Human Telomeric G-quadruplex Research Articles

Involvement of Long-Lived Intermediate States in the Complex Folding Pathway of the Human Telomeric G-Quadruplex.

The energy landscapes of human telomeric G-quadruplexes are complex, and their folding pathways have remained largely unexplored. By using real-time NMR spectroscopy, we investigated the K(+)-induced folding of the human telomeric DNA sequence 5'-TTGGG(TTAGGG)3 A-3'. Three long-lived states were detected during folding: a major conformation (hybrid-1), a previously structurally uncharacterized minor conformation (hybrid-2), and a partially unfolded state. The minor hybrid-2 conformation is formed faster than the more stable hybrid-1 conformation. Equilibration of the two states is slow and proceeds via a partially unfolded intermediate state, which can be described as an ensemble of hairpin-like structures.

Pyridostatins selectively recognize two different forms of the human telomeric G-quadruplex structures and their anti-tumor activities in vitro

G-quadruplex as a therapeutic target to develop novel anti-cancer agents has attracted a growing interest. Among all the ligands of G-quadruplexes, pyridostatin derivatives play a very important role. Here, we first reported the recognition of the fundamental skeleton pyridostatin I, which was simply synthesized. Compared to pyridostatin II comprising terminal amino groups, pyridostatin I selectively stabilized intramolecular anti-parallel telomeric G-quadruplex by raising the melting temperature about 20 °C at 295 nm of H22, while pyridostatin II preferred to stabilize intermolecular parallel telomeric G-quadruplex by raising the melting temperature about 25 °C at 265 nm of H7, maybe due to the suited size measurements between G-quadruplex hosts and pyridostatin guests. MTT assays indicated that pyridostatin II had better cytotoxic effects against HCT-8 and A549 cell lines obviously, indicating positively charged side chains may be required for improving the water solubility and cellular uptake of the apolar central skeleton.

Effects of Salt on the Stability of a G-Quadruplex from the Human c-MYC Promoter

In an atmosphere of potassium ions, a modified c-MYC NHE III1 sequence with two G-to-T mutations (MYC22-G14T/G23T) forms a highly stable parallel-stranded G-quadruplex. The G-quadruplex exhibits a steady increase in its melting temperature, T(M), with an increase in the concentration of the stabilizing cation K(+). On the other hand, an increase in the concentration of nonstabilizing Cs(+) or TMA(+) cations at a constant concentration of K(+) causes a sharp decline in T(M) followed by a leveling off at ∼200 mM Cs(+) or TMA(+). At 51 °C and 600 μM K(+), an increase in Cs(+) concentration from 0 to 800 mM leads to a complete unfolding of the G-quadruplex. These observations are consistent with the picture in which more counterions accumulate in the vicinity of the unfolded state of MYC22-G14T/G23T (nonspecific ion binding) than in that of the G-quadruplex state. We estimate that the unfolded state condenses one extra counterion compared to the G-quadruplex state. Taken together with our earlier results, our data suggest that sodium or potassium cations sequestered inside the central cavity stabilize the G-quadruplex conformation acting as specifically bound ligands. Nonspecifically bound (condensed) counterions may slightly stabilize, exert no influence (human telomeric G-quadruplexes), or strongly destabilize (MYC22-G14T/G23T) the G-quadruplex conformation. We offer a structural rationalization for the enhanced thermal stability of the MYC22-G14T/G23T G-quadruplex.

ATP-dependent G-quadruplex unfolding by Bloom helicase exhibits low processivity.

Various helicases and single stranded DNA (ssDNA) binding proteins unfold G-quadruplex (GQ) structures. However, the underlying mechanisms of this activity have only recently come to focus. We report kinetic studies on Bloom (BLM) helicase and human telomeric GQ interactions using single-molecule Förster resonance energy transfer (smFRET). Using partial duplex DNA (pdDNA) constructs with different 5′ ssDNA overhangs, we show that BLM localizes in the vicinity of ssDNA/double-stranded DNA (dsDNA) junction and reels in the ssDNA overhang in an ATP-dependent manner. A comparison of DNA constructs with or without GQ in the overhang shows that GQ unfolding is achieved in 50–70% of reeling attempts under physiological salt and pH conditions. The unsuccessful attempts often result in dissociation of BLM from DNA which slows down the overall BLM activity. BLM-mediated GQ unfolding is typically followed by refolding of the GQ, a pattern that is repeated several times before BLM dissociates from DNA. BLM is significantly less processive compared to the highly efficient GQ destabilizer Pif1 that can repeat GQ unfolding activity hundreds of times before dissociating from DNA. Despite the variations in processivity, our studies point to possible common patterns used by different helicases in minimizing the duration of stable GQ formation.

