G-quadruplex Recognition Research Articles

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.

G-Quadruplex Structures in the Human Genome as Novel Therapeutic Targets

G-quadruplexes are secondary structures that may form within guanine-rich nucleic acid sequences. Telomeres have received much attention in this regard since they can fold into several distinct intramolecular G-quadruplexes, leading to the rational design and development of G-quadruplex-stabilizing molecules. These ligands were shown to selectively exert an antiproliferative and chemosensitizing activity in in vitro and in vivo tumor models, without appreciably affecting normal cells. Such findings point to them as possible drug candidates for clinical applications. Other than in telomeres, G-quadruplexes may form at additional locations in the human genome, including gene promoters and untranslated regions. For instance, stabilization of G-quadruplex structures within the promoter of MYC, KIT, or KRAS resulted in the down-regulation of the corresponding oncogene either in gene reporter assays or in selected experimental models. In addition, the alternative splicing of a number of genes may be affected for a therapeutic benefit through the stabilization of G-quadruplexes located within pre-mRNAs. It is now emerging that G-quadruplex structures may act as key regulators of several biological processes. Consequently, they are considered as attractive targets for broad-spectrum anticancer therapies, and much effort is being made to develop a variety of ligands with improved G-quadruplex recognition properties. Quarfloxin, a fluoroquinolone derivative designed to target a G-quadruplex within ribosomal DNA and disrupt protein-DNA interactions, has entered clinical trials for different malignancies. This review will provide some hints on the role of G-quadruplex structures in biological processes and will evaluate their implications as novel therapeutic targets.

Recognition of intermolecular G-quadruplexes by full length nucleophosmin. Effect of a leukaemia-associated mutation

Nucleophosmin (NPM) is a nucleolar protein involved in ribosome biogenesis. NPM1 gene is frequently mutated in acute myeloid leukaemia (AML), correlating with aberrant cytoplasmic localization of the protein. NPM attachment to the nucleolus in physiological conditions probably depends on binding to nucleic acids, and this recognition could be altered in AML. NPM associates to guanine-rich DNA sequences, able to fold as “G-quadruplexes”. We have analyzed the interaction of pentameric, full length NPM with G-rich oligonucleotides, finding that the protein binds preferentially high-order G-quadruplexes. AML-associated mutation significantly hampers DNA binding, pointing to a possible mechanism contributing to pathological mislocalization of NPM.

Specific recognition and stabilization of monomeric and multimeric G-quadruplexes by cationic porphyrin TMPipEOPP under molecular crowding conditions

Ligands targeting telomeric G-quadruplexs are considered good candidates for anticancer drugs. However, current studies on G-quadruplex ligands focus exclusively on the interactions of ligands and monomeric G-quadruplexes under dilute conditions. Living cells are crowded with biomacromolecules, and the ∼200-nucleotide G-rich single-stranded overhang of human telomeric DNA has the potential to fold into multimeric G-quadruplex structures containing several G-quadruplex units. Studies on interactions between ligands and multimeric G-quadruplexes under molecular crowding conditions could provide a new route for screening specific telomeric G-quadruplex-targeting ligands. Herein, TMPipEOPP, a cationic porphyrin derivative designed by us, was demonstrated as a promising multimeric telomeric G-quadruplex ligand under molecular crowding conditions. It could highly specifically recognize G-quadruplexes. It could also promote the formation of G-quadruplexes and stabilize them. Detailed studies showed that TMPipEOPP interacted with monomeric G-quadruplexes in sandwich-like end-stacking mode of quadruplex/TMPipEOPP/quadruplex and interacted with multimeric human telomeric G-quadruplexes by intercalating into the pocket between two adjacent G-quadruplex units. The pocket size greatly affected TMPipEOPP binding. A larger pocket was advantageous for the intercalation of TMPipEOPP. This work provides new insights into the ligand-binding properties of multimeric G-quadruplexes under molecular crowding conditions and introduces a new route for screening anticancer drugs targeting telomeric G-quadruplexes.

Dual recognition of the human telomeric G-quadruplex by a neomycin-anthraquinone conjugate.

