Interaction of 9-Methoxyluminarine with Different G-Quadruplex Topologies: Fluorescence and Circular Dichroism Studies.

The interactions of G-quadruplexes of different topologies with highly fluorescent 9-methoxyluminarine ligand 9-MeLM were investigated by fluorescence and circular dichroism spectroscopy. The results showed that 9-methoxyluminarine was able to interact and did not destabilize any investigated molecular targets. The studied compound was selectively quenched by parallel c-MYC G-quadruplex DNA, whereas hybrid and antiparallel G4 topology caused only a negligible decrease in the fluorescence of the ligand. A high decrease of fluorescence of the ligand after binding with c-MYC G-quadruplex suggests that this molecule can be used as a selective probe for parallel G-quadruplexes.

Practical Microwave Synthesis of Carbazole Aldehydes for the Development of DNA-Binding Ligands.

Microwave formylation of carbazole derivatives was investigated and 3-monoaldehydes were obtained in high yield. A potential DNA-binding ligand, 3-[(3-ethyl)-2-vinylbenzothiazolium]-9-N-ethyl carbazole iodide, was synthesized and characterized including spectral properties (UV-Vis absorption and fluorescence spectra). The binding selectivity and affinity of three carbazole ligands for double-stranded and G-quadruplex DNA structures were studied using a competitive dialysis method in sodium- and potassium-containing buffer solutions.

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.

Carbazole Derivatives' Binding to c-KIT G-Quadruplex DNA.

The binding affinities of three carbazole derivatives to the intramolecular G-quadruplex (GQ) DNA formed by the sequence 5'-AGGGAGGGCGCTGGGAGGAGGG-3', derived from the c-KIT 1 oncogene region, were investigated. All carbazole cationic ligands that differed in the substituents on the nitrogen atom were able to stabilize G-quadruplex, as demonstrated using UV-Vis, fluorescence and CD spectroscopic techniques as well as molecular modeling. The spectrophotometric titration results showed spectral features characteristic of these ligands-bathochromic shifts and initial hypochromicity followed by hyperchromicity at higher GQ concentrations. All free carbazole ligands exhibited modest fluorescent properties, but after binding to the DNA the fluorescence intensity increased significantly. The binding affinities of carbazole ligands to the c-KIT 1 DNA were comparable showing values in the order of 105 M−1. Molecular modeling highlights the differences in interactions between each particular ligand and studied G-quadruplex, which potentially influenced binding strength. Obtained results relevant that all three investigated ligands have stabilization properties on studied G-quadruplex.

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.

Interaction of metallacrown complexes with G-quadruplex DNA.

Interactions of the G-quadruplex (GQ) DNA with two pentacoordinate lanthanide (III) metallacrown (MC) complexes containing phenylalanine hydroxamic acid (pheHA) and copper(II) ions of the formula Eu 15-[MCCu,pheHA]-5 (1) and Tb 15-[MCCu,pheHA]-5 (2) were investigated. Binding of both metallacrowns to human telomeric G-quadruplex DNA was followed using CD spectroscopy, DNA melting profiles, and fluorescent intercalator displacement (FID) assay. A new G-quadruplex binding assay based of quenching of Tb(III)-GQ luminescence was proposed and evaluated. All performed tests confirmed interactions of MCs with studied GQ structure. Binding affinities of MCs were appreciable (KMC ~2-5×10(5)M(-1)). Higher concentration of MCs (the ratio of GQ:MC above 2.5) caused destabilization of tetraplex structure of GQ as evidenced by CD spectroscopy, melting temperatures, and Tb(III)-GQ luminescence quenching results.

Biological potential of carbazole derivatives.

Carbazole skeleton is the key structural motif of many biologically active compounds including synthetic and natural products. Over the past several years, a large number of research highlighting the significance of carbazole derivatives has been reported in the literature. The present review focuses on the recent progress, from 2010 until now, in knowledge on various biological properties of classical, tricyclic carbazole derivatives.

N-Methyl-4-hydrazino-7-nitrobenzofurazan: a fluorogenic substrate for peroxidase-like DNAzyme, and its potential application.

Characterization and optimization studies of N-methyl-4-hydrazino-7-nitrobenzofurazan (MNBDH) as a new fluorogenic substrate in the peroxidation reaction catalyzed by DNAzyme are reported. The effects of pH, H2O2 concentration, metal-cation type, and the concentration and type of surfactant on the fluorescence intensity were investigated. The optimized reaction was subsequently used for the development of an assay for DNA detection based on a molecular-beacon probe. The use of a fluorogenic substrate enabled the detection of a single-stranded DNA target with a 1 nmol L(-1) detection limit.

Fluorescence energy transfer probes based on the guanine quadruplex formation for the fluorometric detection of potassium ion.

