All linear double-stranded (ds) DNA viruses package their genome into a preformed procapsid via an ATP-driving motor. The central component of the phi29 DNA-packaging motor is the portal connector composed of twelve copies of the protein gp10, which form a dodecamer channel that acts as a pathway for the translocation of dsDNA. With a diameter of 3.6 nm at its narrowest end, it is one of the largest among typical channel proteins that have been incorporated into lipid membranes. Explicit engineering and pore modifications of the phi29 connector are possible due to its available crystal structure, thus inspiring the 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 of the phi29 DNA packaging motor as a highly sensitive device for capture and fingerprinting of chemicals and biopolymers in real time at single molecule resolution using electrophysiological assays. The connector will be reengineered for added functionality and various approaches will be undertaken to modulate the dimensions of the pore. Alteration of the charge distributions will be made by mutagenesis in a controlled fashion to ensure optimal sensing. The channel will be designed to act as a stochastic biosensor and functionalized with recognition groups to bind various analytes of interest. Methods to increase the stability and lifetime of the lipid/connector complex will be developed to facilitate high throughput electrophysiological measurement of a single pore with provisions for rapid and efficient sample and buffer exchange. The long-term objective is to develop the channel into a robust sensing device for detecting biomolecules or chemicals at extremely low concentrations in the presence of many contaminants for a wide range of applications in biotechnology, earlier disease diagnosis, environmental surveillance, custom quarantine, drug testing and national security.
The membrane embedded nanopore of bacteriophage phi29 biomotor will be reengineered, characterized and developed into a single pore sensor for detection of biomolecules and chemicals. The combination of capture and fingerprinting with multiple identification parameters for single molecule detection in real time will make the system highly sensitive to detect molecules at extremely low concentrations in the presence of many contaminants.
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