NANOPORE FUNCTIONALITY CONTROL
The present invention relates to a method of controlling the functionality of a substrate
containing at least one nanopore and a DNA construct for insertion into a nanopore.
Single nanopores in insulating barriers are under investigation as sensors for single
molecules in solution 1. Since the first experiments on DNA and RNA translocations by
Kasianowicz et al. 2 research activities in nanopore sensing have grown rapidly. Single solidstate
nanopores produced by silicon nanotechnology and ion beam milling were first
employed for ionic current-based detection of DNA by Li et al. 3. DNA folding detection with
nanocapillaries 4, the development of alternative fabrication protocols like transmissionelectron
drilling of solid-state nanopores 5, or the combination of nanopores with other singlemolecule
techniques like optical tweezers 6, magnetic tweezers 7 or single molecule
fluorescence 8 has given the field new impetus.
In a typical application, the nanopore separates two chambers containing an aqueous
solution of positive and negative salt ions. Additionally, one of the reservoirs contains a
molecule of interest. When a potential difference is applied across the membrane an ionic
current can be measured as ions pass through the nanopore. Molecules translocating
through the nanopore transiently block the current. The change in current and dwell time as
the molecule passes through the nanopore can be measured with a sensitive current
detector. The current traces recorded can then be analysed to infer molecular properties.
Two remaining challenges are the control of the exact shape of nanopores and the analytespecific
modification of their cavities. Coating with lipid bilayers9 or polymers 10 provides
means of modifying the surface properties of solid-state nanopores, but the creation of fixed
structures with nanometer scale control of geometry and positioning of functional chemical
motifs has yet to be demonstrated. The recent combination of hybrid biological and solidstate
nanopores 11 crossed the boundary between nanopores extracted from living organisms
and those derived from silicon nanotechnology. While this achievement allows for the design
of nanopores adapted for molecular sensing 12 and sequencing 13 , protein nanopores have
fixed diameters which are often in the single nanometer range. This currently limits the
range of analytes to unfolded proteins or single-stranded DNA chains.
It would be desirable to have a method for forming easily tunable nanopores with diameters
from 1nm to 100nm that could therefore be applied to a much wider range of analytes.
Further, as solid-state nanopores can be high cost items which may be problematic to
manufacture reproducibly (particularly when the nanopores have diameters of 20 n or less),
it would be desirable to improve the usable life and/or reusability of such nanopores. The
present invention is at least partly based on a realisation that control of nanopore size and
functionality in a hybrid nanopore can be achieved by the application of a potential
difference.
Accordingly, in a first aspect, the present invention provides a method of controlling the
functionality of a substrate containing at least one nanopore, the method including the steps
of:
introducing to the substrate a solution containing a molecular construct having a body
formation which defines an aperture and a tail formation extending from the body formation;
applying a potential difference across the substrate to thread the tail formation
through the nanopore thereby docking the molecular construct to the substrate with the
aperture aligned with the nanopore; and
expelling the molecular construct from the substrate by varying the potential
difference.
Thus, advantageously, the molecular construct can be used to control the functionality of the
nanopore (e.g. by controlling the construct's size, geometry, chemical functionality, etc.
particularly at the aperture), but the construct can be removed, allowing the substrate e.g. to
be re-used for a different analyte.
The method of the first aspect may have any one or, to the extent that they are compatible,
any combination of the following optional features.
The variation of the potential difference may typically include a reversal of the potential
across the substrate.
The method may include a further step of flushing the expelled molecular construct away
from the substrate after its expulsion, for example by the use of an appropriate micro-fluidics
arrangement. Indeed, such an arrangement can also be used to introduce the solution
containing the molecular construct to the substrate in the first place.
The method may include further steps after the expelling step of: introducing to the substrate
a solution containing a different molecular construct having a body formation which defines
an aperture and a tail formation extending from the body formation; and re-applying a
potential difference across the substrate to thread the tail formation through the nanopore
thereby clocking the different molecular construct to the substrate with the aperture aligned
with the nanopore. In this way, the functionality of the nanopore can be controllably and
repeatably modified. For example, a range of different molecular constructs can be
successively docked to the substrate to make the nanopore functionally sensitive to a
corresponding range of different analytes.
In a second aspect, the present invention provides a method of detecting the presence of an
analyte in a solution, the method including the steps of:
(a) providing a substrate containing at least one nanopore;
(b) introducing to the substrate a solution which may contain the analyte, and a
molecular construct having a body formation which defines an aperture, and which has a one
or more binding sites for the analyte at the aperture;
(c) applying a potential difference across the substrate to dock the molecular
construct to the substrate with the aperture aligned with the nanopore, and measuring the
corresponding change in ionic current through the nanopore;
(d) comparing the measured change in ionic current to a reference change in ionic
current; and
(e) determining that the analyte is present in the solution when the measured change
in ionic current differs from the reference change in ionic current by more than a
predetermined amount.
Thus the analyte, when present, binds to the molecular construct in the solution and,
because the one or more binding sites are at the aperture, can thereby fully or partially block
the aperture. When the molecular construct docks to the substrate, the blockage affects the
ability of ions to pass through the nanopore and hence affects the measured change in ionic
current.
Advantageously, analyte specificity can thereby be provided by the molecular construct.
Thus if a further solution contains a molecule which is of similar size to the analyte but which
does not bind to the molecular construct, applying steps (b) to (e) to both solutions should
allow a user to distinguish between the solution containing the analyte and the further
solution.
The method of the second aspect may have any one or, to the extent that they are
compatible, any combination of the following optional features.
The applying step (c) may include repeatedly performing the sub-steps of:
(i) applying the potential difference across the substrate to dock the molecular
construct to the substrate with the aperture aligned with the nanopore, and measuring the
corresponding change in ionic current through the nanopore; and
(ii) expelling the molecular construct from the substrate by varying (e.g. reversing) the
potential difference; and
wherein, in the comparing step (d), the measured change in ionic current is an
average measured change derived from the repeated measurements of sub-step (c-i).
Repeating the measurements can help to improve detection accuracy.
Typically, the measured change and the reference change are decreases in ionic current.
The reference change can conveniently be the change in ionic current that would have
occurred if there had been no analyte in the solution. Indeed, the method may further
include the steps of:
(A) providing a substrate containing at least one nanopore;
(B) introducing to the substrate a solution containing the molecular construct in the
absence of the analyte; and
(C) appfying a potential difference across the substrate to dock the molecular
construct, in the absence of the analyte, to the substrate with the aperture aligned with the
nanopore, and measuring the corresponding change in ionic current through the nanopore;
and
wherein, in the comparing step (d), the reference change in ionic current is the
change in current measured at step (C). The solution of step (B) preferably has the same
ionic strength and pH as the solution of step (b). For example, it may contain an identical
concentration of the same dissolved salt and be identically buffered.
The applying step (C) may include repeatedly performing the sub-steps of:
(i) applying the potential difference across the substrate to dock the molecular
construct, in the absence of the analyte, to the substrate with the aperture aligned with the
nanopore, and measuring the corresponding change in ionic current through the nanopore;
and
(ii) expelling the molecular construct from the substrate by varying (e.g. reversing) the
potential difference; and
wherein, in the comparing step (d), the reference change in ionic current is an
average measured change derived from the repeated measurements of sub-step (C-i).
The substrate of step (A) can be a different but equivalent substrate to the substrate of step
(a). Steps (A) to (C) can then be performed before, during or after the performance of steps
(a) to (c).
Another option, however, is for the substrate of step (A) to be the same substrate as the
substrate of step (a). Steps (B) to (C) can then be performed before or after the performance
of steps (b) to (c). However, whichever is performed first, the method may include a further
step of flushing the first solution away from the substrate after expulsion of that solution's
molecular construct, and before the introduction of the second solution. Again, the flushing
can be performed by the use of an appropriate micro-f!uidics arrangement. The arrangement
can also be used to introduce the first and second solutions to the substrate.
The analyte may be an antibody.
The molecular construct may further have a tail formation extending from the body formation,
the tail formation being threaded through the nanopore to dock the molecular construct to the
substrate when the potential difference is applied across the substrate.
The method of the first or second aspect may have any one or, to the extent that they are
compatible, any combination of the following optional features.
The substrate may have plural nanopores, and the apertures of respective molecular
constructs may be aligned with the nanopores by the application of the potential difference
across the substrate. Likewise, the plural molecular constructs may be expelled from the
substrate by varying the potential difference.
The substrate may be a silicon-nitride or silicon-oxide substrate, e.g. with one or more
electron or ion beam drilled nanopores.
Preferably, the body formation has a docking surface which contacts the substrate when the
construct is docked thereto, at least the docking surface being hydrophilic. In a typically
aqueous solution, this can help to prevent the body formation from binding too tightly to a
mating surface of the substrate, whereby variation of the potential difference can more easily
expel the molecular construct from the substrate. In contrast, the a-hemolysin protein pore
disclosed by Hall et a/. 1 is hydrophobic.
Preferably, the body formation can include or be a sleeve formation which defines the
aperture, the application of the potential difference causing the molecular construct to dock to
the substrate such that the sleeve formation lines the nanopore. The above-mentioned
docking surface can include or be the external surface of the sleeve formation.
Preferably, the molecular construct is a DNA construct. Advantageously, DNA is
hydrophilic, which, as discussed above, promotes the exchangeability of the construct.
Further, the DNA construct can conveniently be made by DNA-based self-assembly or
"origami" techniques. DNA-based self-assembly 14 employs the programmabiltty of DNA
sequences to build rationally designed objects of ever increasing complexity. Owing to the
development of DNA origami 15 it is now possible to design and fabricate almost arbitrary
nanosized shapes 16 . The origami method typically uses a 7-8 kb long m13mp18-based
single-strand as a scaffold for the assembly of hundreds of distinct synthetic staple strands.
Each of these staples can potentially be extended with a nucleotide (nt) sequence of interest
or a wide range of chemical modifications 17 . In addition, the use of DNA origami allows for
the addition of functional chemical groups, fluorophores, gold nanoparticles etc. at subnanometre
position accuracy by employing modifications to the oligonucleotide staple
strands used to fold the long DNA single strand25. These modifications have the potential for
the integration of DNA origami and nanopores with techniques such as fluorescence
detection and Raman spectroscopy. Thus DNA is well suited for the synthesis of
programmable constructs with chemically adjustable cavities and custom tailored
geometries.
Indeed, in a third aspect, the present invention provides a DNA construct (e.g. a DNA origami
construct) for docking to a substrate having a nanopore, the construct having a body
formation which defines an aperture, and a tail formation extending from the body formation
for threading through the nanopore to dock the construct to the substrate with the aperture
and nanopore in alignment. The body formation can include or be a sleeve formation which
defines the aperture and which lines the nanopore when the construct is docked to the
substrate. The construct can include functional chemical groups, fluorophores, and/or gold
nanoparticles.
Further, in a fourth aspect, the present invention provides the use of DNA origami to form the
DNA construct of the second aspect.
Further optional features of the invention will now be set out. These are applicable singly or
in any combination with any of the above aspects of the invention.
The nanopore preferably has a diameter of 100 n or less, and more preferably of 50 or 20
nm or less. Nanopores of 20 nm diameter or less are generally particularly difficult to
produce reproducibly, or indeed at all. Thus the use of the molecular construct in relation to
the nanopores of this size can be particularly beneficial, allowing the same nanopore to be
used for repeatedly, e.g. for different analytes. This not only helps to relieve the burden of
producing numbers of nanopores in the first place, but allows the same nanopore to be used
for comparative testing.
The nanopore preferably has a diameter of 1 nm or more, and more preferably of 2 or 5 nm
or more.
Further optional features of the invention are set out below.
Embodiments of the invention will now be described by way of example with reference to the
accompanying drawings in which:
Figure 1: DNA origami design (a) Schematic representation of the DNA origami structure
with the double stranded overhang. Double helices are presented as rods (b) Top: Side
view of the nanopore section of the DNA origami structure with helices shown as cylinders.
The overall length of the nanopore is 150 bp (51 .Onm) with the smallest opening of 7.5 x
7.5 nm2. Length A=40bp (13.6nm), length B=48bp (16.3nm). Bottom: Top view of the
structure, indicating the square shape with 11 by 11 helices with a total area of 27.5 x
27.5 nm2. (c) Transmission electron microscope (TEM) images of assembled DNA origami
constructs negatively stained with uranyl acetate (d) TEM image of upright standing DNA
origami construct, cf. bottom view of (b). Scale bars = 40nm.
Figure 2: Characterization of a single hybrid nanopore. (a) Current as a function of time
through a single solid-state nanopore with a diameter of 13nm. Voltage is switched from
OmV to +100 mV at 2.8 s . At 6.2 s a sudden drop in current indicates insertion of a DNA
origami construct, see arrow (b) Current as a function of time through a single hybrid
nanopore while reducing the voltage in 10 mV steps (c) Current-voltage characteristic of the
hybrid nanopore (line/dots) in comparison with the current-voltage characteristic of the bare
solid-state nanopore (plain line).
Figure 3: Repeated insertion of DNA origami constructs into an 18nm diameter solid-state
nanopore. (a) Top panel: applied voltage as a function of time for insertion of DNA origami
constructs. At +100 mV hybrid nanopores are stable while at -100 mV the DNA origami
construct can be driven out of the solid-state nanopore. Lower panel: corresponding ionic
current trace showing formation of hybrid nanopores, indicated by the arrows (b) Histogram
of the relative current change for 12 insertions, measured in 5 different experiments and
nanopores. The relative current change is the quotient of the hybrid pore current lH b
divided by the open pore current lss . Right: Sketches of the situations with lSs and i -
Figure 4 : l-DNA events through bare solid-state nanopore and hybrid nanopores. (a) Ionic
current as a function of time at +100 mV for an experiment with both DNA origami and l-DNA
in the solution, with 0.5 nM and .0 n concentration respectively. For t<1 1.4 s , DNA
translocations through the bare nanopore (15nm diameter) are observed. After formation of
the hybrid nanopore, events for the hybrid nanopore are observed (b) Typical events for the
bare nanopore shown top and the hybrid nanopore shown bottom (c) Current histograms
indicating DNA translocations for the bare solid-state and hybrid nanopores. The baseline
for both histograms was subtracted for easy comparison, and the current was digitally filtered
at 3 kHz for clarity.
Figure 5: a schematic representation of a complete testing cycle including: (a) the
introduction of a DNA construct having a sleeve formation and tail formation into a reservoir
on one side of a substrate having a nanopore; (b) application of a positive polarity potential
difference to thread the tail formation through the nanopore and seat the sleeve formation in
the nanopore; (c) testing of the current signature through the resulting hybrid nanopore when
an analyte is introduced into the reservoir; and (d) expulsion of the construct on application of
a negative polarity potential difference in readiness e.g. for the introduction of a different
DNA construct into the reservoir and a further testing cycle.
Figure 6 : a schematic representation of: (a) the introduction of a DNA construct to a
substrate having a nanopore, the construct having a body formation in the form of a plate
defining a central aperture, with a tail formation extending from an edge of the aperture; and
(b) the docking of the construct to the substrate, with the tail formation threaded through the
aperture, the flat body formation overlaying the substrate, and the central aperture aligned
with the nanopore.
Figure 7: atomic force microscopy pictures of DNA constructs docked to a substrate (a) and
(b) without streptavidin bound to the constructs, and (c) and (d) with streptavidin bound to the
constructs.
Figure 8 : schematic plots of the change in ionic current expected on docking the constructs
of Figure 7 (a) from a solution without streptavidin and (b) from a solution with streptavidin.
Figure 9 : histograms of actual percentage current decrease for repeated docking
experiments using the constructs of Figure 7 (a) from a solution without streptavidin and (b)
from a solution with streptavidin.
n the following we demonstrate the successful insertion of DNA constructs into solid-state
nanopores. Further, we demonstrate that such constructs can be controllably and repeatedly
inserted into the nanopores and can be used for resistive-pulse sensing. Finally, we
describe a technique for detecting specific analytes.
Materials and Methods
A DNA origami construct was designed using the square-lattice version of the caDNAno
software from Douglas et al. 18 . As the scaffold strand, the 8634 nt-long m13mp18-based 1Sa
single strand was chosen and cut at the EcoRI and BamHI restriction sites. Of the 8613
bases of the cut scaffold, 6264 were incorporated into an origami structure with the help of
42 staple strands and 2349 bases were allowed to form a linear extension of one of the
helices. This part of the single-stranded scaffold was complemented with 49 consecutive
40mers and 48mers to form a 2344 basepairs (bp) long double strand with 48 nicks which
acted as a leash to ensure correctly-oriented trapping of the DNA construct into the solidstate
nanopore.
The constructs were assembled by heating a Tris-HCI (10 mM) - EDTA ( 1 mM) buffer (pH
8.0) containing the cut scaffold (10 nM), the staple strands and the complements of the leash
(100 nM each), and 14 mM MgCI2. The assembled structures were purified from the excess
staple strands by either agarose gel electrophoresis or centrifugation with 10OkDa MWCO
filters.
Nanopores with diameters between 12-18 n were fabricated in silicon-nitride (SiN)
membranes with an FEI Tecnai F20 transmission electron microscope (TEM) equipped with
a field emission gun (FEG) which was operated at an acceleration voltage of 200 kV and an
extraction voltage of 4000V. The SiN membranes were 30 nm thick with 50 x 50 m2
windows (DuraSin, Protochips, USA).
