G-quadruplex Stability Research Articles

Hybrids Based on Graphene Oxide and Porphyrin as Tools for Detection and Stabilization of DNA G-Quadruplexes.

Telomerase inhibition has been an important strategy in cancer therapies, but for which effective drugs are still required. Here, noncovalent hybrid nanoplatforms containing the tetracationic 5,10,15,20-tetrakis(1-methyl-pyridinium-4-yl)porphyrin (TMPyP) and graphene oxide (GO) were prepared for promoting telomerase inhibition through the selective detection and stabilization of DNA guanine-quadruplex (G-Q) structures. Upon binding TMPyP to the GO sheets, the typical absorption bands of porphyrin have been red-shifted and the fluorescence emission was quenched. Raman mapping was used for the first time to provide new insights into the role of the electrostatic and π–π stacking interactions in the formation of such hybrids. The selective recovery of fluorescence observed during the titration of TMPyP@GO with G-Q, resembles a selective “turn-off–on” fluorescence sensor for the detection of G-Q, paving the way for a new class of antitumor drugs.

Loop permutation affects the topology and stability of G-quadruplexes.

G-quadruplexes are unusual DNA and RNA secondary structures ubiquitous in a variety of organisms including vertebrates, plants, viruses and bacteria. The folding topology and stability of intramolecular G-quadruplexes are determined to a large extent by their loops. Loop permutation is defined as swapping two or three of these regions so that intramolecular G-quadruplexes only differ in the sequential order of their loops. Over the past two decades, both length and base composition of loops have been studied extensively, but a systematic study on the effect of loop permutation has been missing. In the present work, 99 sequences from 21 groups with different loop permutations were tested. To our surprise, both conformation and thermal stability are greatly dependent on loop permutation. Loop permutation actually matters as much as loop length and base composition on G-quadruplex folding, with effects on Tm as high as 17°C. Sequences containing a longer central loop have a high propensity to adopt a stable non-parallel topology. Conversely, sequences containing a short central loop tend to form a parallel topology of lower stability. In addition, over half of interrogated sequences were found in the genomes of diverse organisms, implicating their potential regulatory roles in the genome or as therapeutic targets. This study illustrates the structural roles of loops in G-quadruplex folding and should help to establish rules to predict the folding pattern and stability of G-quadruplexes.

Abstract 687: A dGMP fill-in G-quadruplex forms in the PDGFR-β promoter that serves as a unique target for drug design

Abstract Platelet-derived growth factor receptor beta (PDGFR-β) is a cell-surface-receptor tyrosine kinase implicated in platelet-derived growth factor (PDGF) signaling pathways, and overexpression of PDGFR-β is associated with tumor growth, angiogenesis, and migration, making PDGFR-β an attractive target for cancer therapy. The formation of DNA G-quadruplexes in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter has been found to inhibit PDGFR-β transcriptional activity. We previously determined the major G-quadruplex structure formed in the PDGFR-β promoter using a 22-mer sequence of pu22m1, which adopts a folding pattern containing a unique broken G-strand with the 3' tetrad-guanine coming from a distant site connected by a five nucleotide lateral loop. However, extension of the 3' end flanking segment of pu22m1 results in destabilization of the G-quadruplex, suggesting that this structure may be unstable in vivo. Recently, it has been reported that a unique type of intramolecular G-vacancy-bearing G-quadruplex (GVBQ) can be formed with three G3 tracts and one G2 tract and has an incomplete G-tetrad with a G-vacancy. A guanine derivative, such as GMP or GTP, can fill the G-vacancy with Hoogsteen hydrogen bonding and complete the G-tetrad, resulting in G-quadruplex stabilization. The unique broken-strand structure of PDGFR-β pu22m1 suggests a possibility of the formation of a GVBQ structure in this sequence, which can be completed and stabilized by guanine and guanine derivatives. To test this hypothesis, we examined the formation and stability of G-quadruplex formed in pu22m1 variants and the potential of guanine derivatives to fill in the incomplete G-tetrad and stabilize the overall G-quadruplex, using nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy and DMS footprinting experiments. Our results showed that a GVBQ could form in in the PDGFR-β promoter sequence and that dGMP could insert and complete the incomplete G-tetrad to induce the formation of a stable and well-defined dGMP-fill-in-GVBQ in potassium solution. In addition, we discovered two natural plant alkaloids, berberine and chelerythrine, which could stabilize this dGMP-fill-in-GVBQ, with an increase in melting temperature by more than 10 °C. Therefore, it appears that dGMP could interact and stabilize the broken-stranded PDGFR-β promoter G-quadruplex, which may play a role in PDGFR-β transcriptional regulation. Elevated levels of guanine and guanine derivatives/metabolites are found in tumor cells, suggesting that the GVBQs may play important regulatory roles in tumor cell pathological processes, such as PDGFR-β transcriptional regulation. Furthermore, our results suggest that this unique dGMP-fill-in G-quadruplex in the PDGFR-β promoter may serve as a novel anticancer drug target for modulating PDGFR-β gene regulation. Citation Format: Kaibo Wang, Clement Lin, Guanhui Wu, Danzhou Yang. A dGMP fill-in G-quadruplex forms in the PDGFR-β promoter that serves as a unique target for drug design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 687.

