|
|
 |
| |
PRINTABLE VERSION
Our
laboratory has developed a technology that can measure DNA
structure and
dynamics with angstrom-level precision. It is well suited
to analysis of duplex termini, and it is amenable to high throughput
experiments. The approach is based on a nanoscale pore formed
by the alpha-hemolysin channel, which is inserted in a lipid
bilayer.
Using X-ray diffraction to analyze the assembled protein reveals
the shape of the heptameric pore: a 2.6-nm-wide aperture
leading into a slightly wider
vestibule, which then abruptly narrows to 1.5 nm in the transmembrane
region (Figure 1a,b). The 2.6-nm-wide aperture is formed by
a threonine ring (T9), and the 1.5-nm-wide transmembrane region
is formed by a ring of glutamic acid (E113) and lysine residues
(K147).
Figure 1.
Details of the alpha-hemolysin pore. a) Crystal structure
of the assembled heptamer at 1.9 Å resolution
(from Song et al, 1996). b) Dimensions of the heptameric
pore.
The limiting 1.5-nm-diameter aperture near the center is
formed by a ring of glutamic acid (E113) and lysine residues
(K147). The 2.6 nm-diameter opening at the entrance to the
pore vestibule is formed by a threonine ring (T9). c) A scale
model of ssDNA superimposed on the pore. The space filling
residues are K147. Note that the ssDNA molecule is sufficiently
narrow (~1.3 nm diameter) to fit through this limiting aperture.
d) A B-form dsDNA molecule superimposed to scale on the pore.
In this case, the space filling residues are threonine 9.
The duplex can fit into the pore vestibule but it is too
large (~2.2 nm diameter) to fit through the narrow aperture
at K147. |
Single-stranded
DNA (ssDNA) is narrow enough to fit through
the limiting aperture (Figure 1c), but
double-stranded DNA (dsDNA) is too wide,
and it is limited to entry into the pore
vestibule (Figure 1d). In a solution of
1.0M KCl (pH 8.0), a 120 mV applied potential
produces a steady current (Io)
of 120 ± 5 pA at 23 ºC through
the open channel. Translocation of single-stranded
linear DNA (~1.3 nm diameter) reduces
this current to ~14 pA (I/Io=12%).
Each monomer within ssDNA traverses the
length of the 10-nm pore in 1-3 microseconds
at ambient temperature.
|
| |
|
| |
Double-stranded DNA causes a very different current blockade, as revealed by
studies using synthetic oligonucleotide hairpins. We chose DNA
and RNA hairpins as model duplexes, because they can be formed
from short, highly pure oligonucleotides that can be designed
to adopt one base-paired secondary structure in 1.0M salt at
room temperature. The initial experiments involved a well-characterized
DNA hairpin with a six-base-pair stem and a four-deoxythymidine
loop. When captured within an alpha-hemolysin nanopore, molecules
such as this can cause a partial current blockade (or ‘shoulder’)
lasting hundreds of milliseconds (“B” in lower part
of Figure 2) followed by a rapid downward spike (“C” in
lower part of Figure 2). This “shoulder-spike” signature
is consistent with two sequential steps: 1)
capture of a hairpin stem
in the vestibule (“B” in upper part of Figure 2),
where the molecule rattles in place because the duplex stem
cannot fit through the 1.5-nm diameter-limiting aperture of
the pore; and 2) simultaneous dissociation of the six base
pairs in the hairpin stem, thus allowing the extended single-strand
to traverse the channel (“C” in upper part of Figure
2).
Recently we used the nanopore device
to detect single-nucleotide substitutions in dsDNA using hairpin
molecules with longer duplex stems [1]. Unlike current signatures
caused by short hairpins (≤7 bp), individual blockades caused
by 9- and 10-bp DNA hairpins gated between several discrete
conductance states. We used a combination of Hidden Markov
Models and Support Vector Machines to analyze these gating
patterns [2], which allowed us to detect the identity and orientation
of Watson-Crick base pairs at the termini of individual DNA
hairpin molecules. The mechanism underlying these discrete
current transitions involves binding between the protein and
nucleotides of the hairpin loop or terminus and dynamic properties
of the DNA molecule itself. The latter component is the focus
of this study. Preliminary results are discussed in the following
section.
|
|
| |
|
Figure 2. Impedance
of ionic current through the alpha-hemolysin pore by a
6 bp DNA hairpin. The
current trace is shown in the lower panel. Each letter
corresponds to a diagram in the top panel. A) Open channel
current of ~120 pA. B) Capture of the hairpin molecule
in the vestibule reduces the current to ~55 pA. Our data
indicate that the hairpin loop is perched at the mouth
of the vestibule and the stem is inside the vestibule.
