Human telomere, oncogenic promoter and 5'-UTR G-quadruplexes: diverse higher order DNA and RNA targets for cancer therapeutics

Guanine-rich DNA sequences can form G-quadruplexes stabilized by stacked G–G–G–G tetrads in monovalent cation-containing solution. The length and number of individual G-tracts and the length and sequence context of linker residues define the diverse topologies adopted by G-quadruplexes. The review highlights recent solution NMR-based G-quadruplex structures formed by the four-repeat human telomere in K⁺ solution and the guanine-rich strands of c-myc, c-kit and variant bcl-2 oncogenic promoters, as well as a bimolecular G-quadruplex that targets HIV-1 integrase. Such structure determinations have helped to identify unanticipated scaffolds such as interlocked G-quadruplexes, as well as novel topologies represented by double-chain-reversal and V-shaped loops, triads, mixed tetrads, adenine-mediated pentads and hexads and snap-back G-tetrad alignments. The review also highlights the recent identification of guanine-rich sequences positioned adjacent to translation start sites in 5′-untranslated regions (5′-UTRs) of RNA oncogenic sequences. The activity of the enzyme telomerase, which maintains telomere length, can be negatively regulated through G-quadruplex formation at telomeric ends. The review evaluates progress related to ongoing efforts to identify small molecule drugs that bind and stabilize distinct G-quadruplex scaffolds associated with telomeric and oncogenic sequences, and outlines progress towards identifying recognition principles based on several X-ray-based structures of ligand–G-quadruplex complexes.     

Polymorphism of human telomeric quadruplex structures

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). Compounds that can stabilize the intramolecular DNA G-quadruplexes formed in the human telomeric sequence have been shown to inhibit the activity of telomerase and telomere maintenance, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. Knowledge of intramolecular human telomeric G-quadruplex structure(s) formed under physiological conditions is important for structure-based rational drug design and thus has been the subject of intense investigation. This review will give an overview of recent progress on the intramolecular human telomeric G-quadruplex structures formed in K(+) solution. It will also give insight into the structure polymorphism of human telomeric sequences and its implications for drug targeting.     

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.     

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.     

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

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

Sequence specificity of inter- and intramolecular G-quadruplex formation by human telomeric DNA

Human telomeric DNA consists of tandem repeats of the sequence 5'-d(TTAGGG)-3'. Guanine-rich DNA, such as that seen at telomeres, forms G-quadruplex secondary structures. Alternative forms of G-quadruplex structures can have differential effects on activities involved in telomere maintenance. With this in mind, we analyzed the effect of sequence and length of human telomeric DNA on G-quadruplex structures by native polyacrylamide gel electrophoresis and circular dichroism. Telomeric oligonucleotides shorter than four, 5'-d(TTAGGG)-3' repeats formed intermolecular G-quadruplexes. However, longer telomeric repeats formed intramolecular structures. Altering the 5'-d(TTAGGG)-3' to 5'-d(TTAGAG)-3' in any one of the repeats of 5'-d(TTAGGG)(4)-3' converted an intramolecular structure to intermolecular G-quadruplexes with varying degrees of parallel or anti-parallel-stranded character, depending on the length of incubation time and DNA sequence. These structures were most abundant in K(+)-containing buffers. Higher-order structures that exhibited ladders on polyacrylamide gels were observed only for oligonucleotides with the first telomeric repeat altered. Altering the sequence of 5'-d(TTAGGG)(8)-3' did not result in the substantial formation of intermolecular structures even when the oligonucleotide lacked four consecutive telomeric repeats. However, many of these intramolecular structures shared common features with intermolecular structures formed by the shorter oligonucleotides. The wide variability in structure formed by human telomeric sequence suggests that telomeric DNA structure can be easily modulated by proteins, oxidative damage, or point mutations resulting in conversion from one form of G-quadruplex to another.     

Preferential binding of a G-quadruplex ligand to human chromosome ends

The G-overhangs of telomeres are thought to adopt particular conformations, such as T-loops or G-quadruplexes. It has been suggested that G-quadruplex structures could be stabilized by specific ligands in a new approach to cancer treatment consisting in inhibition of telomerase, an enzyme involved in telomere maintenance and cell immortality. Although the formation of G-quadruplexes was demonstrated in vitro many years ago, it has not been definitively demonstrated in living human cells. We therefore investigated the chromosomal binding of a tritiated G-quadruplex ligand, ³H-360A (2,6-N,N′-methyl-quinolinio-3-yl)-pyridine dicarboxamide [methyl-³H]. We verified the in vitro selectivity of ³H-360A for G-quadruplex structures by equilibrium dialysis. We then showed by binding experiments with human genomic DNA that ³H-360A has a very potent selectivity toward G-quadruplex structures of the telomeric 3′-overhang. Finally, we performed autoradiography of metaphase spreads from cells cultured with ³H-360A. We found that ³H-360A was preferentially bound to chromosome terminal regions of both human normal (peripheral blood lymphocytes) and tumor cells (T98G and CEM1301). In conclusion, our results provide evidence that a specific G-quadruplex ligand interacts with the terminal ends of human chromosomes. They support the hypothesis that G-quadruplex ligands induce and/or stabilize G-quadruplex structures at telomeres of human cells.     