Abstract 4839: Targeting glioma stem cells by pharmacologic stabilization of G-quadruplexes

Abstract Among heterogeneous cell population in a tumor, cancer stem cells (or tumor-propagating cells) are defined as a cell fraction that shows self-renewal, multipotency, high tumorigenicity, and resistance to chemo/radiotherapy. Because cancer stem cells are supposed to drive initiation, metastasis, and recurrence of the disease, identification of their therapeutic targets would have a profound implication for eradication of cancer. G-quadruplex (G4) is an atypical four-stranded nucleic acid structure that can be formed at guanine-rich sequences, such as the telomeric TTAGGG repeats. Accumulating evidence supports that G4s are widely distributed through the genome in living cells and affect various intracellular events, including DNA replication, gene expression and translation, whereas pharmacologic stabilization of G4s has been implicated for cancer intervention. However, precise mechanisms for the efficacy and target cancer types remain elusive. Here we demonstrate that G4 stabilization by chemical compounds, called G4 ligands, preferentially inhibits the growth of glioma stem cells in culture and in vivo. While the established glioma stem cells maintain stemness under the serum-free sphere culture conditions, serum stimulation induces their differentiation into non-stem glioma cells. We found that these glioma stem cells are highly sensitive to G4 ligands, such as a natural compound telomestatin and its newly synthesized derivative, and a bisquinolinium compound Phen-DC3, all of which can stabilize G4s. Upon the ligand treatment, glioma stem cells activated the replication stress pathway more potently than non-stem glioma cells, which could partly explain the selectivity of the deleterious effect. Consistently, these ligands induced DNA damage response in glioma stem cells but not in non-stem glioma cells. In glioma stem cells, about 20% to 30% of DNA damage foci were telomeric (i.e., telomere dysfunction-induced foci: TIFs) whereas the rest were non-telomeric, suggesting that the G4 ligands recognized both telomeric and non-telomeric G4s. Temozolomide, a clinical alkylating agent for brain tumors, induced DNA damage response but did not preferentially attack telomeres. As a potential pharmacodynamic biomarker, the immunofluorescence intensities of the nuclear G4 foci were enhanced by G4 ligands. Furthermore, these ligands directly inhibited in vitro transcription and translation of mRNAs that contained G4-forming sequences, suggesting additional mode of action. While G4 ligands inhibit telomerase activity, classical telomerase inhibitors without G4-stabilizing activity had no preferential impact on glioma stem cells. Together, these observations suggest that G4 is a promising target for pinpointing intractable glioma stem cells. Citation Format: Sachiko Okabe, Takahiro Nakamura, Daiki Hasegawa, Reina Kojima, Keiji Okamoto, Ichiro Nakano, Kazuo Shin-ya, Kazuo Nagasawa, Hiroyuki Seimiya. Targeting glioma stem cells by pharmacologic stabilization of G-quadruplexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4839.

Small Molecule Driven Stabilization of Promoter G-Quadruplexes and Transcriptional Regulation of c-MYC.

