Binding Study of the Fluorescent Carbazole Derivative with Human Telomeric G-Quadruplexes

The carbazole ligand 3 was synthesized, characterized and its binding interactions with human telomeric (22HT) G-quadruplex DNA in Na+ and K+-containing buffer were investigated by ultraviolet-visible (UV-Vis) spectrophotometry, fluorescence, circular dichroism (CD) spectroscopy, and DNA melting. The results showed that the studied carbazole ligand interacted and stabilized the intramolecular G-quadruplexes formed by the telomeric sequence in the presence of sodium and potassium ions. In the UV-Vis titration experiments a two-step complex formation between ligand and G-quadruplex was observed. Very low fluorescence intensity of the carbazole derivative in Tris HCl buffer in the presence of the NaCl or KCl increased significantly after addition of the 22HT G4 DNA. Binding stoichiometry of the ligand/G-quadruplex was investigated with absorbance-based Job plots. Carbazole ligand binds 22HT with about 2:1 stoichiometry in the presence of sodium and potassium ions. The binding mode appeared to be end-stacking with comparable binding constants of ~105 M−1 as determined from UV-Vis and fluorescence titrations data. The carbazole ligand is able to induce formation of G4 structure of 22HT in the absence of salt, which was proved by CD spectroscopy and melting studies. The derivative of carbazole 3 shows significantly higher cytotoxicity against breast cancer cells then for non-tumorigenic breast epithelial cells. The cytotoxic activity of ligand seems to be not associated with telomerase inhibition.

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A comprehensive evaluation of a typical plant telomeric G-quadruplex (G4) DNA reveals the dynamics of G4 formation, rearrangement, and unfolding

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012383 ◽

2020 ◽

Vol 295 (16) ◽

pp. 5461-5469 ◽

Cited By ~ 3

Author(s):

Wen-Qiang Wu ◽

Ming-Li Zhang ◽

Chun-Peng Song

Keyword(s):

Single Molecule ◽

Comprehensive Evaluation ◽

Cd Spectroscopy ◽

Telomeric Sequence ◽

Single Molecule Fret ◽

G4 Dna ◽

G Quadruplex ◽

Simple Repeats ◽

Plant Telomeres ◽

Shed Light

Telomeres are specific nucleoprotein structures that are located at the ends of linear eukaryotic chromosomes and play crucial roles in genomic stability. Telomere DNA consists of simple repeats of a short G-rich sequence: TTAGGG in mammals and TTTAGGG in most plants. In recent years, the mammalian telomeric G-rich repeats have been shown to form G-quadruplex (G4) structures, which are crucial for modulating telomere functions. Surprisingly, even though plant telomeres are essential for plant growth, development, and environmental adaptions, only few reports exist on plant telomeric G4 DNA (pTG4). Here, using bulk and single-molecule assays, including CD spectroscopy, and single-molecule FRET approaches, we comprehensively characterized the structure and dynamics of a typical plant telomeric sequence, d[GGG(TTTAGGG)3]. We found that this sequence can fold into mixed G4s in potassium, including parallel and antiparallel structures. We also directly detected intermediate dynamic transitions, including G-hairpin, parallel G-triplex, and antiparallel G-triplex structures. Moreover, we observed that pTG4 is unfolded by the AtRecQ2 helicase but not by AtRecQ3. The results of our work shed light on our understanding about the existence, topological structures, stability, intermediates, unwinding, and functions of pTG4.

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Stabilization of c-KIT G-Quadruplex DNA Structures by the RNA Polymerase I Inhibitors BMH-21 and BA-41

International Journal of Molecular Sciences ◽

10.3390/ijms20194927 ◽

2019 ◽

Vol 20 (19) ◽

pp. 4927 ◽

Cited By ~ 1

Author(s):

Stefania Mazzini ◽

Raimundo Gargallo ◽

Loana Musso ◽

Francesca De Santis ◽

Anna Aviñó ◽

...

Keyword(s):

Gene Expression Regulation ◽

Binding Constants ◽

Binding Mode ◽

Precise Determination ◽

Ligand Molecule ◽

Quadruplex Dna ◽

Dna Structures ◽

G Quadruplex ◽

Human Lymphoma ◽

Polymerase I

The stabilization of G-quadruplex DNA structures by small molecules with affinity to oncogene promoters has emerged as a promising anticancer strategy, due to a potential role in gene expression regulation. We explored the ability of BMH-21 (1) and its analogue BA-41 (2) to bind the G-quadruplex structure present in the c-KIT promoter by biophysical methods and molecular modeling. We provide evidence that both compounds interact with the c-KIT 21-mer sequence. The stable monomeric intramolecular parallel G-quadruplex obtained by the mutation of positions 12 and 21 allowed the precise determination of the binding mode by NMR and molecular dynamics studies. Both compounds form a complex characterized by one ligand molecule positioned over the tetrad at the 3′-end, stabilized by an extensive network of π–π interactions. The binding constants (Kb) obtained with fluorescence are similar for both complexes (around 106 M−1). Compound BA-41 (2) showed significant antiproliferative activity against a human lymphoma cell line, SU-DHL4, known to express substantial levels of c-KIT. However, the partial inhibition of c-KIT expression by Western blot analysis suggested that the interaction of compound 2 with the c-KIT promoter is not the primary event and that multiple effects provide a contribution as determinants of biological activity.

