Single Channel Sensing of dsDNA using the Membrane- Adapted Nanopore of phi29 DNA Packaging Motor

Abstract

The bacteriophage phi29 DNA-packaging motor, geared by six pRNA molecules, contains a truncated cone shape connector channel that is 3.6nm in diameter at its narrow end and 6nm in diameter at its wide end. This channel allows dsDNA to enter and exit the virus procapsid during virus maturation and infection, respectively. We modified the genes that code for the core of the phi29 DNA packaging motor in order to change the amino acid sequence of its protein for membrane incorporation. The modified connector was reconstituted into liposomes and fused into a planar lipid bilayer membrane. Distinctive current jumps were found for each connector insertion. The conductance of each connector channel was measured and found to be uniform (4.8nS in 1M KCl). The membrane embedded connector channel was found to be able to translocate dsDNA. The translocations were recorded as blockage events of the current. The blockages were equal for each of the individual channels, generating a clean, homogenous and uniform signal representing DNA translocation.The connector channel is larger than the previously studied ion channels, which could only let ssDNA pass. The robust property of the connector in ion and dsDNA tranlocation has extensive potentials in microelectromechanical sensing, microreactors, gene delivery, drug loading and DNA sequencing. Single molecule and low concentration sensing can be achieved using this membrane embedded connector system. Additionally, the available crystal structure of the connector protein makes it easy to modify the channel for specific applications and the established large scale purification procedure of the connector will facilitate its practice.