structure in the presence of K(+). Complexes of drugs with the 21-mer in 1?:?1 and 2?:?1 stoichiometry coexist in solution. Association constants were obtained from titration experiments and confirmed by isothermal titration calorimetry. The fluorescence of the drugs was quenched upon complexation. UV circular dichroism (CD) spectra of the complexes were characterized by the G-quadruplex signal and indicated that drug binding influences the equilibrium between quadruplex conformations. The visible CD spectra were exclusively due to the drug and show differences in the complexation modes of the two drugs. Spectroscopic and thermodynamic parameters of the 1?:?1 complexes point to drug stacking with the G-quadruplex top or bottom tetrad. Thermodynamic data suggests that the binding of the second drug molecule in the 2?:?1 complex may occur in a groove. Complexation caused a small increase in the thermal stability of the G-quadruplex main conformation, shifting T(m) from 62 to 67 °C.     

Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular g-quadruplex

Telomestatin is a natural product isolated from Streptomyces anulatus 3533-SV4 and has been shown to be a very potent telomerase inhibitor. The structural similarity between telomestatin and a G-tetrad suggested to us that the telomerase inhibition might be due to its ability either to facilitate the formation of or trap out preformed G-quadruplex structures, and thereby sequester single-stranded d[T(2)AG(3)](n) primer molecules required for telomerase activity. Significantly, telomestatin appears to be a more potent inhibitor of telomerase (5 nM) than any of the previously described G-quadruplex-interactive molecules. In this communication we provide the first experimental evidence that telomestatin selectively facilitates the formation of or stabilizes intramolecular G-quadruplexes, in particular, that produced from the human telomeric sequence d[T(2)AG(3)](4). A simulated annealing (SA) docking approach was used to study the binding interactions of telomestatin with the intramolecular antiparallel G-quadruplex structure. Each intramolecular G-quadruplex molecule was found to bind two telomestatin molecules (unpublished results). A 2:1 model for the telomestatin bound in the external stacking mode in an energy minimized complex with the human telomeric basket-type G-quadruplex was constructed. Our observation that a G-quadruplex-interactive molecule without significant groove interactions is able to reorient in a G-quadruplex structure proints to the importance of core interaction with an asymmetric G-quadruplex structure in producing selective binding. Furthermore, the G-quadruplex interactions of telomestatin are more selective for the intramolecular structure in contrast to other G-quadruplex-interactive agents, such as TMPyP4.     

Formation of (3+1) G-quadruplexes with a long loop by human telomeric DNA spanning five or more repeats

Structural studies of human telomeric repeats represent an active field of research with potential applications toward the development of specific telomeric quadruplex-targeting drugs for anticancer treatment. To date, high-definition structures were limited to DNA sequences containing up to four GGGTTA repeats. Here we investigate the formation of G-quadruplexes in sequences spanning five to seven human telomeric repeats using NMR, UV, and CD spectroscopy. A (3+1) G-quadruplex with a long propeller loop was isolated from a five-repeat sequence utilizing a guanine-to-inosine substitution. A simple approach of selective site-specific labeling of guanine residues was devised to rigorously determine the folding topology of the oligonucleotide. The same scaffold could be extrapolated to six- and seven-repeat sequences. Our results suggest that long human telomeric sequences consisting of five or more GGGTTA repeats could adopt (3+1) G-quadruplex structures harboring one or more repeat(s) within a single loop. We report on the formation of a Watson-Crick duplex within the long propeller loop upon addition of the complementary strand, demonstrating that the long loop could serve as a new recognition motif.     

靶向G-四链体的小分子配体在癌症治疗中应用的研究进展

G-四链体是一类由Hoogsteen氢键维持稳定的,富含鸟嘌呤的DNA或RNA二级结构。人类基因组中存在大量潜在的形成G-四链体的序列,所形成的G-四链体结构能够调控基因组的稳定性、DNA复制和基因表达,其中包括很多与癌症相关基因。因此寻找能够诱导DNA的G富集区域形成G-四链体结构的配体,进而筛选潜在抗癌药物的先导化合物,已成为癌症治疗研究的热点之一。本文对近年来发现和设计的以G-四链体为靶点的小分子配体,按照靶向的G-四链体结构类型和配体的分子结构进行分类,综述了这类化合物在癌症治疗方面的研究进展,分析了相关靶向治疗存在的问题,并对未来的研究方向进行了展望。     

Mass spectrometric studies on effects of counter ions of TMPyP4 on binding to human telomeric DNA and RNA G-quadruplexes

A comparative study on human telomeric DNA G-quadruplex binding of meso-5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) between its two salt forms, i.e., tetratosylate and tetrachloride, was conducted by using ESI-TOF-MS, UV-melting measurement, and molecular modeling methods. Besides cation TMPyP4, the tosyl anion was found to bind to human telomeric DNA G-quadruplex with multiple binding stoichiometries from 1:1 to 3:1 observed in ESI-TOF-MS spectra, indicating that the stabilization activity of TMPyP4 tetratosylate on G-quadruplex is derived from a synergetic effect of both TMPyP4 cation and tosyl anion. A molecular modeling study suggests that a tosyl anion fills up the vacant space between TMPyP4 cation and DNA G-quadruplex and thus stabilizes the complex by 3.8 kcal/mol. Therefore, it is estimated that TMPyP4 tetratosylate’s activity might not reflect the real effect of TMPyP4 cation in some bioassays related to G-quadruplex stabilization. This was verified by the results of less binding affinity of TMPyP4 tetrachloride with DNA G-quadruplex obtained from ESI-TOF-MS measurement, and of 2.27 °C less thermal stabilization of TMPyP4 tetrachloride for DNA G-quadruplex, compared to its tetratosylate under the same conditions. Our study demonstrated the influence of counter ions of TMPyP4 on G-quadruplex binding, which sheds light on the proper usage of TMPyP4 salt in the chemical and biological research associated with G-quadruplex binding. Subsequently, the binding of TMPyP4 tetrachloride to human telomeric RNA G-quadruplexes was studied with ESI-TOF-MS technique. The binding constants of TMPyP4 with human telomeric G-quadruplexes indicated that TMPyP4 binds to human telomeric RNA G-quadruplex one order of magnitude stronger than DNA counterpart. This is a comprehensive mass spectrometric report on binding study of TMPyP4 with human telomeric DNA/RNA G-quadruplexes.