Molecular dynamics and principal components of potassium binding with human telomeric intra-molecular G-quadruplex.

Telomere assumes intra-molecular G-quadruplex that is a significant drug target for inhibiting telomerase maintenance of telomeres in cancer. Metal cations have been recognized as playing important roles in stabilizing G-quadruplex, but their binding processes to human telomeric G-quadruplex remain uncharacterized. To investigate the detailed binding procedures, molecular dynamics simulations were conducted on the hybrid [3 + 1] form-one human telomeric intra-molecular G-quadruplex. We show here that the binding of a potassium ion to a G-tetrad core is mediated by two alternative pathways. Principal component analysis illustrated the dominant concerted motions of G-quadruplex occurred at the loop domains. MM-PBSA calculations revealed that binding was energetically favorable and driven by the electrostatic interactions. The lower binding site was found more constructive favorable for binding. Our data provide useful information on a potassium-mediated stable structure of human telomeric intra-molecular G-quadruplex, implicating in ion disorder associated conformational changes and targeted drug design.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0155-3) contains supplementary material, which is available to authorized users.

Synthesis and antiproliferative activity of 2,7-diamino l0-(3,5-dimethoxy)benzyl-9(10H)-acridone derivatives as potent telomeric G-quadruplex DNA ligands

A novel series of l0-(3,5-dimethoxy)benzyl-9(10H)-acridone derivatives with terminal ammonium substituents at C2 and C7 positions on the acridone ring were successfully synthesized as antiproliferation agents. The biologic activity of the acridone compounds against leukemia CCRF-CEM cells demonstrated that some of the compounds displayed good antiproliferative activity, among which compound 6a containing dimethylamine substituents at the terminal C2 and C7 positions exhibited the highest cytotoxicity with IC50 at 0.3μM. In addition compound 6a showed little toxicity against normal 293T cells proliferation with IC50 more than 100μM. Further study indicated that compound 6a had strong binding activity to human telomeric G-quadruplex DNA, as detected by mass spectrometry, CD spectroscopy, UV absorption, FRET and fluorescence quenching assays. Our data suggested that the activity of 6a might be associated with its stabilization of G-quadruplex DNA, which can be developed as potent antitumor agent.

Selective nuclei accumulation of ruthenium(II) complex enantiomers that target G-quadruplex DNA

Different enantiomers exhibit large differences in their biological activity and/or toxicity, but they rarely involve the relationship of the agents for molecular and cellular imaging with the chiral structure of ruthenium complexes. Here, we report that an enantiomer of a polypyridyl ruthenium complex can selectively accumulate in the nucleus of HepG2 cells. Confocal laser scanning microscopy studies show that this phenomenon occurs via a non-endocytotic, but temperature-dependent, mechanism of cellular uptake in HepG2 cells. DNA oligonucleotides with repetitive tracts of guanine bases that can form G-quadruplex structures have aroused interest as therapeutic agents and as targets for anticancer drug design. Various biophysical techniques show that the Λ-enantiomer of ruthenium complexes can selectively stabilize human telomeric G-quadruplex DNA and has a strong preference for G-quadruplex over duplex DNA. Judged from the NMR results, we speculate that at higher 4:1 ligand/G-quadruplex stoichiometry, complex Λ–Ru is likely to bind with each groove of the tetraplex in a dimeric form or intercalate with the G-tetrad in the 3′ terminal face and coexist with other modes. The molecular modeling analysis is in agreement with the NMR titrations performed in this investigation indicating that ruthenium complexes are actually characterized by a mixed binding mode. The results provide many opportunities for the development of novel agents for living cell-related studies.

All-atomic simulations on human telomeric G-quadruplex DNA binding with thioflavin T.