The authors report the recognition of a G-quadruplex formed by four repeat human telomeric DNA with aminosugar intercalator conjugates. The recognition of the G-quadruplex through dual binding mode ligands significantly increased the affinity of ligands for the G-quadruplex. One such example is a neomycin-anthraquinone conjugate (2) which exhibited nanomolar affinity for the quadruplex, and the affinity of (2) is nearly 1000 fold higher for the human telomeric G-quadruplex DNA than its constituent units, neomycin and anthraquinone.

Shooting for Selective Druglike G-Quadruplex Binders: Evidence for Telomeric DNA Damage and Tumor Cell Death

Targeting of DNA secondary structures, such as G-quadruplexes, is now considered an appealing opportunity for drug intervention in anticancer therapy. So far, efforts made in the discovery of chemotypes able to target G-quadruplexes mainly succeeded in the identification of a number of polyaromatic compounds featuring end-stacking binding properties. Against this general trend, we were persuaded that the G-quadruplex grooves can recognize molecular entities with better drug-like and selectivity properties. From this idea, a set of small molecules was identified and the structural features responsible for G-quadruplex recognition were delineated. These compounds were demonstrated to have enhanced affinity and selectivity for the G-quadruplex over the duplex structure. Their ability to induce selective DNA damage at telomeric level and to induction of apoptosis and senescence on tumor cells is herein experimentally proven.

G-quadruplex recognition activities of E. Coli MutS

BackgroundGuanine quadruplex (G4 DNA) is a four-stranded structure that contributes to genome instability and site-specific recombination. G4 DNA folds from sequences containing tandemly repetitive guanines, sequence motifs that are found throughout prokaryote and eukaryote genomes. While some cellular activities have been identified with binding or processing G4 DNA, the factors and pathways governing G4 DNA metabolism are largely undefined. Highly conserved mismatch repair factors have emerged as potential G4-responding complexes because, in addition to initiating heteroduplex correction, the human homologs bind non-B form DNA with high affinity. Moreover, the MutS homologs across species have the capacity to recognize a diverse range of DNA pairing variations and damage, suggesting a conserved ability to bind non-B form DNA.ResultsHere, we asked if E. coli MutS and a heteroduplex recognition mutant, MutS F36A, were capable of recognizing and responding to G4 DNA structures. We find by mobility shift assay that E. coli MutS binds to G4 DNA with high affinity better than binding to G-T heteroduplexes. In the same assay, MutS F36A failed to recognize G-T mismatched oligonucleotides, as expected, but retained an ability to bind to G4 DNA. Association with G4 DNA by MutS is not likely to activate the mismatch repair pathway because nucleotide binding did not promote release of MutS or MutS F36A from G4 DNA as it does for heteroduplexes. G4 recognition activities occur under physiological conditions, and we find that M13 phage harboring G4-capable DNA poorly infected a MutS deficient strain of E. coli compared to M13mp18, suggesting functional roles for mismatch repair factors in the cellular response to unstable genomic elements.ConclusionsTaken together, our findings demonstrate that E. coli MutS has a binding activity specific for non-B form G4 DNA, but such binding appears independent of canonical heteroduplex repair activation.

A New Cationic Porphyrin Derivative (TMPipEOPP) with Large Side Arm Substituents: A Highly Selective G-Quadruplex Optical Probe

