G-quadruplex DNA Structures Research Articles

Design and synthesis of new benzimidazole-carbazole conjugates for the stabilization of human telomeric DNA, telomerase inhibition, and their selective action on cancer cells.

Cell-permeable small molecules that enhance the stability of the G-quadruplex (G4) DNA structures are currently among the most intensively pursued ligands for inhibition of the telomerase activity. Herein we report the design and syntheses of four novel benzimidazole-carbazole conjugates and demonstrate their high binding affinity to G4 DNA. S1 nuclease assay confirmed the ligand mediated G-quadruplex DNA protection. Additional evidence from Telomeric Repeat Amplification Protocol (TRAP-LIG) assay demonstrated efficient telomerase inhibition activity by the ligands. Two of the ligands showed IC50 values in the sub-micromolar range in the TRAP-LIG assay, which are the best among the benzimidazole derivatives reported so far. The ligands also exhibited cancer cell selective nuclear internalization, nuclear condensation, fragmentation, and eventually antiproliferative activity in long-term cell viability assays. Annexin V-FITC/PI staining assays confirm that the cell death induced by the ligands follows an apoptotic pathway. An insight into the mode of ligand binding was obtained from the molecular dynamics simulations.

Elevated levels of G-quadruplex formation in human stomach and liver cancer tissues.

Four-stranded G-quadruplex DNA secondary structures have recently been visualized in the nuclei of human cultured cells. Here, we show that BG4, a G-quadruplex-specific antibody, can be used to stain DNA G-quadruplex structures in patient-derived tissues using immunohistochemistry. We observe a significantly elevated number of G-quadruplex-positive nuclei in human cancers of the liver and stomach as compared to background non-neoplastic tissue. Our results suggest that G-quadruplex formation can be detected and measured in patient-derived material and that elevated G-quadruplex formation may be a characteristic of some cancers.

Studies on Non-Covalent Interaction of Coumarin Attached Pyrimidine and 1-Methyl Indole 1,2,3 Triazole Analogues with Intermolecular Telomeric G-Quadruplex DNA Using ESI-MS and Spectroscopy

In the present study, electrospray ionization mass spectrometry (ESI-MS) and spectroscopy have been used to evaluate the non-covalent interaction, stoichiometry, and selectivity of two synthetic coumarin-attached nucleoside and non-nucleoside 1,2,3-triazoles, namely, (1-(5-(hydroxymethyl)-4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-furan-2-yl)5-methyl pyrimidine-2,4(1H,3H)-dione (Tr1) and 4-((1-((-1-methyl-1H-indol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (Tr2) with two different human telomeric intermolecular G-quadruplex DNA structures formed by d(T2AG3) and d(T2AG3)2 sequences. ESI-MS studies indicate that Tr1 specifically interacts with four-stranded intermolecular parallel quadruplex complex, whereas Tr2 interacts with two hairpin as well as four-stranded intermolecular parallel quadruplex complexes. UV–Visible spectroscopic studies suggest that Tr1 and Tr2 interact with G-quadruplex structure and unwind them. Job plots show that stoichiometry of ligand:quadruplex DNA is 1:1. Circular dichroism (CD) studies of G-quadruplex DNA and Tr1/Tr2 ligands manifest that they unfold DNA on interaction. Fluorescence studies demonstrate that ligand molecules intercalate between the two stacks of quadruplex DNA and non-radiative energy transfer occurs between the excited ligand molecules (donor) and quadruplex DNA (acceptor), resulting in enhancement of fluorescence emission intensity. Thus, these studies suggest that nucleoside and non-nucleoside ligands efficiently interact with d(T2AG3) and d(T2AG3)2 G-quadruplex DNA but the interaction is not alike with all kinds of quadruplex DNA, this is probably due to the variation in the pharmacophores and structure of the ligand molecules.

