Viruses, cancer cells, and other pathogens rely on DNA polymerization to divide and propagate. More benignly, control over DNA polymerization helped establish and remains central to the biotechnology industry. The first goal of the proposed research is the development of nanoscale electronic circuits with single DNA polymerase molecules wired into a carbon nanotube-based field effect transistor. This architecture provides an exquisitely sensitive method for uncovering the kinetics, dynamics and individual steps required for enzymatic catalysis. The long-term goal of this research is to apply DNA polymerase nanocircuits to examine the enzyme's mechanism along with inventing new methods to investigate drug discovery and drug resistance. The experiments proposed here apply a new tool for single-molecule studies to focus on DNA polymerase, a long-studied enzyme with an extensive toolbox of approaches and reagents for its study. Single molecule studies of DNA polymerase using FRET also provide a firm foundation for the proposed experiments. Tethering individual molecules of DNA polymerase to carbon-based nanocircuits allows observation of the enzyme catalyzing phosphodiester bond formation with unprecedented time resolution (single microseconds) and duration (up to hours). Unexplored with the precision of single molecule studies, mutations to DNA polymerase conferring drug resistance have been identified for both anti-cancer and anti-viral treatments. Understanding how such mutations allow the enzyme to avoid inhibition, yet remain functional, could guide the development of more effective anti-cancer and anti-viral compounds.
The polymerization of DNA is a crucial step in the replication of cancerous cells, viruses, and other pathogens. Thus, DNA polymerase, the protein responsible for catalyzing DNA polymerization with high speed and specificity, presents an important target for a wide variety of therapeutics. This proposal applies a new tool to dissect how DNA polymerase works and can be inhibited in unprecedented detail. Such studies could guide the development of future therapeutics targeting specific steps of the DNA polymerase mechanism and avoiding drug resistance.