Antiparallel G-quadruplex Research Articles

A Universal Base in a Specific Role: Tuning up a Thrombin Aptamer with 5-Nitroindole

In this study we describe new modified analogs of the thrombin binding aptamer (TBA) containing 5-nitroindole residues. It has been shown that all modified TBAs form an anti-parallel G-quadruplex structure and retain the ability to inhibit thrombin. The most advanced TBA variant (TBA-N8) has a substantially increased clotting time and two-fold lower IC50 value compared to the unmodified prototype. Molecular modelling studies suggest that the improved anticoagulant properties of TBA-N8 result from changes in the binding mode of the analog. A modified central loop in TBA-N8 is presumed to participate in the binding of the target protein. Studies of FAM labelled TBA and TBA-N8 showed an improved binding affinity of the modified aptamer and provided evidence of a direct interaction between the modified central loop and thrombin. Our findings have implications for the design of new aptamers with improved binding affinities.

G-Tetraplex-Induced FRET within Telomeric Repeat Sequences Using (Py) A-(Per) A as Energy Donor-Acceptor Pair.

G-tetraplex induced fluorescence resonance energy transfer (FRET) within telomeric repeat sequences has been studied using a nucleoside-tethered FRET pair embedded in the human telomeric G-quadruplex forming sequence (5'-A GGG TT(Py) A GGG TT(Per) A GGG TTA GGG-3', Py=pyrene, Per=perylene). Conformational change from a single strand to an anti-parallel G-quadruplex leads to FRET from energy donor ((Py) A) to acceptor ((Per) A). The distance between the FRET donor/acceptor partners was controlled by changing the number of G-quartet spacer units. The FRET efficiency decreases with increase in G-quartet units. Overall findings indicate that this could be further used for the development of FRET-based sensing and measurement techniques.

Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway.

We present evidence that Tim establishes a physical and functional interaction with DDX11, a super-family 2 iron-sulfur cluster DNA helicase genetically linked to the chromosomal instability disorder Warsaw breakage syndrome. Tim stimulates DDX11 unwinding activity on forked DNA substrates up to 10-fold and on bimolecular anti-parallel G-quadruplex DNA structures and three-stranded D-loop approximately 4–5-fold. Electrophoretic mobility shift assays revealed that Tim enhances DDX11 binding to DNA, suggesting that the observed stimulation derives from an improved ability of DDX11 to interact with the nucleic acid substrate. Surface plasmon resonance measurements indicate that DDX11 directly interacts with Tim. DNA fiber track assays with HeLa cells exposed to hydroxyurea demonstrated that Tim or DDX11 depletion significantly reduced replication fork progression compared to control cells; whereas no additive effect was observed by co-depletion of both proteins. Moreover, Tim and DDX11 are epistatic in promoting efficient resumption of stalled DNA replication forks in hydroxyurea-treated cells. This is consistent with the finding that association of the two endogenous proteins in the cell extract chromatin fraction is considerably increased following hydroxyurea exposure. Overall, our studies provide evidence that Tim and DDX11 physically and functionally interact and act in concert to preserve replication fork progression in perturbed conditions.

Formation and stabilization of the telomeric antiparallel G-quadruplex and inhibition of telomerase by novel benzothioxanthene derivatives with anti-tumor activity.

G-quadruplexes formed in telomeric DNA sequences at human chromosome ends can be a novel target for the development of therapeutics for the treatment of cancer patients. Herein, we examined the ability of six novel benzothioxanthene derivatives S1–S6 to induce the formation of and stabilize an antiparallel G-quadruplex by EMSA, UV-melting and CD techniques and the influence of S1–S6 on A549 and SGC7901 cells through real-time cell analysis, wound healing, trap assay methods. Results show that six compounds could differentially induce 26 nt G-rich oligonucleotides to form the G-quadruplex with high selectivity vs C-rich DNA, mutated DNA and double-stranded DNA, stabilize it with high affinity, promote apoptosis and inhibit mobility and telomerase activity of A549 cells and SGC7901 cells. Especially, S1, S3, S4 displayed stronger abilities, of which S3 was the most optimal with the maximum ΔTm value being up to 29.8 °C for G-quadruplex, the minimum IC50 value being 0.53 μM and the maximum cell inhibitory rate being up to 97.2%. This study suggests that this type of compounds that induce the formation of and stabilize the telomeric antiparallel G-quadruplex, and consequently inhibit telomerase activity, leading to cell apoptosis, can be screened for the discovery of novel antitumor therapeutics.

