title: Nanopore sensing of individual transcription factors bound to DNA.
abstract: Transcription factor (TF)-DNA interactions are the primary control point in regulation of gene expression. Characterization of these interactions is essential for understanding genetic regulation of biological systems and developing novel therapies to treat cellular malfunctions. Solid-state nanopores are a highly versatile class of single-molecule sensors that can provide rich information about local properties of long charged biopolymers using the current blockage patterns generated during analyte translocation, and provide a novel platform for characterization of TF-DNA interactions. The DNA-binding domain of the TF Early Growth Response Protein 1 (EGR1), a prototypical zinc finger protein known as zif268, is used as a model system for this study. zif268 adopts two distinct bound conformations corresponding to specific and nonspecific binding, according to the local DNA sequence. Here we implement a solid-state nanopore platform for direct, label- and tether-free single-molecule detection of zif268 bound to DNA. We demonstrate detection of single zif268 TFs bound to DNA according to current blockage sublevels and duration of translocation through the nanopore. We further show that the nanopore can detect and discriminate both specific and nonspecific binding conformations of zif268 on DNA via the distinct current blockage patterns corresponding to each of these two known binding modes.
authors.affiliation: Department of Biomedical Engineering Boston University Boston, Massachusetts 02215 U.S.A.
authors.forename: Allison
authors.initials: A
authors.lastname: Squires
authors.affiliation: Department of Biomedical Engineering Boston University Boston, Massachusetts 02215 U.S.A.
authors.forename: Evrim
authors.initials: E
authors.lastname: Atas
authors.affiliation: 1] Department of Biomedical Engineering Boston University Boston, Massachusetts 02215 U.S.A. [2] Department of Biomedical Engineering The Technion - Israel Institute of Technology Haifa, Israel, 32000.
authors.forename: Amit
authors.initials: A
authors.lastname: Meller
mesh_terms: D000818:Animals; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051766:Early Growth Response Protein 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D058608:Nanopores; D036103:Nanotechnology / Q000379:methods; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D016335:Zinc Fingers / Q000235:genetics
chemical_list: C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D004247:DNA