The discovery of uncommon DNA structures and speculation about their potential functions in genes has brought attention to specific DNA structure recognition. G-quadruplexes are four-stranded nucleic acid structures formed by G-rich DNA (or RNA) sequences. G-rich sequences with a high potential to form G-quadruplexes have been found in many important genomic regions. Porphyrin derivatives with cationic side arm substituents are important G-quadruplex-binding ligands. For example, 5,10,15,20-Tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin (TMPyP4), interacts strongly with G-quadruplexes, but has poor selectivity for G-quadruplex versus duplex DNA. To increase the G-quadruplex recognition specificity, a new cationic porphyrin derivative, 5,10,15,20-tetra-{4-[2-(1-methyl-1- piperidinyl)ethoxy]phenyl} porphyrin (TMPipEOPP), with large side arm substituents was synthesized, and the interactions between TMPipEOPP and different DNA structures were compared. The results show that G-quadruplexes cause large changes in the UV-Vis absorption and fluorescence spectra of TMPipEOPP, but duplex and single-stranded DNAs do not, indicating that TMPipEOPP can be developed as a highly specific optical probe for discriminating G-quadruplex from duplex and single-stranded DNA. Visual discrimination is also possible. Job plot and Scatchard analysis suggest that a complicated binding interaction occurs between TMPipEOPP and G-quadruplexes. At a low [G-quadruplex]/[TMPipEOPP] ratio, one G-quadruplex binds two TMPipEOPP molecules by end-stacking and outside binding modes. At a high [G-quadruplex]/[TMPipEOPP] ratio, two G-quadruplexes bind to one TMPipEOPP molecule in a sandwich-like end-stacking mode.

Formation and recognition of G-quadruplex relevant for pilin antigenic variation in Neisseria gonorrhoeae

In this research, NMR, ESI-MS, and circular dichroism (CD) spectroscopies were used to investigate the formation and recognition of G-quadruplex by a G-rich sequence that has been demonstrated to be relevant for pilin variation in Neisseria gonorrhoeae . The NMR spectra provided strong evidence that the G-quadruplex structure was formed from this G-rich sequence, and this result was confirmed by CD spectroscopy. In addition, two small-molecule natural products (jatrorrhizine hydrochloride (J) and dehydrocorydaline (D)) were found to bind to this G-quadruplex over the corresponding duplex DNA with high selectivity by ESI-MS.

Selective recognition of G-quadruplex in the vascular endothelial growth factor gene with small-molecule natural products by electrospray ionization (ESI) mass spectrometry and circular dichroism (CD) spectrometry

In this study, electrospray ionization mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy were used to investigate selective recognition of G-quadruplex in the vascular endothelial growth factor (VEGF) gene with 12 small-molecule natural products. We found that kaempferol, a natural flavonol, shows the highest binding affinity among the 12 natural molecules. The results from ESI-MS and CD spectra indicated that kaempferol could enhance the thermal stability of the VEGF–G-quadruplex and showed selective recognition for the G-quadruplex in a solution consisting of the G-quadruplex and the corresponding duplex DNA.

Tuning G-Quadruplex vs Double-Stranded DNA Recognition in Regioisomeric Lysyl-Peptidyl-Anthraquinone Conjugates

Anthraquinone is a versatile scaffold to provide effective DNA binders. This planar system can be easily conjugated to protonable side chains: the nature of the lateral groups and their positions around the tricyclic moiety largely affect the DNA recognition process in terms of binding affinity and mode, as well as sequence and structure of the target nucleic acid. Starting from an anthracenedione system symmetrically functionalized with N-terminal lysyl residues, we incremented the length of side chains by introducing a Gly, Ala, or Phe spacer, characterized by different flexibility, lipophilicity, and bulkiness. Moreover, 2,6, 2,7, 1,8, and 1,5 regioisomers were examined to yield a small bis(lysyl-peptidyl) anthracenedione library. By merging spectroscopic, enzymatic, and cellular results, we showed that the proper combination of a basic aminoacid (Lys) with a more hydrophobic residue (Phe) can provide selective G-quadruplex recognition, in particular when side chains are located at positions 2,6 or 2,7. In fact, while these derivatives effectively bind G-quadruplex structures, they behave at the same time as rather poor double-stranded DNA intercalators. As a result, the Lys-Phe substituted anthraquinones are poorly cytotoxic but still able to promote a senescence mechanism in cancer cells. This combination of chemical and biological properties foresees potentially valuable applications in anticancer medicinal chemistry.

