Biomedical Engineering Reference
In-Depth Information
The total channel length is 10 nm and is comprised of a 5 nm vestibule that
protrudes into the
cis
compartment and a 5 nm transmembrane domain embedded
in the lipid bilayer. Between pH 7-9,
-
hemolysin
forms a relatively stable and
reproducible non-gating channel with less than 2% variation in open pore current
under temperature stabilized conditions [
96
]. The comparable inner channel diam-
eter of
a
-
hemolysin
to ssDNA (diameter ~1.3 nm) suggests that less than one Debye
length (~3
˚
in 1 M KCl) separates the translocating biomolecule from the amino
acid residues in the pore. Although dsDNA is too large to translocate through
a
a
-
hemolysin
, up to a 10 bp fragment can reside in the vestibule. This makes
a
-
hemolysin
a very powerful tool for examining biomolecular interactions and the
binding affinities of individual molecules at the single molecule level.
In a landmark study, Kasianowicz et al. demonstrated the ability to electrically
detect individual ssDNA and ssRNAmolecules using
-
hemolysin
nanopores embed-
ded in planar phospholipid bilayers [
43
]. A plethora of studies have since followed
elucidating the biophysics of single molecule transport
a
through proteinaceous
a
-
hemolysin
. For example, Meller et al. examined the effects of polymer length on
translocation velocity [
61
]. Polymers longer than the pore length were seen to
translocate at constant speed however shorter polymers exhibited a length dependent
velocity. Studies by Mathe et al. revealed that
-
hemolysin
nanopore sensors are
sensitive enough to differentiate between 3
0
and 5
0
threading of ssDNA in the pore
with 5
0
threading resulting in a twofold increase in translocation times relative to 3
0
threading, attributed to the tilt reorientation of bases towards the 5
0
end of the
molecule [
58
]. Brun et al. showed that biomolecule flux through proteinaceous
a
a
-
hemolysin
is highly dependent on the applied voltage with the capture rate of
ssDNA [
60
,
64
] and small polyelectrolytes [
8
], following a simple Van't Hoff-
Arrhenius relationship. Kasianowicz and co-workers further showed that the asym-
metric structure of
a-hemolysin
promotes biomolecule entry from the
cis
side
(side with the vestibule) as opposed to the
trans
side [
32
]. Reduced biomolecule
flux from the
trans
side was attributed to a combination of factors; (1) the high
entropic barrier associated with the highly confined geometry of the
barrel on the
trans
side and (2) electrostatic repulsion of DNA by the negatively charged asparatic
acid residues located on the
trans
side. The unzipping of hair-pin DNA structures
using
a
-
hemolysin
was observed by Vercoutere et al. for sufficiently short hairpins.
Vercoutere demonstrated the ability to discriminate between 3 and 8 bp long hairpins
with single base resolution [
92
]. Meller further demonstrated that sequence specific
information could be derived directly from ssDNA translocation experiments through
biological
b
-
hemolysin
[
60
]. Poly(dA) and Poly(dC) strands exhibited different
translocation times attributed to the strong base stacking of poly(dA) relative to
poly(dC), thereby making the poly(dA) sequence more rigid during translocation.
a
a
-
hemolysin
nanopores also hold tremendous value in the field of DNA sequenc-
ing. Stoddart recently demonstrated the ability to resolve individual nucleotides
located in homopolymeric and heteropolymeric ssDNA immobilized in biological
a
-
hemolysin
[
83
]. Mitchell et al. showed that chemical labels attached to bases
could be used to resolve individual bases in a translocating DNA strand [
62
].
Interestingly blockage durations and amplitudes could be tuned by varying the
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