Interaction of water with the G-quadruplex loop contributes to the binding energy of G-quadruplex to protein

Unlike DNA duplexes that release water upon interaction with protein, the binding of DNA G-quadruplex of the thrombin-binding aptamer (TBA) to thrombin takes up water. Here, to reveal the mechanism of water uptake, we designed four mutants of TBA (ΔT3, ΔT7, ΔT9, ΔT12), in which thymine residues (T3, T7, T9 and T12) were deleted from the loop regions of TBA G-quadruplex. For the mutants the thermodynamics and the osmolyte effects on the interactions with thrombin were investigated. The mutants ΔT3, ΔT9 and ΔT12 decreased the binding constants of the G-quadruplex to thrombin. Furthermore, an osmotic stress analysis indicated that the number of water molecules binding to the complex decreased in the mutants ΔT3 and ΔT9. The decrease in the binding affinity was related to loss of binding of the loop nucleotides to water molecules. Therefore, the interaction between loops of the G-quadruplex and water molecules contributed to the binding energy of G-quadruplex to protein. Our study suggests that water binding is essential for the binding of G-quadruplex to protein.     

Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2'-C-piperazino-UNA monomer

Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2'-C-piperazino-UNA residue and UNA residues incorporated in several positions on thermodynamics, kinetics and biological properties of the aptamer. 2'-C-Piperazino-UNA is characterized by more efficient stabilization of quadruplex structure in comparison to regular UNA and increases thermodynamic stability of TBA by 0.28-0.44 kcal/mol in a position depending manner with retained quadruplex topology and molecularity. The presence of UNA-U in positions U3, U7, and U12 results in the highest stabilization of G-quadruplex structure (ΔΔG(37)(°)=-1.03kcal/mol). On the contrary, the largest destabilization mounting to 1.79 kcal/mol was observed when UNA residues were placed in positions U7, G8, and U9. Kinetic studies indicate no strict correlation between thermodynamic stability of modified variants and their binding affinity to thrombin. Most of the studied variants bind thrombin, albeit with decreased affinity in reference to unmodified TBA. Thrombin time assay studies indicate three variants as being as potent as TBA in fibrin clotting inhibition.     

Site-specific replacement of the thymine methyl group by fluorine in thrombin binding aptamer significantly improves structural stability and anticoagulant activity

Here we report investigations, based on circular dichroism, nuclear magnetic resonance spectroscopy, molecular modelling, differential scanning calorimetry and prothrombin time assay, on analogues of the thrombin binding aptamer (TBA) in which individual thymidines were replaced by 5-fluoro-2′-deoxyuridine residues. The whole of the data clearly indicate that all derivatives are able to fold in a G-quadruplex structure very similar to the ‘chair-like’ conformation typical of the TBA. However, only ODNs TBA-F4 and TBA-F13 have shown a remarkable improvement both in the melting temperature (ΔT_m ≈ +10) and in the anticoagulant activity in comparison with the original TBA. These findings are unusual, particularly considering previously reported studies in which modifications of T4 and T13 residues in TBA sequence have clearly proven to be always detrimental for the structural stability and biological activity of the aptamer. Our results strongly suggest the possibility to enhance TBA properties through tiny straightforward modifications.     

Stability and binding properties of a modified thrombin binding aptamer

Aptamer-based drugs represent an attractive approach in pharmacological therapy. The most studied aptamer, thrombin binding aptamer (TBA), folds into a well-defined quadruplex structure and binds to its target with good specificity and affinity. Modified aptamers with improved biophysical properties could constitute a new class of therapeutic aptamers. In this study we show that the modified thrombin binding aptamer (mTBA), ^3′GGT^5′-^5′TGGTGTGGTTGG^3′, which also folds into a quadruplex structure, is more stable than its unmodified counterpart and shows a higher thrombin affinity. The stability of the modified aptamer was investigated using differential scanning calorimetry, and the energetics of mTBA and TBA binding to thrombin was characterized by means of isothermal titration calorimetry (ITC). ITC data revealed that TBA/thrombin and mTBA/thrombin binding stoichiometry is 1:2 for both interactions. Structural models of the two complexes of thrombin with TBA and with mTBA were also obtained and subjected to molecular dynamics simulations in explicit water. Analysis of the models led to an improvement of the understanding of the aptamer-thrombin recognition at a molecular level.     

The effect of chemical modifications on the thermal stability of different G-quadruplex-forming oligonucleotides

A systematic study of the thermal and conformational properties of chemically modified G-quadruplexes of different molecularities is reported. The effect of backbone charge and atom size, thymine/uracyl substitution as well as the effect of modification at the ribose 2′-position was analyzed by UV spectroscopy. Additional calorimetric studies were performed on different modified forms of the human telomeric sequence. Determination of the differential spectra allowed more insights into the conformational properties of the oligonucleotides. Lack of negative charge at the phosphate backbone yielded to a general destabilization of the G-quadruplex structure. On the other hand, substitution of thymine with uracyl resulted in a moderate or strong stabilization of the structure. Additional modification at the sugar 2′-position gave rise to different effects depending on the molecularity of the quadruplex. In particular, loss of hydrogen bond capacity at the 2′-position strongly affected the conformation of the G-quadruplex. Altogether, these results demonstrate that the effect of some modifications depends on the sequence context, thus providing helpful information for the use of chemically modified quadruplexes as therapeutic agents or as structural elements of supramolecular complexes.     

