Electronic control of DNA polymerase binding and unbinding to single DNA molecules tethered in a nanopore

This work combines computer control with a nanopore sensor to detect and manipulate single DNA molecules and DNA/enzyme complexes captured in the pore. In the setup, a membrane protein self-assembles into a lipid bilayer and an electric potential applied across the membrane creates a measurable ionic current that flows through the pore. A sufficiently large potential produces an electric field capable of capturing DNA molecules and enzyme-bound DNA complexes in the pore. Captured unbound DNA molecules pass through the pore. Because enzymes are too large to enter the pore, enzyme bound to captured DNA must dissociate from the DNA before the DNA translocates through the pore. The basis for detection of translocating polynucleotides, such as DNA, is the measurable reduction in ionic current through the pore. Variations in the current amplitude can differentiate unbound DNA from enzyme-bound DNA in the pore and enable real-time detection of enzyme dissociation from captured DNA. A finite state machine automates detection and reaction to the dissociation of enzymes from captured DNA in real time. Moreover, additional characteristics of the current signal permit identification of the base present at the enzyme's catalytic site, providing a means for single-base sequencing. Combining these capabilities, this research explores methods for repeated control of a single tethered DNA molecule, suspended in the nanopore by voltage control and biochemical alteration. In the tethered configuration, the DNA is available for rapid and repeated binding of enzymes above the pore. The repeated binding and subsequent dissociation of enzymes represents a dramatic advance in the level of control of DNA in a nanopore. With such control of DNA, titration experiments allow investigation into the detectable limits of complex assembly, at the single-molecule level, with statistical significance and provide pre-steady-state biochemical information.