Analysis of Polymerase-DNA Interactions and Polymerase Activity with Electrically Actuated DNA Nanolevers on a Chip

Next generation DNA sequencing technologies are on the verge of revolutionizing research and clinical genomics. An important cornerstone for ever faster, less expensive and more accurate genomic information are highly optimized and error-free DNA polymerases.Here we present a novel method to study DNA polymerases and their interaction with nucleic acids on a chip. DNA molecules, which are end-tethered to microelectrodes on a chip, are set in motion by alternating electric fields, and the molecular dynamics of their oscillation (orientation switching) are measured by fluorescence energy transfer. The association and dissociation of various DNA polymerases are monitored in real-time by analyzing changes in the DNA motion that occur due to polymerase binding. Chemical rate constants of association and dissociation and affinity constants are determined.For the polymerases phi29, Taq, and the Klenow fragment, the influences of temperature, mono- and divalent ion concentration, and presence/absence of dNTPs on polymerase binding kinetics are investigated. The polymerization activity is evaluated and even exonuclease activity can be observed in real-time. Simultaneously, information on the size of the DNA-polymerase complex is obtained with sub-nanometer accuracy and conformational changes in the ternary polymerase-dNTP-DNA complex are revealed (match/mismatch situation).The method is label-free, uses a parallel microelectrode format for multiplexed assays and microfluidics for low sample consumption. It bears great potential as a powerful tool for the characterization of polymerases and facilitates the engineering of polymerases for, e.g., more efficient sequencing technologies.