Advancement in the Structural Polymorphism of G-Quadruplexes

Genomes contain a large number of putative guanine-rich sequences, specifically on promoter regions, untranslated regions (UTR’s) and telomeres etc. that could form guanine-quadruplexes, and may serve as important structural and regulatory elements. They can also be the source of genomic instability which may lead to cancer, aging and human genetic diseases. Four guanines in the same plane, joined together with Hoogsteen hydrogen bonding, and stacked over one another resulting in guanine tetrads, give rise to an incredible class of G-quadruplexes. An extensive range of G-quadruplex structures is well documented, where they differ in number of strands (uni, bi, or tetramolecular), conformations (parallel, antiparallel or mixed), shapes (chair or basket form), or types of loops (edgewise/ lateral, diagonal, double chain reversal/ propeller, or V-shaped loops) etc. With advancements in the techniques, various new multistranded G-rich DNA structures are enriching the DNA structural databases. The most recent ones are (3+1) G-quadruplex, Tri-G-quadruplex, G-triplex DNA etc. which actually add to the diversity of G-quadruplex structures. Exploring their polymorphism with various biophysical and biochemical techniques has hence become extremely important. This review mainly focuses on the discussion of these unusual and comparatively new polymorphic G-quadruplex DNA structures. The robustness of these unique (3+1) G-quadruplex or G-triplex or tri-G-quadruplex structures actually can be exploited for providing a strong foundation for the designing of structure-specific drugs. Recently, G-quadruplex structures have been quantitatively visualized in human cells by engineering structure-specific antibody. Considering these developments, the mapping of G-quadruplex structures in genome may now be possible, with a goal of controlling the gene functions or other cellular processes, which might be involved in diseases like cancer.

Topology of a DNA G-Quadruplex Structure Formed in the HIV-1 Promoter: A Potential Target for Anti-HIV Drug Development

Nucleic acid sequences containing guanine tracts are able to adopt noncanonical four-stranded nucleic acid structures called G-quadruplexes (G4s). These structures are based on the stacking of two or more G-tetrads; each tetrad is a planar association of four guanines held together by eight hydrogen bonds. In this study, we analyzed a conserved G-rich region from HIV-1 promoter that is known to regulate the transcription of the HIV-1 provirus. Strikingly, our analysis of an alignment of 1684 HIV-1 sequences from this region showed a high conservation of the ability to form G4 structures despite a lower conservation of the nucleotide primary sequence. Using NMR spectroscopy, we determined the G4 topology adopted by a DNA sequence from this region (HIV-PRO1: 5' TGGCCTGGGCGGGACTGGG 3'). This DNA fragment formed a stable two G-tetrad antiparallel G4 with an additional Watson-Crick CG base pair. This hybrid structure may be critical for HIV-1 gene expression and is potentially a novel target for anti-HIV-1 drug development.

Targeted Singlet Oxygen Generation Using Different DNA-Interacting Perylene Diimide Type Photosensitizers

Singlet oxygen quantum yields (ΦΔ) of different perylene diimides (PDIs) containing phenyl (PDI-Ph), pyrene (PDI-Pyr), and indole (PDI-In) units in bay positions of the ring were determined using 1,3-diphenylisobenzofuran (DPBF) method in toluene/methanol (99:1) system. Pyrene-substituted PDI were the most efficient singlet oxygen generator among the investigated photosensitizers with a quantum yield of Φ Δ = 0.93 in toluene/methanol. Additionally, their binding affinities to G-quadruplex DNA structure were investigated by steady-state measurements. There were marked red shifts of absorbance bands for PDI-Pyr/DNA strand complexes with respect to the absorption maxima of DNA-free solution of PDI-Pyr in phosphate buffer at pH 6.

The DNA-forming properties of 6-selenoguanine

We present here an exhaustive characterization of the structure and properties of 6-selenoguanine, an isoster of guanine, and the impact of its introduction in DNA. This study reports the results of state-of-the-art quantum mechanical calculations and atomistic molecular dynamics simulations carried out to shed light on the impact of the replacement of guanine (G) by 6-selenoguanine (SeG) in different forms of DNA. The results point out that the G → SeG substitution leads to stable DNA duplex, antiparallel triplex and G-quadruplex structures, though local distortions are also found. These structural changes affect the thermodynamic stability of the mutation leading to a clear destabilization for all studied systems. Interestingly, the lowest effect has been found when the mutation was placed in the triplex-forming oligonucleotide strand in a reverse Hoogsteen orientation, which favours the antiparallel triplex formation regarding the G-tetraplex formation. Detailed QM studies strongly suggest that SeG impacts the HOMO-LUMO gap and accordingly the transfer properties of DNA, opening the way to modulate the conductivity properties of non-natural DNAs.

