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.     

Structure of the human telomere in Na+ solution: an antiparallel (2+2) G-quadruplex scaffold reveals additional diversity

Single-stranded DNA overhangs at the ends of human telomeric repeats are capable of adopting four-stranded G-quadruplex structures, which could serve as potential anticancer targets. Out of the five reported intramolecular human telomeric G-quadruplex structures, four were formed in the presence of K⁺ ions and only one in the presence of Na⁺ ions, leading often to a perception that this structural polymorphism occurs exclusively in the presence of K⁺ but not Na⁺. Here we present the structure of a new antiparallel (2+2) G-quadruplex formed by a derivative of a 27-nt human telomeric sequence in Na⁺ solution, which comprises a novel core arrangement distinct from the known topologies. This structure complements the previously elucidated basket-type human telomeric G-quadruplex to serve as reference structures in Na⁺-containing environment. These structures, together with the coexistence of other conformations in Na⁺ solution as observed by nuclear magnetic resonance spectroscopy, establish the polymorphic nature of human telomeric repeats beyond the influence of K⁺ ions.     

Human telomere d[(TTAGGG)4] undergoes a conformational transition to the Na+-form upon binding with sanguinarine in presence of K+

Guanine-rich telomeric sequences fold into G-quadruplex conformation and are known to bind a variety of ligands including potential drug candidates. By means of CD spectroscopy and fluorescence lifetime measurements we demonstrate that putative anticancer therapeutic sanguinarine (SGR) exhibits two distinct interactions with human telomere d[(TTAGGG)₄] (H24) in presence of K⁺. Up to about 1:2 M ratio of H24:SGR (10 μM H24), two molecules of SGR bind H24. Above this molar ratio, SGR induces a conformational transition in H24 from the K⁺-form to the Na⁺-form. The demonstration of SGR-induced conformational transition in a G-quadruplex formed by a human telomeric sequence could provide new insights into interaction of drugs with quadruplex DNA structure.     

Structure of the intramolecular human telomeric G-quadruplex in potassium solution: a novel adenine triple formation

We report the NMR solution structure of the intramolecular G-quadruplex formed in human telomeric DNA in K⁺. The hybrid-type telomeric G-quadruplex consists of three G-tetrads linked with mixed parallel–antiparallel G-strands, with the bottom two G-tetrads having the same G-arrangement (anti:anti:syn:anti) and the top G-tetrad having the reversed G-arrangement (syn:syn:anti:syn). The three TTA loop segments adopt different conformations, with the first TTA assuming a double-chain-reversal loop conformation, and the second and third TTA assuming lateral loop conformations. The NMR structure is very well defined, including the three TTA loops and the two flanking sequences at 5′- and 3′-ends. Our study indicates that the three loop regions interact with the core G-tetrads in a specific way that defines and stabilizes the unique human telomeric G-quadruplex structure in K⁺. Significantly, a novel adenine triple platform is formed with three naturally occurring adenine residues, A21, A3 and A9, capping the top tetrad of the hybrid-type telomeric G-quadruplex. This adenine triple is likely to play an important role in the formation of a stable human telomeric G-quadruplex structure in K⁺. The unique human telomeric G-quadruplex structure formed in K⁺ suggests that it can be specifically targeted for anticancer drug design.     

A novel chair-type G-quadruplex formed by a Bombyx mori telomeric sequence

Recently, the human telomeric d[TAGGG(TTAGGG)₃] sequence has been shown to form in K⁺ solution an intramolecular (3+1) G-quadruplex structure, whose G-tetrad core contains three strands oriented in one direction and the fourth in the opposite direction. Here we present a study on the structure of the Bombyx mori telomeric d[TAGG(TTAGG)₃] sequence, which differs from the human counterpart only by one G deletion in each repeat. We found that this sequence adopted multiple G-quadruplex structures in K⁺ solution. We have favored a major G-quadruplex form by a judicious U-for-T substitution in the sequence and determined the folding topology of this form. We showed by NMR that this was a new chair-type intramolecular G-quadruplex which involved a two-layer antiparallel G-tetrad core and three edgewise loops. Our result highlights the effect of G-tract length on the folding topology of G-quadruplexes, but also poses the question of whether a similar chair-type G-quadruplex fold exists in the human telomeric sequences.     

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.     