G-quadruplexes have been considered attractive therapeutic targets for the development of anticancer agents. We herein report synthesis of a series of carbazole derivatives by employing modular one-pot Cu(I) catalyzed cycloaddition. These carbazole derivatives are easily synthesizable, soluble in aqueous media, and able to strongly interact with quadruplexes. FRET based melting assay and fluorescence titration experiments suggest that a carbazole derivative, Cz-1, preferentially binds c-MYC quadruplex DNA over other investigated quadruplex and duplex DNAs. The biological studies revealed that Cz-1 inhibits cancer cell proliferation by inducing apoptosis. Moreover, Cz-1 inhibits the expression of c-MYC at transcriptional as well as translational levels. Exon-specific-assay confirms that the downregulation of MYC expression is mainly driven by the binding of Cz-1 with the promoter G-quadruplex structures. Immunocytochemistry, using quadruplex binding antibody BG4, further suggests that Cz-1 induces and stabilizes G-quadruplexes in a cellular system.

APTO-253 Stabilizes G-quadruplex DNA, Inhibits MYC Expression, and Induces DNA Damage in Acute Myeloid Leukemia Cells.

APTO-253 is a phase I clinical stage small molecule that selectively induces CDKN1A (p21), promotes G0-G1 cell-cycle arrest, and triggers apoptosis in acute myeloid leukemia (AML) cells without producing myelosuppression in various animal species and humans. Differential gene expression analysis identified a pharmacodynamic effect on MYC expression, as well as induction of DNA repair and stress response pathways. APTO-253 was found to elicit a concentration- and time-dependent reduction in MYC mRNA expression and protein levels. Gene ontogeny and structural informatic analyses suggested a mechanism involving G-quadruplex (G4) stabilization. Intracellular pharmacokinetic studies in AML cells revealed that APTO-253 is converted intracellularly from a monomer to a ferrous complex [Fe(253)3]. FRET assays demonstrated that both monomeric APTO-253 and Fe(253)3 stabilize G4 structures from telomeres, MYC, and KIT promoters but do not bind to non-G4 double-stranded DNA. Although APTO-253 exerts a host of mechanistic sequelae, the effect of APTO-253 on MYC expression and its downstream target genes, on cell-cycle arrest, DNA damage, and stress responses can be explained by the action of Fe(253)3 and APTO-253 on G-quadruplex DNA motifs. Mol Cancer Ther; 17(6); 1177-86. ©2018 AACR.

Pursuing origins of (poly)ethylene glycol-induced G-quadruplex structural modulations

Molecular crowding conditions provided by high concentration of cosolutes are utilized for characterization of biomolecules in cell-mimicking environment and development of drug-delivery systems. In this context, (poly)ethylene glycols are often used for studying non-canonical DNA structures termed G-quadruplexes, which came into focus by emerging structural biology findings and new therapeutic drug design approaches. Recently, several reports were made arguing against using (poly)ethylene glycols in role of molecular crowding agents due to their direct impact on DNA G-quadruplex stability and topology. However, the available data on structural details underlying DNA interaction is very scarce and thus limits in-depth comprehension. Herein, structural and thermodynamic analyses were strategically combined to assess G-quadruplex-cosolute interactions and address previously reported variances regarding the driving forces of G-rich DNA structural transformations under molecular crowding conditions. With the use of complementary (CD, NMR and UV) spectroscopic methods and model approach we characterized DNA G-quadruplex in the presence of the smallest and one of the largest typically used (poly)ethylene glycols. Dehydration effect is the key contributor to ethylene-glycol-induced increased stability of the G-quadruplex, which is in the case of the large cosolute mainly guided by the subtle direct interactions between PEG 8000 and the outer G-quartet regions.

Alternative splicing of human telomerase reverse transcriptase in gliomas and its modulation mediated by CX-5461

