Membrane-Embedded Channel of Bacteriophage Phi29 DNA-Packaging Motor for Translocation and Sensing of Double-Stranded DNA - Nanopores: Sensing and Fundamental Biological Interactions - page 92

a b

2500

5.5 kbp dsDNA
38 bp dsDNA

40

2000

30

1500

20

1000

10

500

0

0

0 5 10 15 20 25 30 35 40

27 28 29 30 31 32 33 34 35 36

Blockade %

Dwell Time (ms)

Fig. 4.8 (a) A histogram of current blockade percentage induced by linear dsDNA (2 kbp) with
a total 3,264 events in the presence of 1 M NaCl, pH 7.8 under 75 mV holding potential.
(b) Comparison of dwell times for translocation of 38 bp and 5.5 kbp ds-DNA. Figures reproduced
with permissions from: (a) Ref. [ 43 ], # The Royal Society of Chemistry; (b) Ref. [ 42 ], # Nature
Publishing Group

4.5.3 Effect of pH on the Stability and Translocation Behavior
of Connector Channels

The stability of the connector channel was investigated under extreme pH condi-
tions in the presence of a 20 bp DNA in both chambers. Current traces were
recorded under a constant voltage at each of the pH conditions investigated in
presence of 1 M NaCl (Fig 4.9a-c ). The respective conductance values are
2.50 0.11 nS ( N ¼ 6) at pH 2(5 mM phosphoric acid buffer), 2.73 0.07 nS

( N ¼ 6) at pH 7 (5 mM Na 2 HPO 4 buffer) and 2.78 0.12 nS ( N ¼ 6) at pH 12

(5 mM Na 2 HPO 4 7H 2 O buffer). The channel under the extreme pH environments

remained open with well-behaved uniform channel conductance. Furthermore,
DNA translocation events are observed. At pH 2, formation of apurinic acid
occurred leading to short current blockade events (Fig. 4.9a ). Although the structure
of DNA is affected by extreme pH conditions, the phi29 connector retains its
stable channel properties under strong acidic or alkaline conditions.

4.5.4 Possible Artifacts in Current Signals

Artifacts can arise from random noise. The connector channel by itself is excep-
tionally stable; the conductance is uniform; and, does not display voltage gating
properties under the reported conditions of

150 mV to +150 mV. Occasionally,
unspecific blockade events with detectable time very close to the limit of sampling
frequency were observed. These were attributed to interactions of connector
reconstituted liposomes with the planar membrane since these events occurred
in absence of DNA and their frequency was observed to increase after the addition