Note that the current resides at this amplitude for about
100 ms, which is more than 3 orders of magnitude longer
than for a linear strand of similar length. C) When the
duplex stem dissociates, the applied electric field pulls
the resulting ssDNA through the limiting aperture, causing
a transient spike to about 15 pA residual current. |
|
| |
SEQUENCE-SPECIFIC CHANNEL GATING MECHANISMS:
DNA HAIRPINS OF 9 AND 10 BASE PAIRS
Capture of a single 9- or 10-bp DNA hairpin
results in a current signature with discrete steps (Figure 3,
top panel) that are not observed for shorter hairpins. These
current levels include four phenomena: an intermediate level
(IL) that initiates all 9- or-10 bp hairpin events; an upper
conductance level (UL); a lower conductance level (LL) that occurs
only after an upper level (UL) trace; and spikes (S) down from
the lower level that indicate close proximity of the terminal
base pair to the pore limiting aperture. Before the ssDNA extends
into the limiting aperture, another step called the “frayed
state” (F) occurs, but this is too fast for capture on
the current signature.
We have developed a working model to explain
the current transitions caused by hairpin capture (Figure 3,
bottom panel). In our model,
each 9-bp hairpin is captured so that the terminal base pair
can interact with amino acids in the vestibule wall near
the limiting aperture formed by lysine K147 and glutamic acid
E113
of alpha-hemolysin. Circular dichroism assays indicate that
the 9-bp hairpin stem is predominantly a B form duplex in bulk
phase. The length per base pair of B form DNA is 3.4 angstroms,
therefore
the total stem length would be 30.6 angstroms. The distance
between
the narrowest part of the vestibule mouth at the T9 threonine
ring and the pore-limiting aperture at lysine K147/ glutamic
acid
Figure 3. Current
signature caused by capture of a 9bp hairpin in the alpha-hemolysin
pore vestibule.
The upper panel shows the current trace acquired at 10
kHz bandwidth. The four most prominent levels are UL (upper
level), IL (intermediate level), LL (lower level), and
S (spike level). The lower panel (a-e) shows the orientation
and dynamics of the captured DNA hairpin that we propose
to account for the current transitions. The F or ‘frayed’ state
is an essential step prior to ssDNA extension into the
limiting aperture, however it is too fast to observe using
our current electronics. A detailed discussion of the blockade
mechanism is given in the text. |
E113 is 33 angstroms. Therefore,
if the hairpin loop is perched at the ring formed by
the threonine ring, the 9-bp
stem
would reach to ~3 angstroms of the limiting aperture.
Given our uncertainty about the exact position of the hairpin
loop and
the finite precision of the alpha-hemolysin X-ray crystal
structure (1.9 angstroms), the estimated position of the hairpin
terminus
is accurate within ±1 bp or ±3.4 angstroms.
What do the different
conductance states represent? Our data indicate that the intermediate
conductance state (IL) is caused
by orientation and immobilization of the hairpin terminus when
it interacts electrostatically with the 3´ terminal nucleotide
and residues in the vestibule wall (Figure 3a). The dwell time
for this intermediate conductance state is independent of the
5´ nucleotide. The IL state invariably transitions to
the upper conductance state, UL (Figure 3b). In the model,
this state
corresponds to desorption of the terminal base pair from the
protein wall
and thermal motion of the hairpin stem in orientations that
allow greater ion current flow through the limiting aperture.
These
orientations may be angular displacement of the hairpin terminus
away from the channel axis or axial orientation of the molecule
allowing ion current to flow along the major groove of the
duplex stem.
From the UL conductance state,
the hairpin may return to the IL state or it may transition into
a third conductance state, LL (Figure 3c). Residence time in
the LL state depends strongly on the identity of the terminal
5' base pair. The 3´ nucleotide alone has no effect on LL dwell
time; however, it does appear to augment binding when it is base
paired with a 5´ nucleotide. When the duplex end frays
from this bound state (Figure 3d), the 3´ strand may extend
and penetrate
the limiting aperture, resulting in a transient spike (Figure
3e). Based on this model, it is clear that kinetics in several
of the conductance states are strongly influenced by protein-DNA
interactions that are unique to this system. For the UL conductance
state, however, the model suggests that the end of the duplex
stem is not bound to the protein. Recent experiments support
this conclusion and highlight the sensitivity of the UL current
to very subtle changes in stem sequence identity.
References
- Nanopores
and nucleic acids: prospects for ultrarapid
sequencing. Deamer DW,
Akeson M. Trends Biotechnol 2000 Apr;
18(4):147-51.
- Water
transport by the bacterial channel alpha-hemolysin.
Paula S, Akeson M, Deamer D. Biochim BiophysActa. 1999 Apr
14; 1418(1): 117-26.
|
|
| |
 |
 |
 |
Center
for Biomolecular Science & Engineering
Engineering 2, Suite 501, Mail Stop CBSE/ITI
UC Santa Cruz, Santa Cruz, CA 95064
phone (831) 459-1544 • fax
(831) 459-1809
cbseweb@soe.ucsc.edu
Questions about the UCSC Genome Browser? Email genome@soe.ucsc.edu
|
© January 2005,
CBSE
Updated 7/2008
|
|
| |
|
| |
|
