Interaction of hnRNP A1 with telomere DNA G-quadruplex structures studied at the single molecule level

G-rich telomeric DNA sequences can form G-quadruplex structures. The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and a shortened derivative (UP1) are active in telomere length regulation, and it has been reported that UP1 can unwind G-quadruplex structures. Here, we investigate the interaction of hnRNP A1 with G-quadruplex DNA structures containing the human telomere repeat (TTAGGG) by gel retardation assays, ensemble fluorescence energy transfer (FRET) spectroscopy, and single molecule FRET microscopy. Our biochemical experiments show that hnRNP A1 binds well to the G-quadruplex telomeric DNA. Ensemble and single molecule FRET measurements provide further insight into molecular conformation: the telomeric DNA overhang is found to be in a folded state in the absence of hnRNP A1 and to remain predominantly in a compact state when complexed with hnRNP A1. This finding is in contrast to the previously reported crystal structures of UP1-telomere DNA complexes where the DNA oligo within the protein-DNA complex is in a fully open conformation.     

Finding a human telomere DNA–RNA hybrid G-quadruplex formed by human telomeric 6-mer RNA and 16-mer DNA using click chemistry: A protective structure for telomere end

Telomeric repeat-containing RNA is a non-coding RNA molecule newly found in mammalian cells. The telomere RNA has been found to localize to the telomere DNA, but how the newly discovered RNA molecule interacts with telomere DNA is less known. In this study, using the click chemistry we successfully found that a 6-mer human telomere RNA and 16-mer human telomere DNA sequence can form a DNA–RNA hybrid type G-quadruplex structure. Detection of the click-reaction products directly probes DNA–RNA G-quadruplex structures in a complicated solution, whereas traditional methods such as NMR and crystallography may not be suitable. Importantly, we found that formation of DNA–RNA G-quadruplex induced an exonuclease resistance for telomere DNA, indicating that such structures might be important for protecting telomeric DNA from enzyme digestion to avoid telomere DNA shortening. These results provide the direct evidence for formation of DNA–RNA hybrid G-quadruplex structure by human telomere DNA and RNA sequence, suggesting DNA–RNA hybrid G-quadruplex structure associated between telomere DNA and RNA may respond to chromosome end protection and/or present a valuable target for drug design.     

The orientation of the ends of G-quadruplex structures investigated using end-extended oligonucleotides

Human telomere DNA is of intense interest because of its role in the biology of both cancer and aging. The single-stranded telomere terminus can adopt the structure of a G-quadruplex, which is of particular important for anticancer drug discovery many researchers have reported various G-quadruplex structures in the human telomere. Although the human telomere consists of a number of tandem repeats, higher-order G-quadruplex structures are less discussed due to the complexity of the structures. Here we examined the orientation of the ends of the G-quadruplex structures with consideration given to higher-order structures. We prepared end-extended and ^BrG-substituted oligonucleotides. Native PAGE analysis, CD measurements and NMR spectroscopy showed that the ends of stable G-quadruplex structures point in opposite directions. Our results indicate that the human telomere DNA is likely to form rod-like higher-order structures. This may provide important information for understanding telomere structure and the development of telomere G-quadruplex-binding molecules as telomerase inhibitors.     

Bisaryldiketene derivatives: A new class of selective ligands for c-myc G-quadruplex DNA

A series of bisaryldiketene derivatives were designed and synthesized as a new class of specific G-quadruplex ligands. The ligand-quadruplex interactions were further evaluated by FRET, ITC, and PCR stop assay. In contrast to most of the G-quadruplex ligands reported so far, which comprise an extended aromatic ring, these compounds are neither polycyclic nor macrocyclic, but have a non-aromatic and relative flexible linker between two quinoline moieties enabling the conformation of compounds to be flexible. Our results showed that these bisaryldiketene derivatives could selectively recognize G-quadruplex DNA rather than binding to duplex DNA. Moreover, they showed promising discrimination between different G-quadruplex DNA. The primary binding affinity of ligand M2 for c-myc G-quadruplex DNA was over 200 times larger than that for telomere G-quadruplex DNA.     