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.     

Telomeric repeat mutagenicity in human somatic cells is modulated by repeat orientation and G-quadruplex stability

Telomeres consisting of tandem guanine-rich repeats can form secondary DNA structures called G-quadruplexes that represent potential targets for DNA repair enzymes. While G-quadruplexes interfere with DNA synthesis in vitro, the impact of G-quadruplex formation on telomeric repeat replication in human cells is not clear. We investigated the mutagenicity of telomeric repeats as a function of G-quadruplex folding opportunity and thermal stability using a shuttle vector mutagenesis assay. Since single-stranded DNA during lagging strand replication increases the opportunity for G-quadruplex folding, we tested vectors with G-rich sequences on the lagging versus the leading strand. Contrary to our prediction, vectors containing human [TTAGGG]₁₀ repeats with a G-rich lagging strand were significantly less mutagenic than vectors with a G-rich leading strand, after replication in normal human cells. We show by UV melting experiments that G-quadruplexes from ciliates [TTGGGG]₄ and [TTTTGGGG]₄ are thermally more stable compared to human [TTAGGG]₄. Consistent with this, replication of vectors with ciliate [TTGGGG]₁₀ repeats yielded a 3-fold higher mutant rate compared to the human [TTAGGG]₁₀ vectors. Furthermore, we observed significantly more mutagenic events in the ciliate repeats compared to the human repeats. Our data demonstrate that increased G-quadruplex opportunity (repeat orientation) in human telomeric repeats decreased mutagenicity, while increased thermal stability of telomeric G-quadruplexes was associated with increased mutagenicity.     

Effects of a halogenated G-quadruplex ligand from the pyridine dicarboxamide series on the terminal sequence of XpYp telomere in HT1080 cells

Non-canonical four-stranded structures called G-quadruplexes can form among telomere repeats during its replication. Small molecule ligands able to interact and to stabilize G-quadruplexes were shown to disrupt the binding of essential telomeric components, such as POT1 and to trigger a telomeric dysfunction associated with a delayed growth arrest in tumor cells. We describe here the chemical synthesis and the G-quadruplex binding properties of three halogenated analogs of the 360A ligand that belongs to the 2,6 pyridine dicarboxamide series. 360A is now commonly used as a benchmark both for biophysical and cellular assays as this compound was shown to display a potent affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA and to induce delayed growth inhibition in HT1080 tumor cell line. Two biophysical assays indicate that, in most cases, the presence of the halogen atom seems to slightly improve the interaction with the telomeric quadruplex. For stability reasons, the bromo derivative (360A–Br) was selected for the cellular assays. Since POT1 participates to the fine tuning of the C-strand end resection during telomere replication, we investigated the effect of 360A–Br to alter the terminal nucleotide composition of XpYp telomere in HT1080 cells using C-STELA. HT1080 cells treated for up to 24 days with 360A–Br presented some minor but significant variations of C-strand terminal nucleotide composition, also observed with a partial siRNA depletion of POT1. The relevance of these minor modifications of the telomeric C-strand resection induced by 360A–Br in HT1080 cells are discussed.     

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

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

Structural diversity and extreme stability of unimolecular Oxytricha nova telomeric G-quadruplex

Oxytricha nova telomeric DNA contains guanine-rich short-tandem repeat sequences (GGGGTTTT) n and terminates as a single strand at the 3'-end. This single-stranded overhang forms a novel DNA structure, namely, G-quadruplex, comprising four quartets. In this study, we investigated the structures and dynamics of unimolecular Oxytricha nova ( O. nova) telomeric G-quadruplexes by performing single molecule fluorescence resonance energy transfer (FRET) spectroscopy and bulk circular dichroism (CD) measurements. We observed that unimolecular O. nova G-quadruplexes exhibit structural polymorphism according to monovalent cations. In the presence of Na (+), only antiparallel conformation is detected, which was demonstrated in previous studies; however, in the presence of K (+), they fold into two different conformations, a parallel conformation and an antiparallel one different from that induced by Na (+). Furthermore, these G-quadruplexes show extremely high stability in their dynamics when compared with human G-quadruplexes. While human telomeric G-quadruplexes that possess three quartets display fast dynamic behavior (<100 s) at low K (+) concentrations or high temperatures, O. nova G-quadruplexes maintain their conformational state for a long time (>1000 s), even at the lowest K (+) concentration and the highest temperature investigated. This high stability is primarily due to an extra quartet that results in additional cation coordination. In addition to cation coordination, we propose that other factors such as base stacking and the size of the thymine loop may contribute to the stability of O. nova G-quadruplexes; this is based on the fact that the O. nova G-quadruplexes were observed to be more stable than the human ones in the presence of Li (+), which is known to greatly destabilize G-quadruplexes because of imprecise coordination. This extreme stability of four-quartet G-quadruplexes enables telomere protection even in the absence of protective proteins or in the case of abrupt environmental changes, although only a single G-quadruplex structure can be derived from the short single-stranded overhang.     

