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Published on: February 7, 2019
Development of a novel multiplex in vitro binding assay to profile p53-DNA interactions
Walter Goh1, David Lane, Farid Ghadessy
1p53 Laboratory, Immunos, Singapore.
Abstract:
The p53 tumor suppressor plays a critical role in cancer biology, functioning as a transcription factor capable of directing cell fate. It interacts with specific DNA response elements (REs) to regulate the activity of target genes. We describe here a novel, non-radioactive assay to measure p53-DNA binding which involves the sequential use of in vitro transcription/ translation (IVT), immunoprecipitation and real-time PCR. The method reliably enables the detection of sequence-specific DNA binding of full-length p53 at low concentrations of physiologically relevant REs (<5 nM). Furthermore, we demonstrate multiplexing of 4 different REs in a single binding reaction. The use of IVT precludes the requirement for purified protein, enabling rapid characterization of the binding properties of p53 variants. Uniquely, it also offers the opportunity to add compounds during translation that might modulate and activate p53. When compared to prevailing protein-DNA binding assays, this method exhibits comparable or higher sensitivity, in addition to an expansive dynamic range afforded by the use of real-time PCR. A further extrapolation of its utility is demonstrated when the addition of a peptide known to activate p53 increased its binding to a consensus RE, consistent with published data.
Insights
A new assay measures p53-DNA binding using in vitro transcription/translation and real-time PCR. This method is sensitive, adaptable for p53 variants, and allows testing of p53-activating compounds.
Area of Science:
- Molecular Biology
- Cancer Biology
- Biochemistry
Background:
- The p53 tumor suppressor is a critical transcription factor in cancer biology.
- p53 regulates target gene activity through interaction with specific DNA response elements (REs).
- Existing assays for p53-DNA binding can be limited in sensitivity or scope.
Purpose of the Study:
- To develop a novel, non-radioactive assay for measuring p53-DNA binding.
- To enable rapid characterization of p53 variants and their binding properties.
- To provide a platform for screening compounds that modulate p53 activity.
Main Methods:
- The assay combines in vitro transcription/translation (IVT), immunoprecipitation, and real-time PCR.
- It detects sequence-specific DNA binding of full-length p53 to low concentrations of REs.
- Multiplexing allows simultaneous detection of binding to multiple REs.
Main Results:
- The assay reliably detects p53-DNA binding at physiologically relevant RE concentrations (<5 nM).
- It demonstrates the ability to multiplex binding reactions for up to 4 different REs.
- The method shows comparable or higher sensitivity and a broader dynamic range than existing assays.
- Activation of p53 by a peptide was shown to increase its binding to a consensus RE.
Conclusions:
- This novel assay provides a sensitive and versatile tool for studying p53-DNA interactions.
- The method facilitates rapid analysis of p53 variants and the discovery of novel p53 modulators.
- Its non-radioactive nature and adaptability make it suitable for various research applications in cancer biology.