DNA-RNA hybrid G-quadruplex tends to form near the 3′ end of telomere overhang

Telomeric repeat-containing RNA (TERRA) has been suggested to participate in telomere maintenance. TERRA consisting of UUAGGG repeats is capable of forming an intermolecular G-quadruplex (GQ) with single-stranded TTAGGG-repeat DNA in the telomere 3′ overhang. To explore the structural features and potential functions of this DNA-RNA hybrid GQ (HGQ), we used single-molecule FRET to study the folding patterns of DNA with four to seven telomeric tandem repeats annealed with a short RNA consisting of two or five telomeric repeats. Our data highlight that RNA prefers to form DNA-RNA HGQ near the 3′ end of telomeric DNA. Furthermore, the unfolding of secondary structures by a complementary C-rich sequence was observed for DNA GQ but not for DNA-RNA HGQ, which demonstrated the enhanced stability of the telomere 3′ end via hybridization with RNA. These conformational and physical properties of telomeric DNA-RNA HGQ suggest that TERRA might limit access to the 3′ end of the telomeric DNA overhang, which is known to be critical for the interaction with telomerase and other telomere-associated proteins.     

Cloning, localization and differential expression of the Trypanosoma cruzi TcOGNT-2 glycosyl transferase

It is well established that G-quadruplex DNA structures form at ciliate telomeres and their formation throughout the cell-cycle by telomere-end-binding proteins (TEBPs) has been analyzed. During replication telomeric G-quadruplex structure has to be resolved to allow telomere replication by telomerase. It was shown that both phosphorylation of TEBPβ and binding of telomerase are prerequisites for this process, but probably not sufficient to unfold G-quadruplex structure in timely manner to allow replication to proceed. Here we describe a RecQ-like helicase required for unfolding of G-quadruplex structures in vivo. This helicase is highly reminiscent of human RecQ protein-like 4 helicase as well as other RecQ-like helicase found in various eukaryotes and E. coli. In situ analyses combined with specific silencing of either the telomerase or the helicase by RNAi and co-immunoprecipitation experiments demonstrate that this helicase is associated with telomerase during replication and becomes recruited to telomeres by this enzyme. In vitro assays showed that a nuclear extract prepared from cells in S-phase containing both the telomerase as well as the helicase resolves telomeric G-quadruplex structure. This finding can be incorporated into a mechanistic model about the replication of telomeric G-quadruplex structures during the cell cycle.     

Folding dynamics and conformational heterogeneity of human telomeric G-quadruplex structures in Na+ solutions by single molecule FRET microscopy

G-quadruplex structures can occur throughout the genome, including at telomeres. They are involved in cellular regulation and are potential drug targets. Human telomeric G-quadruplex structures can fold into a number of different conformations and show large conformational diversity. To elucidate the different G-quadruplex conformations and their dynamics, we investigated telomeric G-quadruplex folding using single molecule FRET microscopy in conditions where it was previously believed to yield low structural heterogeneity. We observed four FRET states in Na⁺ buffers: an unfolded state and three G-quadruplex related states that can interconvert between each other. Several of these states were almost equally populated at low to medium salt concentrations. These observations appear surprising as previous studies reported primarily one G-quadruplex conformation in Na⁺ buffers. Our results permit, through the analysis of the dynamics of the different observed states, the identification of a more stable G-quadruplex conformation and two transient G-quadruplex states. Importantly these results offer a unique view into G-quadruplex topological heterogeneity and conformational dynamics.     

G-quadruplex preferentially forms at the very 3' end of vertebrate telomeric DNA

Human chromosome ends are protected with kilobases repeats of TTAGGG. Telomere DNA shortens at replication. This shortening in most tumor cells is compensated by telomerase that adds telomere repeats to the 3′ end of the G-rich telomere strand. Four TTAGGG repeats can fold into G-quadruplex that is a poor substrate for telomerase. This property has been suggested to regulate telomerase activity in vivo and telomerase inhibition via G-quadruplex stabilization is considered a therapeutic strategy against cancer. Theoretically G-quadruplex can form anywhere along the long G-rich strand. Where G-quadruplex forms determines whether the 3′ telomere end is accessible to telomerase and may have implications in other functions telomere plays. We investigated G-quadruplex formation at different positions by DMS footprinting and exonuclease hydrolysis. We show that G-quadruplex preferentially forms at the very 3′ end than at internal positions. This property provides a molecular basis for telomerase inhibition by G-quadruplex formation. Moreover, it may also regulate those processes that depend on the structure of the very 3′ telomere end, for instance, the alternative lengthening of telomere mechanism, telomere T-loop formation, telomere end protection and the replication of bulky telomere DNA. Therefore, targeting telomere G-quadruplex may influence more telomere functions than simply inhibiting telomerase.     