The SiN chip containing a nanopore was sealed into polydimethylsiloxane (PDMS)
microfluidic channels. The PDMS (Sylgard 184, Dow Corning) was made by mixing base
and curing agent in a weight ratio of 10:1 and curing in an aluminum mold for 10 minutes at
150°C. The aluminum mold design comprised channels at either side of the pore for
introducing buffer and a required sample volume of only 10 L. The PDMS was plasmabonded
to a glass slide to prevent leaks 19 . Before introducing buffer, the nanopore
containing TEM chip was plasma cleaned for 1 minute. A Gigaohm seal was then formed
between the two reservoirs of the PDMS by painting fresh PDMS around the edge of the chip
and curing on a hot plate for 60 seconds at 120°C. Subsequently, buffer solution was added
to both sides of the chip.
For ionic current measurements, Ag/AgCI electrodes were fabricated by electro-deposition of
a AgCI layer onto 0.2 thick silver wire. The two electrodes were placed in the reservoirs
on either side of the nanopore and connected to the headstage of an Axopatch 200B
amplifier (Molecular Devices, USA). The amplifier headstage and nanopore device were
enclosed in a Faraday cage to reduce electromagnetic interference. All ionic current
recordings were performed with the internal Bessel filter of the amplifier at 10 kHz and
recorded at up to 100 kHz bandwidth. The data was later filtered for analysis. Voltages were
set and currents recorded by custom written LabView software 4, or with ClampEx
(Molecular Devices, USA).
For translocation experiments we used linearized l-DNA (New England Biolabs, USA)
diluted in the measurement buffer prior to all experiments. After purification DNA origami
constructs were diluted to a concentration of approximately 0.5 nM in the measurement
buffer.
DNA Origami Construct
An aim of the design process was to find an accessible geometry that would fit into the
conical form of the solid-state nanopore while allowing for stable insertion in a wide range of
nanopore diameters. A schematic overview of the construct is shown in Figure . Al lengths
are calculated by assuming 0.34 nm helical step size per bp and an average center-to-center
distance of 2.5 nm per helix pair (see Figure 1b)15, 2 .
The geometry has a sleeve formation of staggered double helices whose z-axes are pointing
into the solid-state pore forming four overlapping skirts with a quadratic base, the helices
defining a central aperture. The innermost skirt is formed by 16 helices, 48bp long
(16.3 nm), where each face of this square cylinder is comprised of five parallel helices
resulting in an outer edge length of 12.5 nm and an inner edge length of 7.5 nm, see Figure
1b. The next skirt is built up from 24 helices (7 helices per face) and overlaps with the
innermost skirt over a length of at least 16 bp to a maximum of 40 bp. The third layer
consists of 32 helices and the outermost layer of 40 helices with each layer overlapping over
at least 8 bp to a maximum of 16 bp. For the edge facing the solution (distal) we calculated a
length of 11 x 2.5 nm = 27.5 nm which is large enough to prevent slipping of the assembled
structure into the solid-state pore, which in our experiments has a diameter of typically about
12-18 nm. In order to guide the voltage-driven self-assembly, we added a double stranded
DNA tail to the tip of the sleeve formation. It is believed that the tail threads through the
nanopore to assist and precede the insertion of the sleeve formation therein. The overall
length of the origami structure without the tail is 5 1.0 nm, the outer diagonal diameter is
38.9 nm, while the centre opening is 7.5 x 7.5 nm2. Typical TEM images recorded at 100 kV
(JEM-101 1, JEOL) of the correctly folded structure are shown in Figure 1c and 1d, as side
and top view, respectively. The segmented double stranded DNA tail can be observed in
both images.
Hybrid Nanopore Formation
After a solid-state nanopore was assembled into a microfluidic measurement cell, the
current-voltage characteristic was first tested in a 20nm filtered buffer solution of O.SxTBE,
5.5mM MgCI2, 1M KCI. Figure 2 shows an example trace of the characteristic current
signature observed after adding the DNA origami construct to one side of the solid-state
nanopore. Recording in this experiment started at 0 mV and after 2.8s +100mV was applied
(with positive polarity making the DNA construct move towards the solid-state nanopore). An
initial ionic current of 8.7 nA was measured through the bare solid-state nanopore. After
about 6.6 s we see a sudden drop in the ionic current indicating the correct insertion of a
DNA origami construct into the solid-state nanopore. The newly formed hybrid nanopore
was then tested by recording a current-voltage characteristic shown in Figure 2b. The
voltage was decreased from +100 to -100 mV in steps of -10 mV. The points shown in
Figure 2c are extracted by averaging over each current step. The line for the current-voltage
characteristic of the bare solid-state nanopore was measured before adding the DNA origami
solution. We observe that at positive voltages the current through the hybrid nanopore is
lower than for the bare solid-state nanopore. The bare solid-state nanopore shows a linear
IV curve whereas after the formation of a hybrid nanopore the IV curve shows rectification.