BackgroundGlioma is a heterogeneous, invasive primary brain tumor with a wide range of patient survival and a lack of reliable prognostic biomarkers. Human telomerase reverse transcriptase (hTERT) has been reported in the presence of multiple transcripts in various tumor systems. The biological function and precise regulatory mechanisms of hTERT transcripts remain uncertain.MethodsAlternative splicing of hTERT and telomerase activity were examined in 96 glioma specimens, including 38 glioblastomas (GBMs), 23 oligodendrogliomas (ODMs), and 35 oligoastrocytomas (OAMs). The correlation between telomerase activity or hTERT transcripts and patient clinical characteristics was investigated. We examined the regulation of alternative splicing of hTERT and telomerase activity by G-quadruplex stabilizer CX-5461 in GBM cells. The biological effects of CX-5461 on GBM cell lines, including inhibition of cell proliferation, effects on cell cycle/apoptosis, and telomere DNA damage were further explored.ResultsThe β splicing was verified in human gliomas and hTERT+β was significantly correlated with higher telomerase activity, higher KPS, larger tumor size, and higher tumor grades. Meanwhile, glioma patients lacking hTERT+β expression or telomerase activity showed a significant survival benefit. Notably, CX-5461 altered hTERT splicing patterns, leading to an increase of hTERT-β transcript and a decrease of hTERT+β transcript expression, which inhibits telomerase activity. In addition, CX-5461 had cytotoxic effects on GBM cells and caused telomere DNA damage response, induced G2/M arrest and apoptosis.ConclusionsThe hTERT+β is verified to be correlated with clinical parameters in gliomas, and could serve as a prognostic marker or possibly therapeutic target for gliomas. CX-5461 can regulate the splicing pattern of hTERT, inhibit telomerase activity, and kill GBM cells.

Carbazole ligands as c-myc G-quadruplex binders

The interactions of c-myc G-quadruplex with three carbazole derivatives were investigated by UV–Vis spectrophotometry, fluorescence, CD spectroscopy, and molecular modeling. The results showed that a combination of carbazole scaffold functionalized with ethyl, triazole and imidazole groups resulted in stabilization of the intramolecular G-quadruplex formed by the DNA sequence derived from the NHE III1 region of c-myc oncogene (Pu22). Binding to the G-quadruplex Pu22 resulted in the significant increase in fluorescence intensity of complexed ligands 1–3. All ligands were capable of interacting with G4 DNA with binding stoichiometry indicating that two ligand molecules bind to G-quadruplex with comparable affinity, which agrees with binding model of end-stacking on terminal G-tetrads.

Structural and biophysical insight into dual site binding of the protoberberine alkaloid palmatine to parallel G-quadruplex DNA using NMR, fluorescence and Circular Dichroism spectroscopy

Plant derived small molecules, which interact with and stabilize G-quadruplex DNA, act as inhibitors of telomere elongation and oncogene expression in humans. The inhibition of telomerase enzyme has immense potential since it is over expressed in most cancer cells. Interaction of palmatine, an antitumor alkaloid, to parallel G-quadruplex DNA, [d(TTGGGGT)]4 and [d(TTAGGGT)]4, has been investigated using Nuclear Magnetic Resonance (NMR), fluorescence and Circular Dichroism (CD) spectroscopy. Titrations were monitored by 1H and 31P NMR spectra and solution structure of palmatine-[d(TTGGGGT)]4 complex was obtained by restrained Molecular Dynamics (rMD) simulations using distance restraints from 2D NOESY spectra. Thermal stabilization of DNA was determined by CD, 1H NMR and Differential Scanning Calorimetry (DSC). Binding of palmatine induces 98% enhancement of fluorescence accompanied by blue shift ∼8 nm. CD spectral bands of DNA show minor changes. Diffusion NMR studies confirm formation of a stable complex. Proton NMR signals of palmatine shift upfield upon binding and NOE cross peaks of H10, H3, H28, 5OCH3 protons with T2, A3/G3, G6 and T7 residues reveal dual recognition sites in both G-quadruplex DNA sequences, resulting in thermal stabilization of G-quadruplex by ∼13–17 °C. Restrained molecular dynamics simulations using NOE distance restraints for 2:1 palmatine-[d(TTGGGGT)]4 complex reveal end-stacking of palmatine at G6pT7 step and groove binding along T2pG3 step. Binding to [d(TTAGGGT)]4 takes place at T2pA3pG4 and G6pT7 steps. Structural features of molecular recognition of two different G-quadruplex DNA sequences by palmatine have relevance in rational drug development for anti-cancer therapy.

