Membrane-Embedded Channel of Bacteriophage Phi29 DNA-Packaging Motor for Translocation and Sensing of Double-Stranded DNA - Nanopores: Sensing and Fundamental Biological Interactions - page 95

in the cis -chamber remained undetectable over the 90 min time course ( N ¼ 4

experiments). It was further observed that the blockade events from the current
trace closely correlated with the number of DNA molecules passing through the
pores quantified by Q-PCR (Fig. 4.10b ).
To rule out the possibility that the increase in the DNA copy number in the
cis- chamber was due to membrane leakage, three additional experiments were
carried out under known conditions of BLM or partition leakage. When leaking
occurred, the copy number of DNA per microliter of solution in the cis -chamber
was approximately 10 4 to 10 5 -fold higher than those experiments without leakage.

4.6 Phi29 Motor Channel Exercises a One-Way
DNA Traffic Mechanism

The connector channel exercises a one-way traffic property for dsDNA trans-
location from N-terminal entrance (narrower-end) to C-terminal exit (wider-end),
as demonstrated by voltage ramping, electrode polarity switching, and sedi-
mentation force assessment. This is the first instance of a native wild type protein
channel exhibiting a rectifying behavior with regard to DNA translocation.
The connector channel, however retains a two-way traffic property for ions with
equal conductance under both positive and negative trans- membrane potentials
[ 42 - 44 ].

4.6.1 One-Way Traffic of dsDNA Probed by Ramping
Potential to a Membrane with a Single Channel

As noted earlier, the connector channel is inserted into the BLM via vesicle
fusion of the liposome/connector complex with the planar lipid membrane. The
orientation of the connector channel in the BLM is therefore random, with either
the C-terminus facing the cis -chamber or the N-terminus oriented towards the

cis -chamber.
The application of a ramping potential (2.2 mV s 1 ) from 100 mV to +100 mV

to the BLM containing a single connector channel revealed a unidirectional trans-
location of dsDNA (Fig. 4.11 ). Since the phosphate backbone of DNA is nega-
tively charged, DNA tends to migrate from the negative to the positive potential.
Figure 4.11a shows a control experiment in the absence of DNA. When dsDNA
was pre-mixed in both the cis and trans -chambers at identical concentrations,
DNA translocation was observed at either negative (Fig. 4.11b ) or positive
(Fig. 4.11c ) trans -membrane voltage, depending upon the orientation of the
connector in the BLM. The same phenomena were observed with dsDNA of
varying lengths (from 12 bp to 5,500 bp) as well as for ssDNA.