Aptamer-based single-walled carbon nanohorn sensors for ochratoxin A detection

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus ochraceus and Penicillium verrucosum, is one of the most significant mycotoxins encountered in foods. In this research, a novel fluorescence biosensing strategy for simple and sensitive OTA detection using an aptamer specific for OTA as recognition element and single-walled carbon nanohorns (SWCNHs) as fluorescence quencher was reported. In the absence of target molecule (OTA), FAM (carboxyfluorescein)-modified aptamer was adsorbed onto the SWCNH surface. In this arrangement, the fluorophore and quencher were in close proximity; thus, FAM fluorescence was readily quenched by fluorescence resonance energy transfer from dye to carbon nanohorn. In the presence of target molecules (OTA), the OTA-specific aptamer form an antiparallel G-quadruplex, which is resistant to being wrapped onto SWCNHs. Therefore, the fluorescence of FAM cannot be quenched, and the fluorescent intensity as a function of OTA concentration was correspondingly measured. We obtained a detection limit of 17.2 nM for our sensing platform based on SWCNHs, with a linear detection range of 20–500 nM. The proposed assay system also exhibited high selectivity for OTA against other mycotoxins. The biosensor was verified for real sample application by testing 1% red wine containing buffer solution spiked with a series of OTA concentrations, and there is a good linear relationship (correlation coefficient = 0.990) between the fluorescence intensity and the logarithm of OTA concentration. Utilization of the proposed biosensor for quantitative determination of mycotoxins in food samples indicated the sensor being a useful tool for verifying the effectiveness of mycotoxin control strategies.

The genomic sequences near the mir‐23b‐27b‐24‐1 cluster form G‐quadruplexes and are selectively bound by the natural alkaloid tetrandrine

Although the microRNAs miR-23b, miR-27b and miR-24 are located in the same cluster, their expressions in various pathological states are not always comparable. By searching the genomic sequence around mir23b-27b-24-1 in rat, we identified three potential G-quadruplex sequences (PQS) which can fold into different types of G-quadruplexes, including parallel or antiparallel. Natural alkaloids, tetrandrine (TET), displayed different binding affinity with the three G-quadruplexes which may potentially regulate the expression of mir23b-27b-24-1 cluster members. Both electrospray ionization mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy were utilized to detect the formation of G-quadruplexes. Six small molecules were screened by ESI-MS for their binding affinity with three G-quadruplexes, which were evaluated by their IRa values. The results of ESI-MS and CD experiments confirmed the formation of three G-quadruplexes neighboring the mir23b-27b-24-1 cluster; two of them adopted antiparallel G-quadruplexes, another adopted a parallel G-quadruplex. Screening of small molecules by ESI-MS showed tetrandrine had selective binding affinity for the parallel G-quadruplex. G-quadruplex stabilization by tetrandrine was verified by CD variable temperature measurements. The gene of the mir23b-27b-24-1 cluster harbors three G-quadruplexes with typical sequences and structures. Tetrandrine had a selective binding affinity to the parallel G-quadruplex and stabilized it significantly. Copyright © 2015 John Wiley & Sons, Ltd.

Study on Electrochemical Insulin Sensing Utilizing a DNA Aptamer-Immobilized Gold Electrode.

We investigated an insulin-sensing method by utilizing an insulin-binding aptamer IGA3, which forms an anti-parallel G-quadruplex with folded single strands. Spectroscopic observation indicates that some anti-parallel G-quadruplex bind hemin and show peroxidase activity. In this study, the peroxidase activity of IGA3 with hemin was confirmed by spectrophotometric measurements, i.e., the activity was three-times higher than hemin itself. IGA3 was then immobilized onto a gold electrode to determine its electrochemical activity. The peroxidase activity of the immobilized IGA3-hemin complex was determined by cyclic voltammetry, and a cathodic peak current of the electrode showed a dependence on the concentration of H2O2. The cathodic peak current of the IGA3-hemin complex decreased by binding it to insulin, and this decrease depended on the concentration of insulin.