Non-canonical DNA structures: Comparative quantum mechanical study

A study of relative thermodynamic stability of non-canonical DNA structures (triplexes, G-quadruplexes, i-motifs) for the first time was conducted on the basis of quantum chemical DFT/B3LYP/6-31++G (d) calculations. Results of the calculations completely reproduce the experimental data on stability of G-quadruplexes comparatively Watson-Crick B-DNA. It was discovered that combinations of non-canonical DNA structures were energetically more favorable than separated nitrogenous bases. Supramolecular complexes of the non-canonical DNA structures (NSs) can be considered as a biological drug targets in gene regulation (for example in tumor therapy), in contrast to previous works, where NSs were studied independently.

Synthetic Chlorophyll-A Derivatives Stabilize DNA G-Quadruplex Structures

A DNA G-quadruplex structure is one of the higher-order DNA structures found in a telomere region of chromosomes and suppresses enzymatic telomere-elongation reactions which frequently occur in cancer cells. Therefore, ligand compounds stabilizing such a DNA G-quadruplex structure is expected as a new-type of anti-cancer agents. To evaluate the interaction of the ligand with DNA G-quadruplexes in an aqueous solution, the examined ligands must be highly water-soluble. Such ligands have been usually substituted with hydrophilic substituents in a molecule. Here we report synthesis of several chlorophyll-a derivatives, in which their inner nitrogen is methylated, to enable their complexation and stabilization with a DNA G-quadruplex in an aqueous solution. Their binding constants with the DNA G-quadruplex structure were determined by optical spectroscopic techniques. The values using cationic N-methylated chlorophyll-a derivatives were larger than those using neutral unmethylated analogs. In addition, the thermodynamic stabilities of several G-quadruplexes with the synthetic ligands were analyzed by CD spectroscopy and will be discussed.

Stability of the Na+ Form of the Human Telomeric G-Quadruplex: Role of Adenines in Stabilizing G-Quadruplex Structure.

G-quadruplexes are higher order DNA structures that play significant roles in gene transcription and telomeric maintenance. The formation and stability of the G-quadruplex structures are under thermodynamic control and may be of biological significance for regulatory function of cellular processes. Here, we report the structural influence and energetic contributions of the adenine bases in the loop sequences that flank G-repeats in human telomeric DNA sequence. Spectroscopic and calorimetric techniques are used to measure the thermal stability and thermodynamic contributions to the stability of human telomeric G-quadruplexes that have been designed with systematic changes of A to T throughout the telomeric sequence. These studies demonstrate that the thermal stability of the G-quadruplex structure is directly related to the number and position of the adenines that are present in the telomeric sequence. The melting temperature (Tm) was reduced from 59 °C for the wild-type sequence to 47 °C for the sequence where all four adenines were replaced with thymines (0123TTT). Furthermore, the enthalpy required for transitioning from the folded to unfolded G-quadruplex structure was reduced by 15 kcal/mol when the adenines were replaced with thymines (37 kcal/mol for the wild-type telomeric sequence reduced to 22 kcal/mol for the sequence where all four adenines were replaced with thymines (0123TTT)). The circular dichroism melting studies for G-quadruplex sequences having a single A to T change showed significantly sloping pretransition baselines and their differential scanning calorimetry (DSC) thermograms revealed biphasic melting profiles. In contrast, the deoxyoligonucleotides having sequences with two or more A to T changes did not exhibit sloping baselines or biphasic DSC thermograms. We attribute the biphasic unfolding profile and reduction in the enthalpy of unfolding to the energetic contributions of adenine hydrogen bonding within the loops as well as the adenine stacking to the G-tetrads of the G-quadruplex structure.

Design, synthesis, and antiprotozoal evaluation of new 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline derivatives.

A series of new 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline derivatives was synthesized, and the compounds were screened in vitro against three protozoan parasites (Plasmodium falciparum, Leishmania donovani, and Trypanosoma brucei brucei). Biological results showed antiparasitic activity with IC50 values in the μm range. The in vitro cytotoxicity of these molecules was assessed by incubation with human HepG2 cells; for some derivatives, cytotoxicity was observed at significantly higher concentrations than antiparasitic activity. The 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline 1h was identified as the most potent antimalarial candidate with ratios of cytotoxic-to-antiparasitic activities of 107 and 39 against a chloroquine-sensitive and a chloroquine-resistant strain of P. falciparum, respectively. As the telomeres of the parasite P. falciparum are the likely target of this compound, we investigated stabilization of the Plasmodium telomeric G-quadruplexes by our phenanthroline derivatives through a FRET melting assay. The ligands 1f and 1m were noticed to be more specific for FPf8T with higher stabilization for FPf8T than for the human F21T sequence.

