Abstract
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 design, FRET can provide a real-time measurement of structural integrity and dynamics of biomacromolecules in solution and is particularly suitable for studying G-quadruplex (G4) nucleic acids and their ligand interactions. FRET-based assays are ideally suited for high throughput screening (HTS) methodology because they are simple, sensitive, and easily automated. G4s are stable nucleic acid structures involved in important regulatory roles in gene replication, transcription, and genomic instability. Four-stranded G4s are promising drug targets as these non-canonical structures are enriched in oncogene promoters, 5′ UTRs, and telomeres, and have been linked to regulation of gene expression in cancer and other diseases. Although molecules that bind to G4s, with subsequent influence on gene expression, have been well documented, the identification of new chemical scaffolds that potently and selectively bind to G4s and control specific gene expression are still much less common. Here, we describe a detailed protocol of a FRET-based HTS methodology to identify novel G4 ligands.
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References
Chen Y, Yang D (2012) Sequence, stability, and structure of G-quadruplexes and their interactions with drugs. Curr Protoc Nucleic Acid Chem. Chapter 17: Unit 17.5. https://doi.org/10.1002/0471142700.nc1705s50
Neidle S (2017) Quadruplex nucleic acids as targets for anticancer therapeutics. Nat Rev Chem 1(5):0041
Bochman ML, Paeschke K, Zakian VA (2012) DNA secondary structures: stability and function of G-quadruplex structures. Nat Rev Genet 13(11):770
Hänsel-Hertsch R, Di Antonio M, Balasubramanian S (2017) DNA G-quadruplexes in the human genome: detection, functions and therapeutic potential. Nat Rev Mol Cell Biol 18(5):279
Hänsel-Hertsch R, Beraldi D, Lensing SV, Marsico G, Zyner K, Parry A, Di Antonio M, Pike J, Kimura H, Narita M (2016) G-quadruplex structures mark human regulatory chromatin. Nat Genet 48(10):1267
Biffi G, Tannahill D, McCafferty J, Balasubramanian S (2013) Quantitative visualization of DNA G-quadruplex structures in human cells. Nat Chem 5(3):182
Biffi G, Di Antonio M, Tannahill D, Balasubramanian S (2014) Visualization and selective chemical targeting of RNA G-quadruplex structures in the cytoplasm of human cells. Nat Chem 6(1):75–80
Neidle S (2016) Quadruplex nucleic acids as novel therapeutic targets. J Med Chem 59(13):5987–6011
Balasubramanian S, Hurley LH, Neidle S (2011) Targeting G-quadruplexes in gene promoters: a novel anticancer strategy? Nat Rev Drug Discov 10(4):261
Wang K-B, Li D-H, Hu P, Wang W-J, Lin C, Wang J, Lin B, Bai J, Pei Y-H, Jing Y-K (2016) A series of β-carboline alkaloids from the seeds of Peganum harmala show G-quadruplex interactions. Org Lett 18(14):3398–3401
Amrane S, Kerkour A, Bedrat A, Vialet B, Andreola M-L, Mergny J-L (2014) Topology of a DNA G-quadruplex structure formed in the HIV-1 promoter: a potential target for anti-HIV drug development. J Am Chem Soc 136(14):5249–5252
Felsenstein KM, Saunders LB, Simmons JK, Leon E, Calabrese DR, Zhang S, Michalowski A, Gareiss P, Mock BA, Schneekloth JS Jr (2015) Small molecule microarrays enable the identification of a selective, quadruplex-binding inhibitor of MYC expression. ACS Chem Biol 11(1):139–148
Kang H-J, Cui Y, Yin H, Scheid A, Hendricks WP, Schmidt J, Sekulic A, Kong D, Trent JM, Gokhale V (2016) A pharmacological chaperone molecule induces cancer cell death by restoring tertiary DNA structures in mutant hTERT promoters. J Am Chem Soc 138(41):13673–13692
Qin H, Zhao C, Sun Y, Ren J, Qu X (2017) Metallo-supramolecular complexes enantioselectively eradicate cancer stem cells in vivo. J Am Chem Soc 139(45):16201–16209
Xu H, Di Antonio M, McKinney S, Mathew V, Ho B, O’Neil NJ, Dos Santos N, Silvester J, Wei V, Garcia J (2017) CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours. Nat Commun 8:14432
Főrster T (1959) 10th Spiers Memorial Lecture. Transfer mechanisms of electronic excitation. Discuss Faraday Soc 27:7–17
Masuko M, Ohuchi S, Sode K, Ohtani H, Shimadzu A (2000) Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions. Nucleic Acids Res 28(8):e34–e00
Juskowiak B, Takenaka S (2006) Fluorescence resonance energy transfer in the studies of guanine quadruplexes. In: Fluorescent energy transfer nucleic acid probes. Springer, pp 311–341
Yeung AT, Holloway BP, Adams PS, Shipley GL (2004) Evaluation of dual-labeled fluorescent DNA probe purity versus performance in real-time PCR. BioTechniques 36(2):266–275
Acknowledgments
This research was supported by the National Institutes of Health (R01CA177585 (DY), and P30CA023168 (Purdue Center for Cancer Research)). We thank Dr. Clement Lin, Dr. Buket Onel, and Dr. Jonathan Dickerhoff for helpful discussion and proofreading the manuscript.
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Wang, K., Flaherty, D.P., Chen, L., Yang, D. (2019). High-Throughput Screening of G-Quadruplex Ligands by FRET Assay. In: Yang, D., Lin, C. (eds) G-Quadruplex Nucleic Acids. Methods in Molecular Biology, vol 2035. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9666-7_19
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DOI: https://doi.org/10.1007/978-1-4939-9666-7_19
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