A Biophysical study of the p53-DNA interactions at the single-molecule level

Abstract

The p53 protein has been widely studied since its discovery as a tumour suppressor protein in 1989 with applications in cancer diagnostics and anti-cancer therapy. p53 has been found to be mutated in 50% of human cancers. Therefore, understanding the properties, mechanisms and mutations of the protein is undoubtedly of great importance in the pursuit of understanding and treating cancer.

In this thesis, single molecule methods such as atomic force microscopy (AFM) and solid-state nanopores were applied to investigate the binding interaction between p53 protein and DNA. The focus of this research was to examine p53 binding to different DNA molecules with or without specific binding sites and to distinguish between bare DNA and different p53-DNA complexes in a label-free manner.

Firstly, AFM was utilised to examine DNA and p53 individually to determine the size and characteristics of the analytes separately. Thereafter, successful binding of p53 to DNA was confirmed and statistics were obtained with respect to the size and position of p53 protein along the DNA samples.

Next, solid-state nanopores were employed to attempt ultrafast single molecule sensing without the need for labels or immobilisation of the DNA and DNA-p53 complexes. Successful single molecule detection was achieved with nanopipette sensors. Further experiments using low-noise planar nanopore devices resulted in successful discrimination between bare DNA and DNA-p53 complexes in a label-free manner. This new development in the p53 field provides a novel method of detecting the tumour suppressor protein p53 binding to DNA. However, the nanopore characteristics for DNA-p53 complexes with different DNA sequences were within experimental error of each other. This study provides a framework for future development towards the detection and distinction of different p53 interactions with DNA.