Selection of zinc fingers that bind single-stranded telomeric DNA in the G-quadruplex conformation

There is considerable interest in molecules that bind to telomeric DNA sequences and G-quadruplexes with specificity. Such molecules would be useful to test hypotheses for telomere length regulation, and may have therapeutic potential. The versatility and modular nature of the zinc finger motif makes it an ideal candidate for engineering G-quadruplex-binding proteins. Phage display technology has previously been widely used to screen libraries of zinc fingers for binding to novel duplex DNA sequences. In this study, a three-finger library has been screened for clones that bind to an oligonucleotide containing the human telomeric repeat sequence folded in the G-quadruplex conformation. The selected clones show a strong amino acid consensus, suggesting analogous modes of binding. Binding was found to be both sequence dependent and structure specific. This is the first example of an engineered protein that binds to G-quadruplex DNA, and represents a new type of binding interaction for a zinc finger protein.     

Diffusion of Human Replication Protein A along Single-Stranded DNA

Replication protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA) binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA–DNA interactions, we combined ensemble and single-molecule fluorescence approaches to examine human RPA (hRPA) diffusion along ssDNA and find that an hRPA heterotrimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D₁ ~ 5000 nt² s^− 1 at 37 °C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA.     

FANCJ promotes DNA synthesis through G‐quadruplex structures

Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ^−/− cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes.     

Different loop arrangements of intramolecular human telomeric (3+1) G-quadruplexes in K+ solution

Intramolecular G-quadruplexes formed by the human telomeric G-rich strand are promising anticancer targets. Here we show that four-repeat human telomeric DNA sequences can adopt two different intramolecular G-quadruplex folds in K⁺ solution. The two structures contain the (3+1) G-tetrad core, in which three G-tracts are oriented in one direction and the fourth in the opposite direction, with one double-chain-reversal and two edgewise loops, but involve different loop arrangements. This result indicates the robustness of the (3+1) core G-quadruplex topology, thereby suggesting it as an important platform for structure-based drug design. Our data also support the view that multiple human telomeric G-quadruplex conformations coexist in K⁺ solution. Furthermore, even small changes to flanking sequences can perturb the equilibrium between different coexisting G-quadruplex forms.     

Accelerated assembly of G-quadruplex structures by a small molecule.

In the presence of alkali cations, notably potassium and sodium, DNA oligomers that possess two G-rich repeats associate into either a tetrameric parallel G-quadruplex or a variety of dimeric antiparallel G-quadruplexes. The formation of such structures is normally a very slow process. Some proteins, such as the beta-subunit of the Oxytricha telomere-binding protein, promote the formation of G-quadruplex structures in a chaperone-like manner. In this report, we present data concerning the role of a perylene derivative, PIPER, in the assembly of G-quadruplex structures as the first example of a small ligand behaving as a driver in the assembly of polynucleotide secondary structures. Gel-shift experiments demonstrate that PIPER can dramatically accelerate the association of a DNA oligomer containing two tandem repeats of the human telomeric sequence (TTAGGG) into di- and tetrameric G-quadruplexes. In so doing, PIPER alters the oligomer dimerization kinetics from second to first order. The presence of 10 microM PIPER accelerates the assembly of varied dimeric G-quadruplexes an estimated 100-fold from 2 microM oligomer. These results imply that some biological effects elicited by G-quadruplex-interactive agents, such as the induction of anaphase bridges, may stem from the propensity such compounds have for assembling G-quadruplexes.     

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

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

Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase

O⁶-alkylguanine-DNA alkyltransferase (AGT) is a single-cycle DNA repair enzyme that removes pro-mutagenic O⁶-alkylguanine adducts from DNA. Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood. Here, we examine the functions of this enzyme on O⁶-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex. On a folded 22-nt G-quadruplex substrate, binding saturated at 2 AGT:DNA, significantly less than the ∼5 AGT:DNA found with linear single-stranded DNAs of similar length, and less than the value found with the telomere sequence under conditions that inhibit quadruplex formation (4 AGT:DNA). Despite these differences, AGT repaired 6mG adducts located within folded G-quadruplexes, at rates that were comparable to those found for a duplex DNA substrate under analogous conditions. Repair was kinetically biphasic with the amplitudes of rapid and slow phases dependent on the position of the adduct within the G-quadruplex: in general, adducts located in the top or bottom tetrads of a quadruplex stack exhibited more rapid-phase repair than did adducts located in the inner tetrad. This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs.