The potential of nanopores for
single-molecule, ultra-rapid
sequencing of DNA
Bionanotechnology option Molecular pores in nanotechnology
Candidate no. : 22518
Word count : 3123
The potential of nanopores for single-molecule, ultra-rapid
sequencing of DNA
Introduction
Sequencing the human genome was a project that started 15 years ago using
techniques pioneered by Sanger et al (1) and Maxam and Gilbert (2). The estimated
cost of sequencing a similar mammalian genome five years ago was US$300 million
(3) using equipment can sequence ~30,000 bases per instrument per day at a cost of ~
US$0.50 per nucleotide (4). Today, another human genome could be sequenced in
about six months, at a cost of ~US$30 million (5). However, developments are taking
place investigating the notion that in the future nanopores may be used as a cheaper
way to determine DNA sequences, at rates between 1000 and 10,000 bases per second
(4).
The fundamental principle behind nanopore DNA sequencing is that the
nanopores are behaving as ‘Coulter counters’ – macromolecules carrying a net
electrical charge are electrophoretically driven through the nanopore by an applied
electric potential across the pore. Ions flowing through this pore cause a detectable
current. When macromolecules enter the pore they partially block it and reduce the
flow of ions. This causes measurable transient drops in the current that can be
monitored to determine characteristics of that macromolecule (6). The hypothesis is
that as the DNA is driven through the pore, each nucleotide will have a different
affect on the current. Thus one can determine the sequence from the changes in the
current as the DNA passes through the pore. In order to do this, single nucleotide
resolution is required – it must be possible to distinguish one nucleotide from the next
in the sequence.
α-Hemolysin
The first stage in nanopore DNA sequencing was to find an appropriate
nanopore. An obvious example was the α-Hemolysin (α-HL) protein from
Staphylococcus aureus (figure 1). α-HL has properties that make it an ideal choice for
experimental use; it is wide enough to accommodate passage of a single-stranded (ss)
polynucleotide, it can remain open for a long period of time (up to 24 hours), whilst
remaining stable under a variety of ionic strengths, temperatures approaching 100oC
(7) and up to 65oC in denaturing detergents (8). Also for future investigations, α-HL is
useful as it tolerates radical alterations in its amino acid sequence. This can be utilised
to perhaps engineer a pore that is better equipped for DNA sequencing, indeed α-HL
has already been modified to detect divalent metal ions (9) and small organic
molecules (10).
2
Figure 1 (from (11)). The crystal structure of α-HL is known to 1.9 Å resolution (12) and has an
aperture that is wide enough to accommodate DNA or RNA. α-HL is a heptameric protein that self
assembles in the lipid bilayer. It is comprised of three domains; the cap and the rim (comprising the cis
‘head’), and the (membrane spanning, trans) stem. The trans side of the protein spans the membrane,
whilst the cis side is the mushroom-shaped ‘head’ of the protein. The protein is ~10 nm high, with the
pore running the length of the protein. This aqueous channel ranges in diameter from 1.5 to 4.6 nm,
with the cis entrance being 2.6 nm, the trans entrance being 2 nm. Double-stranded (ds) polynucleic
acids can enter the cis vestibule of the protein, but only ss-polymers can traverse the 1.5 nm diameter
constriction and thread through the narrow stem region.
The first experiment
Kasianowicz et al (13) showed for the first time that a polynucleotide could be
driven through a nanopore. It was reasoned that it should be possible for the
polyanionic polynucleotide to be drawn through a continuously open channel by an
applied trans-membrane voltage (indeed, the initial aim of the experiment was to
prove this, with characterisation of the polymer almost an afterthought). Furthermore,
it was postulated that due to the dimensions of the channel (figure 1), the
polynucleotide would have to pass through in an extended linear chain. This passage
of the polynucleotide could be detected by the partial blockage that it caused to the
ion flow.
The system was set up with a solvent-free bilayer of diphytanoylphosphatidylcholine separating two buffer-filled compartments (1.5 ml of 1 M KCl
and 5 mM Hepes at pH 7.5). Less than 1 μg α-HL protein was added to one
compartment, which then reconstituted into the bilayer. After a channel had formed,
the compartment was washed with fresh buffer to prevent further channel formation.
Upon application of a potential of -120 mV (with the cis side negative), a
steady single channel current ensued. With the addition of poly[U] the current
dropped by 85-100% and lasted for hundreds to thousands of microseconds. Similar
timescale events were seen for poly[C], poly[dT], poly[dC], and poly[dA,dT,dC]. The
blockage times were proportional to nucleotide length and inversely proportional to
the applied voltage.
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A profile of the duration of blockage revealed that the events fell into three
groups (figure 2), one of which was very short time-period. The other two groups
were attributed to the polynucleotide being threaded through the pore in different
orientations, i.e. 5’ first or 3’ first (at the time there was no physical basis to explain
this, but subsequent models (14) speculate this is due the polymers acting in a ratchetlike fashion) .
Figure 2 (from (13)). The lifetimes of the blockages fall into three groups, the first of which (peak 1)
was very short and was explained as the polynucleotide transiently blocking the entrance to the pore
before dissociating again. The other two groups (peaks 2 and 3) represent polynucleotide passage
through the pore, possibly in different orientation.
Deamer and Akeson (4) were sceptical of the notion that a seemingly small
applied potential could capture the ends of individual nucleic acid molecules and draw
them through a 1.5 nm pore. In order to show that it was indeed the polynucleotide
translocation that was causing the blockages, a further experiment was carried out
comparing ssDNA to dsDNA (13). The dsDNA was shown to cause indefinitely long
blockages (suggesting it enters the cis vestibule, but not into the stem), whereas the
ssDNA causes transient decreases in current. PCR analysis of the buffer
compartments showed that the predicted amount of ssDNA (and none of the dsDNA)
had translocated from the cis to trans compartments.
Kasianowicz et al (13) then postulated that characteristics of the DNA could
be determined, and eventually sequencing could occur.
Characterisation of DNA
Earlier experiments had shown that polymers passing through pores could be
characterised and also help to reveal information about the pore (15) (16), and now
work was being carried out to characterise polynucleic acids. The first experiments set
out to try and distinguish different homopolynucleotides (14) (17) (18). It was seen
that simply measuring the speed of translocation could be used to distinguish between
some nucleotide species (poly[U] traverses the pore ~20 times faster than poly[dA]
(14), and about ten times faster than poly[A] (17)). In a seminal experiment, Akeson
et al (18) were the first to show they could use α-HL to distinguish different
polynucleotides from their current blockade characteristics (length of blockage and
degree of blockage). They found that the blockages caused by poly[A] were smaller
than those caused by poly[C] (~85% decrease compared to ~95%), and were also
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longer than poly[C] (22 ± 6 μs compared to 5 ± 2 μs per nucleotide). The current
amplitudes for poly[A] and poly[U] were virtually indistinguishable, but poly[U]
blockades were typically shorter. It was noted that some of the results went against
prior conceptions based upon the size of the purine/pyrimidine. For example, cytosine
is a much smaller moiety than adenine, but causes a larger blockage. This anomaly
was attributed to the secondary structure of the RNA. Poly[A] and poly[C] both form
helices with diameters of 2.1 and 1.3 nm respectively. Thus the poly[A] helix is too
big to fit through the α-HL 1.5 nm aperture, so has to unwind (which explains why
poly[A] transition takes longer). Therefore the helical poly[C] obscures a larger part
of the channel than the extended-chain poly[A]. Another possibility was that cytosine
actually interacted with α-HL in some way. This theory was disproved by also testing
poly[dC]. Poly[dC] cannot form a helix, and thus should cause a lower decrease in the
current, which was observed.
An additional experiment was performed using a polynucleotide that contained 30
adenine bases and 70 cytosine bases. It was hoped that the method would produce a
bilevel current that would indicate the passage of one nucleotide species to the other.
This was indeed the case (figure 3); a ~95% blockage (solid arrow) that reduced to a
~85% blockage (dashed arrow). This also shows that the poly[C] end (3’) entered the
pore first, as would be expected due to its narrow secondary structure helix (5’ end
first transitions were seen, but resulted in permanent blockages). Akeson et al (18)
were the first of many to realise that if traversal time could be slowed, then single
nucleotide detection may be achievable.
Figure 3 (adapted from (18)). The current profile of the experiment carried out using the A(30)C(70)Gp
polynucleotide. Several bilevel blockage events can be seen, with the two levels occurring at ~95 and
~85% blockage. The change in level indicates the transition from poly[C] to poly[A].
Characterisation of the same bases has also occurred in DNA (19). Here the two
species are distinguished using three parameters; the most probable translocation
current, the most probable translocation duration, and the characteristic dispersal
values for individual translocation durations. The translocation durations was plotted
against blockade current to produce ‘event diagrams’, of which poly[dA] and
poly[dC] fall in to two distinct groups with only 1% overlap. This could also be used
to distinguish poly[dAdC]50 and poly[dA50dC50].
These experimental results prove that detailed characterisation of
polynucleotides is possible, and that further developments could lead to single
nucleotide resolution.
5
Single nucleotide resolution
Single nucleotide resolution is the ultimate goal that will lead to sequencing.
The first of several experimental procedures where it was claimed that
polynucleotides with only one base difference were distinguishable was carried out by
Howorka et al (20). Pores were engineered with an oligonucleotide linked to a
cysteine residue on α-HL. The result is a nanopore with a piece of ssDNA covalently
attached within the cis vestibule of α-HL. It was shown that lengths of DNA that were
complimentary to the tethered oligonucleotide could be distinguished from those with
a single-base mis-match from observing their current trace. Furthermore, an
oligonucleotide (of nine bases) where the final three bases (positions seven, eight, and
nine) are unknown was tethered inside the α-HL and the final codon sequenced by
using an array of oligonucleotides (seven bases long) that each had a different base at
position seven. Whichever one of these produced a trace indicative of a
complimentary sequence would reveal which base occupied position seven. The same
was repeated for positions eight and nine. Thus one could say, that α-HL was used for
sequencing of DNA, though it was not ‘single-molecule’, and in no way ‘ultra-rapid’.
This technique was using the duplex-formation properties of the oligonucleotides,
rather than the current-disruption properties of different bases to determine the
sequence. Indeed, the authors proposed that the modified nanopore would be a useful
tool with which to study DNA duplex formation in detail.
A similar (duplex-formation properties) approach was carried out by Deamer
and Branton (21) where they again demonstrated ‘single-base resolution’. This time
the DNA duplex was in the form of a blunt ended hairpin. The hairpin was used in an
attempt to keep the DNA in the α-HL pore for longer, and thus slow down the
translocation. Hairpins were designed so that only intramolecular interactions
occurred (22), and they initially used a six-base-pair stem with a four-T loop (23). As
the ds-hairpin is too wide to pass through the α-HL 1.5 nm aperture, translocation can
only occur upon spontaneous dissociation of all the hydrogen bonds (caused by a
force of ~20 pN exerted by an applied voltage of 125 mV). Blunt-ended DNA
hairpins of stem length varying from three to eight bases were used, and it was
observed that with each base addition the size of blockage was increased. The
duration of the blockage also increased with stem-length, and correlated well with the
free energy of hairpin formation. Hairpins containing a mis-match were produced, and
the blockage duration was decreased from ~1 s to 10 ms. Thus, theoretically this
technique can distinguish two DNA molecules that differ in only one nucleotide,
however the authors do admit that these results do not lead to a method of sequencing.
Further investigations into DNA hairpins (24) (25) have found that the ionic current
signature whilst the hairpin is in the cis vestibule depends on the number of hydrogen
bonds within the terminal base pair, the stacking between the terminal base pair and
its nearest neighbour, and the 5’ vs 3’ orientation (24). Thus all four combinations of
basepairs can be distinguished. Recently, non-blunt-end hairpins have been
investigated (25). These showed that hairpin unzipping times decrease as follows; 8
bp hairpin > 8 bp hairpin with a single mis-match > 7 bp hairpin. The results show
agreement with dissociation timescales of hairpins in bulk solution (26) which
suggests that the hairpins stability is not affected by possible DNA-pore or
electrostatic interactions.
6
Nakane et al (27) also have results that, as they describe it, demonstrated a
‘proof-of-concept’ for a single molecule oligonucleotide sensor capable of
distinguishing short oligonucleotides with single base pair resolution. This involved a
piece of ss-DNA biotinylated on the 5’ end to prevent it completely passing through
the pore (figure 4). This is driven through the α-HL pore to a group of target ssDNAs
on the other side of the membrane. The two will form a duplex, and then the potential
is reversed, causing the withdrawal of the biotinylated DNA back through the pore.
The time taken for the probe to withdraw will depend upon the strength of the duplex
interactions. Thus, again, matched and mis-matched DNA can be discriminated.
Although the authors have ‘proved-their-concept’, it offers little in the advancement
towards sequencing. Furthermore, the potential for target DNA segments to move
back across α-HL (trans to cis) is not accounted for or even mentioned.
Figure 4 (adapted from (27)). The biotinylated ssDNA is used to probe the pieces of ssDNA on the
other side of the membrane. Upon the reversal of the potential, the probe DNA moves back through the
pore, but is hindered by the duplex that is formed. Complete probe DNA withdrawal can only occur
when the duplex has dissociated, thus a completely complementary sequence will require a longer time
to dissociate than a sequence that contains a mis-match.
The authors did, however, proclaim that higher specificity had been achieved
in nanopore-based sensors by incorporating a probe molecule that is permanently
tethered to the interior of the pore (concept has since been used in further experiments
(28) (29)). This counteracts an underlying problem in α-HL pore transduction; the fact
that the DNA is driven through the pore too quickly. Several authors (18) (30) (11) (4)
have recognised that in order to obtain single nucleotide detection the traversal time
needs to be slowed. It has been shown both through Molecular Dynamics (MD)
simulations (31) and experiments (32) that the dynamics of DNA translocation are
sensitive to the magnitude of the applied electric field. However, one cannot simply
reduce the voltage, as it still needs to be maintained high to overcome backward
movement of the polynucleotide. The reason for this problem is that the number of
ions involved in transition between one base and the next is only ~100 ions per
microsecond, and the time interval for a measurement is just few microseconds, so the
difference is lost in the noise (4). One approach to slow down translocation was to use
DNA that formed a hairpin, causing it to remain inside α-HL, as described previously
(21) (24) (25). The other, more recent approach is to use DNA that remains
permanently within the pore (28) (29) by forming a rotaxane (figure 5) (28) and
pseudorotaxane (29).
7
Figure 5 (adapted from (29) and (28)). The ssDNA was prevented from leaving the pore in either
direction, through addition of streptavidin to the PEG region on the trans side and the formation of a
stable DNA hairpin on the cis side (right). Ashkenasy et al (29) produced a pseudorotaxane by
engineering DNA with a stable terminal hairpin that holds the polynucleotide in the α-HL pore (left).
The goal of Ashkenasy et al (29) was to determine which part of the DNA encased in
the α-HL pore gave rise to the signature ionic current. In order to do this, the typical
current of poly[dA] (residual current ‘IR’ of ~22%) and poly[dC] (IR ~31%) was
measured. They knew that the specific region ‘recognised’ by α-HL was ~20
nucleotides away from the end of the hairpin, so poly[dC] was produced with a single
A that varied in position from 18 to 22. Multiple current readings were then taken,
and each event was characterised as either ‘A-type’ (IR < 27%) or ‘C-type’ (IR > 27%)
and the percentage of each type calculated. For A-position 18, 19, 21, and 22, the Ctype events were in the large majority (> 65%), however, for position 20 there was a
majority A-type signal. Thus, it is at position 20 where nucleobase ‘sensing’ occurs.
