Membrane-Embedded Channel of Bacteriophage Phi29 DNA-Packaging Motor for Translocation and Sensing of Double-Stranded DNA - Nanopores: Sensing and Fundamental Biological Interactions - page 78

4.1 Introduction

Viral genomes are enclosed in a protein shell called a capsid. Following biosynthesis,
viral structural proteins and the genome interact with each other to form a complete
virion through a process referred to as DNA or RNA packaging [ 1 , 2 ]. All linear
double-stranded (ds) DNA or RNA viruses, including dsDNA bacteriophages [ 1 ],
adenoviruses [ 3 ], poxviruses [ 4 ], human cytomegaloviruses (HCMV) [ 5 ], herpes
simplex viruses (HSV) [ 6 ], and dsRNA bacteriophages [ 7 ], share a common feature
in that their genome is packaged into a preformed procapsid. This energetically
unfavorable process is accomplished by a packaging motor that harvests energy
from ATP [ 1 , 8 - 10 ]. One particularly attractive example of DNA-packaging
machinery, in which both protein and RNA are involved, is the Bacillus subtilis
bacteriophage phi29 DNA-packaging motor [ 9 , 11 - 14 ].
The bacteriophage phi29 DNA-packaging motor is comprised of a 12-subunit
gp10 connector [ 15 , 16 ], six copies of ATP-binding DNA packaging RNA
(pRNA) [ 11 , 17 , 18 ] and an ATPase protein gp16 [ 19 , 20 ] to provide the chemical
energy required for DNA translocation (Fig. 4.1a, b ). These purified components
can be combined in vitro and assembled into one of the most powerful nano-
machines known to date [ 21 ]. A central channel, 6.0 nm at the wide end and
3.6 nm at the narrow end (Fig. 4.1c, d ), is formed by three long a -helices from
each of the 12 gp10 subunits of the connector. The cross-sectional area of the
channel is 10 nm 2 at the narrow end and 28 nm 2 at its wider end. The mode of
connector insertion and anchoring within the viral capsid is mediated via hydropho-
bic domains and protein-protein interactions. The portal proteins of many viruses
are morphologically similar sharing the common feature of DNA translocation, but
exhibit large variations in sequence homologies and molecular weights [ 22 ].
Development of a highly sensitive detection system is important in many areas,
including, but not limited to pathogen identification, disease diagnosis, environmen-
tal monitoring, and national security. A wide range of processes such as transport of
DNA, RNA, peptides, proteins, polymers, and pharmaceutical agents have been
studied using electrophysiological measurements [ 23 - 27 ]. Using the well studied
alpha-hemolysin (

-HL) channel from Staphylococcus aureus , the length of single-
stranded (ss) DNA or RNA was determined [ 28 ]. Subsequently, DNA hairpin
molecules have been used to decelerate the DNA translocation rate through the
a

a

-HL pore, thereby demonstrating that the nanopore device can distinguish single
nucleotide polymorphisms [ 29 ]. A hybridization sensor developed by covalent
attachment of individual DNA oligonucleotides within the lumen of the pore was
used to identify single base pair differences [ 30 ]. Detection of base pair stacking
and strand orientation within the pore have also been reported [ 31 ]. Other protein
channels that have been investigated include alamethicin for detection of poly-
ethylene glycol [ 32 ] and reengineered MspA protein from M. smegmatis for trans-
location of single-stranded DNA (ssDNA) [ 33 ].
Due to limitation of the pore size, to date, most of the single pore studies have
focused on the translocation of ssDNA. While other studies have shown the