COUPLING METHOD
Field of the invention
The invention relates to a new method of determining the presence, absence or
characteristics of an analyte. The analyte is coupled to a membrane. The invention also relates
to nucleic acid sequencing.
Background of the invention
There is currently a need for rapid and cheap nucleic acid (e.g. DNA or R A) sequencing
technologies across a wide range of applications. Existing technologies are slow and expensive
mainly because they rely on amplification techniques to produce large volumes of nucleic acid
and require a high quantity of specialist fluorescent chemicals for signal detection.
Nanopores have great potential as direct, electrical biosensors for polymers and a variety
of small molecules. In particular, recent focus has been given to nanopores as a potential DNA
sequencing technology. Two methods for DNA sequencing have been proposed; 'Exonuclease
Sequencing', where bases are processively cleaved from the polynucleotide by an exonuclease
and are then individually identified by the nanopore and also 'Strand Sequencing', where a
single DNA strand is passed through the pore and nucleotides are directly identified. Strand
Sequencing may involve the use of a DNA handling enzyme to control the movement of the
polynucleotide through the nanopore.
When a potential is applied across a nanopore, there is a drop in the current flow when an
analyte, such as a nucleotide, resides transiently in the barrel for a certain period of time.
Nanopore detection of the analyte gives a current blockade of known signature and duration.
The concentration of an analyte can then be determined by the number of blockade events per
unit time to a single pore.
For nanopore applications, such as DNA Sequencing, efficient capture of analyte from
solution is required. For instance, in order to give the DNA handling enzyme used in DNA
Sequencing a sufficiently high duty cycle to obtain efficient sequencing, the number of
interactions between enzyme and polynucleotide needs to be maximal, so that a new
polynucleotide is bound as soon as the present one is finished. Therefore, in DNA Sequencing, it
is preferred to have the polynucleotide at as high a concentration as is possible so that, as soon as
an enzyme finishes processing one, the next is readily available to be bound. This becomes a
particular problem as the concentration of polynucleotide, such as DNA, becomes limiting, e.g.
DNA from cancer cell samples for epigenetics. The more dilute the sample then the longer
between sequencing runs, up to the point where binding the first polynucleotide is so limiting
that it is unfeasible.
The limits of nanopore detection have been estimated for various analytes. Capture of a
92-nucleotide synthetic piece of single strand DNA (ssDNA) by a protein nanopore (hemolysin)
was determined to be at a frequency of 3.0±0.2 s uM 1 (Maglia, Restrepo et al. 2008, Proc Natl
Acad Sci U S A 105(50): 19720-5). Capture could be increased -10 fold by the addition of a
ring of positive charges at the entrance to the hemolysin barrel (23.0±2 s 1 uM 1) . To put this
into context, 1 uM of 92 nucleotide ssDNA is equivalent to 3 1 ug of DNA required per single
channel recording, assuming a cis chamber volume of 1ml. The market leading genomic DNA
purification kit from human blood (Qiagen's PAXgene Blood DNA Kit) currently gives
expected yields of between 150 - 500 ug of genomic from 8.5 ml of human whole blood.
Therefore, this disclosed increase in analyte detection is still well short of the step change
required for ultra-sensitive detection and delivery.
Summary of the invention
The inventors have surprisingly demonstrated ultra low concentration analyte delivery by
coupling the analyte to a membrane in which the relevant detector is present. This lowers by
several orders of magnitude the amount of analyte required in order to be detected. The extent to
which the amount of analyte needed is reduced could not have been predicted.
In particular, the inventors surprisingly report an increase in the capture of single
stranded DNA by ~4 orders of magnitude over that previously reported. As both the detector
and analyte are now on the same plane, then ~103 M s more interactions occur per second, as
diffusion of both molecules is in two dimensions rather than three dimensions. This has
dramatic implications on the sample preparation requirements that are of key concern for
diagnostic devices such as next-generation sequencing systems.
In addition, coupling the analyte to a membrane has added advantages for various
nanopore-enzyme sequencing applications. In Exonuclease Sequencing, when the DNA analyte
is introduced the pore may become blocked permanently or temporarily, preventing the detection
of individual nucleotides. When one end of the DNA analyte is localised away from the pore,
for example by coupling or tethering to the membrane, surprisingly it was found that this
temporary or permanent blocking is no longer observed. By occupying one end of the DNA by
coupling it to the membrane it also acts to effectively increase the analyte concentration over the
detector and so increase the sequencing systems duty cycle. This is discussed in more detail
below.
Accordingly, the invention provides a method for determining the presence, absence or
characteristics of an analyte, comprising (a) coupling the analyte to a membrane and (b) allowing
the analyte to interact with a detector present in the membrane and thereby determining the
presence, absence or characteristics of the analyte.
The invention also provides:
a method of sequencing an analyte which is a target polynucleotide, comprising:
(a) coupling the target polynucleotide to a membrane;
(b) allowing the target polynucleotide to interact with a detector present in the
membrane, wherein the detector comprises a transmembrane pore and an
exonuclease, such that the exonuclease digests an individual nucleotide from one end
of the target polynucleotide;
(c) allowing the nucleotide to interact with the pore;
(d) measuring the current passing through the pore during the interaction and thereby
determining the identity of the nucleotide; and
(e) repeating steps (b) to (d) at the same end of the target polynucleotide and thereby
determining the sequence of the target polynucleotide;
a method of sequencing an analyte which is a target polynucleotide, comprising:
(a) coupling the target polynucleotide to a membrane;
(b) allowing the target polynucleotide to interact with a detector present in the
membrane, wherein the detector comprises a transmembrane pore, such that the target
polynucleotide moves through the pore; and
(c) measuring the current passing through the pore as the target polynucleotide moves
with respect to the pore and thereby determining the sequence of the target
polynucleotide;
a kit for sequencing an analyte which is a target polynucleotide comprising (a) a
transmembrane pore, (b) a polynucleotide binding protein and (c) means to couple the target
polynucleotide to a membrane; and
an apparatus for sequencing an analyte which is a target polynucleotide, comprising (a) a
membrane, (b) a plurality of transmembrane pores in the membrane, (c) a plurality of
polynucleotide binding proteins and (d) a plurality of target polynucleotides coupled to the
membrane.
Description of the Figures
Fig. 1 shows nanopore sensing of an analyte. A) Shows a nanopore with the direction of
the current flow indicated by grey arrows. A predicted current trace is shown below. B) Shows a
nanopore with an analyte translocating through the pore. The direction of analyte movement is
indicated by arrow 1 and the direction of the current flow by the grey arrows. A predicted
current trace is shown below showing how the current changes as the analyte translocates
through the pore.
Fig. 2 shows a method for tethering DNA nanopore interactions. Sections A and B show
transient tethered ssDNA and how the current trace changes as the ssDNA translocates through
the pore. Sections C and D show stable tethered ssDNA and how the current trace changes as
the ssD A is captured by the pore.
Fig. 3 shows capture of a DNA-enzyme complex, followed by dissociation of the DNA
and the enzyme, and subsequent DNA de-hybridisation.
Fig. 4 shows the experimental setup for Example 2. Comparison between (1) a
primer/template DNA analyte in solution (A - top) where the concentrations of material are in
the high nanomolar range (400 nM DNA used and 800 nN enzyme used) and (2) a tethered
system (B - bottom) where the amount of material is sub-nanomolar ( 1 nM DNA used and 5 nN
enzyme used).
Fig. 5 shows KF binding times on top of the nanopore for non-tethered analyte (DNA) in
the absence of KF (DNA concentration = 400 nM).
Fig. 6 shows KF binding times on top of the nanopore for non-tethered analyte (DNA) in
the presence of KF (DNA concentration = 400 nM, KF concentration = 800 nM). KF binding
was 1-100 ms.
Fig. 7 shows KF binding times on the top of the nanopore for tethered analyte (DNA) in
the absence of KF (DNA concentration = 1nM).
Fig. 8 shows KF binding times on top of the nanopore for tethered analyte (DNA) in the
presence of KF (DNA concentration = 1 nM, KF concentration = 5 nM). KF binding was
0.1-10 s .
Fig. 9 shows an example of a Phi29 DNA polymerase mediated unzipping event of
transiently tethered dsDNA. The drop in current from the open pore level is thought to be a
blockade caused by capturing a DNA:protein complex. This captured complex resides on the
nanopore for ~5 seconds giving a constant current level before rapidly changing between levels
and then finally returning to the open pore level. This is thought to be a pause before unzipping
is initiated and a single A moves through the reader head so giving the oscillation in current.
When the duplex has been fully unzipped the target strand translocates, the primer and
polymerase dissociate and so the current returns to the open pore level.
Fig. 10 shows an example of a Phi29 DNA polymerase mediated unzipping event of
solution dsDNA. The drop in current from the open pore level is thought to be a blockade
caused by capturing a DNA:protein complex. This captured complex resides on the nanopore for
-12 seconds giving a constant current level before rapidly changing between levels and then
finally returning to the open pore level. This is thought to be a pause before unzipping is
initiated and as the single A moves through the reader head so giving the oscillation in current.
When the duplex has been fully unzipped the target strand translocates, the primer and
polymerase dissociate and so the current returns to the open pore level.
Fig. 11 shows an example of event sequences from one unzipping run for non-tethered
dsDNA analyte. The number of levels observed as well as the level and duration for these are
broadly consistent with the tethered experiments.
Fig. 12 shows an example of event sequences from one unzipping run for tethered
dsDNA analyte. The number of levels observed as well as the level and duration for these are
broadly consistent with the solution (non-tethered) DNA experiments.
Fig. 13 shows a plasmid map of tethered strand sequencing analytes from genomic DNA.
Primers were designed complementary to PhiX 174 genomic DNA. The same sense primer was
used for all and contained a 5'-50polyT region followed by 4 abasic sites before the
complementary region. The hybridisation sites for the antisense primers were varied according
to the desired fragment size. Each antisense primer contained a '-cholesterol group.