Structure of a two-G-tetrad intramolecular G-quadruplex formed by a variant human telomeric sequence in K+ solution: insights into the interconversion of human telomeric G-quadruplex structures

Human telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. The telomeric sequence shows intrinsic structure polymorphism. Here we report a novel intramolecular G-quadruplex structure formed by a variant human telomeric sequence in K⁺ solution. This sequence forms a basket-type intramolecular G-quadruplex with only two G-tetrads but multiple-layer capping structures formed by loop residues. While it is shown that this structure can only be detected in the specifically truncated telomeric sequences without any 5′-flanking residues, our results suggest that this two-G-tetrad conformation is likely to be an intermediate form of the interconversion of different telomeric G-quadruplex conformations.     

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.     

Structural basis of sodium–potassium exchange of a human telomeric DNA quadruplex without topological conversion

Understanding the mechanism of Na⁺/K⁺-dependent spectral conversion of human telomeric G-quadruplex (G4) sequences has been limited not only because of the structural polymorphism but also the lack of sufficient structural information at different stages along the conversion process for one given oligonucleotide. In this work, we have determined the topology of the Na⁺ form of Tel23 G4, which is the same hybrid form as the K⁺ form of Tel23 G4 despite the distinct spectral patterns in their respective nuclear magnetic resonance (NMR) and circular dichroism spectra. The spectral difference, particularly the well-resolved imino proton NMR signals, allows us to monitor the structural conversion from Na⁺ form to K⁺ form during Na⁺/K⁺ exchange. Time-resolved NMR experiments of hydrogen–deuterium exchange and hybridization clearly exclude involvement of the global unfolding for the fast Na⁺/K⁺ spectral conversion. In addition, the K⁺ titration monitored by NMR reveals that the Na⁺/K⁺ exchange in Tel23 G4 is a two-step process. The addition of K⁺ significantly stabilizes the unfolding kinetics of Tel23 G4. These results offer a possible explanation of rapid spectral conversion of Na⁺/K⁺ exchange and insight into the mechanism of Na⁺/K⁺ structural conversion in human telomeric G4s.     

Effect of sequence and metal ions on UVB-induced anti cyclobutane pyrimidine dimer formation in human telomeric DNA sequences

Irradiation of G-quadruplex forming human telomeric DNA with ultraviolet B (UVB) light results in the formation of anti cyclobutane pyrimidine dimers (CPDs) between loop 1 and loop 3 in the presence of potassium ions but not sodium ions. This was unexpected because the sequences involved favor the nonphotoreactive hybrid conformations in K⁺ solution, whereas a potentially photoreactive basket conformation is favored in Na⁺ solution. To account for these contradictory results, it was proposed that the loops are too far apart in the basket conformation in Na⁺ solution but close enough in a two G-tetrad basket-like form 3 conformation that can form in K⁺ solution. In the current study, Na⁺ was still found to inhibit anti CPD formation in sequences designed to stabilize the form 3 conformation. Furthermore, anti CPD formation in K⁺ solution was slower for the sequence previously shown to exist primarily in the proposed photoreactive form 3 conformation than the sequence shown to exist primarily in a nonphotoreactive hybrid conformation. These results suggest that the form 3 conformation is not the principal photoreactive conformation, and that G-quadruplexes in K⁺ solution are dynamic and able to access photoreactive conformations more easily than in Na⁺ solution.     

Interaction of [Ru(bpy)2(dppz)] with human telomeric DNA: Preferential binding to G-quadruplexes over i-motif

Inspired by the enormous importance attributed to the structure and function of human telomeric DNA, we focus our attention on the interaction of [Ru(bpy)₂(dppz)]²⁺ with the guanine-rich single-strand oligomer 5′-AGGGTTAGGGTTAGGGTTAGGG-3′ (22AG) and the complementary cytosine-rich strand (22CT). In Na⁺ buffer, 22AG may adopt an antiparallel basket quadruplex, whereas, it favours a mixed parallel/antiparallel structure in K⁺ buffer. 22CT may self-associate at acidic pH into an i-motif. In this paper, the interaction between [Ru(bpy)₂(dppz)]²⁺ and each unusual DNA was evaluated. It was interesting that [Ru(bpy)₂(dppz)]²⁺ could promote the human telomeric repeat 22AG to fold into intramolecular antiparallel G-quadruplex without any other cations. What's more, [Ru(bpy)₂(dppz)]²⁺ was found to have a strong preference for binding to G-quadruplexes that were induced through either Na⁺ or K⁺, while weak binding to i-motif was observed. The results also indicated that [Ru(bpy)₂(dppz)]²⁺ could serve as a prominent molecular “light switch” for both G-quadruplexes, revealing a potential application of the title complex in luminescent signaling of G-quadruplex DNA.     