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Chiral metallo-supramolecular complexes selectively induce human telomeric G-quadruplex formation under salt-deficient conditions

Chiral molecular recognition of human telomeric DNA is important for rational drug design and developing structural probes of G-quadruplexes. Here we report that a chiral supramolecular complex can selectively induce human telomeric G-quadruplex formation and discriminate different G-quadruplex sequences under salt-deficient conditions studied by circular dichroism (CD), UV meltings, stopped-flow spectroscopy, fluorescence resonance energy transfer, enzyme cleavage, and gel electrophoresis. P-enantiomer induced G-quadruplex formation is fast and does not require a large excess of P enantiomer. More importantly, this chiral compound induces loop sequence-dependent G-quadruplex formation.     

Molecular Recognition of the Hybrid‐2 Human Telomeric G‐Quadruplex by Epiberberine: Insights into Conversion of Telomeric G‐Quadruplex Structures

Human telomeres can form DNA G‐quadruplex (G4), an attractive target for anticancer drugs. Human telomeric G4s bear inherent structure polymorphism, challenging for understanding specific recognition by ligands or proteins. Protoberberines are medicinal natural‐products known to stabilize telomeric G4s and inhibit telomerase. Here we report epiberberine (EPI) specifically recognizes the hybrid‐2 telomeric G4 predominant in physiologically relevant K⁺ solution and converts other telomeric G4 forms to hybrid‐2, the first such example reported. Our NMR structure in K⁺ solution shows EPI binding induces extensive rearrangement of the previously disordered 5′‐flanking and loop segments to form an unprecedented four‐layer binding pocket specific to the hybrid‐2 telomeric G4; EPI recruits the (?1) adenine to form a “quasi‐triad” intercalated between the external tetrad and a T:T:A triad, capped by a T:T base pair. Our study provides structural basis for small‐molecule drug design targeting the human telomeric G4.     

Identifying G-quadruplex-binding ligands using DNA-functionalized gold nanoparticles

The G-rich overhang of human telomere tends to form a G-quadruplex structure, and G-quadruplex formation can effectively inhibit telomerase activity in most cancer cells. Therefore, it is important to identify the formation and properties of the G-quadruplex, with the particular aim of selecting G-quadruplex-binding ligands that could potentially lead to the development of anticancer therapeutic agents. With this goal in mind, we report a fluorescence resonance energy transfer (FRET) assay system for the identification of G-quadruplex ligands using DNA-functionalized gold nanoparticles (DNA-GNPs) as the fluorescence quencher and a carboxyfluorescein (FAM)-tagged human telomeric sequence (F-GDNA) as the recognition probe. A thiolated complementary strand of human telomeric DNA (cDNA), which first adheres to the surface of the GNPs and then hybridizes with F-GDNA, results in the fluorescence quenching of F-GDNA by the GNPs. However, fluorescence is restored when single-stranded F-GDNA folds into a G-quadruplex structure upon the binding of quadruplex ligands, leading to the release of F-GDNA from the surface of the GNPs. Combined data from fluorescence measurements and CD spectroscopy indicated that ligands selected by this FRET method could induce GDNA to form a G-quadruplex. Therefore, this FRET G-quadruplex assay is a simple and effective approach to identify quadruplex-binding ligands, and, as such, it promises to provide a solid foundation for the development of novel anticancer therapeutic agents.     

Methods for investigating G-quadruplex DNA/ligand interactions

DNA is considered an important target for drug design and development. Until recently, the focus was on double-stranded (duplex) DNA structures. However, it has now been shown that single stranded DNA can fold into hairpin, triplex, i-motif and G-quadruplex structures. The more interesting G-quadruplex DNA structures comprise four strands of stacked guanine (G)-tetrads formed by the coplanar arrangement of four guanines, held together by Hoogsteen bonds. The DNA sequences with potential to form G-quadruplex structures are found at the chromosomal extremities (i.e. the telomeres) and also at the intra-chromosomal region (i.e. oncogenic promoters) in several important oncogenes. The formation of G-quadruplex structures is considered to have important consequences at the cellular level and such structures have been evoked in the control of expression of certain genes involved in carcinogenesis (c-myc, c-kit, K-ras etc.) as well as in the perturbation of telomeric organization. It has been shown that the formation of quadruplexes inhibits the telomere extension by the telomerase enzyme, which is up-regulated in cancer cells. Therefore, G-quadruplex structures are an important target for drug design and development and there is a huge interest in design and development of small molecules (ligands) to target these structures. A large number of so-called G-quadruplex ligands, displaying varying degrees of affinity and more importantly selectivity (i.e. the ability to interact only with quadruplex-DNA and not duplex-DNA), have been reported. Access to efficient and robust in vitro assays is needed to effectively monitor and quantify the G-quadruplex DNA/ligand interactions. This tutorial review provides an overview of G-quadruplex ligands and biophysical techniques available to monitor such interactions.