Phi29 Motor Nanopore for Single Molecule Sensing

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DESCRIPTION (provided by applicant): A wide variety of highly-ordered structures of macromolecules and organized nanomachines exist in biological systems for transporting polynucleic acids and polypeptides. The ingenious design of the bacteriophage phi29 DNA packaging motor is an intriguing element in this category. All linear dsDNA viruses package their genome into a preformed procapsid via an ATP-driving motor involving two nonstructural enzymes or ATPases. The central component of the phi29 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 double-stranded DNA. 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 in lipid membranes. Explicit engineering and pore modifications of the phi29 connector are possible due to its available crystal structure, thus inspiring its biomedical applications. The short-term objective of this application is to demonstrate the utility of the lipid-embedded channel of the phi29 DNA packaging motor as a highly sensitive device for characterizing biopolymers at single molecule resolution using electrophysiological assays. The connector will first be reengineered for added functionality and various approaches to modulate the conductance of the connector will be systematically investigated. The dimensions of the pore and charge distributions will then be altered by mutagenesis for specific pore modifications in a controlled fashion to ensure optimal sensing. Finally, the channel will be designed to act as a stochastic biosensor and functionalized with modifying groups to recognize nucleotide bases of double stranded DNA for potential sequencing applications. The long-term objective is to develop the channel into a robust sensing device for detecting pathogens and chemicals at extremely low concentrations for disease diagnosis and environmental monitoring, and to utilize it as a high throughput nanopore-based apparatus for the next generation sequencing of double stranded DNA. The channel can further be used as a tool to assay the structure and function of biological molecules and complexes as well as future avenues for therapeutic DNA loading, packaging, and gene delivery to specific cells for the treatment of cancers, viral infections, and genetics diseases. PUBLIC HEALTH RELEVANCE: The membrane adapted bacteriophage phi29 portal motor nanopore protein will be modified, characterized and developed for highly sensitive detection of pathogens and chemicals at extremely low concentrations as well as for characterizing double-stranded DNA at the single molecule level. The channel can further be developed as a tool for therapeutic DNA packaging and gene delivery to specific cells for the treatment of cancers, viral infections, and genetics diseases.