G-quadruplex formation in double strand DNA probed by NMM and CV fluorescence.

G-quadruplexes (GQs) are alternative DNA secondary structures that can form throughout the human genome and control the replication and transcription of important regulatory genes. Here, we established an ensemble fluorescence assay by employing two GQ-interacting compounds, N-methyl mesoporphyrin IX (NMM) and Crystal Violet (CV). This enables quantitative measurement of the GQ folding propensity and conformation specificity in both single strand (ss) and double strand (ds) DNA. Our GQ mapping indicates that the likelihood of GQ formation is substantially diminished in dsDNA, likely due to the competition from the Watson–Crick base pairing. Unlike GQ folding sequence in ssDNA which forms both parallel and antiparallel GQs, dsDNA displays only parallel folding. Additionally, we employed single molecule FRET to obtain a direct quantitation of stably formed-, weakly folded and unfolded GQ conformations. The findings of this study and the method developed here will enable identifying and classifying potential GQ-forming sequences in human genome.

Double stranded promoter region of BRAF undergoes to structural rearrangement in nearly physiological conditions

The folding of oncogene promoters into non-canonical DNA secondary structures is considered a strategy to control gene expression. Herein, we focused on a 30 bases sequence located upstream of the transcription start site of BRAF (Braf-176) that contains 80% of guanines. We analyzed the structural behavior of the G- and C-rich strands. By the use of spectroscopic and electrophoretic techniques we confirmed that they actually fold into a predominant antiparallel G-quadruplex and into an i-motif, respectively, and that they can coexist at nearly physiological conditions. Finally, the influence of several factors (KCl, pH, PEG200) on the conversion of the double stranded form of the oncogene promoter into the two above mentioned non-canonical structures has been explored.

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

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

Selective recognition of specific G-quadruplex vs. duplex DNA by a phenanthroline derivative

A key problem in designing G-quadruplex ligand is how to discriminate quadruplex DNA specifically from other DNAs, searching ligands targeted at special G-quadruplex structure with high selectivity is a major challenge. Herein, a phenanthrolin-dicarboxylate ester (PD) is proved to exhibit selectivity toward parallel and hybrid G-quadruplex structures with propeller and edge-wise loops, over duplex DNA and antiparallel quadruplex structure with diagonal loop. Such preferred binding of PD to these special G-quadruplex structures is possibly resulted from steric hindrance of: (1) the four substituent groups in PD molecule which prevent close interaction with duplex DNA and (2) the diagonal loop above the G-tetrads in antiparallel G-quadruplex which hinder face-to-face stacking of planar phenanthrolin ring to G-tetrads. In line with its stabilizing ability of G-quadruplex, PD molecule exhibit a inhibitory ability telomerase activity, as well as the potent cytotoxic activity against several human cancer cell lines, which makes it an interesting anti-tumor drug lead.

Two-quartet G-quadruplexes formed by DNA sequences containing four contiguous GG runs.

The DNA sequence containing four contiguous GG runs (G2NxG2NyG2NzG2, G2 sequence) has the potential to form a two-quartet G-quadruplex. However, the prevalence, structure, and function of G2 sequences have not been well-studied. Here, bioinformatics analysis reveals the abundance of G2 sequences in the human genome and their enrichment in promoter regions. The density of G2 sequences in the genome and promoters is much higher than that of the G3 sequence (G3NxG3NyG3NzG3). Experiments show that the conformations and thermal stabilities of the two-quartet G-quadruplexes of G2 sequences are highly sensitive to the length and composition of the loops. Among the two-quartet G-quadruplexes, the parallel G-quadruplex with a loop length of 1 and the antiparallel G-quadruplex with a loop length of 3 show high thermal stabilities. Additionally, the stable parallel G-quadruplexes are stacked into intermolecular higher-order structures. This work determines the prevalence of G2 sequences in the human genome and demonstrates that the G-quadruplex structures for certain loop lengths and compositions may be stable in vivo. Thus, more attention should be paid to the structure and function of the two-quartet G-quadruplex.