Nonlinear optical dye TSQ1 as an efficiently selective fluorescent probe for G-quadruplex DNA

A squarylium dye TSQ1 shows a remarkable fluorescence enhancement selectivity for the G-quadruplex DNA structure against duplex DNA, it can be distinguished even by the naked eye. What is more important, TSQ1 did not induce the G-rich sequence folding into G-quadruplex structure which means TSQ1 can be used to detect G-quadruplex structures in cells.

Visualization and selective chemical targeting of RNA G-quadruplex structures in the cytoplasm of human cells

Following extensive evidence for the formation of four-stranded DNA G-quadruplex structures in vitro, DNA G-quadruplexes have been observed within human cells. Although chemically distinct, RNA can also fold in vitro into G-quadruplex structures that are highly stable because of the 2'-hydroxyl group. However, RNA G-quadruplexes have not yet been reported in cells. Here, we demonstrate the visualization of RNA G-quadruplex structures within the cytoplasm of human cells using a G-quadruplex structure-specific antibody. We also demonstrate that small molecules that bind to G-quadruplexes in vitro can trap endogenous RNA G-quadruplexes when applied to cells. Furthermore, a small molecule that exhibits a preference for RNA G-quadruplexes rather than DNA G-quadruplexes in biophysical experiments also shows the same selectivity within a cellular context. Our findings provide substantive evidence for RNA G-quadruplex formation in the human transcriptome, and corroborate the selectivity and application of stabilizing ligands that target G-quadruplexes within a cellular context.

Development of a new biosensor based on functionalized SBA-15 modified screen-printed graphite electrode as a nano-reactor for Gquadruplex recognition

Gquadruplex is an active target for therapeutic purposes because of the evidence which suggest that G-rich region of the human genome may form Gquadruplex structure. The electrochemical biosensor was prepared by modifying screen-printed graphite electrode (SPE) with synthesized SBA-N-propylpipyrazine-N-(2-mercaptopropane-1-one) (SBA@NPPNSH) mesoporous structure to investigate the Gquadruplex DNA structure (G4DNA). Ascorbic acid (AA) is known as an antioxidant agent that induces reductive properties. It is also important for some therapeutic purposes. In this study, AA was used as the model ligand and its ability to interact with Gquadruplex structure was examined. The pore of SBA@NPPNSH structure can act as a nano-reactor and the interaction of G4DNA/AA is accomplished inside these channels. The structure of SBA@NPPNSH was characterized by transmission electron microscope (TEM), X-ray powder diffraction (XRD), thermo gravimetric analysis (TGA) methods and atomic force microscopy (AFM). The interaction of AA with G4DNA was studied in Tris-HCl buffer and also in the presence of [Fe(CN)6](3-) as a redox label using the CV method. CV current decreases with the increasing concentrations of AA due to the interaction of G4DNA/AA. Circular dichroism (CD) spectroscopy was used to examine the ability of AA to form Gquadruplexes from short and long complementary G4DNA strands. Studying the selectivity using different dsDNA sequences revealed that AA could stabilize G4DNA. Thus, the proposed biosensor can distinguish G4DNA structure from other dsDNA structures.

Effect of Pressure on Thermal Stability of G-Quadruplex DNA and Double-Stranded DNA Structures

Pressure is a thermodynamic parameter that can induce structural changes in biomolecules due to a volumetric decrease. Although most proteins are denatured by pressure over 100 MPa because they have the large cavities inside their structures, the double-stranded structure of DNA is stabilized or destabilized only marginally depending on the sequence and salt conditions. The thermal stability of the G-quadruplex DNA structure, an important non-canonical structure that likely impacts gene expression in cells, remarkably decreases with increasing pressure. Volumetric analysis revealed that human telomeric DNA changed by more than 50 cm3 mol−1 during the transition from a random coil to a quadruplex form. This value is approximately ten times larger than that for duplex DNA under similar conditions. The volumetric analysis also suggested that the formation of G-quadruplex DNA involves significant hydration changes. The presence of a cosolute such as poly(ethylene glycol) largely repressed the pressure effect on the stability of G-quadruplex due to alteration in stabilities of the interactions with hydrating water. This review discusses the importance of local perturbations of pressure on DNA structures involved in regulation of gene expression and highlights the potential for application of high-pressure chemistry in nucleic acid-based nanotechnology.

Identification of new natural DNA G-quadruplex binders selected by a structure-based virtual screening approach.