Ligand-stabilized human telomeric G-quadruplex DNA is believed to be an anticancer agent, as it can impede the continuous elongation of telomeres by telomerase in cancer cells. In this study, five well-established human telomeric G-quadruplex DNA models were probed on their binding behaviors with thioflavin T (ThT) via both conventional molecular dynamics (MD) and well-tempered metadynamics (WT-MetaD) simulations. Novel dynamics and characteristic binding patterns were disclosed by the MD simulations. It was observed that the K(+) promoted parallel and hybridized human telomeric G-quadruplex conformations pose higher binding affinities to ThT than the Na(+) and K(+) promoted basket conformations. It is the end, sandwich, and base stacking driven by π-π interactions that are identified as the major binding mechanisms. As the most energy favorable binding mode, the sandwich stacking observed in (3 + 1) hybridized form 1 G-quadruplex conformation is triggered by reversible conformational change of the G-quadruplex. To further examine the free energy landscapes, WT-MetaD simulations were utilized on G-quadruplex-ThT systems. It is found that all of the major binding modes predicted by the MD simulations are confirmed by the WT-MetaD simulations. The results in this work not only accord with existing experimental findings, but also reinforce our understanding on the dynamics of G-quadruplexes and aid future drug developments for G-quadruplex stabilization ligands.

DNA G-quadruplex detection system employing a protein fibril ligand

Thioflavin T (ThT), a typical probe of protein fibrils, also binds human telomeric G-quadruplexes with high specificity for other DNA structures. Upon binding, ThT fluoresces brightly, making it a promising G-quadruplex probe. Because ThT was originally used for detecting protein fibrils, false-positive detection and imaging of DNA G-quadruplexes is possible in the presence of proteins, such as in living cells. We therefore developed a system for the specific discrimination of DNA G-quadruplexes from protein fibrils through measurement of fluorescence resonance energy transfer (FRET) from ThT to a DNA duplex probe. We designed a complementary DNA (cDNA) that hybridizes with the single-stranded region near the DNA G-quadruplex–forming region. Hexidium iodide (HI), a typical DNA duplex fluorescent intercalator, was utilized as an acceptor for FRET from ThT. FRET occurred only when ThT bound the G-quadruplex region and HI bound the duplex region of the cDNA. Fluorescence analysis showed that this system can detect DNA G-quadruplexes even in the presence of protein fibrils.

Human telomeric RNA G-quadruplex response to point mutation in the G-quartets.

Many putative G-quadruplex forming sequences have been predicted to exist in the human genome and transcriptome. As these sequences are subject to point mutations or SNPs (single nucleotide polymorphisms) during the course of evolution, we attempt to understand impact of these mutations in context of RNA G-quadruplex formation using human telomeric RNA (TERRA) as a model sequence. Our studies suggest that G-quadruplex stability is sensitive to substitution of the guanines comprising G-quartets. While central G-quartet plays a crucial role in maintaining the DNA G-quadruplex stability as evident in literature, there is equal importance of three G-quartets in the stability of RNA quadruplex structure. The work here highlights the alterations in the G-quartet are detrimental to the integrity of overall RNA G-quadruplex structure. Furthermore, TmPyP4 molecules are shown to exhibit similar binding behavior toward telomeric RNA G-quadruplex harboring base substitutions employing CD titrations and isothermal titration calorimetry; well indicating that mutation does not influence TmPyP4 recognition ability as it affects the stability of RNA G-quadruplex. Thus, our study implicates that mutation in G-quartets causes destabilization of RNA G-quadruplex without affecting its trans factor binding ability.

Positional impact of fluorescently modified G-tetrads within polymorphic human telomeric G-quadruplex structures.

Emissive C8-aryl-2'-deoxyguanosines placed within G-tetrads of G-quadruplex structures are useful probes for distinguishing G-quadruplexes from duplex structures using fluorescence spectroscopy. Here, we report the positional impact of C8-furyl-dG ((Fur)dG) on G-quadruplex folding in the human telomere 22-mer oligonucleotide (HTelo22, (d[AG3(T2AG3)3])). The (Fur)dG probe was inserted into four different positions within the three unique G-tetrads of HTelo22, and G-quadruplex folding was monitored by UV-vis thermal denaturation, circular dichroism, and fluorescence spectroscopy. Our studies demonstrate the impacts of C8-aryl-dG adduct formation on G-quadruplex polymorphism in K(+) solution and in the presence of the additives and cosolutes, CH3CN, polyethylene glycol (PEG-600), and N-methyl mesoporphyrin IX (NMM). Our experiments predict that C8-aryl-dG derivatives can serve as useful tools for various in vitro studies aimed at understanding both the implications of DNA adduct formation within G-quadruplex structures and the unique implications imposed by various folding topologies on biological function/recognition.

