title: Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces.
abstract: The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.
authors.affiliation: 1] Dynamic Biosensors GmbH, Lochhamer Str. 15, 82152 Martinsried, Germany [2] Technische Universität München, 85748 Garching, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Langer
authors.affiliation: Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schräml
authors.affiliation: Dynamic Biosensors GmbH, Lochhamer Str. 15, 82152 Martinsried, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Strasser
authors.affiliation: 1] Dynamic Biosensors GmbH, Lochhamer Str. 15, 82152 Martinsried, Germany [2] Technische Universität München, 85748 Garching, Germany.
authors.forename: Herwin
authors.initials: H
authors.lastname: Daub
authors.affiliation: Roche Molecular Systems Inc., 4300 Hacienda Dr Pleasanton, CA 94588, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Myers
authors.affiliation: Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Heindl
authors.affiliation: Dynamic Biosensors GmbH, Lochhamer Str. 15, 82152 Martinsried, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Rant
mesh_terms: D015374:Biosensing Techniques; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D055664:Electrochemical Techniques; D007700:Kinetics; D008839:Microelectrodes; D011485:Protein Binding; D013816:Thermodynamics
chemical_list: D004247:DNA; D004259:DNA-Directed DNA Polymerase