This behavior is due to the asymmetry and the highly charged DNA surface of the hybrid
nanopore22 , and is an important indication that the DNA origami structure is correctly
assembled into the solid-state nanopore.
Repeated Construct Insertions
Our DNA origami constructs can be ejected from the solid-state nanopore by sudden
reversal of the applied potential. This is an important feature allowing for error correction if a
pore did not insert correctly or for exchanging different pores within the same experiment.
The top part of Figure 3a shows the applied voltage as a function of time switching between
+100 and -100 mV. The corresponding ionic current trace is shown in the lower part of
Figure 3a. Initially, at +100mV, a hybrid nanopore is present and a current of 10.2 nA is
observed. Upon switching the voltage to -100 mV the current changes to - 11.8 nA, but
switching back to +100 mV leads to an increase of the current to + 11.6 nA indicating the
original open pore current and the disassembly of the hybrid nanopore. At this voltage the
same or another construct is quickly inserted into the solid-state nanopore at 11.0 s (see
arrow in Figure 3a). The current stabilizes at approximately the same level as for the
previous hybrid nanopore. At t=21 .6 s the voltage is switched again to - 100 mV to eject the
origami structure. Upon switching back to +100 mV at 26.6 s the ionic current returns quickly
to the open nanopore current only to fall back to the hybrid nanopore current of 10.0 nA at
3 1.0 s , indicating another successful assembly.
The histogram in Figure 3b shows the relative current change defined as the hybrid
nanopore current divided by the solid-state nanopore current at +100 mV for 12 insertions
taken for 5 different nanopores. Insertion of the origami construct reduced the solid-state
nanopore current to around 80% of its initial value. This is expected as counter ions in the
vicinity of the DNA are mobile23. We see a consistent change in our experiments with solidstate
nanopores with a range of diameters which points to a well-controlled insertion of the
DNA origami structure.
Our results show that we can controllably and repeatedly insert and eject DNA origami
constructs.
Detection of DNA with Hybrid Nanopores
We are able to detect l-DNA strands in our hybrid nanopores. In this experiment DNA
origami constructs with a concentration of 0.5 nM were added in addition to 1 nM of l-DNA
solution. As shown in Figure 4a, we observe the typical signatures for long DNA molecules
translocating a bare solid-state nanopore, indicating DNA forming a tight hairpin and folding
onto itself4 , 2 . After insertion of a DNA origami construct at 11.4 s (Figure 4a), the current
stabilizes after a few seconds albeit with a higher level of noise. Since the l-DNA is present
in the background, l-DNA interactions with the hybrid nanopore will occur. Three typical
events are presented for DNA translocations in Figure 4b for the bare and hybrid nanopores,
respectively. The corresponding current histograms are shown in Figure 4c. We observe
typical DNA translocation events as peaks in the bare solid-state pore current histogram
(Figure 4c) with peaks at - 100 pA and -200 pA. The data is shown after the baseline was
subtracted. The peaks indicate that DNA folding is readily observable in our bare solid-state
nanopore prior to hybrid nanopore formation. For the hybrid nanopore current histogram, we
observe a clear peak at roughly -60 pA, while we also detect a number of folded events
although with lower frequency (Figure 4b and 4c). This can be explained by the considerably
smaller diameter of the hybrid nanopore.
Figure 5 shows schematically a complete testing cycle including: (a) the introduction of a
DNA construct 1 having a sleeve formation 2 and tail formation 3 into a reservoir 4 on one
side of a substrate 5 having a nanopore 6; (b) application of a positive polarity potential
difference to thread the tail formation through the nanopore and thereby docking the
construct to the substrate with the sleeve formation seated in the nanopore and the central
aperture of the sleeve aligned with the nanopore; (c) testing of the current signature through
the resulting hybrid nanopore when an analyte 7 is introduced into the reservoir; and (d)
ejection of the construct on application of a negative polarity potential difference in readiness
e.g. for the introduction of a different DNA construct into the reservoir and a further testing
cycle.
