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

Scientific Reports
Walter L GohYin Nah Teo

Abstract

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.

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Citations

May 29, 2019·Chemical Communications : Chem Comm·Pichandi AshokkumarAndrey S Klymchenko
Aug 14, 2019·The Journal of Physical Chemistry. B·Bhupesh KumarSukhendu Nath

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Methods Mentioned

BETA
electrophoretic mobility shift assay
ELISA
surface plasmon resonance
Fluorescence
fluorescence correlation spectroscopy
column chromatography
gel filtration

Software Mentioned

GraphPad Prism
GraphPad
AO
R

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