Nanopores have shown great promise for DNA sequencing, and more recently as instruments for probing interactions between DNA and DNA-binding enzymes. The broad aim of the proposed research is to develop a nanopore-based instrument for single molecule analysis of polynucleotide-binding proteins, using the Klenow fragment (KF) of Escherichia coli DNA polymerase I as the model enzyme. There are two specific aims:
Aim 1 : Reduce the diameter of the lipid bilayer that houses the nanopore channel in steps, approaching a bilayer-free channel in a silicon nitride support. Significance: Bilayer diameter reduction will increase the stability and lifetime of the nanopore, from hours to days. Smaller bilayers will also shorten the duration of capacitive transients that are superimposed on the ionic current measurements following a change in applied voltage from milliseconds to microseconds. Reducing the transient settling time will in turn enable sub-millisecond detection of variations in the measured current, caused by changes in the captured macromolecular complex, that arise during or near a voltage change.
Aim 2 : Design and implement filtering and control logic to increase the control authority over the lifetime of the DNA-KF complex, regulating the availability of DNA for binding above the nanopore and the unbinding of KF from DNA by voltage-promoted dissociation. Implement the mathematical modeling framework of the Fokker-Planck equation with Bayesian statistical inference to construct the potential profile of dissociation for enzyme-DNA complexes. Significance: The proposed control approach will dramatically increase the throughput of DNA-KF dissociation time measurements under varying voltage patterns. The modeling framework uses the resulting distribution of dissociation time measurements to construct the shape of the free energy landscape as a function of the reaction coordinate, while permitting arbitrary voltage changing patterns. The framework is more general than Kramers'approximation. Kramers'approximation has been used to estimate the height, width and rate of escape of the potential profile, and assumes constant or slowly changing voltage patterns. Our framework does not assume constant or slowly changing voltage patterns, and uses statistical inference to assign uncertainty to profile model parameters. Advances in bilayer diameter reduction (Aim 1) will increase the range of voltage changing frequencies that can be used to dissociate the enzyme, while reliably detecting dissociation. Public Health Relevance Statement: Novel approaches for single molecule measurement, manipulation and modeling are required to uncover with high resolution the dynamics and function of biological macromolecules. The proposed instrument and modeling tools will provide details, previously not achieved, of the shape of the free energy curve as a function of the reaction coordinate describing the dissociation mechanics of the Klenow fragment of Escherichia coli DNA polymerase I from DNA. Using appropriately designed nucleic acid targets, this instrument has broad potential for reliable detection of DNA and RNA binding proteins, in both laboratory and clinical settings.
| Wang, Hongyun; Hurt, Nicholas; Dunbar, William B (2013) Measuring and modeling the kinetics of individual DNA-DNA polymerase complexes on a nanopore. ACS Nano 7:3876-86 |