Thermodynamics of binding of di- and tetrasubstituted naphthalene diimide ligands to DNA G-quadruplex.

Naphthalene diimide ligands have the potential to stabilize human telomeric G-quadruplex DNA via noncovalent interactions. Stabilization of G-quadruplex high order structures has become an important strategy to develop novel anticancer therapeutics. In this study four naphthalene diimide based ligands were analyzed in order to elucidate the principal factors determining contributions to G-quadruplex-ligand binding. Three possible modes of binding and their respective Gibbs free energies for two naphthalene diimide based di-N-alkylpyridinium substituted ligands have been determined using a molecular docking technique and compared to experimental results. The structures obtained from the molecular docking calculations, were analyzed using the ab initio based fragment molecular orbital (FMO) method in order to determine the major enthalpic contributions to the binding and types of interactions between the ligand and specific residues of the G-quadruplex. A computational methodology for the efficient and inexpensive ligand optimization as compared to fully ab initio methods based on the estimation of binding affinities of the naphthalene diimide derived ligands to G-quadruplex is proposed.

A SERS/fluorescence dual-mode nanosensor based on the human telomeric G-quadruplex DNA: Application to mercury (II) detection.

DNA-metal nanoparticle conjugates have been increasingly exploited for sensing purposes over the past decades. However, most of the existing strategies are operated with canonical DNA structures, such as single-stranded forms, stem-loop structures, and double helix structures. There is intense interest in the development of nano-system based on high order DNA secondary structures. Herein, we propose a SERS/fluorescence dual-mode nanosensor, where the signal transduction mechanism is based on the conformational switching of the human telomeric G-quadruplex DNA. The nanosensor exhibits excellent SERS/fluorescence responses to the complementary strands of G-quadruplexes. Based on T-Hg(2+)-T coordination chemistry, this sensor is effectively applied to determination of Hg(2+) in buffer solution and real samples. It achieves a limit of detection (LOD) as low as 1ppt, which is ~100 times more sensitive than conventional optical sensors. We anticipate that the proposed G-quadruplex-based nanosensor could be applied to the analysis of other metal ions and small molecules in environmental samples and biological systems.

Specific binding of human telomeric G-quadruplex by homobarringtonie: An alkaloid from traditional Chinese medicine

In our study, electrospray ionization mass spectrometry (ESI–MS) was used to investigate the formation and recognition of human telomere G-quadruplex. Among the six small molecules studied, homobarringtonie (shortened as P1), one of the antineoplastic alkaloids separated from Chinese cephalotaxus fortunei, was examined having the highest binding ability with the human telomere G-quadruplex. And the most superiority of P1 was the good binding preference for the G-quadruplex than the duplex DNA. CD melting experiment results implied that P1 could enhance the thermo stability of the G-quadruplex. Moreover, Autodock analysis gave the result that P1 has the biggest docked energy to the telomere G-quadruplex, which was consistent with the result of ESI–MS. The high specificity and binding affinity of P1 to the telomere G-quadruplex make it potential for drug design and cancer relief.

Human Telomeric G-quadruplex DNA sequence (TTAGGGT)4 complexed with Flavonoid Quercetin

Human Telomeric G-quadruplex DNA sequence (TTAGGGT)4 complexed with Flavonoid Quercetin

Ligand-induced conformational changes with cation ejection upon binding to human telomeric DNA G-quadruplexes.

