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Single-Molecule Studies of Unlabeled Full-Length p53 Protein Binding to DNA
Philippa Nuttall1, Kidan Lee2, Pietro Ciccarella3
1Imperial College London , Department of Chemistry, Exhibition Road, London SW7 2AZ, United Kingdom.
Abstract:
p53 is an antitumor protein that plays an important role in apoptosis, preserving genomic stability and preventing angiogenesis, and it has been implicated in a large number of human cancers. For this reason it is an interesting target for both fundamental studies, such as the mechanism of interaction with DNA, and applications in biosensing. Here, we report a comprehensive study of label-free, full length p53 (flp53) and its interaction with engineered double-stranded DNA in vitro, at the single-molecule level, using atomic force microscopy (AFM) imaging and solid-state nanopore sensing. AFM data show that dimeric and tetrameric p53 bind to the DNA in a sequence-specific manner, confirming previously reported relative binding affinities. The statistical significance is tested using both the Grubbs test and stochastic simulations. For the first time, ultralow noise solid-state nanopore sensors are employed for the successful differentiation between bare DNA and p53/DNA complexes. Furthermore, translocation statistics reflect the binding affinities of different DNA sequences, in accordance with AFM data. Our results thus highlight the potential of solid-state nanopore sensors for single-molecule biosensing, especially when labeling is either not possible or at least not a viable option.
Insights
This study reveals how the antitumor protein p53 interacts with DNA at the single-molecule level. Solid-state nanopore sensors successfully differentiate p53/DNA complexes, showing potential for label-free biosensing applications.
Area of Science:
- Biophysics
- Molecular Biology
- Nanotechnology
Background:
- The p53 protein is a crucial tumor suppressor involved in apoptosis, genomic stability, and angiogenesis.
- Dysregulation of p53 is implicated in numerous human cancers, making it a significant target for research and therapeutic strategies.
- Understanding p53's interaction with DNA is vital for both fundamental biological studies and the development of novel biosensing technologies.
Purpose of the Study:
- To comprehensively investigate the label-free, single-molecule interaction between full-length p53 (flp53) and engineered double-stranded DNA in vitro.
- To evaluate the utility of atomic force microscopy (AFM) and solid-state nanopore sensing for characterizing these interactions.
- To demonstrate the potential of nanopore sensing for label-free biosensing applications.
Main Methods:
- Single-molecule imaging using Atomic Force Microscopy (AFM) to visualize p53 binding to DNA.
- Solid-state nanopore sensing with ultralow noise to detect and differentiate DNA and p53/DNA complexes.
- Statistical analysis, including Grubbs test and stochastic simulations, to validate binding affinities and sequence specificity.
Main Results:
- AFM imaging confirmed sequence-specific binding of dimeric and tetrameric p53 to DNA, consistent with known binding affinities.
- Solid-state nanopore sensors successfully distinguished between bare DNA and p53/DNA complexes at the single-molecule level.
- Translocation statistics from nanopore sensing correlated with AFM data, reflecting sequence-specific binding affinities.
Conclusions:
- Solid-state nanopore sensors offer a promising label-free approach for single-molecule biosensing of protein-DNA interactions.
- This technology is particularly advantageous in scenarios where molecular labeling is challenging or not feasible.
- The study provides a foundation for utilizing nanopore sensing in fundamental research and clinical diagnostics involving p53 and other DNA-binding proteins.