Fig. 14 shows PCR generation of tethered strand sequencing analytes from genomic
DNA. Primers were designed complementary to PhiX 1 4 genomic DNA. The same sense
primer was used for all and contained a 5'-50polyT region followed by 4 abasic sites before the
complementary region. The hybridisation sites for the antisense primers were varied according
to the desired fragment size. Each antisense primer contained a 5'-cholesterol group. To confirm
presence of the 50polyT region to the 5' of the sense strand, fragments were digested with the 5'-
3' single strand specific RecJ exonuclease (NEB) and this was analysed on a gel. Lane 1 contains
50nt ssDNA, 235 bp dsDNA only. Lane 2 contains 50nt ssDNA, 235 bp dsDNA which has been
digested with the '-3' single strand specific RecJ exonuclease (NEB). Lane 3 contains 50nt
ssDNA, 400 bp dsDNA only. Lane 4 contains 50nt ssDNA, 400 bp dsDNA which has been
digested with the 5'-3' single strand specific RecJ exonuclease (NEB). Lane 5 contains 50nt
ssDNA, 835 bp dsDNA only. Lane 6 contains 50nt ssDNA, 835 bp dsDNA which has been
digested with the 5'-3 ' single strand specific RecJ exonuclease (NEB).
Fig. 15 shows unzipping events from the 800 bp PhiX 174 amplified fragment. This 800
bp sequence corresponds to the sequence between points 1 and 3 in the plasmid map shown.
Fig. 16 shows unzipping events from the 200 bp PhiX 174 amplified fragment. This 200
bp sequence corresponds to the sequence between points 1 and 2 in the plasmid map shown. The
200mer is aligned against the 800mer sequences shown in Fig. 15 with zero leading and trailing
gap penalties (i.e. it is free to start anywhere, but "internal" gaps are penalised). As expected, the
200mer sections align with the front of the 800mer.
Fig. 17 shows analyte tethering schemes for solid state nanopores. A) Shows tethering
into a modified surface (tethering in a layer). B) Shows tethering to a modified surface
(interaction with the surface). C) Shows tethering to a lipid monolayer on a modified surface. D)
Shows tethering to a lipid bilayer on a modified surface.
Fig. 18 shows methods for coupling double stranded polynucleotides to a lipid
membrane. A) Shows a single tethered dsDNA binding protein interacting with dsDNA analyte.
B) Shows multiple tethered dsDNA binding proteins interacting with a single dsDNA analyte.
C) Shows a single tethered chemical group interacting with dsDNA analyte.
Fig. 19 shows methods for coupling single stranded polynucleotide analytes to lipid
membranes. A) Shows a single tethered ssDNA binding protein interacting with ssDNA. B)
Shows multiple tethered ssDNA binding proteins interacting with a single ssDNA. C) Shows a
single tethered chemical group interacting with ssDNA.
Fig. 20 shows a schematic of one way of using a polynucleotide binding protein to
control DNA movement through a nanopore employing a dsDNA binding protein to couple the
DNA to the membrane. A) A DNA analyte (consisting of a ssDNA leader (grey region) attached
to a dsDNA region) is coupled to the membrane using a tethered dsDNA binding protein,
resulting in a concentration enhancement at the membrane surface. A polynucleotide binding
protein capable of controlling polynucleotide movement is added to the cis compartment where it
binds to the 4 bp overhang. B) Under an applied voltage, the DNA analyte is captured by the
nanopore via the ' leader section (grey region) on the DNA. C) Under the force of the applied
field the DNA is pulled into the pore until the bound polynucleotide binding protein contacts the
top of the pore and prevents further uncontrolled translocation. In this process the antisense
strand is stripped from the DNA strand, therefore, resulting in the detachment of the dsDNA
binding protein from the strand. D) In the presence of appropriate cofactors, the polynucleotide
binding protein on top of the pore moves along the DNA and controls the translocation of the
DNA through the pore. The movement of the polynucleotide binding protein, along the DNA in
a 3' to 5' direction, pulls the threaded DNA out of the pore against the applied field back to the
cis compartment. The last section of DNA to pass through the nanopore is the 5'-leader. The
arrow indicates the direction of DNA movement.
Fig. 2 1 shows a schematic of one way of using a polynucleotide binding protein to
control DNA movement through a nanopore employing a hybridised tether. A) A DNA analyte
(consisting of a ssDNA leader (grey region) attached to a dsDNA region) is coupled to the
membrane using a hybridised tether, resulting in a concentration enhancement at the membrane
surface. A polynucleotide binding protein capable of controlling DNA movement is added to the
cis compartment where it binds to the 4 bp overhang. B) Under an applied voltage, the DNA
analyte is captured by the nanopore via the ' leader section (grey region) on the DNA. C) Under
the force of the applied field the DNA is pulled into the pore until the bound polynucleotide
binding protein contacts the top of the pore and prevents further uncontrolled translocation. In
this process the polynucleotide which is tethered to the membrane (dashed line) is stripped off to
be sequenced (black strand with grey leader region). D) In the presence of appropriate cofactors,
the polynucleotide binding protein on top of the pore moves along the DNA and controls the
translocation of the DNA through the pore. The movement of the polynucleotide binding
protein, along the DNA in a 3' to 5' direction, pulls the threaded DNA out of the pore against the
applied field back to the cis compartment. The last section of DNA to pass through the nanopore
is the 5'-leader. The arrow indicates the direction of DNA movement.
Fig. 22 shows a schematic of one way of using a polynucleotide binding protein to
control DNA movement through a nanopore employing a hybridised tether. A) A DNA analyte
(consisting of ssDNA (black line with the leader sequence shown in grey) hybridised to a ssDNA
tether (dashed line)) is coupled to the membrane using a hybridised tether, resulting in a
concentration enhancement at the membrane surface. A polynucleotide binding protein capable
of controlling DNA movement is added to the cis compartment where it binds to the 4 bp
overhang. B) Under an applied voltage, the DNA analyte is captured by the nanopore via the '
leader section (grey region) on the DNA. C) Under the force of the applied field the DNA is
pulled into the pore until the bound polynucleotide binding protein contacts the top of the pore
and prevents further uncontrolled translocation. In this process the strand which is tethered to the
membrane (dashed line) is stripped off the ssDNA strand to be sequenced (black strand with grey
leader region). D) In the presence of appropriate cofactors, the polynucleotide binding protein on
top of the pore moves along the DNA and controls the translocation of the DNA through the
pore. The movement of the polynucleotide binding protein, along the DNA in a 3' to 5'
direction, pulls the threaded DNA out of the pore against the applied field back to the cis
compartment. The last section of DNA to pass through the nanopore is the 5'-leader. The arrow
indicates the direction of DNA movement.
Fig. 23 shows several methods of tethering a probe, which can be employed for the
detection of microRNA, to a membrane. A) The probe can be permanently tethered to the
membrane. In this instance the region of the probe that hybridises to the microRNA is in the
middle of the probe. The barcoded region (dotted region) of the probe, which is used to identify
the probe, is located at the opposite end of the strand to the tether. Bi and ii) The probe can be
transiently tethered to the membrane by internal hybridisation. In this example the region of the
probe that hybridises to the microRNA is attached to one end of the strand. The barcoding region
(dotted region), which is used to identify the probe, is located directly above the tether and below
the microRNA hybridisation region. In Bii) the hybridisation region of the tether to the probe is
inverted in its binding direction in comparison to Bi). Ci and ii) The probe can be transiently
tethered to the membrane by hybridisation to one end of the probe. In this example the region of
the probe that hybridises to the microRNA is located in the middle of the strand. The barcoding
region (dotted region), which is used to detect the presence or absence of the microRNA, is
located below the microRNA hybridisation region at the opposite end of the probe to the tether.
In Cii) the hybridisation region of the tether to the probe is inverted in its binding direction in
comparison to Ci).
Description of the Sequence Listing
SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encoding the NNNRRK
mutant MspA monomer.
SEQ ID NO: 2 (also referred to as "Bl") shows the amino acid sequence of the mature
form of the NNN-RRK mutant of the MspA monomer. The mutant lacks the signal sequence
and includes the following mutations: D90N, D91N, D93N, Dl 18R, D134R and E139K. These
mutations allow DNA transition through the MspA pore.
SEQ ID NO: 3 shows the polynucleotide sequence encoding one subunit of a-hemolysin-
Mll lR (a-HL-R).
SEQ ID NO: 4 shows the amino acid sequence of one subunit of a-HL-R.
SEQ ID NO: 5 shows the codon optimised polynucleotide sequence encoding the Phi29
DNA polymerase.
SEQ ID NO: 6 shows the amino acid sequence of the Phi29 DNA polymerase.
SEQ ID NO: 7 shows the codon optimised polynucleotide sequence derived from the
sbcB gene from E. coli. It encodes the exonuclease I enzyme (EcoExo I) from E. coli.
SEQ ID NO: 8 shows the amino acid sequence of exonuclease I enzyme (EcoExo I) from
E. coli.
SEQ ID NO: 9 shows the codon optimised polynucleotide sequence derived from the
xthA gene from E. coli. It encodes the exonuclease III enzyme fromii. coli.
SEQ ID NO: 10 shows the amino acid sequence of the exonuclease III enzyme from E.
coli. This enzyme performs distributive digestion of 5' monophosphate nucleosides from one
strand of double stranded DNA (dsDNA) in a 3' - 5' direction. Enzyme initiation on a strand
requires a 5' overhang of approximately 4 nucleotides.
SEQ ID NO: 11 shows the codon optimised polynucleotide sequence derived from the
recJ gene from T. thermophilus. It encodes the RecJ enzyme from T. thermophilus (TthRecJcd).
SEQ ID NO: 12 shows the amino acid sequence of the RecJ enzyme from T.
thermophilus ( ί/zRecJ-cd). This enzyme performs processive digestion of 5' monophosphate
nucleosides from ssDNA in a 5' - 3' direction. Enzyme initiation on a strand requires at least 4
nucleotides.
SEQ ID NO: 13 shows the codon optimised polynucleotide sequence derived from the
bacteriophage lambda exo (redX) gene. It encodes the bacteriophage lambda exonuclease.
SEQ ID NO: 14 shows the amino acid sequence of the bacteriophage lambda
exonuclease. The sequence is one of three identical subunits that assemble into a trimer. The
enzyme performs highly processive digestion of nucleotides from one strand of dsDNA, in a 5'-
3'direction (http://www.neb.com/nebecomm/products/productM0262.asp). Enzyme initiation on
a strand preferentially requires a 5' overhang of approximately 4 nucleotides with a 5'
phosphate.
SEQ ID NOs: 15 to 17 show the amino acid sequences of the mature forms of the MspB,
C and D mutants respectively. The mature forms lack the signal sequence.
SEQ ID NOs: 18 to 32 show the sequences used in the Examples.
SEQ ID NO: 33 shows the polynucleotide sequence encoding one subunit of a-HL-Q.
SEQ ID NO: 34 shows the amino acid sequence of one subunit of a-HL-Q.
SEQ ID NO: 35 shows the polynucleotide sequence encoding one subunit of a-HLE287C-
QC-D5FLAGH6.
SEQ ID NO: 36 shows the amino acid sequence of one subunit of a-HL-E287C-QCD5FLAGH6.
SEQ ID NO: 37 shows the polynucleotide sequence encoding one subunit of a -
hemolysin-ElllN/K147N (a-HL-NN; Stoddart et al, PNAS, 2009; 106(19): 7702-7707).
SEQ ID NO: 38 shows the amino acid sequence of one subunit of a-HL-NN.
SEQ ID NO: 39 shows the sequence used in Example 5 .
SEQ ID NO: 40 and 4 1 show the sequences used in Example 6.
Detailed description of the invention
It is to be understood that different applications of the disclosed products and methods
may be tailored to the specific needs in the art. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments of the invention only, and is
not intended to be limiting.
In addition as used in this specification and the appended claims, the singular forms "a",
"an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for
example, reference to "an analyte" includes two or more analytes, reference to "a detector"
includes two or more such detectors, reference to "a pore" includes two or more such pores,
reference to "a nucleic acid sequence" includes two or more such sequences, and the like.
All publications, patents and patent applications cited herein, whether supra or infra, are
hereby incorporated by reference in their entirety.
Methods of the invention
The invention provides a method for determining the presence, absence or characteristics
of an analyte. The method comprises coupling the analyte to a membrane and allowing the
analyte to interact with a detector present in the membrane. The presence, absence or
characteristics of the analyte is thereby determined. In one embodiment, the invention provides
a method for determining the presence or absence of an analyte, comprising (a) coupling the
analyte to a membrane and (b) allowing the analyte to interact with a detector present in the
membrane and thereby determining the presence or absence of the analyte.
As discussed above, coupling the analyte to a membrane containing the detector lowers
by several orders of magnitude the amount of analyte required. The method is of course
advantageous for detecting analytes that are present at low concentrations. The method
preferably allows the presence or characteristics of the analyte to be determined when the analyte
is present at a concentration of from about O.OOlpM to about InM, such as less than O.OlpM, less
than O.lpM, less than lpM, less than IOrM or less than IOOrM.
The method of the invention is particularly advantageous for nucleic acid sequencing
because, as discussed above, only small amounts of purified nucleic acid can be obtained from
human blood. The method preferably allows estimating the sequence of, or allows sequencing
of, a target polynucleotide that is present at a concentration of from about O.OOlpM to about
InM, such as less than O.OlpM, less than O.lpM, less than lpM, less than IOrM or less than
IOOrM.
Coupling one end of a polynucleotide to the membrane (even temporarily) also means
that the end will be prevented from interfering with the nanopore-based sequencing process.
This is discussed in more detail below with reference to the Exonuclease Sequencing method of
the invention.
The method of the invention may comprise determining or measuring one or more
characteristics of an analyte, such as a polynucleotide. The method may involve determining or
measuring two, three, four or five or more characteristics of the analyte, such as a
polynucleotide. For polynucleotides, the one or more characteristics are preferably selected from
(i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the
sequence of the target polynucleotide, (iv) the secondary structure of the target polynucleotide
and (v) whether or not the target polynucleotide is modified. Any combination of (i) to (v) may
be determined or measured in accordance with the invention. The method preferably comprises
estimating the sequence of or sequencing a polynucleotide.
Analyte
The analyte can be any substance. Suitable analytes include, but are not limited to, metal
ions, inorganic salts, polymers, such as a polymeric acids or bases, dyes, bleaches,
pharmaceuticals, diagnostic agents, recreational drugs, explosives and environmental pollutants.
The analyte can be an analyte that is secreted from cells. Alternatively, the analyte can
be an analyte that is present inside cells such that the analyte must be extracted from the cells
before the invention can be carried out.
The analyte is preferably an amino acid, peptide, polypeptide, a protein or a
polynucleotide. The amino acid, peptide, polypeptide or protein can be naturally-occurring or
non-naturally-occurring. The polypeptide or protein can include within it synthetic or modified
amino acids. A number of different types of modification to amino acids are known in the art.
For the purposes of the invention, it is to be understood that the analyte can be modified by any
method available in the art.
The protein can be an enzyme, antibody, hormone, growth factor or growth regulatory
protein, such as a cytokine. The cytokine may be selected from an interleukin, preferably IFN-1,
IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 or IL-13, an interferon, preferably IL-g or other cytokines
such as TNF-a. The protein may be a bacterial protein, fungal protein, virus protein or parasitederived
protein. Before it is contacted with the pore or channel, the protein may be unfolded to
form a polypeptide chain.
The analyte is most preferably a polynucleotide, such as a nucleic acid. Polynucleotides
are discussed in more detail below. A polynucleotide may be coupled to the membrane at its '
end or 3' end or at one or more intermediate points along the strand. The polynucleotide can be
single stranded or double stranded as discussed below. The polynucleotide may be circular. The
polynucleotide may be an aptamer, a probe which hybridises to microRNA or microRNA itself
(Wang, Y. et al, Nature Nanotechnology, 201 1, 6, 668-674).
When the analyte is a probe which hybridises to microRNA, the probe may be coupled
permanently (Fig. 23A) or transiently (Fig. 23 B and C) to the membrane. The probe itself may
be adapted to couple directly to the membrane or may hybridise to a complementary
polynucleotide which has been adapted to couple to the membrane. The analyte may be a
complex of microRNA hybridised to a probe where the probe has distinctive sequences or
barcodes enabling it to be identified unambiguously.
When the analyte is an aptamer, the aptamer may be coupled permanently or transiently
to the membrane. The aptamer itself may be adapted to couple directly to the membrane or may
hybridise to a complementary polynucleotide which has been adapted to couple to the
membrane. The aptamer may be bound or unbound to a protein analyte and the ultimate purpose
of detecting the aptamer may be to detect the presence, absence or characteristics of a protein
analyte to which it binds.
The analyte is present in any suitable sample. The invention is typically carried out on a
sample that is known to contain or suspected to contain the analyte. The invention may be
carried out on a sample that contains one or more analytes whose identity is unknown.
Alternatively, the invention may be carried out on a sample to confirm the identity of one or
more analytes whose presence in the sample is known or expected.
The sample may be a biological sample. The invention may be carried out in vitro on a
sample obtained from or extracted from any organism or microorganism. The organism or
microorganism is typically archaean, prokaryotic or eukaryotic and typically belongs to one the
five kingdoms: plantae, animalia, fungi, monera and protista. The invention may be carried out
in vitro on a sample obtained from or extracted from any virus. The sample is preferably a fluid
sample. The sample typically comprises a body fluid of the patient. The sample may be urine,
lymph, saliva, mucus or amniotic fluid but is preferably blood, plasma or serum. Typically, the
sample is human in origin, but alternatively it may be from another mammal animal such as from
commercially farmed animals such as horses, cattle, sheep or pigs or may alternatively be pets
such as cats or dogs. Alternatively a sample of plant origin is typically obtained from a
commercial crop, such as a cereal, legume, fruit or vegetable, for example wheat, barley, oats,
canola, maize, soya, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils,
sugar cane, cocoa, cotton.
The sample may be a non-biological sample. The non-biological sample is preferably a
fluid sample. Examples of a non-biological sample include surgical fluids, water such as
drinking water, sea water or river water, and reagents for laboratory tests.
The sample is typically processed prior to being assayed, for example by centrifugation
or by passage through a membrane that filters out unwanted molecules or cells, such as red blood
cells. The sample may be measured immediately upon being taken. The sample may also be
typically stored prior to assay, preferably below -70°C.
Membrane
Any membrane may be used in accordance with the invention. Suitable membranes are
well-known in the art. The membrane is preferably an amphiphilic layer. An amphiphilic layer
is a layer formed from amphiphilic molecules, such as phospholipids, which have both
hydrophilic and lipophilic properties. The amphiphilic molecules may be synthetic or naturally
occurring. Non-naturally occurring amphiphiles and amphiphiles which form a monolayer are
known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir,
2009, 25, 10447-10450). Block copolymers are polymeric materials in which two or more
monomer sub-units that are polymerized together to create a single polymer chain. Block
copolymers typically have properties that are contributed by each monomer sub-unit. However,
a block copolymer may have unique properties that polymers formed from the individual subunits
do not possess. Block copolymers can be engineered such that one of the monomer subunits
is hydrophobic (i.e. lipophilic), whilst the other sub-unit(s) are hydrophilic whilst in
aqueous media. In this case, the block copolymer may possess amphiphilic properties and may
form a structure that mimics a biological membrane. The block copolymer may be a diblock
(consisting of two monomer sub-units), but may also be constructed from more than two
monomer sub-units to form more complex arrangements that behave as amphipiles. The
copolymer may be a triblock, tetrablock or pentablock copolymer.
Archaebacterial bipolar tetraether lipids are naturally occurring lipids that are constructed
such that the lipid forms a monolayer membrane. These lipids are generally found in
extremophiles that survive in harsh biological environments, thermophiles, halophiles and
acidophiles. Their stability is believed to derive from the fused nature of the final bilayer. It is
straightforward to construct block copolymer materials that mimic these biological entities by
creating a triblock polymer that has the general motif hydrophilic-hydrophobic-hydrophilic.
This material may form monomelic membranes that behave similarly to lipid bilayers and
encompasse a range of phase behaviours from vesicles through to laminar membranes.
Membranes formed from these triblock copolymers hold several advantages over biological lipid
membranes. Because the triblock copolymer is synthesized, the exact construction can be
carefully controlled to provide the correct chain lengths and properties required to form
membranes and to interact with pores and other proteins.
Block copolymers may also be constructed from sub-units that are not classed as lipid
sub-materials; for example a hydrophobic polymer may be made from siloxane or other nonhydrocarbon
based monomers. The hydrophilic sub-section of block copolymer can also possess
low protein binding properties, which allows the creation of a membrane that is highly resistant
when exposed to raw biological samples. This head group unit may also be derived from nonclassical
lipid head-groups.
Triblock copolymer membranes also have increased mechanical and environmental
stability compared with biological lipid membranes, for example a much higher operational
temperature or pH range. The synthetic nature of the block copolymers provides a platform to
customize polymer based membranes for a wide range of applications.
In a preferred embodiment, the invention provides a method for determining the
presence, absence or characteristics of an analyte, comprising (a) coupling the analyte to a
membrane comprising a triblock copolymer, optionally wherein the membrane is modified to
facilitate the coupling, and (b) allowing the analyte to interact with a detector present in the
membrane and thereby determining the presence, absence or characteristics of the analyte. As
discussed above, a triblock copolymer is a polymer formed from three different monomer subunits.