This recognition site is near the trans opening of α-HL, and is consistent with results
where tethered DNA was used to probe the interior of the pore (16) and MD (33). It is
worth noting that this recognition site is not the 1.5 nm limiting aperture at the trans
end of the stem, as one might expect. These results indicate that if the translocation
rate of the DNA can be slowed (in this case stopped), then single base discrimination
and thus sequencing may be possible.
The future
In one of the earliest papers to describe DNA sequencing using nanopores
(13), a list of conditions that must be met for sequencing was proposed; each base
must produce a different characteristic blockage, the limiting aperture must reflect
presence of one base at a time, ionic flow measurements must exceed rate of
nucleotide movement, backward movement of nucleotides must be minimal, and the
system must be able to withstand treatments to reduce secondary structure. To fulfil
these criteria, it has been proposed that a single nucleotide aperture or high affinity
contact site could be engineered into the α-HL cis vestibule (30), or slow the
translocation of DNA by using a polymerase to allow the slow stepwise synthesis of a
ss-polynucleotide into the pore. However, there are intrinsic problems with using αHL; Deamer and Akeson (4) note that the cis vestibule can accommodate 10-15
nucleotides that can contribute to the amplitude of blockages. It was also argued (11)
8
that as a long-term sequencing system the α-HL protein (and even the lipid bilayer)
was fragile, and unlikely to be used as a commercial device. The most likely approach
in the future is to use artificial nanopores.
There are several methods that can be used to produce pores of the appropriate
dimensions. Track-etched polycarbonate membranes (34) are readily available with
diameters of tens of nm. Gold nanotubules (35) can be produced by gold plating
track-etched membranes to obtain pores with molecular dimensions (<2 nm) (36) that
can detect molecular complexes (37). The main problem with the use of artificial
nanopores is the irregularity and uncertainty in the pore dimensions. A step towards
regular (and adequate) pore size has come from feedback-controlled ion-sputtering
system where Si3N4 membranes with a 2-5 nm pore have been made (38) using Ar+
ion beams. This system counts the ions transmitted through the gradually opening
pore and extinguished the ion-sputtering erosion at the appropriate time. It can
routinely produce pores of 2, 3, and 4 nm. Si3N4 (39) and carbon nanotube (40)
membranes have been simulated using MD, and results indicate that if a suitable
artificial nanopore can be produced, the prospect of DNA sequencing may indeed
become a reality.
However, all is not lost for α-HL. Whilst pioneering the way for ultra-rapid
DNA sequencing, several novel uses for the protein have arisen. The simple
homopolynucleotide system can be used to measure hydrolysis levels (13); upon
addition of a ribonuclease, the frequency of current blockades increases, and can be
measured. Hairpin analysis (25) may permit studies of multiple hairpin domains, and
examination of secondary and tertiary structures in the ribosome. Finally, Deamer and
Akeson (4) proposed the utilisation of different ss-polynucleotides as TargetedMolecular-Bar-Codes (TMBCs) for fast analysis of surface antigens on a cell. Each
TMBC is covalently attached to a FAB that recognises a different surface protein.
These are all exposed to the selected cell, and any un-bound TMBC-FABs are then
washed away. The TMBCs are then chemically detached, and identified (thus surface
proteins identified) using α-HL.
I believe that rapid DNA sequencing using α-HL is still a long way off. There
are many intrinsic problems that must be resolved, such as DNA translocating too
rapidly. Even if a reliable artificial nanopore can be produced, there are already
developments into a new technique that use a cell’s DNA replication technology (41)
to hopefully sequence a genome quickly at a cost of $100,000 in the short-term
($1000 long-term!). There are also commercial sequencing machines that will be
available by 2007 that can (apparently) sequence a whole human genome start to
finish in three days for a cost of $5,000 (5). However, this works using the current
human genome as a template, and would be less effective for sequencing other
mammalian genomes. Thus although many experiments have been successfully
carried out that derive information about the DNA, I think they are merely interesting
curiosities of the abilities of nanopores, rather than their potential applications.
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