The G-quadruplex DNA structures are mainly present at the terminal portion of telomeres and can be stabilized by ligands able to recognize them in a specific manner. The recognition process is usually related to the inhibition of the enzyme telomerase indirectly involved and over-expressed in a high percentage of human tumors. There are several ligands, characterized by different chemical structures, already reported in the literature for their ability to bind and stabilize the G-quadruplex structures. Using the structural and biological information available on these structures; we performed a high throughput in silico screening of commercially natural compounds databases by means of a structure-based approach followed by docking experiments against the human telomeric sequence d[AG3(T2AG3)3]. We identified 12 best hits characterized by different chemical scaffolds and conformational and physicochemical properties. All of them were associated to an improved theoretical binding affinity with respect to that of known selective G-binders. Among these hits there is a chalcone derivative; structurally very similar to the polyphenol butein; known to remarkably inhibit the telomerase activity.

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.

Specialization among Iron-Sulfur Cluster Helicases to Resolve G-quadruplex DNA Structures That Threaten Genomic Stability

G-quadruplex (G4) DNA, an alternate structure formed by Hoogsteen hydrogen bonds between guanines in G-rich sequences, threatens genomic stability by perturbing normal DNA transactions including replication, repair, and transcription. A variety of G4 topologies (intra- and intermolecular) can form in vitro, but the molecular architecture and cellular factors influencing G4 landscape in vivo are not clear. Helicases that unwind structured DNA molecules are emerging as an important class of G4-resolving enzymes. The BRCA1-associated FANCJ helicase is among those helicases able to unwind G4 DNA in vitro, and FANCJ mutations are associated with breast cancer and linked to Fanconi anemia. FANCJ belongs to a conserved iron-sulfur (Fe S) cluster family of helicases important for genomic stability including XPD (nucleotide excision repair), DDX11 (sister chromatid cohesion), and RTEL (telomere metabolism), genetically linked to xeroderma pigmentosum/Cockayne syndrome, Warsaw breakage syndrome, and dyskeratosis congenita, respectively. To elucidate the role of FANCJ in genomic stability, its molecular functions in G4 metabolism were examined. FANCJ efficiently unwound in a kinetic and ATPase-dependent manner entropically favored unimolecular G4 DNA, whereas other Fe-S helicases tested did not. The G4-specific ligands Phen-DC3 or Phen-DC6 inhibited FANCJ helicase on unimolecular G4 ∼1000-fold better than bi- or tetramolecular G4 DNA. The G4 ligand telomestatin induced DNA damage in human cells deficient in FANCJ but not DDX11 or XPD. These findings suggest FANCJ is a specialized Fe-S cluster helicase that preserves chromosomal stability by unwinding unimolecular G4 DNA likely to form in transiently unwound single-stranded genomic regions.

Synthesis, G‐Quadruplex Stabilisation, Docking Studies, and Effect on Cancer Cells of Indolo[3,2‐b]quinolines with One, Two, or Three Basic Side Chains

G-quadruplex (G4) DNA structures in telomeres and oncogenic promoter regions are potential targets for cancer therapy, and G4 ligands have been shown to modulate telomerase activity and oncogene transcription. Herein we report the synthesis and G4 thermal stabilisation effects, determined by FRET melting assays, of 20 indolo[3,2-b]quinolines mono-, di-, and trisubstituted with basic side chains. Molecular modelling studies were also performed in an attempt to rationalise the ligands' binding poses with G4. Overall, the results suggest that ligand binding and G4 DNA thermal stabilisation increase with an N5-methyl or a 7-carboxylate group and propylamine side chains, whereas selectivity between G4 and duplex DNA appears to be modulated by the number and relative position of basic side chains. From all the indoloquinoline derivatives studied, the novel trisubstituted compounds 3 d and 4 d, bearing a 7-(aminoalkyl)carboxylate side chain, stand out as the most promising compounds; they show high G4 thermal stabilisation (ΔTm values between 17 and 8 °C) with an inter-G4 ΔTm trend of Hsp90A>KRas21R≈F21T>c-Kit2, 10-fold selectivity for G4 over duplex DNA, and 100-fold selectivity for the HCT116 cancer cell line (IC50 and IC90: <10 μM) over primary rat hepatocytes. Compounds 3 d and 4 d also decreased protein expression levels of Hsp90 and KRas in HCT116 cancer cells.