Dinuclear Ruthenium(II) Complexes That Induce and Stabilise G‐Quadruplex DNA

A series of dinuclear ruthenium(II) complexes were synthesised, and the complexes were determined to be new highly selective compounds for binding to telomeric G-quadruplex DNA. The interactions of these complexes with telomeric G-quadruplex DNA were studied by using circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET) melting assays, isothermal titration calorimetry (ITC) and molecular modelling. The results showed that the complexes 1, 2 and 4 induced and stabilised the formation of antiparallel G-quadruplexes of telomeric DNA in the absence of salt or in the presence of 100 mM K(+)-containing buffer. Furthermore, complexes 1 and 2 strongly bind to and effectively stabilise the telomeric G-quadruplex structure and have significant selectivity for G-quadruplex over duplex DNA. In comparison, complex 3 had a much lesser effect on the G-quadruplex, suggesting that possession of a suitably sized plane for good π-π stacking with the G-quadruplets is essential for the interaction of the dinuclear ruthenium(II) complexes with the G-quadruplex. Moreover, telomerase inhibition by the four complexes and their cellular effects were studied, and complex 1 was determined to be the most promising inhibitor of both telomerase and HeLa cell proliferation.

Construction of anti-parallel G-quadruplexes through sequential templated click.

Biologically relevant DNA sequences were assembled onto addressable cyclopeptide scaffolds through sequential oxime and CuAAc reactions. The resulting conjugates are able to fold into well-defined anti-parallel DNA G-quadruplex structures, which exhibit high stability and reduced polymorphism.

Unimolecular antiparallel G-quadruplex folding topology of 2'-5'-isoTBA sequences remains unaltered by loop composition.

A 2'-5'-linked isoTBA 15 mer sequence with (232) loop composition formed stable antiparallel quadruplex structures similar to the SELEX derived 15 mer TBA sequence with (232) loop composition. A parallel versus antiparallel topology of 3'-5'-G-quadruplexes is largely dictated by the loop length, and it is known that the truncated loops favour parallel quadruplexes. In contrast to TBA, systematic reduction of the loop length in isoTBA from (232) to (222), (131) or even (111) did not alter the antiparallel topology of the resulting 14 mer, 13 mer and 11 mer G-rich modified isoTBA-like sequences.

The structure of G-quadruplex thrombine-binding DNA aptamer RA36

The structure of the 31-meric aptameric DNA oligonucleotide RA36 was studied. This aptamer inhibits the coagulant activity of thrombin more effectively compared with the widely known aptamer 15TGT (thrombin-binding aptamer). RA36 aptamer has a two-pattern structure, which includes two G-rich regions capable of forming a G-quadruplex. We showed by circular dichroism that the aptamer RA36 forms an anti-parallel G-quadruplex similar to the G-quadruplex of 15TGT. The thermal stability of G-quadruplex RA36 is significantly lower than that of 15TGT under physiological conditions (concentration of the stabilizing cation 5 mM). The double-quadruplex structure of RA36 is confirmed by the CD spectra of deletion mutants; i.e., G-quadruplex can be formed both by the first and the second G-rich site of aptamer RA36.

Interdependence of pyrene interactions and tetramolecular G4-DNA assembly.

Controlling the arrangement of organic chromophores in supramolecular architectures is of primary importance for the development of novel functional molecules. Insertion of a twisted intercalating nucleic acid (TINA) moiety, containing phenylethynylpyren-1-yl derivatives, into a G-rich DNA sequence alters G-quadruplex folding, resulting in supramolecular structures with defined pyrene arrangements. Based on CD, NMR and ESI-mass-spectra, as well as TINA excited dimer (excimer) fluorescence emission we propose that insertion of the TINA monomer in the middle of a dTG4T sequence (i.e. dTGGXGGT, where X is TINA) converts a parallel tetramolecular G-quadruplex into an assembly composed of two identical antiparallel G-quadruplex subunits stacked via TINA-TINA interface. Kinetic analysis showed that TINA-TINA association controls complex formation in the presence of Na(+) but barely competes with guanine-mediated association in K(+) or in the sequence with the longer G-run (dTGGGXGGGT). These results demonstrate new perspectives in the design of molecular entities that can kinetically control G-quadruplex formation and show how tetramolecular G-quadruplexes can be used as a tuneable scaffold to control the arrangement of organic chromophores.

