Viral genomes are enclosed in a protein shell called capsid. During last step of replication and morphogenesis, double-stranded DNA viruses such, as bacteriophage phi29 (Dwight Anderson AND Bernard Reilly) that infects bacteria Bacillus subtilis, uses a nanomotor to package its genome into a preformed procapsid, or proheads for bacteriophages (Lee Taejin AND Peixuan Guo, the books). After synthesis by separate machinery, viral structural proteins and the genome must interact with each other to form a complete virion through a process referred to as DNA or RNA packaging. However, all linear double-stranded (ds)DNA or dsRNA viruses, including dsDNA bacteriophages (Guo, 1994), adenoviruses (Zhang and Imperiale, 2000), poxviruses (Koonin et al., 1993), human cytomegaloviruses (HCMV) (Scheffczik et al., 2002), herpes simplex viruses (HSV) (Salmon and Baines, 1998) and dsRNA bacteriophages (Olkkonen et al., 1990), possess a common feature in that their genome is packaged into a preformed procapsid. This entropically unfavourable process is accomplished by an ATP-driven packaging motor (Earnshaw and Casjens, 1980).
The phi29 DNA-packaging nanomotor (Fig. 1, Guo et al, Science 1987, PNAS ) is one of the strongest biological motors known. A key challenge is to contextualize to human systems our understanding of the unique phi29 nanomotor and its components, and to manipulate this nanomachine in an artificial environment. Our center will continue our studies of the phi29 motor to characterize its physical properties, and we will also rebuild the motor for therapeutic purposes, possibly for drug delivery. We plan to re-engineer the motor to function in lipid bilayers and other polymers, continue our detailed mechanistic studies of the re-engineered motor, and develop arrays of motors for single molecule sensing of chemicals, biomarkers, diseased cells, pathogens as well as for single pore sequencing of DNA.
Single Pore Sensing can be readily described by its name, insertion of a singular biological nanopore into a membrane for the purpose of sensing polynucleotides or proteins, all with or without chemical modifications. Since its conception in the 1980s with the first demonstration of its potential by Kasianowicz et al., the researchers were able to definitively show that ssRNA and ssDNA (ss – Single Stranded) could be translocated or moved through an α-Hemolysin nanopore [1]. During the past 2 decades scientists have improved upon insertion techniques and the variety of pores to be inserted. Recent interest has been devoted to using bacteriophage nanopores to improve upon the limitations from α-Hemolysin. However, some features have remained constant throughout the development process such as usage of a Lipid Bilayer Membrane and electrical currents to drive the analytes through the inserted nanopore. Some of the most prominently used nanopores pores have been α-Hemolysin, Aerolysin, and Phi29[2]. The crucial feature in the use of all these nanopores has been the diameter of their inner channel, Aerolysin with 2.6 nm, α-Hemolysin with 1.2 nm and Phi29 with 3.6 nm [3,4,5]. The Phi29 and Aerolysin connectors with an inner channel diameter above 2nm, the diameter of dsDNA, make them more amenable to a variety of uses in Single Pore Sensing.
Viral genomes are enclosed in a protein shell called capsid. During last step of replication and morphogenesis, double-stranded DNA viruses such, as bacteriophage phi29 (Dwight Anderson AND Bernard Reilly) that infects bacteria Bacillus subtilis, uses a nanomotor to package its genome into a preformed procapsid, or proheads for bacteriophages (Lee Taejin AND Peixuan Guo, the books). After synthesis by separate machinery, viral structural proteins and the genome must interact with each other to form a complete virion through a process referred to as DNA or RNA packaging. However, all linear double-stranded (ds)DNA or dsRNA viruses, including dsDNA bacteriophages (Guo, 1994), adenoviruses (Zhang and Imperiale, 2000), poxviruses (Koonin et al., 1993), human cytomegaloviruses (HCMV) (Scheffczik et al., 2002), herpes simplex viruses (HSV) (Salmon and Baines, 1998) and dsRNA bacteriophages (Olkkonen et al., 1990), possess a common feature in that their genome is packaged into a preformed procapsid. This entropically unfavourable process is accomplished by an ATP-driven packaging motor (Earnshaw and Casjens, 1980).
The phi29 DNA-packaging nanomotor (Fig. 1, Guo et al, Science 1987, PNAS ) is one of the strongest biological motors known. A key challenge is to contextualize to human systems our understanding of the unique phi29 nanomotor and its components, and to manipulate this nanomachine in an artificial environment. Our center will continue our studies of the phi29 motor to characterize its physical properties, and we will also rebuild the motor for therapeutic purposes, possibly for drug delivery. We plan to re-engineer the motor to function in lipid bilayers and other polymers, continue our detailed mechanistic studies of the re-engineered motor, and develop arrays of motors for single molecule sensing of chemicals, biomarkers, diseased cells, pathogens as well as for single pore sequencing of DNA.
Single Pore Sensing can be readily described by its name, insertion of a singular biological nanopore into a membrane for the purpose of sensing polynucleotides or proteins, all with or without chemical modifications. Since its conception in the 1980s with the first demonstration of its potential by Kasianowicz et al., the researchers were able to definitively show that ssRNA and ssDNA (ss – Single Stranded) could be translocated or moved through an α-Hemolysin nanopore [1]. During the past 2 decades scientists have improved upon insertion techniques and the variety of pores to be inserted. Recent interest has been devoted to using bacteriophage nanopores to improve upon the limitations from α-Hemolysin. However, some features have remained constant throughout the development process such as usage of a Lipid Bilayer Membrane and electrical currents to drive the analytes through the inserted nanopore. Some of the most prominently used nanopores pores have been α-Hemolysin, Aerolysin, and Phi29[2]. The crucial feature in the use of all these nanopores has been the diameter of their inner channel, Aerolysin with 2.6 nm, α-Hemolysin with 1.2 nm and Phi29 with 3.6 nm [3,4,5]. The Phi29 and Aerolysin connectors with an inner channel diameter above 2nm, the diameter of dsDNA, make them more amenable to a variety of uses in Single Pore Sensing.