Relations between the loop transposition of DNA G-quadruplex and the catalytic function of DNAzyme

The structures of DNA G-quadruplexes are essential for their functions in vivo and in vitro. Our present study revealed that sequential order of the three G-quadruplex loops, that is, loop transposition, could be a critical factor to determinate the G-quadruplex conformation and consequently improved the catalytic function of G-quadruplex based DNAzyme. In the presence of 100 mM K⁺, loop transposition induced one of the G-quadruplex isomers which shared identical loops but differed in the sequential order of loops into a hybrid topology while the others into predominately parallel topologies. ¹D NMR spectroscopy and mutation analysis suggested that the hydrogen bonding from loops residues with nucleotides in flanking sequences may be responsible for the stabilization of the different conformations. A well-known DNAzyme consisting of G-quadruplex and hemin (Ferriprotoporphyrin IX chloride) was chosen to test the catalytic function. We found that the loop transposition could enhance the reaction rate obviously by increasing the hemin binding affinity to G-quadruplex. These findings disclose the relations between the loop transposition, G-quadruplex conformation and catalytic function of DNAzyme.     

Study of the interaction between the G-quadruplex-forming thrombin-binding aptamer and the porphyrin 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)-21,23H-porphyrin tetratosylate

The G-quadruplex DNA structure has been suggested to be a potential target for anticancer therapies. Therefore, there is increasing interest in the development of drugs that could modulate the stability of G-quadruplex structures. In the current work, the interaction between the thrombin-binding aptamer (TBA, 5′-GGT TGG TGT GGT TGG-3′), which can form an intramolecular G-quadruplex structure, and the porphyrin 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)-21,23H-porphyrin tetratosylate (TmPyP4) was studied. The application of a high-performance liquid chromatography-photodiode array (HPLC-PDA) detector-based method to study this kind of interaction was tested. Molecular absorption data recorded along the chromatographic runs were analyzed by means of multivariate data analysis methods. Moreover, biospecific interaction analysis (BIA) by surface plasmon resonance (SPR) and melting and mole ratio experiments monitored by UV-visible molecular absorption and circular dichroism spectroscopies, were applied to confirm and expand the chromatographic studies. The results showed the formation of an interaction complex with a stoichiometry 1:1 (TmPyP4/TBA) and logarithm of the equilibrium constant equal to 5.7 ± 0.2. Melting and circular dichroism data reflected that the initial G-quadruplex structure of TBA is stabilized in the interaction complex, being slightly distorted by the presence of the ligand.     

A structure-activity relationship of a thrombin-binding aptamer containing LNA in novel sites

In this report, structural characterization, aptamer stability and thrombin of a new modified thrombin-ligand complex binding aptamer (TBA) containing anti-guanine bases and a loop position locked nucleic acid (LNA) are presented. NMR, circular dichroic spectroscopy and molecular modeling were used to characterize the three-dimensional structure of two G-quadruplexes. LNA-modification of the anti-guanosines yields G-quadruplexes that show affinity and inhibitory activity toward thrombin, whereas LNA-modification of a thymine nucleotide in the TGT loop increases the thermal stability of TBA. As assessed by denatured PAGE electrophoresis, all modified aptamers display an increase in environmental stability. The prothrombin time assay and fibrinogen assay showed that the aptamers still had good inhibitory activity, and 15 of them had the longest PT time. Therefore, the LNA modification is well suited to improve the physicochemical and biological properties of the native thrombin-binding aptamer.     

G-rich VEGF aptamer with locked and unlocked nucleic acid modifications exhibits a unique G-quadruplex fold

The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K⁺ rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes exhibits several unique structural features. Five consecutive guanine residues are all involved in G-quartet formation and occupy positions in adjacent DNA strands, which are bridged with a no-residue propeller-type loop. A two-residue D-shaped loop facilitates inclusion of an isolated guanine residue into the vacant spot within the G-quartet. The remaining two G-rich tracts of three residues each adopt parallel orientation and are linked with edgewise and propeller loops. Both 5′ with 3 nt and 3′ with 4 nt overhangs display well-defined conformations, with latter adopting a basket handle topology. Locked residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex. Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general.     

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.     

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.     