Interaction of polycationic Ni(II)-salophen complexes with G-quadruplex DNA.

A series of nine Ni(II) salophen complexes involving one, two, or three alkyl-imidazolium side-chains was prepared. The lengths of the side-chains were varied from one to three carbons. The crystal structure of one complex revealed a square planar geometry of the nickel ion. Fluorescence resonance energy transfer melting of G-quadruplex structures in the presence of salophen complex were performed. The G-quadruplex DNA structures were stabilized in the presence of the complexes, but a duplex DNA was not. The binding constants of the complexes for parallel and antiparallel G-quadruplex DNA, as well as hairpin DNA, were measured by surface plasmon resonance. The compounds were selective for G-quadruplex DNA, as reflected by equilibrium dissociation constant KD values in the region 0.1-1 μM for G-quadruplexes and greater than 2 μM for duplex DNA. Complexes with more and shorter side-chains had the highest binding constants. The structural basis for the interaction of the complexes with the human telomeric G-quadruplex DNA was investigated by computational studies: the aromatic core of the complex stacked over the last tetrad of the G-quadruplex with peripherical cationic side chains inserted into opposite grooves. Biochemical studies (telomeric repeat amplification protocol assays) indicated that the complexes significantly inhibited telomerase activity with IC50 values as low as 700 nM; the complexes did not significantly inhibit polymerase activity.

Structure variations of TBA G-quadruplex induced by 2'-O-methyl nucleotide in K+ and Ca2+ environments.

Thrombin binding aptamer (TBA), a 15-mer oligonucleotide of d(GGTTGGTGTGGTTGG) sequence, folds into a chair-type antiparallel G-quadruplex in the K(+) environment, and each of two G-tetrads is characterized by a syn-anti-syn-anti glycosidic conformation arrangement. To explore its folding topology and structural stability, 2'-O-methyl nucleotide (OMe) with the C3'-endo sugar pucker conformation and anti glycosidic angle was used to selectively substitute for the guanine residues of G-tetrads of TBA, and these substituted TBAs were characterized using a circular dichroism spectrum, thermally differential spectrum, ultraviolet stability analysis, electrophoresis mobility shift assay, and thermodynamic analysis in K(+) and Ca(2+) environments. Results showed that single substitutions for syn-dG residues destabilized the G-quadruplex structure, while single substitutions for anti-dG residues could preserve the G-quadruplex in the K(+) environment. When one or two G-tetrads were modified with OMe, TBA became unstructured. In contrast, in Ca(2+) environment, the native TBA appeared to be unstructured. When two G-tetrads were substituted with OMe, TBA seemed to become a more stable parallel G-4 structure. Further thermodynamic data suggested that OMe-substitutions were an enthalpy-driven event. The results in this study enrich our understanding about the effects of nucleotide derivatives on the G-quadruplex structure stability in different ionic environments, which will help to design G-quadruplex for biological and medical applications.

Selective recognition of parallel and anti-parallel thrombin-binding aptamer G-quadruplexes by different fluorescent dyes.

G-quadruplexes (G4) have been found increasing potential in applications, such as molecular therapeutics, diagnostics and sensing. Both Thioflavin T (ThT) and N-Methyl mesoporphyrin IX (NMM) become fluorescent in the presence of most G4, but thrombin-binding aptamer (TBA) has been reported as the only exception of the known G4-forming oligonucleotides when ThT is used as a high-throughput assay to identify G4 formation. Here, we investigate the interactions between ThT/NMM and TBA through fluorescence spectroscopy, circular dichroism and molecular docking simulation experiments in the absence or presence of cations. The results display that a large ThT fluorescence enhancement can be observed only when ThT bind to the parallel TBA quadruplex, which is induced to form by ThT in the absence of cations. On the other hand, great promotion in NMM fluorescence can be obtained only in the presence of anti-parallel TBA quadruplex, which is induced to fold by K+ or thrombin. The highly selective recognition of TBA quadruplex with different topologies by the two probes may be useful to investigate the interactions between conformation-specific G4 and the associated proteins, and could also be applied in label-free fluorescent sensing of other biomolecules.