Not so crystal clear: the structure of the human telomere G-quadruplex in solution differs from that present in a crystal

The structure of human telomere DNA is of intense interest because of its role in the biology of both cancer and aging. The sequence [5′-AGGG(TTAGGG)₃] has been used as a model for telomere DNA in both NMR and X-ray crystallographic studies, the results of which show dramatically different structures. In Na⁺ solution, NMR revealed an antiparallel G-quadruplex structure that featured both diagonal and lateral TTA loops. Crystallographic studies in the presence of K⁺ revealed a flattened, propeller-shaped structure featuring a parallel-stranded G-quadruplex with symmetrical external TTA loops. We report the results of biophysical experiments in solution and computational studies that are inconsistent with the reported crystal structure, indicating that a different structure exists in K⁺ solutions. Sedimentation coefficients were determined experimentally in both Na⁺ and K⁺ solutions and were compared with values calculated using bead models for the reported NMR and crystal structures. Although the solution NMR structure accurately predicted the observed S-value in Na⁺ solution, the crystal structure predicted an S-value that differed dramatically from that experimentally observed in K⁺ solution. The environments of loop adenines were probed by quantitative fluorescence studies using strategic and systematic single-substitutions of 2-aminopurine for adenine bases. Both fluorescence intensity and quenching experiments in K⁺ yielded results at odds with quantitative predictions from the reported crystal structure. Circular dichroism and fluorescence quenching studies in the presence of the crowding agent polyethylene glycol showed dramatic changes in the quadruplex structure in K⁺ solutions, but not in Na⁺ solutions, suggesting that the crystal environment may have selected for a particular conformational form. Molecular dynamics simulations were performed to yield model structures for the K⁺ quadruplex form that are consistent with our biophysical results and with previously reported chemical modification studies. These models suggest that the biologically relevant structure of the human telomere quadruplex in K⁺ solution is not the one determined in the published crystalline state.     

Modelling the regulation of telomere length: the effects of telomerase and G-quadruplex stabilising drugs

Telomeres are guanine-rich sequences at the end of chromosomes which shorten during each replication event and trigger cell cycle arrest and/or controlled death (apoptosis) when reaching a threshold length. The enzyme telomerase replenishes the ends of telomeres and thus prolongs the life span of cells, but also causes cellular immortalisation in human cancer. G-quadruplex (G4) stabilising drugs are a potential anticancer treatment which work by changing the molecular structure of telomeres to inhibit the activity of telomerase. We investigate the dynamics of telomere length in different conformational states, namely t-loops, G-quadruplex structures and those being elongated by telomerase. By formulating deterministic differential equation models we study the effects of various levels of both telomerase and concentrations of a G4-stabilising drug on the distribution of telomere lengths, and analyse how these effects evolve over large numbers of cell generations. As well as calculating numerical solutions, we use quasicontinuum methods to approximate the behaviour of the system over time, and predict the shape of the telomere length distribution. We find those telomerase and G4-concentrations where telomere length maintenance is successfully regulated. Excessively high levels of telomerase lead to continuous telomere lengthening, whereas large concentrations of the drug lead to progressive telomere erosion. Furthermore, our models predict a positively skewed distribution of telomere lengths, that is, telomeres accumulate over lengths shorter than the mean telomere length at equilibrium. Our model results for telomere length distributions of telomerase-positive cells in drug-free assays are in good agreement with the limited amount of experimental data available.     

Oligonucleotide Models of Telomeric DNA and RNA Form a Hybrid G-quadruplex Structure as a Potential Component of Telomeres

Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends.     