The “Janus face” of the thrombin binding aptamer: Investigating the anticoagulant and antiproliferative properties through straightforward chemical modifications

BackgroundThe thrombin binding aptamer (TBA) is endowed with both anticoagulant and antiproliferative activities. Its chemico-physical and/or biological properties can be tuned by the site-specific replacement of selected residues.MethodsFour oligodeoxynucleotides (ODNs) based on the TBA sequence (5′-GGTTGGTGTGGTTGG-3′) and containing 2′-deoxyuridine (U) or 5-bromo-2′-deoxyuridine (B) residues at positions 4 or 13 have been investigated by NMR and CD techniques. Furthermore, their anticoagulant (PT assay) and antiproliferative properties (MTT assay) have been tested and compared with two further ODNs containing 5-hydroxymethyl-2′-deoxyuridine (H) residues in the same positions, previously investigated.ResultsThe CD and NMR data suggest that all the investigated ODNs are able to form G-quadruplexes strictly resembling that of TBA. The introduction of B residues in positions 4 or 13 increases the melting temperature of the modified aptamers by 7 °C. The replacement of thymidines with U in the same positions results in an enhanced anticoagulant activity compared to TBA, also at low ODN concentration. Although all ODNs show antiproliferative properties, only TBA derivatives containing H in the positions 4 and 13 lose the anticoagulant activity and remarkably preserve the antiproliferative one.ConclusionsAll ODNs have shown antiproliferative activities against two cancer cell lines but only those with U and B are endowed with anticoagulant activities similar or improved compared to TBA.General significance:The appropriate site-specific replacement of the residues in the TT loops of TBA with commercially available thymine analogues is a useful strategy either to improve the anticoagulant activity or to preserve the antiproliferative properties by quenching the anticoagulant ones.     

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.     

Recognition of different base tetrads by RHAU (DHX36): X-ray crystal structure of the G4 recognition motif bound to the 3′-end tetrad of a DNA G-quadruplex

G-quadruplexes (G4) are secondary structures of nucleic acids that can form in cells and have diverse biological functions. Several biologically important proteins interact with G-quadruplexes, of which RHAU (or DHX36) – a helicase from the DEAH-box superfamily, was shown to bind and unwind G-quadruplexes efficiently. We report a X-ray co-crystal structure at 1.5 Å resolution of an N-terminal fragment of RHAU bound to an exposed tetrad of a parallel-stranded G-quadruplex. The RHAU peptide folds into an L-shaped α-helix, and binds to a G-quadruplex through π-stacking and electrostatic interactions. X-ray crystal structure of our complex identified key amino acid residues important for G-quadruplex-peptide binding interaction at the 3′-end G•G•G•G tetrad. Together with previous solution and crystal structures of RHAU bound to the 5′-end G•G•G•G and G•G•A•T tetrads, our crystal structure highlights the occurrence of a robust G-quadruplex recognition motif within RHAU that can adapt to different accessible tetrads.     

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.     

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.     

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.     

Structure and Stability of Human Telomeric G-Quadruplex with Preclinical 9-Amino Acridines

G-quadruplexes are higher-order DNA structures formed from guanine-rich sequences, and have been identified as attractive anticancer drug targets. Elucidating the three-dimensional structure of G-quadruplex with 9-amino acridines and the specific interactions involved in binding selectivity are the key to understanding their mechanism of action. Fluorescence titration assays, competitive dialysis and NMR studies have been used to study the binding specificity of 9-amino acridines to DNA. Structural models of the complexes with the telomeric DNA G-quadruplex based on NMR measurements were developed and further examined by molecular dynamics simulations and free energy calculations. Selective binding of 9-amino acridines for G-quadruplex sequences were observed. These compounds bind between A and G-tetrads, involving significant π-π interactions and several strong hydrogen bonds. The specific interactions between different moieties of the 9-amino acridines to the DNA were examined and shown to play a significant role in governing the overall stabilities of DNA G-quadruplex complexes. Both 9-amino acridines, with similar binding affinities to the G-quadruplex, were shown to induce different level of structural stabilization through intercalation. This unique property of altering structural stability is likely a contributing factor for affecting telomerase function and, subsequently, the observed differences in the anticancer activities between the two 9-amino acridines.     

Circular dichroism spectra demonstrate formation of the thrombin-binding DNA aptamer G-quadruplex under stabilizing-cation-deficient conditions

It is noteworthy that the formation of the DNA G-quadruplex is induced by factors other than stabilizing cations because this event probably occurs in living cells. Previous studies have shown that thrombin-binding DNA aptamer (TBA) forms a chair-type intramolecular G-quadruplex structure that binds with thrombin protein in the absence of stabilizing cations. Here, we used circular dichroism (CD) spectroscopy to confirm G-quadruplex formation in the presence of thrombin without stabilizing cations. We obtained characteristic CD spectra that demonstrated that TBA forms the distinctive G-quadruplex structure. Additionally, we investigated G-quadruplex formation induced by change of solvent environment: the influence of low-temperature conditions and molecular crowding.