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ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress

Nature Communications ◽

10.1038/s41467-021-24206-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yu-Ching Teng ◽

Aishwarya Sundaresan ◽

Ryan O’Hara ◽

Vincent U. Gant ◽

Minhua Li ◽

...

Keyword(s):

Dna Sequences ◽

Genome Stability ◽

Helicase Domain ◽

Quadruplex Dna ◽

Heterochromatin Formation ◽

Replicative Stress ◽

G4 Dna ◽

Mutation Burden ◽

G Quadruplex ◽

Dna Replication Stress

AbstractATRX is a tumor suppressor that has been associated with protection from DNA replication stress, purportedly through resolution of difficult-to-replicate G-quadruplex (G4) DNA structures. While several studies demonstrate that loss of ATRX sensitizes cells to chemical stabilizers of G4 structures, the molecular function of ATRX at G4 regions during replication remains unknown. Here, we demonstrate that ATRX associates with a number of the MCM replication complex subunits and that loss of ATRX leads to G4 structure accumulation at newly synthesized DNA. We show that both the helicase domain of ATRX and its H3.3 chaperone function are required to protect cells from G4-induced replicative stress. Furthermore, these activities are upstream of heterochromatin formation mediated by the histone methyltransferase, ESET, which is the critical molecular event that protects cells from G4-mediated stress. In support, tumors carrying mutations in either ATRX or ESET show increased mutation burden at G4-enriched DNA sequences. Overall, our study provides new insights into mechanisms by which ATRX promotes genome stability with important implications for understanding impacts of its loss on human disease.

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Subtle structural alterations in G-quadruplex DNA regulate site specificity of fluorescence light-up probes

Nucleic Acids Research ◽

10.1093/nar/gkz1205 ◽

2020 ◽

Vol 48 (3) ◽

pp. 1108-1119 ◽

Cited By ~ 5

Author(s):

Rajendra Kumar ◽

Karam Chand ◽

Sudipta Bhowmik ◽

Rabindra Nath Das ◽

Snehasish Bhattacharjee ◽

...

Keyword(s):

Rational Design ◽

Dna Structure ◽

Biological Processes ◽

Chemical Research ◽

Quadruplex Dna ◽

Dna Structures ◽

Future Studies ◽

G4 Dna ◽

G Quadruplex ◽

Fluorescence Light

Abstract G-quadruplex (G4) DNA structures are linked to key biological processes and human diseases. Small molecules that target specific G4 DNA structures and signal their presence would therefore be of great value as chemical research tools with potential to further advance towards diagnostic and therapeutic developments. However, the development of these types of specific compounds remain as a great challenge. In here, we have developed a compound with ability to specifically signal a certain c-MYC G4 DNA structure through a fluorescence light-up mechanism. Despite the compound's two binding sites on the G4 DNA structure, only one of them result in the fluorescence light-up effect. This G-tetrad selectivity proved to originate from a difference in flexibility that affected the binding affinity and tilt the compound out of the planar conformation required for the fluorescence light-up mechanism. The intertwined relation between the presented factors is likely the reason for the lack of examples using rational design to develop compounds with turn-on emission that specifically target certain G4 DNA structures. However, this study shows that it is indeed possible to develop such compounds and present insights into the molecular details of specific G4 DNA recognition and signaling to advance future studies of G4 biology.

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Characterization of a G-quadruplex from hepatitis B virus and its stabilization by binding TMPyP4, BRACO19 and PhenDC3

Scientific Reports ◽

10.1038/s41598-021-02689-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Orsolya Réka Molnár ◽

András Végh ◽

Judit Somkuti ◽

László Smeller

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Binding Constants ◽

Temperature Stability ◽

Cd Spectroscopy ◽

Parallel Structure ◽

Hybrid Form ◽

B Virus ◽

G Quadruplex

AbstractSpecific guanine rich nucleic acid sequences can form non-canonical structures, like the four stranded G-quadruplex (GQ). We studied the GQ-forming sequence (named HepB) found in the genome of the hepatitis B virus. Fluorescence-, infrared- and CD-spectroscopy were used. HepB shows a hybrid form in presence of K+, but Na+, Li+, and Rb+ induce parallel structure. Higher concentrations of metal ions increase the unfolding temperature, which was explained by a short thermodynamic calculation. Temperature stability of the GQ structure was determined for all these ions. Na+ has stronger stabilizing effect on HepB than K+, which is highly unusual. The transition temperatures were 56.6, 53.8, 58.5 and 54.4 °C for Na+, K+, Li+, and Rb+ respectively. Binding constants for Na+ and K+ were 10.2 mM and 7.1 mM respectively. Study of three ligands designed in cancer research for GQ targeting (TMPyP4, BRACO19 and PhenDC3) showed unequivocally their binding to HepB. Binding was proven by the increased stability of the bound form. The stabilization was higher than 20 °C for TMPyP4 and PhenDC3, while it was considerably lower for BRACO19. These results might have medical importance in the fight against the hepatitis B virus.

