A novel molecular rotor facilitates detection of p53-DNA interactions using the Fluorescent Intercalator Displacement Assay

We have investigated the use of fluorescent molecular rotors as probes for detection of p53 binding to DNA. These are a class of fluorophores that undergo twisted intramolecular charge transfer (TICT). They are non-fluorescent in a freely rotating conformation and experience a fluorescence increase...

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Bibliographic Details
Published in:Scientific reports 2018-08, Vol.8 (1), p.12946-13, Article 12946
Main Authors: Goh, Walter L., Lee, Min Yen, Lim, Ting Xiang, Chua, Joy S., Brenner, Sydney, Ghadessy, Farid J., Teo, Yin Nah
Format: Article
Language:English
Subjects:
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Acridine orange
Apoptosis
Base pairs
Cancer
Deoxyribonucleic acid
DNA
DNA probes
Embryos
Fluorescence
Fluorophores
Humanities and Social Sciences
multidisciplinary
Nucleotide sequence
p53 Protein
Probes
Regulatory sequences
Rotors
Science
Science (multidisciplinary)
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Summary:We have investigated the use of fluorescent molecular rotors as probes for detection of p53 binding to DNA. These are a class of fluorophores that undergo twisted intramolecular charge transfer (TICT). They are non-fluorescent in a freely rotating conformation and experience a fluorescence increase when restricted in the planar conformation. We hypothesized that intercalation of a molecular rotor between DNA base pairs would result in a fluorescence turn-on signal. Upon displacement by a DNA binding protein, measurable loss of signal would facilitate use of the molecular rotor in the fluorescent intercalator displacement (FID) assay. A panel of probes was interrogated using the well-established p53 model system across various DNA response elements. A novel, readily synthesizable molecular rotor incorporating an acridine orange DNA intercalating group (AO-R) outperformed other conventional dyes in the FID assay. It enabled relative measurement of p53 sequence-specific DNA interactions and study of the dominant-negative effects of cancer-associated p53 mutants. In a further application, AO-R also proved useful for staining apoptotic cells in live zebrafish embryos.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-018-31197-9