Conclusions
We have experimentally demonstrated the formation of hybrid nanopores comprising 3D
DNA origami structures inserted into a solid-state nanopore. The hybrid nanopores can be
repeatedly assembled by reversing the applied potential, which enables the functionality of a
nanopore sensor to be changed during an experiment. Our measurements of DNA detection
show that DNA origami constructs can be used as resistive-pulse sensors. The constructs
offer the possibility to adapt the diameter, shape and surface functionality of hybrid
However, although the concept of construct insertion and expulsion has been demonstrated
using a DNA construct, it could also be performed using constructs based on other suitable
molecules. In particular, a molecular construct which, like DNA, can provide a hydrophilic
external surface, may similarly help to prevent the construct from binding too tightly to the
mating surface of the nanopore.
Further, although we have used a construct having a sleeve formation (which shows
particular promise for stable docking and for controlling the functionality of the nanopore),
other types of construct can be adopted. For example, Figure 6 shows schematically (a) the
introduction of a DNA construct 11 to a substrate 15 having a nanopore 16, the DNA
construct having a body formation in the form of a flat plate 12 defining a central aperture 18,
with a tail formation 13 extending from an edge of the aperture, and (b) the docking of the
construct to the substrate, with the tail formation threaded through the aperture, the flat body
formation overlaying the substrate, and the central aperture aligned with the nanopore.
Specific Analyte Detection
The approach described above involves measuring changes in ionic current as molecules
translocate through the nanopore. Molecules of different type but similar molecular mass
tend to generate similar changes in ionic current as the molecules translocate through the
nanopore. Thus we next describe an approach which provides detection specificity for
particular analytes.
More particularly, a molecular construct such as a DNA origami construct described above,
can be used to trap an analyte of interest at the mouth of the nanopore by introducing one or
more binding sites for the particular analyte at the aperture in the construct. Each time a
construct is docked to the nanopore a current decrease due to the construct partially blocking
the ionic current flow (Figure 1) through the pore can be measured. However, when the
analyte is present in the solution and therefore bound at the aperture, the current decrease is
typically greater than when the analyte is absent from the solution.
This detection method can be adapted to many biomolecules of interest, such as antibodies,
since it on y requires the provision of binding sites on the construct, which in the case of DNA
constructs can be achieved readily with oligonucleotide modifications offered by DNA
synthesis companies.
The system is reversible. For example, in the case of DNA constructs and a SiN chip
containing nanopores, a high positive voltage can be applied to dock the constructs to the
pores and a high negative voltage can be applied to force them away from the pores. The
step change in ionic current upon construct docking can be measured many times in one
experiment (e.g. over hundreds of voltage reversals) to build up robust statistics. Further,
since only a step change in current is being measured, low bandwidth electronics can be
used.
As a proof of principle, a flat square DNA origami construct with a central aperture and four
binding sites at the mouth of the aperture for the protein streptavidin was designed. A
solution was prepared containing O.SxTBE, 5.5mM MgCI2, 1M KCI. The constructs were then
added to the solution, which was introduced to a SiN chip similar to those described above.
A positive potential was applied to dock the constructs to the nanopores of the chip.
Although the DNA constructs did not have tail formations, the apertures of the constructs
were nonetheless able to align to the pores of the chips. Figures 7(a) and (b) show atomic
force microscopy pictures of the docked constructs.
The procedure was then repeated but with streptavidin added to the solution. Figures 7(c)
and (d) show atomic force microscopy pictures of the docked constructs, with bound
streptavidin clearly visible at the apertures.
Figure 8 shows schematically the change in ionic current that would be expected on docking
of the constructs (a) from the solution without streptavidin and (b) from the solution with
streptavidin. The expected decrease in current is greater for (b) than (a). The solution
without streptavidin thus provides a reference decrease against which the decrease from a
further solution which may contain streptavidin can be compared. If the decrease associated
with the further solution differs from the reference by an amount similar to the amount by
which (b) differs from (a), then that is an indication that the further solution contains
streptavidin.
Figures 9(a) and (b) are histograms of percentage current decrease for actual experiments
using the flat square DNA origami construct and streptavidin system of Figures 7(a) to (d).
For the histogram of Figure 9(a) the current decrease for the construct docking process using
the reference system without any protein attached (Figures 7(a) and 7(b)) was repeatedly
measured, whereas for the histogram of Figure 9(b) the current decrease for the construct
docking process using the system with the protein attached (Figures 7(c) and 7(d)) was
repeatedly measured. As expected, the reference system shows a lower reduction in current
than the system with the streptavidin bound to the construct. To ensure that the histograms
were directly comparable, both the reference and the streptavidin-bound constructs were
docked to the same nanopore-containing chip.
While the invention has been described in conjunction with the exemplary embodiments
described above, many equivalent modifications and variations will be apparent to those
skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the
invention set forth above are considered to be illustrative and not limiting. Various changes
to the described embodiments may be made without departing from the spirit and scope of
the invention.