NMR based structure reveals groove binding of mitoxantrone to two sites of [d-(TTAGGGT)]4 having human telomeric DNA sequence leading to thermal stabilization of G-quadruplex

Interaction of mitoxantrone (MTX) with G-quadruplex, leading to inhibition of telomerase enzyme and anticancer action, is not understood. Titrations of MTX with [d-(TTAGGGT)]4, comprising human telomere single repeat sequence, have been monitored by fluorescence and 1H/31P NMR spectroscopy. Binding induces chemical shift changes in GNH (~0.3ppm), T2/T7/A3 base protons and sequence specific shifts in 31P resonances. Absence of large downfield shifts in 31P signals and presence of all sequential NOEs show that classical intercalation of drug between base quartets does not occur. The upfield shift (~0.53ppm) in 2/3H, 1/4OH and minor shift in 6/7H aromatic protons of MTX in complex rule out end stacking as a possible mode of interaction. The 26 short inter molecular contacts of 1/4OH, 11NH, 6/7H and 12CH2 protons of MTX with T1, T2, G6, G7 protons in the structure of complex obtained by restrained Molecular Dynamics simulations show binding at grooves accompanied by 4 hydrogen bonds and partial stacking of MTX with base pairs. Interaction causes thermal stabilization, ΔTm=35°C, which may be attributed to telomerase inhibition. The present study is the first report on solution structure of mitoxantrone-[d-(TTAGGGT)]4 complex and is significant for structure based designing of anthraquinone derivatives as future drugs.

Electrochemical and AFM Characterization of G-Quadruplex Electrochemical Biosensors and Applications.

Guanine-rich DNA sequences are able to form G-quadruplexes, being involved in important biological processes and representing smart self-assembling nanomaterials that are increasingly used in DNA nanotechnology and biosensor technology. G-quadruplex electrochemical biosensors have received particular attention, since the electrochemical response is particularly sensitive to the DNA structural changes from single-stranded, double-stranded, or hairpin into a G-quadruplex configuration. Furthermore, the development of an increased number of G-quadruplex aptamers that combine the G-quadruplex stiffness and self-assembling versatility with the aptamer high specificity of binding to a variety of molecular targets allowed the construction of biosensors with increased selectivity and sensitivity. This review discusses the recent advances on the electrochemical characterization, design, and applications of G-quadruplex electrochemical biosensors in the evaluation of metal ions, G-quadruplex ligands, and other small organic molecules, proteins, and cells. The electrochemical and atomic force microscopy characterization of G-quadruplexes is presented. The incubation time and cations concentration dependence in controlling the G-quadruplex folding, stability, and nanostructures formation at carbon electrodes are discussed. Different G-quadruplex electrochemical biosensors design strategies, based on the DNA folding into a G-quadruplex, the use of G-quadruplex aptamers, or the use of hemin/G-quadruplex DNAzymes, are revisited.

Controlling anticancer drug mediated G-quadruplex formation and stabilization by a molecular container.

Controlling of ligand mediated G-quadruplex DNA (GQ-DNA) formation and stabilization is an important and challenging aspect due to its active involvement in many biologically important processes such as DNA replication, transcription, etc. Here, we have demonstrated that topotecan (TPT), a potential anticancer drug, can instigate the formation and stabilization of GQ-DNA (H24 → GQ-DNA) in the absence of Na+/K+ ions via circular dichroism, fluorescence, NMR, UV melting and molecular dynamics (MD) simulation studies. The primary binding mode of TPT to GQ was found to be stacking at the terminal rather than binding to the groove. We have also reverted this conformational transition (GQ-DNA → H24) using a molecular container, cucurbit[7]uril (CB7), by means of the translocation of the drug (TPT) from GQ-DNA to its nanocavity. Importantly, we have carried out the detection of these conformational transitions using the fluorescence color switch of the drug, which is more direct and simple than some of the other methods that involve sophisticated and complex detection techniques.

