title: Single-Molecule Studies of Unlabeled Full-Length p53 Protein Binding to DNA.
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.
authors: Imperial College London , Department of Chemistry, Exhibition Road, London SW7 2AZ, United Kingdom.
authors: Philippa
authors: P
authors: Nuttall
authors: Department of Materials Science and Engineering, Seoul National University , Seoul 151-742, Korea.
authors: Kidan
authors: K
authors: Lee
authors: Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano , P.za Leonardo da Vinci 32, Milano, Italy.
authors: Pietro
authors: P
authors: Ciccarella
authors: Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano , P.za Leonardo da Vinci 32, Milano, Italy.
authors: Marco
authors: M
authors: Carminati
authors: Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano , P.za Leonardo da Vinci 32, Milano, Italy.
authors: Giorgio
authors: G
authors: Ferrari
authors: Department of Materials Science and Engineering, Seoul National University , Seoul 151-742, Korea.
authors: Ki-Bum
authors: KB
authors: Kim
authors: Imperial College London , Department of Chemistry, Exhibition Road, London SW7 2AZ, United Kingdom.
authors: Tim
authors: T
authors: Albrecht
mesh_terms: D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D016159:Tumor Suppressor Protein p53; D004247:DNA