FRET‐MC: A fluorescence melting competition assay for studying G4 structures in vitro

Luo, Yu; Granzhan, Anton; Verga, Daniela; Mergny, Jean‐Louis

doi:10.1002/bip.23415

Cited by 29 publications

(27 citation statements)

References 49 publications

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“…In a very recent report, Luo et al also explored the competitive nature of FRET-melting and studied whether the interaction between a fluorescent G4-forming oligonucleotide and PhenDC3 is modified by a competitor sequence added in excess [ 202 ]. Sixty-five sequences with a known structure were tested to validate this FRET-melting competition (FRET-MC) assay.…”

Section: Methods To Characterize G4/ligand Interactionsmentioning

confidence: 99%

G-Quadruplexes and Their Ligands: Biophysical Methods to Unravel G-Quadruplex/Ligand Interactions

et al. 2021

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Progress in the design of G-quadruplex (G4) binding ligands relies on the availability of approaches that assess the binding mode and nature of the interactions between G4 forming sequences and their putative ligands. The experimental approaches used to characterize G4/ligand interactions can be categorized into structure-based methods (circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography), affinity and apparent affinity-based methods (surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and mass spectrometry (MS)), and high-throughput methods (fluorescence resonance energy transfer (FRET)-melting, G4-fluorescent intercalator displacement assay (G4-FID), affinity chromatography and microarrays. Each method has unique advantages and drawbacks, which makes it essential to select the ideal strategies for the biological question being addressed. The structural- and affinity and apparent affinity-based methods are in several cases complex and/or time-consuming and can be combined with fast and cheap high-throughput approaches to improve the design and development of new potential G4 ligands. In recent years, the joint use of these techniques permitted the discovery of a huge number of G4 ligands investigated for diagnostic and therapeutic purposes. Overall, this review article highlights in detail the most commonly used approaches to characterize the G4/ligand interactions, as well as the applications and types of information that can be obtained from the use of each technique.

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Section: Methods To Characterize G4/ligand Interactionsmentioning

confidence: 99%

G-Quadruplexes and Their Ligands: Biophysical Methods to Unravel G-Quadruplex/Ligand Interactions

et al. 2021

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“…FRET melting curves ( Figure 3 ) confirmed that BPBA is able to stabilize the G4 structure formed by telomeric RNA (Δ T m = 3.4 (±0.1) °C). Moreover, to further confirm the selectivity of BPBA for G4 over the duplex, a competition FRET melting experiment was carried out in the presence of a large excess of a duplex model, i.e., a 27-mer hairpin duplex-forming DNA ( Hrp 27 ) [ 61 , 62 , 63 , 64 ]. The results of this experiment clearly showed that the stabilizing effect of BPBA on TERRA G4 was not affected by the presence of the duplex competitor ( Table 1 ), meaning that this compound preferably binds to G4s.…”

Section: Resultsmentioning

confidence: 99%

“…The oligonucleotide was dissolved in water at 1 mM, diluted at 1 μM using 5 mM potassium phosphate buffer (pH 7.0) containing 20 mM KCl, and lastly annealed by heating to 90 °C for 5 min, followed by cooling to room temperature overnight and storage at 4 °C for 24 h before data acquisition. Experiments were performed in sealed quartz cuvettes with a path length of 1 cm by using 0.2 μM of prefolded F-TERRA-T G4 target, the ligand at 2 μM, and the Hrp 27 duplex competitor at 0, 6, and 20 μM final concentrations [ 61 , 62 , 63 , 64 ]. In addition, a blank with no compound or competitor was also analyzed.…”

Section: Methodsmentioning

confidence: 99%

Targeting of Telomeric Repeat-Containing RNA G-Quadruplexes: From Screening to Biophysical and Biological Characterization of a New Hit Compound

Marzano

Pagano

Iaccarino

et al. 2021

IJMS

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DNA G-quadruplex (G4) structures, either within gene promoter sequences or at telomeres, have been extensively investigated as potential small-molecule therapeutic targets. However, although G4s forming at the telomeric DNA have been extensively investigated as anticancer targets, few studies focus on the telomeric repeat-containing RNA (TERRA), transcribed from telomeres, as potential pharmacological targets. Here, a virtual screening approach to identify a library of drug-like putative TERRA G4 binders, in tandem with circular dichroism melting assay to study their TERRA G4-stabilizing properties, led to the identification of a new hit compound. The affinity of this compound for TERRA RNA and some DNA G4s was analyzed through several biophysical techniques and its biological activity investigated in terms of antiproliferative effect, DNA damage response (DDR) activation, and TERRA RNA expression in high vs. low TERRA-expressing human cancer cells. The selected hit showed good affinity for TERRA G4 and no binding to double-stranded DNA. In addition, biological assays showed that this compound is endowed with a preferential cytotoxic effect on high TERRA-expressing cells, where it induces a DDR at telomeres, probably by displacing TERRA from telomeres. Our studies demonstrate that the identification of TERRA G4-targeting drugs with potential pharmacological effects is achievable, shedding light on new perspectives aimed at discovering new anticancer agents targeting these G4 structures.

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“…G-quadruplexes (G4) are structures formed by guanine-rich areas and were named after the π-π interactions among the stacks, each of which contains four guanine bases. The principle of FRET has been applied to distinguishing inter-and intramolecular G4 in bulk solution since 2002 [105] and is still an efficient way to test the formation process of G4 in various conditions [106,107].…”

Section: G-quadruplex Of Telomeresmentioning

confidence: 99%

Single-Molecular Förster Resonance Energy Transfer Measurement on Structures and Interactions of Biomolecules

et al. 2021

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Single-molecule Förster resonance energy transfer (smFRET) inherits the strategy of measurement from the effective “spectroscopic ruler” FRET and can be utilized to observe molecular behaviors with relatively high throughput at nanometer scale. The simplicity in principle and configuration of smFRET make it easy to apply and couple with other technologies to comprehensively understand single-molecule dynamics in various application scenarios. Despite its widespread application, smFRET is continuously developing and novel studies based on the advanced platforms have been done. Here, we summarize some representative examples of smFRET research of recent years to exhibit the versatility and note typical strategies to further improve the performance of smFRET measurement on different biomolecules.

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FRET‐MC: A fluorescence melting competition assay for studying G4 structures in vitro

Cited by 29 publications

References 49 publications

G-Quadruplexes and Their Ligands: Biophysical Methods to Unravel G-Quadruplex/Ligand Interactions

G-Quadruplexes and Their Ligands: Biophysical Methods to Unravel G-Quadruplex/Ligand Interactions

Targeting of Telomeric Repeat-Containing RNA G-Quadruplexes: From Screening to Biophysical and Biological Characterization of a New Hit Compound

Single-Molecular Förster Resonance Energy Transfer Measurement on Structures and Interactions of Biomolecules

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