All references are hereby incorporated by reference.
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CLAIMS
1. A DNA construct ( 1 , 11) for docking to a substrate (5, 15) having a nanopore (6, 16),
the construct having a body formation which defines an aperture (18), and a tail formation (3,
13) extending from the body formation for threading through the nanopore to dock the
construct to the substrate with the aperture and nanopore in alignment.
2 . A DNA construct according to claim 1, wherein the body formation includes a sleeve
formation (2) which defines the aperture and which lines the nanopore when the construct is
docked to the substrate.
3 . Use of DNA origami to form the DNA construct of claim 1 or 2 .
4 . A substrate (5, 15) containing one or more nanopores to which are docked respective
DNA constructs according to claim 1 or 2 .
5 . A method of controlling the functionality of a substrate (5, 15) containing at least one
nanopore (6, 16), the method including the steps of:
introducing to the substrate a solution containing a molecular construct (1, 11) having
a body formation which defines an aperture (18) and a tail formation (3, 13) extending from
the body formation;
applying a potential difference across the substrate to thread the tail formation
through the nanopore thereby docking the molecular construct to the substrate with the
aperture aligned with the nanopore; and
expelling the molecular construct from the substrate by varying the potential
difference.
6. A method according to claim 5 , wherein the body formation includes a sleeve
formation (2) which defines the aperture, and the application of the potential difference
causes the molecular construct to dock to the substrate such that the sleeve formation lines
the nanopore.
7 . A method according to claim 5 or 6, wherein the body formation has a docking
surface which contacts the substrate when the construct is docked thereto, at least the
docking surface being hydrophilic.
8 . A method according to any one of claims 5 to 7 , wherein the molecular construct is a
DNA construct.
9 . A method according to any one of claims 5 to 8, including further steps after the
expelling step of:
introducing to the substrate a solution containing a different molecular construct
having a body formation which defines an aperture and a tail formation extending from the
body formation; and
re-applying a potential difference across the substrate to thread the tail formation
through the nanopore thereby docking the different molecular construct to the substrate with
the aperture aligned with the nanopore.
0. A method of detecting the presence of an analyte in a solution, the method including
the steps of:
(a) providing a substrate containing at least one nanopore;
(b) introducing to the substrate a solution which may contain the analyte, and a
molecular construct having a body formation which defines an aperture, and which has a one
or more binding sites for the analyte at the aperture;
(c) applying a potential difference across the substrate to dock the molecular
construct to the substrate with the aperture aligned with the nanopore, and measuring the
corresponding change in ionic current through the nanopore;
(d) comparing the measured change in ionic current to a reference change in ionic
current; and
(e) determining that the analyte is present in the solution when the measured change
in ionic current differs from the reference change in ionic current by more than a
predetermined amount.
11. A method according to claim 10, wherein the applying step (c) includes repeatedly
performing the sub-steps of.
(i) applying the potential difference across the substrate to dock the molecular
construct to the substrate with the aperture aligned with the nanopore, and measuring the
corresponding change in ionic current through the nanopore; and
(ii) expelling the molecular construct from the substrate by varying the potential
difference; and
wherein, in the comparing step (d), the measured change in ionic current is an
average measured change derived from the repeated measurements of sub-step (c-i).
12. A method according to claim 10 or 1 , further including the steps of:
(A) providing a substrate containing at least one nanopore;
(B) introducing to the substrate a solution containing the molecular construct in the
absence of the analyte; and
(C) applying a potential difference across the substrate to dock the molecular
construct, in the absence of the analyte, to the substrate with the aperture aligned with the
nanopore, and measuring the corresponding change in ionic current through the nanopore;
and
wherein, in the comparing step (d), the reference change in ionic current is the
change in current measured at step (C).
13. A method according to claim 12, wherein the applying step (C) includes repeatedly
performing the sub-steps of:
(i) applying the potential difference across the substrate to dock the molecular
construct, in the absence of the analyte, to the substrate with the aperture aligned with the
nanopore, and measuring the corresponding change in ionic current through the nanopore;
and
(ii) expelling the molecular construct from the substrate by varying the potential
difference; and
wherein, in the comparing step (d), the reference change in ionic current is an
average measured change derived from the repeated measurements of sub-step (C-i).
14. A method according to any one of claims 10 to 13, wherein the analyte is an antibody.
15. A method according to any one of claims 10 to 15, wherein the molecular construct is
a DNA construct.