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G-quadruplex DNA drives genomic instability and represents a targetable molecular abnormality in ATRX-deficient malignant glioma

10.1101/347542 ◽

2018 ◽

Author(s):

Yuxiang Wang ◽

Jie Yang ◽

Wei Wu ◽

Rachna Shah ◽

Carla Danussi ◽

...

Keyword(s):

Dna Damage ◽

Malignant Glioma ◽

Model Systems ◽

Copy Number Alterations ◽

Molecular Alteration ◽

Quadruplex Dna ◽

G4 Dna ◽

G Quadruplex

AbstractMutational inactivation of ATRX (α-thalassemia mental retardation X-linked) represents a defining molecular alteration in large subsets of malignant glioma. Yet the pathogenic consequences of ATRX deficiency remain unclear, as do tractable mechanisms for its therapeutic targeting. Here we report that ATRX loss in isogenic glioma model systems induces replication stress and DNA damage by way of G-quadruplex (G4) DNA secondary structure. Moreover, these effects are associated with the acquisition of disease-relevant copy number alterations over time. We then demonstrate, both in vitro and in vivo, that ATRX deficiency selectively enhances DNA damage and cell death following chemical G4 stabilization. Finally, we show that G4 stabilization synergizes with other DNA-damaging therapies, including ionizing radiation, in the ATRX-deficient context. Our findings reveal novel pathogenic mechanisms driven by ATRX deficiency in glioma, while also pointing to tangible strategies for drug development.

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Oxadiazole/Pyridine-Based Ligands: A Structural Tuning for Enhancing G-Quadruplex Binding

Molecules ◽

10.3390/molecules23092162 ◽

2018 ◽

Vol 23 (9) ◽

pp. 2162 ◽

Cited By ~ 5

Author(s):

Filippo Doria ◽

Valentina Pirota ◽

Michele Petenzi ◽

Marie-Paule Teulade-Fichou ◽

Daniela Verga ◽

...

Keyword(s):

Nucleic Acid ◽

Structural Transition ◽

Binding Mode ◽

Telomeric Sequence ◽

Recognition Element ◽

New Family ◽

G Quadruplex ◽

Preferential Binding ◽

High Cytotoxicity ◽

Nucleic Acid Structures

Non-macrocyclic heteroaryls represent a valuable class of ligands for nucleic acid recognition. In this regard, non-macrocyclic pyridyl polyoxazoles and polyoxadiazoles were recently identified as selective G-quadruplex stabilizing compounds with high cytotoxicity and promising anticancer activity. Herein, we describe the synthesis of a new family of heteroaryls containing oxadiazole and pyridine moieties targeting DNA G-quadruplexes. To perform a structure–activity analysis identifying determinants of activity and selectivity, we followed a convergent synthetic pathway to modulate the nature and number of the heterocycles (1,3-oxazole vs. 1,2,4-oxadiazole and pyridine vs. benzene). Each ligand was evaluated towards secondary nucleic acid structures, which have been chosen as a prototype to mimic cancer-associated G-quadruplex structures (e.g., the human telomeric sequence, c-myc and c-kit promoters). Interestingly, heptapyridyl-oxadiazole compounds showed preferential binding towards the telomeric sequence (22AG) in competitive conditions vs. duplex DNA. In addition, G4-FID assays suggest a different binding mode from the classical stacking on the external G-quartet. Additionally, CD titrations in the presence of the two most promising compounds for affinity, TOxAzaPy and TOxAzaPhen, display a structural transition of 22AG in K-rich buffer. This investigation suggests that the pyridyl-oxadiazole motif is a promising recognition element for G-quadruplexes, combining seven heteroaryls in a single binding unit.

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Visible-light photoswitching of ligand binding mode suggests G-quadruplex DNA as a target for photopharmacology

Chemical Communications ◽

10.1039/d0cc01581d ◽

2020 ◽

Vol 56 (38) ◽

pp. 5186-5189 ◽

Cited By ~ 1

Author(s):

Michael P. O’Hagan ◽

Javier Ramos-Soriano ◽

Susanta Haldar ◽

Sadiyah Sheikh ◽

Juan C. Morales ◽

...

Keyword(s):

Ligand Binding ◽

Visible Light ◽

Binding Mode ◽

Quadruplex Dna ◽

G Quadruplex

A pyridinium-decorated photoresponsive dithienylethene selectively targets G-quadruplex DNA, allowing binding mode and toxicity to be controlled exclusively with visible light.

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