All linear double-stranded (ds) DNA viruses package their genome into a preformed procapsid via anATP-driving motor. The central component of the phi29 DNA-packaging motor is the portal connectorcomposed of twelve copies of the protein gp10, which form a dodecamer channel that acts as a pathwayfor the translocation of dsDNA. With a diameter of 3.6 nm at its narrowest end, it is one of the largestamong typical channel proteins that have been incorporated into lipid membranes. Explicit engineering andpore modifications of the phi29 connector are possible due to its available crystal structure, thus inspiringthe use of such a system as a module in biomedical applications or nanodevices. The short-term objective of this proposal is to demonstrate the utility of the lipid-embedded channel ofthe phi29 DNA packaging motor as a highly sensitive device for capture and fingerprinting of chemicals andbiopolymers in real time at single molecule resolution using electrophysiological assays. The connector willbe reengineered for added functionality and various approaches will be undertaken to modulate thedimensions of the pore. Alteration of the charge distributions will be made by mutagenesis in a controlledfashion to ensure optimal sensing. The channel will be designed to act as a stochastic biosensor andfunctionalized with recognition groups to bind various analytes of interest. Methods to increase the stabilityand lifetime of the lipid/connector complex will be developed to facilitate high throughputelectrophysiological measurement of a single pore with provisions for rapid and efficient sample and bufferexchange. The long-term objective is to develop the channel into a robust sensing device for detectingbiomolecules or chemicals at extremely low concentrations in the presence of many contaminants for awide range of applications in biotechnology, earlier disease diagnosis, environmental surveillance, customquarantine, drug testing and national security.
The membrane embedded nanopore of bacteriophage phi29 biomotor will be reengineered; characterizedand developed into a single pore sensor for detection of biomolecules and chemicals. The combination ofcapture and fingerprinting with multiple identification parameters for single molecule detection in real time willmake the system highly sensitive to detect molecules at extremely low concentrations in the presence of manycontaminants.
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