Membrane-Embedded Channel of Bacteriophage Phi29 DNA-Packaging Motor for Translocation and Sensing of Double-Stranded DNA - Nanopores: Sensing and Fundamental Biological Interactions - page 102

DNA-packaging intermediates, (with partial dsDNA within the channel and partial
dsDNA outside the channel) were treated with DNase I or RNase A, or both, the
partially packaged dsDNA stayed inside the procapsid and did not show a reverse
exit in the gradient under highest centrifugation force (Fig. 4.15 ). The data strongly
support the conclusion that the motor channel in an active motor also exercises
the one-way traffic property.
The studies reported here provide direct evidence to prove the pushing or
valving model proposed recently by Guo and Lee [ 9 ]. In this model, the connector
remains static; DNA translocation is induced by a DNA packaging enzyme
or terminase, which pushes a certain length of DNA into the procapsid, and then
shifts to bind to a far distal region of the DNA and inserts an additional section.
This model does not exclude the socket wrench rotating motion by the enzyme.
The one-way traffic makes the DNA enter the procapsid and does not allow it to
come out, similar to the pumping of blood into the heart and the use of valves
to control the flow of blood.

4.7.2 Nanopore Based Stochastic Sensing

Nanopore-based stochastic sensing is an emerging analytical technique that
enables measurements of analytes at the single molecule level. When a molecule
of interest passes through the channel, the ionic current through the pores would
reflect the amplitude, the duration and the rate of the resultant blockade events.
By statistical analysis, information on concentration and identity of molecular
species can be derived. The DNA translocation experiments with the robust phi29
connectors have revealed its potential for nanopore analysis.
The connector has a larger diameter (~3.6 nm) at its narrowest constriction as
compared to some well-established channels, such as the 1.4-nm -haemolysin

channel which only allows the passage of ssDNA. More importantly, for the
development of the phi29 connector based target-selective stochastic sensors, the
larger channel of phi29 connector would provide more flexibility in the choice of
bulky ligands covalently bound inside the channel. Since its crystal structure is
known, a systematic and engineering-based approach can potentially be applied
to develop a system with enhanced analytical capabilities.
There are many bacterial outer membrane porins with larger pores that can be
potentially used as stochastic sensors. However, the gating of these channel proteins
induced by voltage or solution conditions, such as protons, anions or cations, may
cause transient current blockades in single channel recording that would interfere
with detection of analytes. For example, OmpG porins show spontaneous gating
under

a

40 mV [ 55 ]. In contrast, the phi29 connector channel showed stable
channel properties in the voltage range of 150 mV to +150 mV, even at extreme

pH conditions. Therefore, the phi29 connector channel would be an excellent
candidate for biological pore based stochastic sensors.