Electrophysiological Method for Quantification of the Number of phi29 DNA Packaging Nanopore in Planar Bilayer Membrane

Abstract

Bacterial virus phi29 uses one of the strongest DNA packaging nanomotors to package its micron-length genomes into a pro-capsid. After re-engineering, whether the DNA nano-motor can be used to pump drugs, DNA, RNA or other therapeutic molecules into specifically targeted cells represent a great challenge in nanomedicine. We have recently successfully embedded the connector, a core component of phi29 DNA package motor, into a planar bilayer membrane (BLM). Under an electric field, double-stranded DNA translocated through the connector channel.The application of phi29 connector array as a stochastic sensor requires knowledge of the number of channels on each membrane. We herein report a method for precise counting of the number of channels on each membrane by electrophysiological approach. Generally, the number of channels is determined by the conductance of total channels and conductance per single channel. Using a derived empirical equation, we can calculate the conductance per single channel at any salt concentration using conductivity of respective conducting buffer. The total conductance of total channels can be measured by ionic current through all the channels under an applied ramp voltage. Comparison of calculated and true values established this as a feasible, reliable and reproducible approach.