Dominant Driving Forces in Human Telomere Quadruplex Binding-Induced Structural Alterations

Recently various pathways of human telomere (ht) DNA folding into G-quadruplexes and of ligand binding to these structures have been proposed. However, the key issue as to the nature of forces driving the folding and recognition processes remains unanswered. In this study, structural changes of 22-mer ht-DNA fragment (Tel22), induced by binding of ions (K⁺, Na⁺) and specific bisquinolinium ligands, were monitored by calorimetric and spectroscopic methods and by gel electrophoresis. Using the global model analysis of a wide variety of experimental data, we were able to characterize the thermodynamic forces that govern the formation of stable Tel22 G-quadruplexes, folding intermediates, and ligand-quadruplex complexes, and then predict Tel22 behavior in aqueous solutions as a function of temperature, salt concentration, and ligand concentration. On the basis of the above, we believe that our work sets the framework for better understanding the heterogeneity of ht-DNA folding and binding pathways, and its structural polymorphism.     

Kinetic evidence for interaction of TMPyP4 with two different G-quadruplex conformations of human telomeric DNA

Background

Stabilization of G-quadruplex helices by small ligands has attracted growing attention because they inhibit the activity of the enzyme telomerase, which is overexpressed in > 80% cancer cells. TMPyP4, one of the most studied G-quadruplex ligands, is used as a model to show that the ligands can exhibit different binding features with different conformations of a human telomeric specific sequence.

Single-molecule detection of folding and unfolding of the G-quadruplex aptamer in a nanopore nanocavity

Guanine-rich nucleic acids can form G-quadruplexes that are important in gene regulation, biosensor design and nano-structure construction. In this article, we report on the development of a nanopore encapsulating single-molecule method for exploring how cations regulate the folding and unfolding of the G-quadruplex formed by the thrombin-binding aptamer (TBA, GGTTGGTGTGGTTGG). The signature blocks in the nanopore revealed that the G-quadruplex formation is cation-selective. The selectivity sequence is K⁺ > NH₄⁺ ~ Ba²⁺ > Cs⁺ ~ Na⁺ > Li⁺, and G-quadruplex was not detected in Mg²⁺ and Ca²⁺. Ba²⁺ can form a long-lived G-quadruplex with TBA. However, the capability is affected by the cation–DNA interaction. The cation-selective formation of the G-quadruplex is correlated with the G-quadruplex volume, which varies with cation species. The high formation capability of the K⁺-induced G-quadruplex is contributed largely by the slow unfolding reaction. Although the Na⁺- and Li⁺-quadruplexes feature similar equilibrium properties, they undergo radically different pathways. The Na⁺-quadruplex folds and unfolds most rapidly, while the Li⁺-quadruplex performs both reactions at the slowest rates. Understanding these ion-regulated properties of oligonucleotides is beneficial for constructing fine-tuned biosensors and nano-structures. The methodology in this work can be used for studying other quadruplexes and protein–aptamer interactions.     

Human telomeric G-quadruplex formed from duplex under near physiological conditions: Spectroscopic evidence and kinetics

The structural interconversion between the G-quadruplex and duplex in vivo is an important subject. In the present study, we used human telomeric DNA duplex composed of GGG(TTAGGG)₃/CCC(TAACCC)₃ as a model system to investigate its properties under near physiological conditions by spectroscopic methods. Circular dichroism and fluorescence spectra demonstrated that G-quadruplex structure can be formed from duplex at near physiological pH (pH 7.4), salt concentration (150 mM K⁺), and temperature (37 °C) in the presence of molecular crowding agent PEG (400 g/l), whereas the G-quadruplex structure cannot be formed at 25 °C in buffer containing 150 mM K⁺ in the presence of PEG. It is found that the formation rate of G-quadruplex structure depends on the temperature and the concentrations of both PEG and K⁺. This work suggests that human telomeric G-quadruplex structure may be potentially formed from Watson–Crick duplex in vivo.     

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.     