Formation and recognition of G-quadruplex in promoter of c-myb oncogene by electrospray ionization mass spectrometry

In this study, electrospray ionization mass spectrometry (ESI-MS) is used to study the formation of G-quadruplex by d(GGAGGAGGAGGA) which locates at the promoter region of c-myb gene. In addition, a natural small molecule, dehydrocorydaline from a Chinese herb, is found to have the highest binding affinity with the G-quadruplex in nine natural small molecules studied, and the binding selectivity of this natural molecule toward the c-myb G-quadruplex with respect to corresponding duplex DNA is significantly higher than that of the broad-spectrum G-quadruplex-ligand TMPyP4. The result from ESI-MS indicates that the gas-phase kinetic stability of the G-quadruplex can be enhanced by binding of dehydrocorydaline. To further investigate the binding properties of dehydrocorydaline to the G-quadruplex, Autodock3 is used to calculate the docked sites and docked energies of small molecules binding to the G-quadruplex and the result shows that the docked energy of dehydrocorydaline is the biggest in the nine small molecules used, consistent with the result from ESI-MS.

Formation, recognition and bioactivities of a novel G-quadruplex in the STAT3 gene

G-Quadruplexes have been proved to exist in 5'-untranslated region (5'-UTR), promoter, intron and exon regions of many human genes. Here we report an intramolecular G-quadruplex formed by a G-rich sequence in the 3'-flanking region of signal transducers and activators of transcription 3 (STAT3) gene. The results showed that this G-rich sequence could affect the expression of STAT3. When the STAT3 G-quadruplex was stabilized by a novel non-planar ligand Cepharanthine (CEP), the decreased expression of STAT3 was observed in primary cultured cardiomyocytes. We also demonstrated that the down-regulation of STAT3 was most likely occurred at the transcriptional level. Our results provide a new clue for studying the G-quadruplex formation, recognition and function in the 3'-flanking region of gene.

Nucleic acids targeted to drugs: SELEX against a quadruplex ligand

A number of small molecules demonstrate selective recognition of G-quadruplexes and are able to stabilize their formation. In this work, we performed the synthesis of two biotin-tagged G4 ligands and analyzed their interactions with DNA by two complementary techniques, FRET and FID. The compound that exhibited the best characteristics (a biotin pyridocarboxamide derivative with high stabilization of an intramolecular quadruplex and excellent duplex–quadruplex specificity) was used as bait for in vitro selection (SELEX). Among 80 DNA aptamer sequences selected, only a small minority (5/80) exhibited G4-prone motifs. Binding of consensus candidates was confirmed by SPR. These results indicate that G4 ligands that appear highly specific when comparing affinities or stabilization for one quadruplex against one duplex, do not only bind quadruplex sequences but may also recognize other nucleic motifs as well. This observation may be relevant when whole genome or transcriptome analysis of binding sites is seeked for, as unexpected binding sites may also be present.

The evolving world of protein-G-quadruplex recognition: A medicinal chemist’s perspective

The physiological and pharmacological role of nucleic acids structures folded into the non canonical G-quadruplex conformation have recently emerged. Their activities are targeted at vital cellular processes including telomere maintenance, regulation of transcription and processing of the pre-messenger or telomeric RNA. In addition, severe conditions like cancer, fragile X syndrome, Bloom syndrome, Werner syndrome and Fanconi anemia J are related to genomic defects that involve G-quadruplex forming sequences. In this connection G-quadruplex recognition and processing by nucleic acid directed proteins and enzymes represents a key event to activate or deactivate physiological or pathological pathways. In this review we examine protein-G-quadruplex recognition in physiologically significant conditions and discuss how to possibly exploit the interactions’ selectivity for targeted therapeutic intervention.

Fishing potential antitumor agents from natural plant extracts pool by dialysis and G-quadruplex recognition

Screening G-quadruplex ligands from natural plants is important because the ligands may be potential antitumor drugs. A new screening strategy is proposed based on the combination of dialysis and G-quadruplex recognition technique which could separates G-quadruplex ligand from natural extracts and elucidate the structure of this ligand. This result offers a novel approach to obtain active antitumor compounds.

