High-Throughput Screening of G-Quadruplex Ligands by FRET Assay
Fluorescence resonance energy transfer (FRET) is a distance-dependent process by which energy is transferred from an excited donor fluorophore to an acceptor molecule when the donor and acceptor are in close proximity to each other. Depending on the assay
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Introduction Four-stranded G-quadruplexes (G4s) are a family of nucleic acid secondary structures consisting of stacked G-tetrad planes stabilized by Hoogsteen hydrogen bonds and monovalent cations such as Na+ and K+ [1, 2]. There is growing evidence indicating that G4-forming sequences are concentrated at biologically relevant regions and play important regulatory roles in gene replication, transcription, and genomic instability [3, 4]. More recently, G4s have been visualized both in DNA and RNA of human cells, and further marked in human regulatory chromatin [5–7]. Such structures have been implicated in the regulation of genes, some of which are necessary for disease pathogenesis, leading to increased attention as potential novel targets for anticancer and anti-HIV
Danzhou Yang and Clement Lin (eds.), G-Quadruplex Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2035, https://doi.org/10.1007/978-1-4939-9666-7_19, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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agents [8–11]. To date, molecules that bind to G4s and stabilize the G4 secondary structure have been shown to significantly influence transcription and translation of the G4 associated genes [8–14]. The results illustrate the utility of such molecules for disease therapy and emphasize the importance of continued discovery of new chemical scaffolds that potently and selectively for specific G4s [2, 4, 12–15]. Fluorescence resonance energy transfer (FRET) is a dipoledipole coupling process by which the excited-state energy of a fluorescent donor molecule is non-radiatively transferred to an acceptor molecule [16, 17]. As this effect is distance dependent, generally limited to 10–80 A˚, optimization is necessary for placement of donor and acceptor fluorophores to maximize the FRET signal. However, once this step has been performed the assay provides a powerful tool in biomedical research and drug discovery to monitor integrity and dynamics of the target system in real-time [17, 18]. FRET is especially useful for tracking G4 folding and unfolding processes, which can be used to discover G4-interactive small-molecule ligands. For these experiments, a G4-forming nucleic acid is labeled at the 50 and 30 ends by a donor and an acceptor fluorophore, with the requirement that the fluorescence emission spectrum of the donor probe overlaps the excitation spectrum of the acceptor probe. Common FRET pairs include a 6-carboxyfluorescein (FAM) donor and the Black Hole Quencher 1 (BHQ1) or 6-carboxy tetramethylrhodamine (TAMRA) as acceptors. Alternative FRET pairs that display desired spectroscopic properties also include FAM-Cy3, Cy3-Cy5, and FAM-rhodamine dyes. The fluorescence intensity of the probe depends on the distance from the fluorophore to the quencher (or from the FRET acceptor) and this distance is a function of the G-quadruplex folding/unfolding process. In solution, without G4-favorable cations K+ or Na+, the G4-forming nucleic acid mainly exists as a single-strand, but the popul
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