The amphiphilic molecules may be chemically-modified or functionalised to facilitate
coupling of the analyte.
The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is
typically planar. The amphiphilic layer may be curved.
Amphiphilic membranes are typically naturally mobile, essentially acting as two
dimensional fluids with lipid diffusion rates of approximately 10 8 cm s-1. This means that the
detector and coupled analyte can typically move within an amphiphilic membrane.
The membrane is preferably a lipid bilayer. Lipid bilayers are models of cell membranes
and serve as excellent platforms for a range of experimental studies. For example, lipid bilayers
can be used for in vitro investigation of membrane proteins by single-channel recording.
Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of
substances. The lipid bilayer may be any lipid bilayer. Suitable lipid bilayers include, but are
not limited to, a planar lipid bilayer, a supported bilayer or a liposome. The lipid bilayer is
preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in International
Application No. PCT/GB08/000563 (published as WO 2008/102121), International Application
No. PCT/GB08/004127 (published as WO 2009/077734) and International Application No.
PCT/GB2006/001057 (published as WO 2006/100484).
Methods for forming lipid bilayers are known in the art. Suitable methods are disclosed
in the Example. Lipid bilayers are commonly formed by the method of Montal and Mueller
(Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566), in which a lipid monolayer is carried on
aqueous solution/air interface past either side of an aperture which is perpendicular to that
interface. The lipid is normally added to the surface of an aqueous electrolyte solution by first
dissolving it in an organic solvent and then allowing a drop of the solvent to evaporate on the
surface of the aqueous solution on either side of the aperture. Once the organic solvent has
evaporated, the solution/air interfaces on either side of the aperture are physically moved up and
down past the aperture until a bilayer is formed. Planar lipid bilayers may be formed across an
aperture in a membrane or across an opening into a recess.
The method of Montal & Mueller is popular because it is a cost-effective and relatively
straightforward method of forming good quality lipid bilayers that are suitable for protein pore
insertion. Other common methods of bilayer formation include tip-dipping, painting bilayers and
patch-clamping of liposome bilayers.
Tip-dipping bilayer formation entails touching the aperture surface (for example, a pipette
tip) onto the surface of a test solution that is carrying a monolayer of lipid. Again, the lipid
monolayer is first generated at the solution/air interface by allowing a drop of lipid dissolved in
organic solvent to evaporate at the solution surface. The bilayer is then formed by the Langmuir-
Schaefer process and requires mechanical automation to move the aperture relative to the solution
surface.
For painted bilayers, a drop of lipid dissolved in organic solvent is applied directly to the
aperture, which is submerged in an aqueous test solution. The lipid solution is spread thinly over
the aperture using a paintbrush or an equivalent. Thinning of the solvent results in formation of a
lipid bilayer. However, complete removal of the solvent from the bilayer is difficult and
consequently the bilayer formed by this method is less stable and more prone to noise during
electrochemical measurement.
Patch-clamping is commonly used in the study of biological cell membranes. The cell
membrane is clamped to the end of a pipette by suction and a patch of the membrane becomes
attached over the aperture. The method has been adapted for producing lipid bilayers by clamping
liposomes which then burst to leave a lipid bilayer sealing over the aperture of the pipette. The
method requires stable, giant and unilamellar liposomes and the fabrication of small apertures in
materials having a glass surface.
Liposomes can be formed by sonication, extrusion or the Mozafari method (Colas et al.
(2007) Micron 38:841-847).
In a preferred embodiment, the lipid bilayer is formed as described in International
Application No. PCT/GB08/004127 (published as WO 2009/077734). Advantageously in this
method, the lipid bilayer is formed from dried lipids. In a most preferred embodiment, the lipid
bilayer is formed across an opening as described in WO2009/077734 (PCT/GB08/004127).
A lipid bilayer is formed from two opposing layers of lipids. The two layers of lipids are
arranged such that their hydrophobic tail groups face towards each other to form a hydrophobic
interior. The hydrophilic head groups of the lipids face outwards towards the aqueous
environment on each side of the bilayer. The bilayer may be present in a number of lipid phases
including, but not limited to, the liquid disordered phase (fluid lamellar), liquid ordered phase,
solid ordered phase (lamellar gel phase, interdigitated gel phase) and planar bilayer crystals
(lamellar sub-gel phase, lamellar crystalline phase).
Any lipid composition that forms a lipid bilayer may be used. The lipid composition is
chosen such that a lipid bilayer having the required properties, such surface charge, ability to
support membrane proteins, packing density or mechanical properties, is formed. The lipid
composition can comprise one or more different lipids. For instance, the lipid composition can
contain up to 100 lipids. The lipid composition preferably contains 1 to 10 lipids. The lipid
composition may comprise naturally-occurring lipids and/or artificial lipids.
The lipids typically comprise a head group, an interfacial moiety and two hydrophobic
tail groups which may be the same or different. Suitable head groups include, but are not limited
to, neutral head groups, such as diacylglycerides (DG) and ceramides (CM); zwitterionic head
groups, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin
(SM); negatively charged head groups, such as phosphatidylglycerol (PG); phosphatidylserine
(PS), phosphatidylinositol (PI), phosphatic acid (PA) and cardiolipin (CA); and positively
charged headgroups, such as trimethylammonium-Propane (TAP). Suitable interfacial moieties
include, but are not limited to, naturally-occurring interfacial moieties, such as glycerol-based or
ceramide-based moieties. Suitable hydrophobic tail groups include, but are not limited to,
saturated hydrocarbon chains, such as lauric acid (n-Dodecanolic acid), myristic acid (n-
Tetradecononic acid), palmitic acid (n-Hexadecanoic acid), stearic acid (n-Octadecanoic) and
arachidic («-Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid (cis-9-
Octadecanoic); and branched hydrocarbon chains, such as phytanoyl. The length of the chain
and the position and number of the double bonds in the unsaturated hydrocarbon chains can vary.
The length of the chains and the position and number of the branches, such as methyl groups, in
the branched hydrocarbon chains can vary. The hydrophobic tail groups can be linked to the
interfacial moiety as an ether or an ester.
The lipids can also be chemically-modified. The head group or the tail group of the
lipids may be chemically-modified. Suitable lipids whose head groups have been chemicallymodified
include, but are not limited to, PEG-modified lipids, such as l,2-Diacyl-sn-Glycero-3-
Phosphoethanolamine-N -[Methoxy(Polyethylene glycol)-2000]; functionalised PEG Lipids,
such as l,2-Distearoyl-sn-Glycero-3 Phosphoethanolamine-N-[Biotinyl(Polyethylene
Glycol)2000]; and lipids modified for conjugation, such as l,2-Dioleoyl-sn-Glycero-3-
Phosphoethanolamine-N-(succinyl) and 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-
(Biotinyl). Suitable lipids whose tail groups have been chemically-modified include, but are not
limited to, polymerisable lipids, such as l,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-
Phosphocholine; fluorinated lipids, such as l-Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero-3-
Phosphocholine; deuterated lipids, such as l,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine;
and ether linked lipids, such as l,2-Di-0-phytanyl-sn-Glycero-3-Phosphocholine. The lipids
may be chemically-modified or functionalised to facilitate coupling of the analyte.
The amphiphilic layer, for example the lipid composition, typically comprises one or
more additives that will affect the properties of the layer. Suitable additives include, but are not
limited to, fatty acids, such as palmitic acid, myristic acid and oleic acid; fatty alcohols, such as
palmitic alcohol, myristic alcohol and oleic alcohol; sterols, such as cholesterol, ergosterol,
lanosterol, sitosterol and stigmasterol; lysophospholipids, such as l-Acyl-2-Hydroxy-sn-
Glycero-3-Phosphocholine; and ceramides.
In another preferred embodiment, the membrane is a solid state layer. A solid-state layer
is not of biological origin. In other words, a solid state layer is not derived from or isolated from
a biological environment such as an organism or cell, or a synthetically manufactured version of
a biologically available structure. Solid state layers can be formed from both organic and
inorganic materials including, but not limited to, microelectronic materials, insulating materials
such as Si3N4, A1203, and SiO, organic and inorganic polymers such as polyamide, plastics
such as Teflon® or elastomers such as two-component addition-cure silicone rubber, and glasses.
The solid state layer may be formed from graphene. Suitable graphene layers are disclosed in
International Application No. PCT/US2008/0 10637 (published as WO 2009/035647).
Coupling
The analyte may be coupled to the membrane using any known method. If the membrane
is an amphiphilic layer, such as a lipid bilayer, the analyte is preferably coupled to the membrane
via a polypeptide present in the membrane or a hydrophobic anchor present in the membrane.
The hydrophobic anchor is preferably a lipid, fatty acid, sterol, carbon nanotube, polypeptide,
protein or amino acid, for example cholesterol, palmitate or tocopherol. In preferred
embodiments, the analyte is not coupled to the membrane via the detector.
The components of the membrane, such as the amphiphilic molecules or lipids, may be
chemically-modified or functionalised to facilitate coupling of the analyte to the membrane
either directly or via one or more linkers. Examples of suitable chemical modifications and
suitable ways of functionalising the components of the membrane are discussed in more detail
below. Any proportion of the membrane components may be functionalized, for example at least
0.01%, at least 0.1%, at least 1%, at least 10%, at least 25%, at least 50% or 100%.
The analyte may be coupled directly to the membrane. The analyte may be coupled
directly to the membrane at one or more, such as 2, 3, 4 or more, points.
The analyte is preferably coupled to the membrane via a linker. The analyte may be
coupled to the membrane via one or more, such as 2, 3, 4 or more, linkers. One linker may
couple more than one, such as 2, 3, 4 or more, analytes to the membrane.
The analyte may be coupled to the membrane directly at one or more points and via one
or more linkers.
Preferred linkers include, but are not limited to, polymers, such as polynucleotides,
polyethylene glycols (PEGs), polysaccharides and polypeptides. These linkers may be linear,
branched or circular. For instance, the linker may be a circular polynucleotide. If the analyte is
itself a polynucleotide, it may hybridize to a complementary sequence on the circular
polynucleotide linker.
Functionalised linkers and the ways in which they can couple molecules are known in the
art. For instance, linkers functionalised with maleimide groups will react with and attach to
cysteine residues in proteins. In the context of this invention, the protein may be present in the
membrane, may be the analyte itself or may be used to bind to the analyte. This is discussed in
more detail below.
Crosslinkage of analytes can be avoided using a "lock and key" arrangement. Only one
end of each linker may react together to form a longer linker and the other ends of the linker each
react with the analyte or membrane respectively. Such linkers are described in International
Application No. PCT/GB 10/000 132 (published as WO 2010/086602).