Dual-labeled oligonucleotide derivative, FAT-0, carrying 6-carboxyfluorescein (FAM) and 6-carboxy-tetramethylrhodamine (TAMRA) labels at 5'- and 3'-termini of thrombin-binding aptamer (TBA) sequence 5'-GGTTGGTGTGGTTGG-3' and its derivatives, FAT-n (n=3, 5, and 7) were designed and synthesized. FAT-n derivatives contained a T(m)A spacer (m=2, 4, and 6, respectively) at 5'-end of TBA sequence. The probes were developed to estimate the spacer effect on FRET efficiency and to identify the best probe for sensing of K(+). Circular dichroism (CD), UV-vis absorption, and fluorescence studies revealed that all FAT-n probes could form the intramolecular tetraplex structures after binding K(+). Association constants of particular K(+)/FAT-n complexes were determined using different experimental approaches. Suitability of particular probes for sensitive monitoring of K(+) in intra- and extracellular conditions was examined and discussed. Calibration graphs of fluorescence ratio were linear in the K(+) concentration range of 2-10 mM for extracellular conditions showing sensitivity of 1.2% mM(-1) K(+) and for intracellular conditions in the range of 100-200 mM with sensitivity of 0.49% mM(-1) K(+).

Luminescence study of G-quadruplex formation in the presence of Tb3+ ion.

The interactions of Tb3+ with the quadruplex-forming oligonucleotide bearing human telomeric repeat sequence d(G(3)T(2)AG(3)T(2)AG(3)T(2)AG(3)), (htel21), have been studied using luminescence spectroscopy and circular dichroism (CD). Enhanced luminescence of Tb3+, resulting from energy transfer from guanines, indicated encapsulation of Tb3+ ion in the central cavity of quadruplex core. The ability of lanthanide ions (Eu3+ and Tb3+) to mediate formation of quadruplex structure has been further evidenced by the fluorescence energy transfer measurements with the use of oligonucleotide probe labeled with fluorescein and rhodamine FRET partners, FAM-htel21-TAMRA. The CD spectra revealed that Tb3+/htel21 quadruplex possesses antiparallel strand orientation, similarly as sodium quadruplex. Tb3+ binding equilibria have been investigated in the absence and the presence of competing metal cations. At low Tb3+ concentration (8 microM) Tb3+/htel21 quadruplex stability is very high (5 x 10(6) M(-1)) and stoichiometry of 5-7 Tb3+ ions per one quadruplex molecule is observed. Luminescence and CD titration experiments suggested that the cavity of quadruplex accommodates two Tb3+ ions and the remaining Tb3+ ions bind probably to TTA loops of quadruplex. Higher concentration of Tb3+ (above 10 microM) results in the excessive binding of Tb3+ ions that finally destabilizes quadruplex, which undergoes transformation into differently organized assemblies. Such assemblies (probably possessing multiple positive charge) exhibit kinetic stability, which is manifested by a very slow kinetics of displacement of Tb3+ ion by competing cations (Li+, Na+, K+).

Fluorescence anisotropy and FRET studies of G-quadruplex formation in presence of different cations.

Results of the steady-state fluorescence, anisotropy and FRET measurements of G-quadruplex formation in the presence of selected cations (Li(+), Na(+), K(+), NEt(4)(+) and Mg(2+)) are reported. Three different fluorescent oligonucleotides with human telomeric sequence labeled with fluorescein (FAM) and tetramethylrhodamine (TAMRA) were investigated: a dual-labeled 21-mer denoted as PSO (Potassium Sensing Oligonucleotide) and two 5'- and 3'- single-labeled probes, FAM-21 and 21-TAMRA, respectively. The fluorescence signal of FAM-21 increased significantly for all systems and the fluorescence enhancement was comparable in magnitude for monovalent cations but it was more pronounced for Mg(2+) cation. This phenomenon was attributed to the protolytic equilibria of FAM affected by the variation in ionic strength. On the other hand, fluorescence of TAMRA was enhanced selectively by Na(I) cation that was explained by the dequenching of TAMRA emission originated from the peculiarity of the basket-type structure of Na(I)-quadruplex. Anisotropy of FAM-21 (but not 21-TAMRA) appeared to be sensitive to the G-quadruplex formation, showing significant increase with an increase in cation concentration and indicating some restrictions in rotational depolarization of FAM. FRET experiments revealed that all tested cations caused quenching of FAM fluorescence in PSO, but only Na(+) and K(+) ions produced sensitized emission of TAMRA acceptor. Higher FRET efficiency observed in the presence of sodium ion was attributed to the specific spectral factor and steric interactions in the basket-type Na(I)-quadruplex.