title: Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore.
abstract: Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules. Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates. In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme-polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/polymerase/deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.
authors.forename: Seico
authors.initials: S
authors.lastname: Benner
authors.forename: Roger J A
authors.initials: RJ
authors.lastname: Chen
authors.forename: Noah A
authors.initials: NA
authors.lastname: Wilson
authors.forename: Robin
authors.initials: R
authors.lastname: Abu-Shumays
authors.forename: Nicholas
authors.initials: N
authors.lastname: Hurt
authors.forename: Kate R
authors.initials: KR
authors.lastname: Lieberman
authors.forename: David W
authors.initials: DW
authors.lastname: Deamer
authors.forename: William B
authors.initials: WB
authors.lastname: Dunbar
authors.forename: Mark
authors.initials: M
authors.lastname: Akeson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001681:Biological Assay / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D046911:Macromolecular Substances / Q000737:chemistry; D008969:Molecular Sequence Data; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure*; D036103:Nanotechnology / Q000379:methods*; D016062:Porosity; D011485:Protein Binding
chemical_list: D046911:Macromolecular Substances; D004247:DNA; D004259:DNA-Directed DNA Polymerase