The rational design of ligands targeting human telomeric DNA G-quadruplexes is a complex problem due to the structural polymorphism that these sequences can adopt in physiological conditions. Moreover, the ability of ligands to switch conformational equilibria between different G-quadruplex structures is often overlooked in docking approaches. Here, we demonstrate that three of the most potent G-quadruplex ligands (360A, Phen-DC3, and pyridostatin) induce conformational changes of telomeric DNA G-quadruplexes to an antiparallel structure (as determined by circular dichroism) containing only one specifically coordinated K(+) (as determined by electrospray mass spectrometry) and, hence, presumably only two consecutive G-quartets. Control ligands TrisQ, known to bind preferentially to hybrid than to antiparallel structures, and L2H2-6M(2)OTD, known not to disrupt the hybrid-1 structure, did not show such K(+) removal. Instead, binding of the cyclic oxazole L2H2-6M(2)OTD was accompanied by the uptake of one additional K(+). Also contrasting with telomeric G-quadruplexes, the parallel-stranded Pu24-myc G-quadruplex, to which Phen-DC3 is known to bind by end-stacking, did not undergo cation removal upon ligand binding. Our study therefore evidences that very affine ligands can induce conformational switching of the human telomeric G-quadruplexes to an antiparallel structure and that this conformational change is accompanied by removal of one interquartet cation.

Binding modes of a core-extended metalloporphyrin to human telomeric DNA G-quadruplexes.

The molecular recognition of human telomeric G-quadruplexes by a novel cationic π-extended Ni(II)-porphyrin (Ni(II)-TImidP4) is studied in aqueous solutions via (chir)optical spectroscopy, Fluorescence Resonance Energy Transfer (FRET) melting assay, and computational molecular modeling. The results are systematically compared with the recognition by a conventional meso-substituted Ni(II)-porphyrin (Ni(II)-TMPyP4), which allows us to pinpoint the differences in binding modes depending on the G-quadruplex topology. Importantly, FRET melting assays show the higher selectivity of Ni(II)-TImidP4 towards human telomeric G4 than that of Ni(II)-TMPyP4.

The oxidative damage to the human telomere: effects of 5-hydroxymethyl-2′-deoxyuridine on telomeric G-quadruplex structures

As part of the genome, human telomeric regions can be damaged by the chemically reactive molecules responsible for oxidative DNA damage. Considering that G-quadruplex structures have been proven to occur in human telomere regions, several studies have been devoted to investigating the effect of oxidation products on the properties of these structures. However only investigations concerning the presence in G-quadruplexes of the main oxidation products of deoxyguanosine and deoxyadenosine have appeared in the literature. Here, we investigated the effects of 5-hydroxymethyl-2'-deoxyuridine (5-hmdU), one of the main oxidation products of T, on the physical-chemical properties of the G-quadruplex structures formed by two human telomeric sequences. Collected calorimetric, circular dichroism and electrophoretic data suggest that, in contrast to most of the results on other damage, the replacement of a T with a 5-hmdU results in only negligible effects on structural stability. Reported results and other data from literature suggest a possible protecting effect of the loop residues on the other parts of the G-quadruplexes.

Identification of novel interactors of human telomeric G-quadruplex DNA.

A chemoproteomic-driven approach was used to investigate the interaction network between human telomeric G-quadruplex DNA and nuclear proteins. We identified novel G-quadruplex binding partners, able to recognize these DNA structures at chromosome ends, suggesting a possible, and so far unknown, role of these proteins in telomere functions.

The molecular mechanism of ligand unbinding from the human telomeric G-quadruplex by steered molecular dynamics and umbrella sampling simulations.

G-quadruplexes are attractive drug targets in cancer therapy. Understanding the mechanisms of the binding-unbinding processes involving biomolecules and molecular recognition is essential for designing new drugs of G-quadruplexes. We performed steered molecular dynamics and umbrella sampling simulations to investigate the molecular mechanism and kinetics of ligand unbinding processes of the basket, propeller and hybrid G-quadruplex structures. Our studies of the ligand charge effect showed that Coulomb interaction plays a significant role in stabilizing the G-quadruplex structure in the unbinding process. The free energy profiles were carried out and the free energy changes associated with the unbinding process were computed quantitatively, whereas these results could help to identify accessible binding sites and transient interactions. The dynamics of the hydration shell water molecules around the G-quadruplex exhibits an abnormal Brownian motion, and the thickness and free energy of the hydration shell were estimated. A two-step relaxation scheme was theoretically developed to describe the kinetic reaction of BMVC and G-quadruplex interactions. Our computed results fall in a reasonable range of experimental data. The present investigation could be helpful in the structure-based drug design.