The use of a linker is preferred in the sequencing embodiments discussed below. If a
polynucleotide analyte is permanently coupled directly to the membrane, then some sequence
data will be lost as the sequencing run cannot continue to the end of the polynucleotide due to
the distance between the membrane and the detector. If a linker is used, then the polynucleotide
analyte can be processed to completion.The coupling may be permanent or stable. In other
words, the coupling may be such that the analyte remains coupled to the membrane during the
method. The coupling may be transient. In other words, the coupling may be such that the
analyte decouples from the membrane during the method. For certain applications, such as
aptamer detection, the transient nature of the coupling is preferred. If a permanent or stable
linker is attached directly to either the ' or 3' end of a polynucleotide and the linker is shorter
than the distance between the bilayer and the nanopore's channel or the polynucleotide binding
protein's active site, then some sequence data will be lost as the sequencing run cannot continue
to the end of the polynucleotide. If the coupling is transient, then when the coupled end
randomly becomes free of the bilayer, then the polynucleotide can be processed to completion.
Chemical groups that form permanent/stable or transient links with the membrane are discussed
in more detail below. The analyte may be transiently coupled to an amphiphilic layer or lipid
bilayer using cholesterol or a fatty acyl chain. Any fatty acyl chain having a length of from 6 to
30 carbon atom, such as hexadecanoic acid, may be used.
In preferred embodiments, a polynucleotide analyte, such as a nucleic acid, is coupled to
an amphiphilic layer such as a lipid bilayer. Coupling of nucleic acids to synthetic lipid bilayers
has been carried out previously with various different tethering strategies. These are summarised
in Table 3 below.
Table 3
Synthetic polynucleotide analytes or linkers may be functionalised using a modified
phosphoramidite in the synthesis reaction, which is easily compatible for the direct addition of
suitable coupling moieties, such as cholesterol, tocopherol or palmitate, as well as for reactive
groups, such as thiol, cholesterol, lipid and biotin groups. These different attachment chemistries
give a suite of options for attachment to target polynucleotides. Each different modification
group tethers the polynucleotide in a slightly different way and coupling is not always permanent
so giving different dwell times for the analyte to the bilayer. The advantages of transient
coupling are discussed above.
Coupling of polynucleotides to a linker or to a functionalised membrane can also be
achieved by a number of other means provided that a complementary reactive group or a tether
can be added to the target polynucleotide. The addition of reactive groups to either end of DNA
has been reported previously. A thiol group can be added to the 5' of ssDNA or dsDNA using
T4 polynucleotide kinase and ATPyS (Grant, G. P. and P. Z. Qin (2007). "A facile method for
attaching nitroxide spin labels at the 5' terminus of nucleic acids." Nucleic Acids Res 35(10):
e77). An azide group could be added to the 5'-phosphate of ssDNA or dsDNA using T4
polynucleotide kinase and 7-[2-Azidoethyl]-ATP or y-[6-Azidohexyl]-ATP. Using thiol or Click
chemistry a tether, containing either a thiol, iodoacetamide OPSS or maleimide group (reactive
to thiols) or a DIBO (dibenzocyclooxtyne) or alkyne group (reactive to azides), can be covalently
attached to the analyte . A more diverse selection of chemical groups, such as biotin, thiols and
fluorophores, can be added using terminal transferase to incorporate modified oligonucleotides
to the 3' of ssDNA (Kumar, A., P. Tchen, et al. (1988). "Nonradioactive labeling of synthetic
oligonucleotide probes with terminal deoxynucleotidyl transferase." Anal Biochem 169 (2) : 376-
82). Example 3 below describes how DNA can be coupled to a lipid bilayer using
streptavidin/biotin. Streptavidin/biotin coupling may be used for any other analyte. It may also
be possible that tethers could be directly added to target polynucleotides using terminal
transferase with suitably modified nucleotides (eg. cholesterol or palmitate).
Alternatively, the reactive group or tether could be considered to be the addition of a
short piece of polynucleotide, such as DNA, complementary to one already coupled to the
bilayer, so that attachment can be achieved via hybridisation. In this case, the reactive group
may be a single strand or double strand polynucleotide. The reactive group may be ligated to a
single strand or double strand polynucleotide analyte. Ligation of short pieces of ssDNA have
been reported using T4 RNA ligase I (Troutt, A. B., M. G. McHeyzer-Williams, et al. (1992).
"Ligation-anchored PCR: a simple amplification technique with single-sided specificity." Proc
Natl Acad Sci U S A 89(20): 9823-5). Alternatively, either ssDNA or dsDNA could be ligated
to native analyte dsDNA and then the two strands separated by thermal or chemical denaturation.
To native dsDNA, it is possible to add either a piece of ssDNA to one or both of the ends of the
duplex, or dsDNA to one or both ends. For addition of single stranded nucleic acids to the native
DNA this can be achieved using T4 RNA ligase I as for ligation to other regions of single
stranded nucleic acids. For addition of dsDNA to native duplex DNA then ligation can be
"blunt-ended", with complementary 3' dA / dT tails on the native DNA and adapter respectively
(as is routinely done for many sample prep applications to prevent concatemer or dimer
formation) or using "sticky-ends" generated by restriction digestion of the native DNA and
ligation of compatible adapters. Then, when the duplex is melted, each single strand will have
either a ' or 3' modification if ssDNA was used for ligation or a modification at the ' end, the
3' end or both if dsDNA was used for ligation. If the polynucleotide is a synthetic strand, the
coupling chemistry can be incorporated during the chemical synthesis of the polynucleotide. For
instance, the polynucleotide can be synthesised using a primer having a reactive group attached
to it.
Adenylated nucleic acids (AppDNA) are intermediates in ligation reactions, where an
adenosine-monophostate is attached to the 5'-phosphate of the nucleic acid. Various kits are
available for generation of this intermediate, such as the 5' DNA Adenylation Kit from NEB.
By substituting ATP in the reaction for a modifided nucleotide triphosphate, then addition of
reactive groups (such as thiols, amines, biotin, azides, etc) to the 5' of DNA should be possible.
It may also be possible that tethers could be directly added to target polynucleotides using a '
DNA adenylation kit with suitably modified nucleotides (e.g. cholesterol or palmitate).
A common technique for the amplification of sections of genomic DNA is using
polymerase chain reaction (PCR). Here, using two synthetic oligonucleotide primers, a number
of copies of the same section of DNA can be generated, where for each copy the 5' of each
strand in the duplex will be a synthetic polynucleotide. By using an antisense primer single or
multiple nucleotides can be added to 3' end of single or double stranded DNA by employing a
polymerase. Examples of polymerases which could be used include, but are not limited to,
Terminal Transferase, Klenow and E. coli Poly(A) polymerase). By substituting ATP in the
reaction for a modified nucleotide triphosphate then reactive groups, such as a cholesterol, thiol,
amine, azide, biotin or lipid, can be incorporated into the DNA. Therefore, each copy of the
target amplified DNA will contain a reactive group for coupling.
Ideally, the analyte is coupled to the membrane without having to functionalise the
analyte. This can be achieved by anchoring a binding group, such as a polynucleotide binding
protein or a chemical group, to the membrane and allowing the binding group to interact with the
analyte or by functionalizing the membrane. The binding group may be coupled to the membrane
by any of the methods described herein. In particular, the binding group may be coupled to the
membrane using one or more linkers, such as maleimide functionalised linkers.
In this embodiment, the analyte is typically RNA, DNA, PNA, TNA or LNA and may be
double or single stranded. This embodiment is particularly suited to genomic DNA analytes.
The binding group can be any group that interacts with single or double stranded nucleic
acids, specific nucleotide sequences within the analyte or patterns of modified nucleotides within
the analyte, or any other ligand that is present on the polynucleotide.
Suitable binding proteins include E. coli single stranded binding protein, P5 single
stranded binding protein, T4 gp32 single stranded binding protein, the TOPO V dsDNA binding
region, human histone proteins, E. coli HU DNA binding protein and other archaeal, prokaryotic
or eukaryotic single- or double-stranded nucleic acid binding proteins, including those listed
below.
The specific nucleotide sequences could be sequences recognised by transcription factors,
ribosomes, endonucleases, topoisomerases or replication initiation factors. The patterns of
modified nucleotides could be patterns of methylation or damage.
The chemical group can be any group which intercalates with or interacts with a
polynucleotide analyte. The group may intercalate or interact with the polynucleotide analyte
via electrostatic, hydrogen bonding or Van der Waals interactions. Such groups include a lysine
monomer, poly-lysine (which will interact with ssDNA or dsDNA), ethidium bromide (which
will intercalate with dsDNA), universal bases or universal nucleotides (which can hybridise with
any polynucleotide analyte) and osmium complexes (which can react to methylated bases). A
polynucleotide analyte may therefore be coupled to the membrane using one or more universal
nucleotides attached to the membrane. Each universal nucleotide residue may be attached to the
membrane using one or more linkers. Examples of universal bases include inosine, 3-
nitropyrrole, 5-nitroindole, 4-nitroindole, 6-nitroindole, 3,4-dihydro-pyrimido[4,5-c][l,2]oxazin-
7-one (dP), 2-dimethylaminomethyleneamino-6-methyoxyaminopurine (dK), deoxy inosine,
deoxy nebularine.
In this embodiment at least 1%, at least 10%, at least 25%, at least 50%> or 100% of the
membrane components may be functionalized.
Where the binding group is a protein, it may be able to anchor directly into the membrane
without further functonalisation, for example if it already has an external hydrophobic region
which is compatible with the membrane. Examples of such proteins include transmembrane
proteins. Alternatively the protein may be expressed with a genetically fused hydrophobic
region which is compatible with the membrane. Such hydrophobic protein regions are know in
the art
The binding group is preferably mixed with the analyte before contacting with the
membrane, but the binding group may be contacted with the membrane and subsequently
contacted with the analyte.
In another aspect the analyte may be functionalised, using methods described above, so
that it can be recognised by a specific binding group. Specifically the analyte may be
functionalised with a ligand such as biotin (for binding to streptavidin), amylose (for binding to
maltose binding protein or a fusion protein), Ni-NTA (for binding to poly-histidine or polyhistidine
tagged proteins) or a peptides (such as an antigen),
According to a further aspect, the binding group may be used to couple polynucleotide
analyte to the membrane when the analyte has bound to a polynucleotide adapter. Specifically
the analyte binds to an adaptor which comprises a leader sequence designed to preferentially
thread into a detector such as a nanopore. Such a leader sequence may comprise a
homopolymeric polynucleotide or an abasic region. The adaptor typically is designed to
hybridise to a linker and to ligate to or hybridise to the analyte. This creates competition between
the analyte and the adaptor to enter the detector. If the linker comprises a binding group, the
greater length of the analyte compared to the adapter means that several linkers can bind to the
analyte simultaneously, thus increasing the concentration of analyte relative to that of the
adapter.