Toward the design of new DNA G-quadruplex ligands through rational analysis of polymorphism and binding data

Human telomeres play a key role in protecting chromosomal ends from fusion events; they are composed of d(TTAGGG) repeats, ranging in size from 3 to 15 kb. They form G-quadruplex DNA structures, stabilized by G-quartets in the presence of cations, and are involved in several biological processes. In particular, a telomere maintenance mechanism is provided by a specialized enzyme called telomerase, a reverse transcriptase able to add multiple copies of the 5′-GGTTAG-3′ motif to the end of the G-strand of the telomere and which is over-expressed in the majority of cancer cells. The central cation has a crucial role in maintaining the stability of the structure. Based on its nature, it can be associated with different topological telomeric quadruplexes, which depend also on the orientation of the DNA strands and the syn/anti conformation of the guanines. Such a polymorphism, confirmed by the different structures deposited in the Protein Data Bank (PDB), prompted us to apply a computational protocol in order to investigate the conformational properties of a set of known G-quadruplex ligands and their molecular recognition against six different experimental models of the human telomeric sequence d[AG3(T2AG3)3]. The average AutoDock correlation between theoretical and experimental data yielded an r2 value equal to 0.882 among all the studied models. Such a result was always improved with respect to those of the single folds, with the exception of the parallel structure (r2 equal to 0.886), thus suggesting a key role of this G4 conformation in the stacking interaction network. Among the studied binders, a trisubstituted acridine and a dibenzophenanthroline derivative were well recognized by the parallel and the mixed G-quadruplex structures, allowing the identification of specific key contacts with DNA and the further design of more potent or target specific G-quadruplex ligands.

Single molecule FRET data analysis procedures for FRET efficiency determination: Probing the conformations of nucleic acid structures

Single molecule FRET microscopy is an attractive technique for studying structural dynamics and conformational diversity of nucleic acid structures. Some of its strengths are that it can follow structural changes on a fast time scale and identify conformation distributions arising from dynamic or static population heterogeneity. Here, we give a description of the experiment and data analysis procedures of this method and detail what parameters are needed for FRET efficiency calculation. Using single molecule FRET data obtained on G-quadruplex DNA structures that exhibit large conformation diversity, we illustrate that the shape of the FRET distribution changes depending on what parameters are included in the data analysis procedure.

High-Resolution Insight into G-Overhang Architecture

NMR and fluorescence spectroscopy were used to address the effect of intracellular molecular crowding and related hydration on a model telomeric G-quadruplex (G4) DNA structure (d(AG(3)(TTAGGG)(3))). d(AG(3)(TTAGGG)(3)) prevalently adopted the hybrid-1 conformation in vivo, ex vivo, and in dilute potassium-based solution, while it formed the parallel propeller fold in water-depleted potassium-based solution, a commonly used model system for studying intracellular molecular crowding. The dilute potassium-based solution appeared to imitate the properties of the cellular environment required for d(AG(3)(TTAGGG)(3)) folding under in vivo and ex vivo conditions. High-resolution NMR investigations of site-specifically (15)N-labeled G4 units in native-like single-stranded telomeric DNA revealed that the 3'-terminal and internal G4 unit predominantly coexist in 2-tetrad antiparallel basket and hybrid-2 structures that are arranged in "beads-on-a-string"-like fashion. Our data provide the first high-resolution insight into the telomeric G-overhang architecture under essentially physiological conditions and identify the 2-tetrad antiparallel basket and hybrid-2 topologies as the structural targets for the development of telomere-specific G4 ligands.

Research shows four-stranded DNA in human cancer cells

Researchers have for the first time identified an increase in four-stranded G-quadruplex DNA structures at a crucial point in DNA replication in human cancer cells. These structures could be a new target for cancer drugs.

Quantitative visualization of DNA G-quadruplex structures in human cells

Four-stranded G-quadruplex nucleic acid structures are of great interest as their high thermodynamic stability under near-physiological conditions suggests that they could form in cells. Here we report the generation and application of an engineered, structure-specific antibody employed to quantitatively visualize DNA G-quadruplex structures in human cells. We show explicitly that G-quadruplex formation in DNA is modulated during cell-cycle progression and that endogenous G-quadruplex DNA structures can be stabilized by a small-molecule ligand. Together these findings provide substantive evidence for the formation of G-quadruplex structures in the genome of mammalian cells and corroborate the application of stabilizing ligands in a cellular context to target G-quadruplexes and intervene with their function.