348 Targeting pancreatic cancers with a G-quadruplex binding small molecule

The tumor suppressor protein p53 is a key factor in genome stability and one of the most studied of DNA binding proteins. This is the first study on the interaction of wild-type p53 with guanine quadruplexes formed by the human telomere sequence. Using electromobility shift assay and ELISA, we show that p53 binding to telomeric G-quadruplexes increases with the number of telomeric repeats. Further, p53 strongly favors G-quadruplexes folded in potassium over those formed in sodium, thus indicating the telomeric G-quadruplex conformational selectivity of p53. The presence of the quadruplex-stabilizing ligand, N-methyl mesoporphyrin IX (NMM), increases p53 recognition of G-quadruplexes in potassium. Using deletion mutants and selective p53 core domain oxidation, both p53 DNA binding domains are shown to be crucial for telomeric G-quadruplex recognition.

Selective recognition of oncogene promoter G-quadruplexes by Mg2+

Mg2+ is one of the most important cations in cells, affecting the structures and functions of the proteins and nucleic acids. It should be noted that Mg2+ is indispensable in DNA transcription, where G-quadruplex is believed to be actively involved. Therefore, it is important to investigate the influence of Mg2+ on G-quadruplex. Here we studied the effect of Mg2+ on G-quadruplex DNA with CD, FRET, EMSA, and PCR-stop assay. We found that various G-quadruplexes could be differentiated through simultaneous addition of both K+ and Mg2+, which could be used for selective identification of G-quadruplexes in promoter oncogene but not in telomere. Mg2+ at physiological relevant concentration not only greatly enhanced the thermostability of oncogene G-quadruplexes but also efficiently protected them from unfolding by their complementary strands, which revealed the great impact of Mg2+ on the equilibrium between promoter G-quadruplex and duplex DNA. The PCR-stop assay further confirmed that Mg2+ could affect gene transcription by stabilizing promoter G-quadruplex. The above studies were carried out for various G-quadruplexes of varying sequences in promoter oncogenes and telomeric region. Our results suggest that Mg2+ may be a key regulator for G-quadruplexes of oncogene promoter, which can subsequently affect the expression of related genes.

Parallel G-quadruplex Recognition and Stabilization by Oxazine750

The bindings of small molecular oxazine750 to d[TG4T]4,d[G4T4G4]2 and c-myc sequences was characterized by means of circular dichroism,thermal denaturation,and so on.Oxazine750 was found to bind preferentially and strongly to parallel G-quadruplex.It can effectively stabilize the parallel G-quadruplex and destabilize the antiparallel G-quadruplex.The melting temperature of d[TG4T]4 or c-myc is increased by about 11 ℃ or 40 ℃ by oxazine750,respectively.Under the same condition,oxazine750 decreased the melting temperature of d[G4T4G4]2 by about 8 ℃.

Bis-phenanthroline derivatives as suitable scaffolds for effective G-quadruplex recognition

Selective recognition of DNA folding is central to multiple biological and pharmacological applications aimed at precise targeting of distinct genomic regions. Here, we focused on the recognition of physiologically relevant G-quadruplex (G-4) structures by bis-phenanthroline (bis-Phen) ligands containing two Phen moieties covalently linked through an amine or thioether bond. The transition metal ions Mn(2+), Ni(2+), Cu(2+), and the biologically relevant Mg(2+) and Zn(2+) efficiently form 1 : 1 bis-Phen complexes characterised by a large planar structure fit to successfully recognise G-quartet arrangements.Interestingly, metal ion complexation dramatically affects ligand-stabilising effects on G-quadruplex, the melting temperature of the folded structure being increased up to 30 degrees C at ligand concentrations as low as 1 microM in the presence of Ni(2+) and Cu(2+). In addition, the test complexes were able to induce G-4 formation from essentially unfolded G-rich sequences even in the absence of K(+) ions as shown by gel shift and circular dichroism experiments. In line with their G-4 stabilising properties bis-Phen complexes are effective inhibitors of telomerase activity, Ni(II) complexes being effective in the sub-micromolar range. This is combined with lack of unselective DNA-damaging activity and short-term cellular toxicity, which makes the novel compounds (above all their Ni(II) complexes) interesting antiproliferative drug leads.