Any of the methods discussed above for coupling polynucleotides to amphiphilic layers,
such as lipid bilayers, can of course be applied to other analyte and membrane combinations. In
some embodiments, an amino acid, peptide, polypeptide or protein is coupled to a lipid bilayer.
Various methodologies for the chemical attachment of such analytes are available. An example
of a molecule used in chemical attachment is EDC (l-ethyl-3-[3-
dimethylaminopropyljcarbodiimide hydrochloride). Reactive groups can also be added to the 5'
of DNA using commercially available kits (Thermo Pierce, Part No. 22980). Suitable methods
include, but are not limited to, transient affinity attachment using histidine residues and Ni-NTA,
as well as more robust covalent attachment by reactive cysteines, lysines or non natural amino
acids.
Detector
The detector can be any structure that provides a readable signal in response to the
presence, the absence or the characteristics of the analyte. The detector can be any structure that
provides a readable signal in response to the presence or the absence of the analyte. Suitable
detectors are known in the art. They include, but are not limited to transmembrane pores,
tunnelling electrodes, classis electrodes, nanotubes, FETs (field-effect transistors) and optical
detectors, such as atomic force microscopes (AFMs) and scanning tunneling microscopes
(STMs).
In preferred embodiments, the detector detects the analyte using electrical means.
Electrical measurements may be made using standard single channel recording
equipment as describe in Stoddart D et al, Proc Natl Acad Sci, 12;106(19):7702-7, Lieberman
KR et al, J Am Chem Soc. 2010;132(50) :1796 1-72, and International Application
WO-2000/28312. Alternatively, electrical measurements may be made using a multi-channel
system, for example as described in International Application WO-2009/077734 and
International Application WO-201 1/067559.
In other preferred embodiments, the detector does not detect the analyte using fluorescent
means.
The detector preferably comprises a transmembrane pore. A transmembrane pore is a
structure that permits hydrated ions driven by an applied potential to flow from one side of the
membrane to the other side of the membrane.
The transmembrane pore is preferably a transmembrane protein pore. A transmembrane
protein pore is a polypeptide or a collection of polypeptides that permits hydrated ions, such as
analyte, to flow from one side of a membrane to the other side of the membrane. In the present
invention, the transmembrane protein pore is capable of forming a pore that permits hydrated
ions driven by an applied potential to flow from one side of the membrane to the other. The
transmembrane protein pore preferably permits analyte such as nucleotides to flow from one side
of the membrane, such as a lipid bilayer, to the other. The transmembrane protein pore
preferably allows a polynucleotide or nucleic acid, such as DNA or RNA, to be move through
the pore.
The transmembrane protein pore may be a monomer or an oligomer. The pore is
preferably made up of several repeating subunits, such as 6, 7 or 8 subunits. The pore is more
preferably a heptameric or octameric pore.
The transmembrane protein pore typically comprises a barrel or channel through which
the ions may flow. The subunits of the pore typically surround a central axis and contribute
strands to a transmembrane b barrel or channel or a transmembrane a-helix bundle or channel.
The barrel or channel of the transmembrane protein pore typically comprises amino acids
that facilitate interaction with analyte, such as nucleotides, polynucleotides or nucleic acids.
These amino acids are preferably located near a constriction of the barrel or channel. The
transmembrane protein pore typically comprises one or more positively charged amino acids,
such as arginine, lysine or histidine, or aromatic amino acids, such as tyrosine or tryptophan.
These amino acids typically facilitate the interaction between the pore and nucleotides,
polynucleotides or nucleic acids. The nucleotide detection can be facilitated with an adaptor.
This is discussed in more detail below.
Transmembrane protein pores for use in accordance with the invention can be derived
from b-barrel pores or a-helix bundle pores b-barrel pores comprise a barrel or channel that is
formed from b-strands. Suitable b-barrel pores include, but are not limited to, b-toxins, such as
a-hemolysin, anthrax toxin and leukocidins, and outer membrane proteins/porins of bacteria,
such as Mycobacterium smegmatis porin (Msp), for example MspA, MspB, MspC or MspD,
outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane
phospholipase A and Neisseria autotransporter lipoprotein (NalP). a-helix bundle pores
comprise a barrel or channel that is formed from a-helices. Suitable a-helix bundle pores
include, but are not limited to, inner membrane proteins and a outer membrane proteins, such as
WZA and ClyA toxin. The transmembrane pore may be derived from Msp or from a-hemolysin
(a-HL).
For Strand Sequencing, the transmembrane protein pore is preferably derived from Msp,
preferably from MspA. Such a pore will be oligomeric and typically comprises 7, 8, 9 or 10
monomers derived from Msp. The pore may be a homo-oligomeric pore derived from Msp
comprising identical monomers. Alternatively, the pore may be a hetero-oligomeric pore
derived from Msp comprising at least one monomer that differs from the others. The pore may
also comprise one or more constructs which comprise two or more covalently attached
monomers derived from Msp. Suitable pores are disclosed in International Application No.
PCT/GB2012/050301 (claiming priority from US Provisional Application No. 61/441,718).
Preferably the pore is derived from MspA or a homolog or paralog thereof.
A monomer derived from Msp comprises the sequence shown in SEQ ID NO: 2 or a
variant thereof. SEQ ID NO: 2 is the NNN-RRK mutant of the MspA monomer. It includes the
following mutations: D90N, D91N, D93N, Dl 18R, D134R and E139K. A variant of SEQ ID
NO: 2 is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 2
and which retains its ability to form a pore. The ability of a variant to form a pore can be
assayed using any method known in the art. For instance, the variant may be inserted into a lipid
bilayer along with other appropriate subunits and its ability to oligomerise to form a pore may be
determined. Methods are known in the art for inserting subunits into membranes, such as lipid
bilayers. For example, subunits may be suspended in a purified form in a solution containing a
lipid bilayer such that it diffuses to the lipid bilayer and is inserted by binding to the lipid bilayer
and assembling into a functional state. Alternatively, subunits may be directly inserted into the
membrane using the "pick and place" method described in M.A. Holden, H. Bayley. J . Am.
Chem. Soc. 2005, 127, 6502-6503 and International Application No. PCT/GB2006/001057
(published as WO 2006/100484).
Preferred variants are disclosed in International Application No. PCT/GB2012/050301
(claiming priority from US Provisional Application No. 61/441,718). Particularly preferred
variants include, but are not limited to, those comprising the following substitution(s): L88N;
L88S; L88Q; L88T; D90S; D90Q; D90Y; I105L; I105S; Q126R; G75S; G77S; G75S, G77S,
L88N and Q126R; G75S, G77S, L88N, D90Q and Q126R; D90Q and Q126R; L88N, D90Q and
Q126R; L88S and D90Q; L88N and D90Q; E59R; G75Q; G75N; G75S; G75T; G77Q; G77N;
G77S; G77T; I78L; S81N; T83N; N86S; N86T; I87F; I87V; I87L; L88N; L88S; L88Y; L88F;
L88V; L88Q; L88T; I89F; I89V; I89L; N90S; N90Q; N90L; N90Y; N91S; N91Q; N91L;
N91M; N91I; N91A; N91V; N91G; G92A; G92S; N93S; N93A; N93T; I94L; T95V; A96R;
A96D; A96V; A96N; A96S; A96T; P97S; P98S; F99S; G100S; L101F; N102K; N102S; N102T;
S103A; S103Q; S103N; S103G; S103T; VI 041; I105Y; I105L; I105A; I105Q; I105N; I105S;
I105T; T106F; T106I; T106V; T106S; N108P; N108S; D90Q and I105A; D90S and G92S;
L88T and D90S; I87Q and D90S; I89Y and D90S; L88N and I89F; L88N and I89Y; D90S and
G92A; D90S and I94N; D90S and V104I; L88D and I105K; L88N and Q126R; L88N, D90Q
and D91R; L88N, D90Q and D91S; L88N, D90Q and I105V; D90Q, D93S and I105A; N91Y;
N90Y and N91G; N90G and N91Y; N90G and N91G; 105G; N90R; N91R; N90R and N91R;
N90K; N91K; N90 and 91 ; N90Q andN91G; N90G andN91Q; N90Q andN91Q; R118N;
N91C; N90C; N90W; N91W; N90K; N91K; N90R; N91R; N90S andN91S; N90Y and I105A;
N90G and I105A; N90Q and I105A; N90S and I I05A; L88A and I105A; L88S and I105S; L88N
and I105N; N90G and N93G; N90G; N93G; N90G and N91A; I 105 ; I105R; I I05V; I105P;
I105W; L88R; L88A; L88G; L88N; N90R and I105A; N90S and I105A; L88A and I105A; L88S
and I105S; L88N and I105N; L88C; S103C; and I105C.
In addition to the specific mutations discussed above, the variant may include other
mutations. Over the entire length of the amino acid sequence of SEQ ID NO: 2, a variant will
preferably be at least 50% homologous to that sequence based on amino acid identity. More
preferably, the variant may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99%
homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 2 over the
entire sequence. There may be at least 80%, for example at least 85% , 90% or 95%, amino acid
identity over a stretch of 100 or more, for example 125, 150, 175 or 200 or more, contiguous
amino acids ("hard homology").
Standard methods in the art may be used to determine homology. For example the
UWGCG Package provides the BESTFIT program which can be used to calculate homology, for
example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-
395). The PILEUP and BLAST algorithms can be used to calculate homology or line up
sequences (such as identifying equivalent residues or corresponding sequences (typically on their
default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300;
Altschul, S.F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/).
SEQ ID NO: 2 is the NNN-RRK mutant of the MspA monomer. The variant may
comprise any of the mutations in the MspB, C or D monomers compared with MspA. The
mature forms of MspB, C and D are shown in SEQ ID NOs: 15 to 17. In particular, the variant
may comprise the following substitution present in MspB: A138P. The variant may comprise
one or more of the following substitutions present in MspC: A96G, N102E and A138P. The
variant may comprise one or more of the following mutations present in MspD: Deletion of Gl,
L2V, E5Q, L8V, D13G, W21A, D22E, K47T, I49H, I68V, D91G, A96Q, N102D, S103T,
V104I, S136K and G141A. The variant may comprise combinations of one or more of the
mutations and substitutions from Msp B, C and D.
Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 2 in
addition to those discussed above, for example up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions.
Conservative substitutions replace amino acids with other amino acids of similar chemical
structure, similar chemical properties or similar side-chain volume. The amino acids introduced
may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge
to the amino acids they replace. Alternatively, the conservative substitution may introduce
another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or
aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be
selected in accordance with the properties of the 20 main amino acids as defined in Table 4
below. Where amino acids have similar polarity, this can also be determined by reference to the
hydropathy scale for amino acid side chains in Table 5.
Table 4 - Chemical properties of amino acids
Table 5 - Hydropathy scale
Side Chain Hydropathy
e
Leu 3.8
P e 2.8
Cys 2.5
Met 1.9
Ala 1.8
Gly -0.4
Thr -0.7
Ser -0.8
Trp -0.9
Tyr -1.3
Pro -1.6
His -3.2
Glu -3.5
Gin -3.5
Asp -3.5
Asn -3.5
Lys -3.9
Ar -4.5
One or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 may
additionally be deleted from the polypeptides described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30
residues may be deleted, or more.
Variants may include fragments of SEQ ID NO: 2. Such fragments retain pore forming
activity. Fragments may be at least 50, 100, 150 or 200 amino acids in length. Such fragments
may be used to produce the pores. A fragment preferably comprises the pore forming domain of
SEQ ID NO: 2 . Fragments must include one of residues 88, 90, 91, 105, 118 and 134 of SEQ ID
NO: 2. Typically, fragments include all of residues 88, 90, 91, 105, 118 and 134 of SEQ ID NO:
2.
One or more amino acids may be alternatively or additionally added to the polypeptides
described above. An extension may be provided at the amino terminal or carboxy terminal of the
amino acid sequence of SEQ ID NO: 2 or polypeptide variant or fragment thereof. The
extension may be quite short, for example from 1 to 10 amino acids in length. Alternatively, the
extension may be longer, for example up to 50 or 100 amino acids. A carrier protein may be
fused to an amino acid sequence according to the invention. Other fusion proteins are discussed
in more detail below.
As discussed above, a variant is a polypeptide that has an amino acid sequence which
varies from that of SEQ ID NO: 2 and which retains its ability to form a pore. A variant
typically contains the regions of SEQ ID NO: 2 that are responsible for pore formation. The
pore forming ability of Msp, which contains a b-barrel, is provided by b-sheets in each subunit.
A variant of SEQ ID NO: 2 typically comprises the regions in SEQ ID NO: 2 that form b-sheets.
One or more modifications can be made to the regions of SEQ ID NO: 2 that form b-sheets as
long as the resulting variant retains its ability to form a pore. A variant of SEQ ID NO: 2
preferably includes one or more modifications, such as substitutions, additions or deletions,
within its a-helices and/or loop regions.
The monomers derived from Msp may be modified to assist their identification or
purification, for example by the addition of histidine residues (a hist tag), aspartic acid residues
(an asp tag), a streptavidin tag or a flag tag, or by the addition of a signal sequence to promote
their secretion from a cell where the polypeptide does not naturally contain such a sequence. An
alternative to introducing a genetic tag is to chemically react a tag onto a native or engineered
position on the pore. An example of this would be to react a gel-shift reagent to a cysteine
engineered on the outside of the pore. This has been demonstrated as a method for separating
hemolysin hetero-oligomers (Chem Biol. 1997 Jul;4(7):497-505).
The monomer derived from Msp may be labelled with a revealing label. The revealing
label may be any suitable label which allows the pore to be detected. Suitable labels include, but
are not limited to, fluorescent molecules, radioisotopes, e.g. 51, S, enzymes, antibodies,
antigens, polynucleotides and ligands such as biotin.
The monomer derived from Msp may also be produced using D-amino acids. For
instance, the monomer derived from Msp may comprise a mixture of L-amino acids and Damino
acids. This is conventional in the art for producing such proteins or peptides.
The monomer derived from Msp contains one or more specific modifications to facilitate
nucleotide discrimination. The monomer derived from Msp may also contain other non-specific
modifications as long as they do not interfere with pore formation. A number of non-specific
side chain modifications are known in the art and may be made to the side chains of the
monomer derived from Msp. Such modifications include, for example, reductive alkylation of
amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidination with
methylacetimidate or acylation with acetic anhydride.
The monomer derived from Msp can be produced using standard methods known in the
art. The monomer derived from Msp may be made synthetically or by recombinant means. For
example, the pore may be synthesised by in vitro translation and transcription (IVTT). Suitable
methods for producing pores are discussed in International Application Nos. PCT/GB09/001690
(published as WO 2010/004273), PCT/GB09/001679 (published as WO 2010/004265) or
PCT/GB 10/000 133 (published as WO 2010/086603). Methods for inserting pores into
membranes are discussed below.
For Exonuclease Sequencing, the transmembrane protein pore is preferably derived from
a-hemolysin (a-HL). The wild type a-HL pore is formed of seven identical monomers or
subunits (i.e. it is heptameric). The sequence of one monomer or subunit of a-hemolysin Ml 13R
is shown in SEQ ID NO: 4 . The transmembrane protein pore preferably comprises seven
monomers each comprising the sequence shown in SEQ ID NO: 4 or a variant thereof. Amino
acids 1, 7 to 21, 3 1 to 34, 45 to 51, 63 to 66, 72, 92 to 97, 104 to 111, 124 to 136, 149 to 153,
160 to 164, 173 to 206, 210 to 213, 217, 218, 223 to 228, 236 to 242, 262 to 265, 272 to 274, 287
to 290 and 294 of SEQ ID NO: 4 form loop regions. Residues 113 and 147 of SEQ ID NO: 4
form part of a constriction of the barrel or channel of a-HL.
In such embodiments, a pore comprising seven proteins or monomers each comprising
the sequence shown in SEQ ID NO: 4 or a variant thereof are preferably used in the method of
the invention. The seven proteins may be the same (homoheptamer) or different
(heteroheptamer).
A variant of SEQ ID NO: 4 is a protein that has an amino acid sequence which varies
from that of SEQ ID NO: 4 and which retains its pore forming ability. The ability of a variant to
form a pore can be assayed using any method known in the art. For instance, the variant may be
inserted into a lipid bilayer along with other appropriate subunits and its ability to oligomerise to
form a pore may be determined. Methods are known in the art for inserting subunits into
membranes, such as lipid bilayers. Suitable methods are discussed above.
The variant may include modifications that facilitate covalent attachment to or interaction
with a nucleic acid binding protein. The variant preferably comprises one or more reactive
cysteine residues that facilitate attachment to the nucleic acid binding protein. For instance, the
variant may include a cysteine at one or more of positions 8, 9, 17, 18, 19, 44, 45, 50, 51, 237,
239 and 287 and/or on the amino or carboxy terminus of SEQ ID NO: 4 . Preferred variants
comprise a substitution of the residue at position 8, 9, 17, 237, 239 and 287 of SEQ ID NO: 4
with cysteine (A8C, T9C, N17C, K237C, S239C or E287C). The variant is preferably any one
of the variants described in International Application No. PCT/GB09/001690 (published as WO
2010/004273), PCT/GB09/001679 (published as WO 2010/004265) or PCT/GB 10/000 133
(published as WO 2010/086603).
The variant may also include modifications that facilitate any interaction with nucleotides
or facilitate orientation of a molecular adaptor as discussed below. The variant may also contain
modifications that facilitate covalent attachment of a molecular adaptor.
In particular, the variant preferably contains a glutamine at position 139 of SEQ ID NO:
4. The variant preferably has a cysteine at position 119, 121 or 135 of SEQ ID NO: 4. A variant
of SEQ ID NO: 4 may have the wild-type methionine reintroduced at position 113.
Preferred variants of SEQ ID NO: 4 have a methionine at position 113 (Rl 13M), a
cysteine at position 135 (L135C) and a glutamine at position 139 (N139Q). Other preferred
variants of SEQ ID NO: 4 have a methionine at position 113 (Rl 13M) and a glutamine at
position 139 (N139Q). One such variant is shown in SEQ ID NO: 34. A preferred
transmembrane protein pore for use in Exonuclease Sequencing comprises (a) one monomer
comprising a variant of SEQ ID NO: 4 having a methionine at position 113 (Rl 13M), a cysteine
at position 135 (L135C) and a glutamine at position 139 (N139Q) and (b) six monomers each
comprising a variant of SEQ ID NO: 4 having a methionine at position 113 (Rl 13M) and a
glutamine at position 139 (N139Q). The six monomers in (b) each preferably comprise the
sequence shown in SEQ ID NO: 34.
The variant may be a naturally occurring variant which is expressed naturally by an
organism, for instance by a Staphylococcus bacterium. Alternatively, the variant may be
expressed in vitro or recombinantly by a bacterium such as Escherichia coli. Variants also
include non-naturally occurring variants produced by recombinant technology. Over the entire
length of the amino acid sequence of SEQ ID NO: 4, a variant will preferably be at least 50%
homologous to that sequence based on amino acid identity. More preferably, the variant
polypeptide may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous
based on amino acid identity to the amino acid sequence of SEQ ID NO: 4 over the entire
sequence. There maybe at least 80%, for example at least 85%, 90% or 95%, amino acid
identity over a stretch of 200 or more, for example 230, 250, 270 or 280 or more, contiguous
amino acids ("hard homology"). Homology can be determined as discussed above.
Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 4 in
addition to those discussed above, for example up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions.
Conservative substitutions may be made as discussed above.
One or more amino acid residues of the amino acid sequence of SEQ ID NO: 4 may
additionally be deleted from the polypeptides described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30
residues may be deleted, or more.
Variants may be fragments of SEQ ID NO: 4. Such fragments retain pore-forming
activity. Fragments may be at least 50, 100, 200 or 250 amino acids in length. A fragment
preferably comprises the pore-forming domain of SEQ ID NO: 4 . Fragments typically include
residues 119, 121, 135. 113 and 139 of SEQ ID NO: 4.
One or more amino acids may be alternatively or additionally added to the polypeptides
described above. An extension may be provided at the amino terminus or carboxy terminus of
the amino acid sequence of SEQ ID NO: 4 or a variant or fragment thereof. The extension may
be quite short, for example from 1 to 10 amino acids in length. Alternatively, the extension may
be longer, for example up to 50 or 100 amino acids. A carrier protein may be fused to a subunit
or variant.
