title: Single-Molecule Characterization of DNA-Protein Interactions Using Nanopore Biosensors.
abstract: Detection and characterization of nucleic acid-protein interactions, particularly those involving DNA and proteins such as transcription factors, enzymes, and DNA packaging proteins, remain significant barriers to our understanding of genetic regulation. Nanopores are an extremely sensitive and versatile sensing platform for label-free detection of single biomolecules. Analyte molecules are drawn to and through a nanoscale aperture by an electrophoretic force, which acts upon their native charge while in the sensing region of the pore. When the nanopore's diameter is only slightly larger than the biopolymer's cross section (typically a few nm); the latter must translocate through the pore in a linear fashion due to the constricted geometry in this region. These features allow nanopores to interrogate protein-nucleic acids in multiple sensing modes: first, by scanning and mapping the locations of binding sites along an analyte molecule, and second, by probing the strength of the bond between a protein and nucleic acid, using the native charge of the nucleic acid to apply an electrophoretic force to the complex while the protein is geometrically prevented from passing through the nanopore. In this chapter, we describe progress toward nanopore sensing of protein-nucleic acid complexes in the context of both mapping binding sites and performing force spectroscopy to determine the strength of interactions. We conclude by reviewing the strengths and challenges of the nanopore technique in the context of studying DNA-protein interactions.
authors.affiliation: Stanford University, Stanford, CA, United States.
authors.forename: A H
authors.initials: AH
authors.lastname: Squires
authors.affiliation: The Technion, Haifa, Israel.
authors.forename: T
authors.initials: T
authors.lastname: Gilboa
authors.affiliation: The Technion, Haifa, Israel.
authors.forename: C
authors.initials: C
authors.lastname: Torfstein
authors.affiliation: Boston University, Boston, MA, United States.
authors.forename: N
authors.initials: N
authors.lastname: Varongchayakul
authors.affiliation: The Technion, Haifa, Israel; Boston University, Boston, MA, United States. Electronic address: ameller@technion.ac.il.
authors.forename: A
authors.initials: A
authors.lastname: Meller
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D058608:Nanopores; D036103:Nanotechnology; D009690:Nucleic Acid Conformation; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D004268:DNA-Binding Proteins
keywords: DNA–protein interactions; Force spectroscopy; Low-stress silicon nitride; Nanopore; Protein–nucleic acid complexes; Single molecule; Single-stranded DNA