G-quadruplex motifs arranged in tandem occurring in telomeric repeats and the insulin-linked polymorphic region

To date, various G-quadruplex structures have been reported in the human genome. There are numerous studies focusing on quadruplex-forming sequences in general, but few studies have focused on two or more quadruplexes in the same molecule, which are most commonly found in telomeric DNA and other tandem repeats, e.g., insulin-linked polymorphic region (ILPR). Although the human telomere consists of a number of repeats, higher-order G-quadruplex structures are discussed less often because of the complexity of the structures. In this study, sequences consisting of 4-12 repeats of d(G(4)TGT), d(G(3)T(2)A), and/or d(G(4)T(2)A) have been studied by circular dichroism, ultraviolet spectroscopy, and temperature-gradient gel electrophoresis. These sequences serve as a model for the arrangement of quadruplexes in the telomere and ILPR in solution. Our major findings are as follows. (i) The number of G-rich repeats has a great influence on G-quadruplex stability. (ii) The evidence of quadruplex-quadruplex interaction is confirmed. (iii) For the first time, we directly observed the melting behavior of different conformers in a single experiment. Our results agree with other calorimetric and spectroscopic data and data obtained by single-molecule studies, atomic force microscopy, and mechanical unfolding by optical tweezers. We propose that the end of telomeres can be formed by only a few tandem quadruplexes (fewer than three). Our findings improve our understanding of the mechanism of G-quadruplex formation in long repeats in G-rich-regulating parts of genes and telomere ends.     

The effect of the TRF2 N-terminal and TRFH regions on telomeric G-quadruplex structures

The sequence of human telomeric DNA consists of tandem repeats of 5′-d(TTAGGG)-3′. This guanine-rich DNA can form G-quadruplex secondary structures which may affect telomere maintenance. A current model for telomere protection by the telomere-binding protein, TRF2, involves the formation of a t-loop which is stabilized by a strand invasion-like reaction. This type of reaction may be affected by G-quadruplex structures. We analyzed the influence of the arginine-rich, TRF2 N-terminus (TRF2^B), as well as this region plus the TRFH domain of TRF2 (TRF2^BH), on the structure of G-quadruplexes. Circular dichroism results suggest that oligonucleotides with 4, 7 and 8 5′-d(TTAGGG)-3′ repeats form hybrid structures, a mix of parallel/antiparallel strand orientation, in K⁺. TRF2^B stimulated the formation of parallel-stranded structures and, in some cases, intermolecular structures. TRF2^BH also stimulated intermolecular but not parallel-stranded structures. Only full-length TRF2 and TRF2^BH stimulated uptake of a telomeric single-stranded oligonucleotide into a plasmid containing telomeric DNA in the presence of K⁺. The results in this study suggest that G-quadruplex formation inhibits oligonucleotide uptake into the plasmid, but the inhibition can be overcome by TRF2. This study is the first analysis of the effects of TRF2 domains on G-quadruplex structures and has implications for the role of G-quadruplexes and TRF2 in the formation of t-loops.     

Fluorescence-based melting assays for studying quadruplex ligands

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomeres and telomerase are relevant targets in oncology, and telomere ligands and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analysed the FRET method used to measure the stabilization and selectivity of quadruplex ligands towards the human telomeric G-quadruplex. The stabilization value depends on the nature of the fluorescent tags, the incubation buffer, and the method chosen for T(m) calculation, complicating a direct comparison of the results obtained by different laboratories.     

Structure of two intramolecular G-quadruplexes formed by natural human telomere sequences in K+ solution

Intramolecular G-quadruplexes formed by human telomere sequences are attractive anticancer targets. Recently, four-repeat human telomere sequences have been shown to form two different intramolecular (3 + 1) G-quadruplexes in K(+) solution (Form 1 and Form 2). Here we report on the solution structures of both Form 1 and Form 2 adopted by natural human telomere sequences. Both structures contain the (3 + 1) G-tetrad core with one double-chain-reversal and two edgewise loops, but differ in the successive order of loop arrangements within the G-quadruplex scaffold. Our results provide the structural details at the two ends of the G-tetrad core in the context of natural sequences and information on different loop conformations. This structural information might be important for our understanding of telomere G-quadruplex structures and for anticancer drug design targeted to such scaffolds.     