Solution structure of the major G-quadruplex formed in the human VEGF promoter in K+: insights into loop interactions of the parallel G-quadruplexes

Vascular endothelial growth factor (VEGF) proximal promoter region contains a poly G/C-rich element that is essential for basal and inducible VEGF expression. The guanine-rich strand on this tract has been shown to form the DNA G-quadruplex structure, whose stabilization by small molecules can suppress VEGF expression. We report here the nuclear magnetic resonance structure of the major intramolecular G-quadruplex formed in this region in K⁺ solution using the 22mer VEGF promoter sequence with G-to-T mutations of two loop residues. Our results have unambiguously demonstrated that the major G-quadruplex formed in the VEGF promoter in K⁺ solution is a parallel-stranded structure with a 1:4:1 loop-size arrangement. A unique capping structure was shown to form in this 1:4:1 G-quadruplex. Parallel-stranded G-quadruplexes are commonly found in the human promoter sequences. The nuclear magnetic resonance structure of the major VEGF G-quadruplex shows that the 4-nt middle loop plays a central role for the specific capping structures and in stabilizing the most favored folding pattern. It is thus suggested that each parallel G-quadruplex likely adopts unique capping and loop structures by the specific middle loops and flanking segments, which together determine the overall structure and specific recognition sites of small molecules or proteins.LAY SUMMARY: The human VEGF is a key regulator of angiogenesis and plays an important role in tumor survival, growth and metastasis. VEGF overexpression is frequently found in a wide range of human tumors; the VEGF pathway has become an attractive target for cancer therapeutics. DNA G-quadruplexes have been shown to form in the proximal promoter region of VEGF and are amenable to small molecule drug targeting for VEGF suppression. The detailed molecular structure of the major VEGF promoter G-quadruplex reported here will provide an important basis for structure-based rational development of small molecule drugs targeting the VEGF G-quadruplex for gene suppression.     

Single-Molecule TPM Studies on the Conversion of Human Telomeric DNA

Human telomere contains guanine-rich (G-rich) tandem repeats of single-stranded DNA sequences at its 3′ tail. The G-rich sequences can be folded into various secondary structures, termed G-quadruplexes (G4s), by Hoogsteen basepairing in the presence of monovalent cations (such as Na⁺ and K⁺). We developed a single-molecule tethered particle motion (TPM) method to investigate the unfolding process of G4s in the human telomeric sequence AGGG(TTAGGG)₃ in real time. The TPM method monitors the DNA tether length change caused by formation of the G4, thus allowing the unfolding process and structural conversion to be monitored at the single-molecule level. In the presence of its antisense sequence, the folded G4 structure can be disrupted and converted to the unfolded conformation, with apparent unfolding time constants of 82 s and 3152 s. We also observed that the stability of the G4 is greatly affected by different monovalent cations. The folding equilibrium constant of G4 is strongly dependent on the salt concentration, ranging from 1.75 at 5 mM Na⁺ to 3.40 at 15 mM Na⁺. Earlier spectral studies of Na⁺- and K⁺-folded states suggested that the spectral conversion between these two different folded structures may go through a structurally unfolded intermediate state. However, our single-molecule TPM experiments did not detect any totally unfolded intermediate within our experimental resolution when sodium-folded G4 DNA molecules were titrated with high-concentration, excess potassium ions. This observation suggests that a totally unfolding pathway is likely not the major pathway for spectral conversion on the timescale of minutes, and that interconversion among folded states can be achieved by the loop rearrangement. This study also demonstrates that TPM experiments can be used to study conformational changes in single-stranded DNA molecules.     

Improving the affinity of naphthalene diimide ligand to telomeric DNA by incorporating Zn2+ ions into its dipicolylamine groups

N,N′-bis[3-[3-(2,2′-dipicolyl)methylaminopropyl]-methylaminopropyl]naphthalene-1,4,5,8-tetracarboxylic acid diimide, 1, and its complex with zinc ions, 2, were investigated against telomeric sequences, [TAGGG(TTAGGG)₃] and [AGGG(TTAGGG)₃], which reveal different G-quadruplex structures depending on the conditions. Spectrophotometric, SPR, and CD techniques revealed that both ligands showed large binding constants to hybrid-type G-quadruplexes formed in the presence of K⁺ ions. Moreover, 2 revealed higher affinity to investigated oligonucleotides suggesting that complex of naphthalene diimide derivative with Zn²⁺, comparing to 1, provided additional electrostatic or coordination interactions between positively charged zinc ions and condensed negative charged phosphate anions from G4 DNA.