Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces

• DOI: 10.1038/srep12066
• Publication Date: 2015-07-15T11:56:45Z
• Authors: Andreas Langer; Michael Schräml; Ralf Strasser; Herwin Daub; Thomas Myers; Dieter Heindl; Ulrich Rant
• Keywords: DNA clamp

Abstract The engineering of high-performance enzymes for future sequencing and PCR technologies as well the development many anticancer drugs requires a detailed analysis DNA/RNA synthesis processes. However, due to complex molecular interplay involved, real-time methodologies have not been available obtain comprehensive information on both binding parameters enzymatic activities. Here we introduce chip-based method investigate polymerases their interactions with nucleic acids, which employs an electrical actuation DNA templates microelectrodes. Two measurement modes track dynamics induced switching process extension simultaneously quantitate kinetics, dissociation constants thermodynamic energies. high sensitivity reveals previously unidentified tight states Taq Pol I (KF) polymerases. Furthermore, incorporation label-free nucleotides can be followed in changes polymerase conformation (finger closing) during activity are observable.