G-quadruplex formation at the 3' end of telomere DNA inhibits its extension by telomerase, polymerase and unwinding by helicase

Telomere G-quadruplex is emerging as a promising anti-cancer target due to its inhibition to telomerase, an enzyme expressed in more than 85% tumors. Telomerase-mediated telomere extension and some other reactions require a free 3' telomere end in single-stranded form. G-quadruplex formation near the 3' end of telomere DNA can leave a 3' single-stranded tail of various sizes. How these terminal structures affect reactions at telomere end is not clear. In this work, we studied the 3' tail size-dependence of telomere extension by either telomerase or the alternative lengthening of telomere (ALT) mechanism as well as telomere G-quadruplex unwinding. We show that these reactions require a minimal tail of 8, 12 and 6 nt, respectively. Since we have shown that G-quadruplex tends to form at the farthest 3' distal end of telomere DNA leaving a tail of no more than 5 nt, these results imply that G-quadruplex formation may play a role in regulating reactions at the telomere ends and, as a result, serve as effective drug target for intervening telomere function.     

Molecular engineering of G-quadruplex ligands based on solvent effect of polyethylene glycol

Because various non-parallel G-quadruplexes of human telomeric sequences in K(+) solution can be converted to a parallel G-quadruplex by adding polyethylene glycol (PEG) as a co-solvent, we have taken advantage of this property of PEG to study the covalent attachment of a PEG unit to a G-quadruplex ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC). The hybrid ligand with the PEG unit, BMVC-8C3O or BMVC-6C2O by substituting either the tetraethylene glycol or the triethylene glycol terminated with a methyl-piperidinium cation in N-9 position of BMVC, not only induces structural change from different non-parallel G-quadruplexes to a parallel G-quadruplex but also increases the melting temperature of human telomeres in K(+) solution by more than 45°C. In addition, our ligand work provides further confidence that the local water structure plays the key to induce conformational change of human telomere.     

Amide bond direction modulates G-quadruplex recognition and telomerase inhibition by 2,6 and 2,7 bis-substituted anthracenedione derivatives

G-quadruplex structures of DNA represent a potentially useful target for anticancer drugs. Stabilisation of this arrangement at the ends of chromosomes may inhibit the action of telomerase, an enzyme involved in immortalization of cancer cells. Appropriately substituted amido anthracenediones are effective G-quadruplex stabilizers, but no information is available as yet on the possible modulation of G-quadruplex recognition and telomerase inhibition produced by the direction of the amide bond. To understand the basis of amido anthracenedione selectivity, we have synthesized a number of derivatives bearing the -CO-NH- or -NH-CO- group linked to the planar anthraquinone (AQ) moiety at 2,6 and 2,7 positions. The various isomers were tested in terms of telomerase inhibition, determined by the TRAP assay, G-quadruplex stabilisation measured by the increase in melting temperature of the appropriately folded oligonucleotide using FRET, and conformational and G4 binding properties examined by molecular modelling techniques. In all cases, enzymatic inhibition and G-quadruplex stabilization were directly related, which strongly supports the proposed molecular mechanism of telomerase interference. Interestingly, the AQ-NH-CO- arrangement performs invariantly better than the AQ-CO-NH- arrangement, showing a clear preference among isomeric derivatives. Theoretical calculations suggest that the former amide arrangement is co-planar with the aromatic system, whereas the latter is tilted by about 30 degrees when considering the most stable conformation. A more extended planar surface would allow more efficient stacking interactions with the quadruplex structure, hence more effective telomerase inhibition.     

Human replication protein A unfolds telomeric G-quadruplexes

G-quadruplex structures inhibit telomerase activity and must be disrupted for telomere elongation during S phase. It has been suggested that the replication protein A (RPA) could unwind and maintain single-stranded DNA in a state amenable to the binding of telomeric components. We show here that under near-physiological in vitro conditions, human RPA is able to bind and unfold G-quadruplex structures formed from a 21mer human telomeric sequence. Analyses by native gel electrophoresis, cross-linking and fluorescence resonance energy transfer indicate the formation of both 1:1 and 2:1 complexes in which G-quadruplexes are unfolded. In addition, quadruplex opening by hRPA is much faster than observed with the complementary DNA, demonstrating that this protein efficiently unfolds G-quartets. A two-step mechanism accounting for the binding of hRPA to G-quadruplexes is proposed. These data point to the involvement of hRPA in regulation of telomere maintenance.