Assessment of selectivity of G-quadruplex ligands via an optimised FRET melting assay

Rache, Aurore De; Mergny, Jean‐Louis

doi:10.1016/j.biochi.2015.06.002

Cited by 99 publications

(90 citation statements)

References 56 publications

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“…Our results are in line with previous studies, but the ability to quantitatively determine the individual K D values for the first and second potassium binding event (Equations 2 and 3, Table 1) refines the interpretation of equilibrium binding data obtained from calorimetric or spectroscopic studies. Similar effects have been reported when terminal thymine residues are added to synthetic sequences (81), or when fluorescent probes increase the K + binding cooperativity and modify the folding thermodynamics (31). …”

Section: Discussionsupporting

confidence: 67%

“…The structure of 21G (d((GGGTTA) 3 GGG)) has not been solved by NMR, most likely because it is also polymorphic. It is the shortest possible human telomeric sequence containing four tracks of guanines, and it is frequently used in FRET (Förster Resonance Energy Transfer) melting assays with the FAM and TAMRA dyes at the 5′ and 3′ ends (31). 21G, with and without the fluorescent tags, have also been included in the present study, as well as the FRET homologue of 26TTA.…”

Section: Introductionmentioning

confidence: 99%

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Folding and misfolding pathways of G-quadruplex DNA

Marchand¹,

Gabelica²

2016

Nucleic Acids Res

151

245

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G-quadruplexes adopt various folding topologies, but information on their folding pathways remains scarce. Here, we used electrospray mass spectrometry to detect and quantify the specifically bound potassium ions, and circular dichroism to characterize the stacking topology of each ensemble. For human telomeric (hTel) sequences containing the d((GGGTTA)3GGG) core, K+ binding affinity and cooperativity strongly depends on the chosen construct. The shortest sequences bind only one K+ at low KCl concentration, and this 2-quartet G-quadruplex is antiparallel. Flanking bases increase the K+ binding cooperativity. To decipher the folding pathways, we investigated the kinetics of K+ binding to telomeric (hybrid) and c-myc (parallel) G-quadruplexes. G-quadruplexes fold via branched pathways with multiple parallel reactions. Up to six states (one ensemble without K+, two ensembles with 1-K+ and three ensembles with 2-K+) are separated based on their formation rates and ion mobility spectrometry. All G-quadruplexes first form long-lived misfolded structures (off-pathway compared to the most stable structures) containing one K+ and two quartets in an antiparallel stacking arrangement. The results highlight the particular ruggedness of G-quadruplex nucleic acid folding landscapes. Misfolded structures can play important roles for designing artificial G-quadruplex based structures, and for conformational selection by ligands or proteins in a biological context.

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Section: Discussionsupporting

confidence: 67%

Section: Introductionmentioning

confidence: 99%

Folding and misfolding pathways of G-quadruplex DNA

Marchand¹,

Gabelica²

2016

Nucleic Acids Res

151

245

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“…We further investigated the structural selectivity of 1-4 by comparing the DT 1/2 values for various quadruplex topologies (Figure 2). [18] Herein we limit our discussion to 3 and 4, which harbor three cationic arms. Using the F21CTAT and FBom17T sequences (known as displaying an anti-parallel fold) we found the DT 1/2 value for 3 to be similaro rs lightly higher than for 4 (differences of 0-3 8C).…”

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confidence: 99%

Efficient Inhibition of Telomerase by Nickel–Salophen Complexes

et al. 2016

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Four nickel(II)-salophen complexes containing alkyl-imidazolium chains connected at the ortho or meta positions were prepared: N,N'-bis(2-hydroxy-4-methyl-3H-imidazol-1-iumbenzylideneamino)phenylenediamine (1), N,N'-bis(2-hydroxy-3-methyl-3H-imidazol-1-iumbenzylideneamino)phenylenediamine (2), N,N'-bis(2-hydroxy-3-methyl-3H-imidazol-1-iumbenzylideneamino)methyl-3H-imidazol-1-iumphenylenediamine (3), and N,N'-bis(2-hydroxy-4-methyl-3H-imidazol-1-iumbenzylideneamino)methyl-3H-imidazol-1-iumphenylenediamine (4). They protect G-quadruplex DNA (G4 -DNA) against thermal denaturation and show KA values in the range of 7.4×10(5) to 4×10(7) m(-1) for G4 -DNA models. Complex 4 exhibits an IC50 value of 70 nm for telomerase inhibition.