Destabilization of DNA G-Quadruplexes by Chemical Environment Changes during Tumor Progression Facilitates Transcription.

DNA G-quadruplex formation is highly responsive to surrounding conditions, particularly K+ concentration. Malignant cancer cells have a much lower K+ concentration than normal cells because of overexpression of a K+ channel; thus, G-quadruplexes may be unstable in cancer cells. Here, we physicochemically investigated how changes in intracellular chemical environments in vitro and in cells influence G-quadruplex formation and transcription during tumor progression. In vitro, the stable G-quadruplex formation inhibits transcription in a solution containing 150 mM KCl (normal condition). As K+ concentration decreases, which decreases G-quadruplex stability, transcript production from templates with G-quadruplex-forming potential increases. In normal cells, the trend in transcript productions was similar to that in in vitro experiments, with transcription efficiency inversely correlated with G-quadruplex stability. Interestingly, higher transcript levels were produced from templates with G-quadruplex-forming potential in Ras-transformed and highly metastatic breast cancer cells (MDA-MB-231) than in nontransformed and control MCF-7 cells. Moreover, the amount of transcript produced from G-quadruplex-forming templates decreased upon addition of siRNA targeting KCNH1 mRNA, which encodes a potassium voltage-gated channel subfamily H member 1 (KV10.1). Importantly, G-quadruplex dissociation during tumor progression was observed by immunofluorescence using a G-quadruplex-binding antibody in cells. These results suggest that in normal cells, K+ ions attenuate the transcription of certain oncogenes by stabilizing G-quadruplex structures. Our findings provide insight into the novel mechanism of overexpression of certain G-rich genes during tumor progression.

Structure-Property Relationships in CuII -Binding Tetramolecular G-Quadruplex DNA.

A series of artificial metal-base tetrads composed of a CuII cation coordinating to four pyridines, covalently attached to the ends of tetramolecular G-quadruplex DNA strands [LA-D d(G4 )]4 (LA-D =ligand derivatives), was systematically studied. Structurally, the square-planar [Cu(pyridine)4 ] complex behaves analogously to the canonical guanine quartet. Copper coordination to all studied ligand derivatives was found to increase G-quadruplex thermodynamic stability, tolerating a great variety of ligand linker lengths (1-5 atoms) and thus demonstrating the robustness of the chosen ligand design. Only at long linker lengths, the stabilizing effect of copper binding is compensated by the loss of conformational freedom. A previously reported ligand LE with chiral backbone enables incorporation at any oligonucleotide position. We show that ligand chirality distinctly steers CuII -induced G-quadruplex stabilization. 5'-End formation of two metal-base tetrads by tetramolecular G-quadruplex [LE2 d(G)4 ]4 shows that stabilization in the presence of CuII is not additive. All results are based on UV/Vis thermal denaturation, thermal difference, circular dichroism experiments and molecular dynamics simulations.

Investigating Pharmacological Targeting of G-Quadruplexes in the Human Malaria Parasite.

The unique occurrence of G-quadruplexes in the AT-rich genome of human malaria parasite Plasmodium falciparum provides hints about their critical roles in parasite survival, pathogenesis, and host immune evasion. An intriguing question is whether these noncanonical structures can serve as molecular targets for small molecule-based interventions against malaria. In this study, we have investigated the pharmacological targeting of G-quadruplexes for parasite inhibition. We observed that bisquinolinium derivatives of 1,8-naphthyridine and pyridine affected the stability and molecular recognition properties of G-quadruplexes in telomeric and subtelomeric regions in P. falciparum. Parasite inhibition and cytotoxicity assays revealed that these ligands effectively inhibit parasite growth with minimal toxic effects in human cells. G-quadruplex interacting ligands caused degeneration and shortening of parasite telomeres. Ligand-induced perturbations in telomere homeostasis also affected transcriptional state of the subtelomeric region harboring antigenic variation genes. Taken together, our results suggest that quadruplex-ligand interaction disturbs telomeric/subtelomeric chromatin organization and induces DNA damage that consequently leads to parasite death. Our findings also draw attention to the striking differences in telomere dynamics in the protozoan parasite and human host that can be exploited for selective targeting of the telomeric quadruplex of the parasite as a potential antimalarial strategy.