One or more amino acids may be alternatively or additionally added to the polypeptides
described above. An extension may be provided at the amino terminus or carboxy terminus of
the amino acid sequence of SEQ ID NO: 4 or a variant or fragment thereof. The extension may
be quite short, for example from 1 to 10 amino acids in length. Alternatively, the extension may
be longer, for example up to 50 or 100 amino acids. A carrier protein may be fused to a pore or
variant.
As discussed above, a variant of SEQ ID NO: 4 is a subunit that has an amino acid
sequence which varies from that of SEQ ID NO: 4 and which retains its ability to form a pore. A
variant typically contains the regions of SEQ ID NO: 4 that are responsible for pore formation.
The pore forming ability of a-HL, which contains a b-barrel, is provided by b-strands in each
subunit. A variant of SEQ ID NO: 4 typically comprises the regions in SEQ ID NO: 4 that form
b-strands. The amino acids of SEQ ID NO: 4 that form b-strands are discussed above. One or
more modifications can be made to the regions of SEQ ID NO: 4 that form b-strands as long as
the resulting variant retains its ability to form a pore. Specific modifications that can be made to
the b-strand regions of SEQ ID NO: 4 are discussed above.
A variant of SEQ ID NO: 4 preferably includes one or more modifications, such as
substitutions, additions or deletions, within its a-helices and/or loop regions. Amino acids that
form a-helices and loops are discussed above.
The variant may be modified to assist its identification or purification as discussed above.
A particularly preferred pore for use in Exonuclease Sequencing comprises one subunit
shown in SEQ ID NO: 36 (i.e. a-HL-E287C-QC-D5FLAGH6) and six subunits shown in SEQ
ID NO: 34 (i.e. a-HL-Q).
Pores derived from a-HL can be made as discussed above with reference to pores derived
fromMsp.
In some embodiments, the transmembrane protein pore is chemically modified. The pore
can be chemically modified in any way and at any site. The transmembrane protein pore is
preferably chemically modified by attachment of a molecule to one or more cysteines (cysteine
linkage), attachment of a molecule to one or more lysines, attachment of a molecule to one or
more non-natural amino acids, enzyme modification of an epitope or modification of a terminus.
Suitable methods for carrying out such modifications are well-known in the art. The
transmembrane protein pore may be chemically modified by the attachment of any molecule.
For instance, the pore may be chemically modified by attachment of a dye or a fluorophore.
Any number of the monomers in the pore may be chemically modified. One or more,
such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the monomers is preferably chemically modified as
discussed above.
In some embodiments, the transmembrane protein pore comprises a molecular adaptor
that facilitates detection of the analyte. Pores for use in Exonuclease Sequencing typically
comprise a molecular adaptor.
The molecular adaptor may directly facilitate detection of the analyte by mediating an
interaction between the pore and the analyte. In such embodiments, the presence of the adaptor
improves the host-guest chemistry of the pore and the analyte and thereby improves the ability of
the pore to detect the analyte. The principles of host-guest chemistry are well-known in the art.
The adaptor has an effect on the physical or chemical properties of the pore that improves its
interaction with the analyte. The adaptor may alter the charge of the barrel or channel of the
pore or specifically interact with or bind to the analyte thereby facilitating its interaction with the
pore.
In other embodiments, the molecular adaptor indirectly facilitates detection of the analyte
by mediating an interaction between the pore and a product, such as a fragment, formed from
processing of the analyte. For instance, for Exonuclease Sequencing, the molecular adaptor
facilitates an interaction between the pore and individual nucleotides digested from the
polynucleotide analyte. In such embodiments, the presence of the adaptor improves the hostguest
chemistry of the pore and the individual nucleotides and thereby improves the ability of the
pore to detect the individual nucleotides. The adaptor has an effect on the physical or chemical
properties of the pore that improves its interaction with the individual nucleotides. The adaptor
may alter the charge of the barrel or channel of the pore or specifically interact with or bind to
the individual nucleotides thereby facilitating their interaction with the pore.
The molecular adaptor is preferably a cyclic molecule such as a cyclodextrin, a species
that is capable of hybridization, a DNA binder or interchelator, a peptide or peptide analogue, a
synthetic polymer, an aromatic planar molecule, a small positively-charged molecule or a small
molecule capable of hydrogen-bonding.
The adaptor may be cyclic. A cyclic adaptor preferably has the same symmetry as the
pore. The adaptor preferably has eight-fold symmetry if the pore is derived from Msp since Msp
typically has eight subunits around a central axis. The adaptor preferably has seven-fold
symmetry if the pore is derived from a-HL since a-HL typically has seven subunits around a
central axis. This is discussed in more detail below.
The adaptor typically interacts with the analyte via host-guest chemistry. The adaptor is
typically capable of interacting with a nucleotide or polynucleotide. The adaptor comprises one
or more chemical groups that are capable of interacting with the analyte, such as the nucleotide
or polynucleotide. The one or more chemical groups preferably interact with the analyte,
nucleotide or polynucleotide by non-covalent interactions, such as hydrophobic interactions,
hydrogen bonding, Van der Waal's forces, p-cation interactions and/or electrostatic forces. The
one or more chemical groups that are capable of interacting with the nucleotide or
polynucleotide are preferably positively charged. The one or more chemical groups that are
capable of interacting with the nucleotide or polynucleotide more preferably comprise amino
groups. The amino groups can be attached to primary, secondary or tertiary carbon atoms. The
adaptor even more preferably comprises a ring of amino groups, such as a ring of 6, 7 or 8 amino
groups. The adaptor most preferably comprises a ring of seven or eight amino groups. A ring of
protonated amino groups may interact with negatively charged phosphate groups in the
nucleotide or polynucleotide.

CLAIMS
1. A method for determining the presence, absence or characteristics of an analyte,
comprising (a) coupling the analyte to a membrane and (b) allowing the analyte to interact with a
detector present in the membrane and thereby determining the presence, absence or
characteristics of the analyte.
2. A method according to claim 1, wherein the membrane is an amphiphilic layer or a
solid state layer.
3. A method according to claim 1 or 2, wherein the membrane is a lipid bilayer.
4. A method according to any one of the preceding claims, wherein the analyte is
coupled to the membrane via a polypeptide or a hydrophobic anchor.
5. A method according to claim 4, wherein the hydrophobic anchor is a lipid, fatty acid,
sterol, carbon nanotube or amino acid.
6. A method according to any one of the preceding claims, wherein the analyte is
coupled to the membrane via a linker.
7. A method according to any one of the preceding claims wherein the analyte is
coupled transiently or permanently to the membrane.
8. A method according to any one of the preceding claims, wherein the detector detects
the analyte via electrical means.
9. A method according to any one of the preceding claims, wherein the detector
comprises a transmembrane pore.
10. A method according to claim 9, wherein the transmembrane pore is a transmembrane
protein pore.
11. A method according to claim 10, wherein the transmembrane protein pore is derived
from Msp or a-hemolysin (a-HL).
12. A method according to any one of claims 9 to 11, wherein the pore comprises a
molecular adaptor that facilitates detection of the analyte.
13. A method according to any one of the preceding claims, wherein the detector
comprises a polynucleotide binding protein, optionally an exonuclease or a polymerase.
14. A method according to any one of claims 9 to 13, wherein the method comprises:
(a) allowing the analyte to interact with the detector; and
(b) measuring the current passing through the pore during the interaction and thereby
determining the presence, absence or characteristics of the analyte.
15. A method according to any one of the preceding claims, wherein the method is for
identifying the analyte.
16. A method according to any one of the preceding claims, wherein the method is for
estimating the sequence of or sequencing a target polynucleotide.
17. A method according to claim 16, wherein the method comprises digesting a target
polynucleotide to provide a fragment and the fragment is detected.
18. A method of sequencing an analyte which is a target polynucleotide, comprising:
(a) coupling the target polynucleotide to a membrane;
(b) allowing the target polynucleotide to interact with a detector present in the membrane,
wherein the detector comprises a transmembrane pore and an exonuclease, such that the
exonuclease digests an individual nucleotide from one end of the target polynucleotide;
(c) allowing the nucleotide to interact with the pore;
(d) measuring the current passing through the pore during the interaction and thereby
determining the identity of the nucleotide; and
(e) repeating steps (b) to (d) at the same end of the target polynucleotide and thereby
determining the sequence of the target polynucleotide.
19. A method of sequencing an analyte which is a target polynucleotide, comprising:
(a) coupling the target polynucleotide to a membrane;
(b) allowing the target polynucleotide to interact with a detector present in the membrane,
wherein the detector comprises a transmembrane pore, such that the target polynucleotide moves
through the pore; and
(c) measuring the current passing through the pore as the target polynucleotide moves
with respect to the pore and thereby determining the sequence of the target polynucleotide.
20. A method according to claim 19, wherein the method comprises:
(a) coupling the target polynucleotide to a membrane;
(b) allowing the target polynucleotide to interact with a detector present in the membrane,
wherein the detector comprises a transmembrane pore and a polynucleotide binding protein, such
that the protein controls the movement of the target polynucleotide through the pore and
nucleotides in the target polynucleotide interact with the pore; and
(c) measuring the current passing through the pore as the target polynucleotide moves
with respect to the pore and thereby determining the sequence of the target polynucleotide.
2 1. A kit for sequencing an analyte which is a target polynucleotide comprising (a) a
transmembrane pore, (b) a polynucleotide binding protein and (c) means to couple the target
polynucleotide to a membrane.
22. A kit according to claim 21, wherein the polynucleotide binding protein is an
exonuclease and the kit further comprises a molecular adaptor that facilitates an interaction
between the pore and one or more nucleotides in the target polynucleotide.
23. An apparatus for sequencing an analyte which is a target polynucleotide, comprising (a) a
membrane, (b) a plurality of transmembrane pores in the membrane, (c) a plurality of
polynucleotide binding proteins and (d) a plurality of target polynucleotides coupled to the
membrane.
24. An apparatus according to claim 23, wherein the analysis apparatus comprises:
a sensor device that is capable of supporting the membrane and plurality of pores and
being operable to perform polynucleotide sequencing using the pores; and
at least one reservoir for holding material for performing the sequencing.
25. An apparatus according to claim 23 or 24, wherein the apparatus comprises:
a sensor device that is capable of supporting the membrane and plurality of pores and
being operable to perform polynucleotide sequencing using the pores;
at least one reservoir for holding material for performing the sequencing;
a fluidics system configured to controllably supply material from the at least one
reservoir to the sensor device; and
one or more containers for receiving respective samples, the fluidics system being
configured to supply the samples selectively from the one or more containers to the sensor
device.