A novel molecular rotor facilitates detection of p53-DNA interactions using the Fluorescent Intercalator Displacement Assay
| Autor: | Farid J. Ghadessy, Ting Xiang Lim, Joy S. Chua, Walter Goh, Yin Nah Teo, Sydney Brenner, Min Yen Lee |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Base pair Intercalation (chemistry) Mutant lcsh:Medicine Article 03 medical and health sciences chemistry.chemical_compound Humans A-DNA lcsh:Science Fluorescent Dyes Multidisciplinary Chemistry lcsh:R Acridine orange DNA Fluorescence Intercalating Agents Spectrometry Fluorescence 030104 developmental biology Biophysics lcsh:Q Tumor Suppressor Protein p53 P53 binding |
| Zdroj: | Scientific Reports, Vol 8, Iss 1, Pp 1-13 (2018) Scientific Reports |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/s41598-018-31197-9 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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