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“…However in presence of the ligands the signature remained unchanged except for a slightly stronger intensity of the maxima at 288 nm and a small decrease in maxima at 271 than the telo-21G alone except for chelerythrine. The strongest changes were observed in presence of chelerythrine followed by modest changes for berberine, chelidonine, 50 concentration of chelerythrine showed 60% inhibition of telomerase in the qTRAP assay, although it was able to inhibit telomerase even at a very low concentration in a dose-dependent manner. Telomerase activity was strongly inhibited at very low concentrations of chelidonine or sanguinarine and progressed to almost complete inhibition at their respective LD 50 .…”

Section: Discussionmentioning

confidence: 93%

“…The absorbance was read at 570 nm using a plate reader (BioTek, USA) after dissolving the formazan product in dimethylsulfoxide containing 10% SDS and 1% acetic acid. The tests were repeated at least 3 times each in triplicates and the LD 50 values were calculated from dose-response curves using Gen5 version1.06 software.…”

Section: Cell Culture and Cytotoxicitymentioning

confidence: 99%

Selectivity of major isoquinoline alkaloids from Chelidonium majus towards telomeric G-quadruplex: A study using a transition-FRET (t-FRET) assay

Noureini

Esmaeili

Abachi

et al. 2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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BackgroundNatural bioproducts are invaluable resources in drug discovery. Isoquinoline alkaloids of Chelidonium majus constitute a structurally diverse family of natural products that are of great interest, one of them being their selectivity for human telomeric G-quadruplex structure and telomerase inhibition. MethodsThe study focuses on the mechanism of telomerase inhibition by stabilization of telomeric Gquadruplex structures by berberine, chelerythrine, chelidonine, sanguinarine and papaverine. Telomerase activity and mRNA levels of hTERT were estimated using quantitative telomere repeat amplification protocol and qPCR, in MCF-7 cells treated with different groups of alkaloids. The selectivity of the main isoquinoline alkaloids of Chelidonium majus towards telomeric Gquadruplex forming sequences were explored using a sensitive modified thermal FRET-melting measurement in the presence of the complementary oligonucleotide CT22. We assessed and monitored G-quadruplex topologies using circular dichroism (CD) methods, and compared spectra to previously well-characterized motifs, either alone or in the presence of the alkaloids, Molecular modeling was performed to rationalize ligand binding to the G-quadruplex structure. ResultsThe results highlight strong inhibitory effects of chelerythrine, sanguinarine and berberine on telomerase activity, most likely through substrate sequestration. These isoquinoline alkaloids interacted strongly with telomeric sequence G-quadruplex. In comparison, chelidonine and papaverine had no significant interaction with the telomeric quadruplex, while they strongly inhibited telomerase at transcription level of hTERT. Altogether, all of the studied alkaloids showed various levels and mechanisms of telomerase inhibition. ConclusionsWe report on a comparative study of anti-telomerase activity of the isoquinoline alkaloids of Chelidonium majus. Chelerythrine was most effective in inhibiting telomerase activity by substrate sequesteration through G-quadruplex stabilization. General SignificanceUnderstanding structural and molecular mechanisms of anti-cancer agents can help in developing new and more potent drugs with fewer side effects. Isoquinolines are the most biologically active agents from Chelidonium majus, which have shown to be telomeric G-quadruplex stabilizers and potent telomerase inhibitors.

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Assessment of selectivity of G-quadruplex ligands via an optimised FRET melting assay

Cited by 99 publications

References 56 publications

Folding and misfolding pathways of G-quadruplex DNA

Folding and misfolding pathways of G-quadruplex DNA

Efficient Inhibition of Telomerase by Nickel–Salophen Complexes

Selectivity of major isoquinoline alkaloids from Chelidonium majus towards telomeric G-quadruplex: A study using a transition-FRET (t-FRET) assay

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