ENZYME MUTANT
Field of the invention
The invention relates to constructs comprising a nucleic acid binding protein and a
surface. At least one native accessible cysteine residue is removed from the binding protein.
The binding protein is attached to the surface via one or more accessible cysteine residues.
The removal of other accessible cysteine residues from the protein allows control attachment
to the surface. The constructs can be used to generate transmembrane pores having a nucleic
acid binding protein attached thereto. Such pores are particularly useful for sequencing
nucleic acids. The enzyme handles the nucleic acid in such a way that the pore can detect
each of its component nucleotides by stochastic sensing.
Background of the invention
Stochastic detection is an approach to sensing that relies on the observation of
individual binding events between analyte molecules and a receptor. Stochastic sensors can be
created by placing a single pore of nanometer dimensions in an insulating membrane and
measuring voltage-driven ionic transport through the pore in the presence of analyte
molecules. The frequency of occurrence of fluctuations in the current reveals the
concentration of an analyte that binds within the pore. The identity of an analyte is revealed
through its distinctive current signature, notably the duration and extent of current block
(Braha, O., Walker, B., Cheley, S., Kasianowicz, J. J., Song, L., Gouaux, J. E., and Bayley, H.
(1997) Chem.Biol. 4, 497-505; and Bayley, H., and Cremer, P. S. (2001) Nature 413, 226-
230).
Engineered versions of the bacterial pore forming toxin a-hemolysin (a-HL) have
been used for stochastic sensing of many classes of molecules (Bayley, H., and Cremer, P. S.
(2001) Nature 413. 226-230; Shin, S., H., Luchian, T., Cheley, S., Braha, O., and Bayley, H.
(2002) Angew.Chem.Int.Ed. 41, 3707-3709; and Guan, X., Gu, L.-Q., Cheley, S., Braha, O.,
and Bayley, H. (2005) ChemBioChem 6, 1875-1881). In the course of these studies, it was
found that attempts to engineer a-HL to bind small organic analytes directly can prove taxing,
with rare examples of success (Guan, X., Gu, L.-Q., Cheley, S., Braha, O., and Bayley, H.
(2005) ChemBioChem 6, 1875-1881). Fortunately, a different strategy was discovered, which
utilised non-covalently attached molecular adaptors, notably cyclodextrins (Gu, L.-Q., Braha,
O., Conlan, S., Cheley, S., and Bayley, H. (1999) Nature 398, 686-690), but also cyclic
peptides (Sanchez-Quesada, J.. Ghadiri, M. R., Bayley, H., and Braha, O. (2000)

J.Am.Chem.Soc. 122, 11758-11766) and cucurbiturils (Braha, O., Webb, J., Gu, L.-Q., Kim,
K., and Bayley, H. (2005) ChemPhysChem 6, 889-892). Cyclodextrins become transiently
lodged in the α-HL pore and produce a substantial but incomplete channel block. Organic
analytes, which bind within the hydrophobic interiors of cyclodextrins, augment this block
allowing analyte detection (Gu, L.-Q., Braha, O., Conlan, S., Cheley, S., and Bayley, H.
(1999) Nature 398, 686-690).
There is currently a need for rapid and cheap DNA or RNA 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. Stochastic
sensing has the potential to provide rapid and cheap DNA sequencing by reducing the quantity
of nucleotide and reagents required.
Summary of the invention
The inventors have surprisingly demonstrated that a nucleic acid binding protein
remains functional if all or all but one its native accessible cysteine residues are removed by
substitution. The inventors have also shown that generation of a nucleic acid binding protein
with only one or more accessible cysteine residues allows the controlled attachment of the
protein to a surface. The one or more accessible cysteine residues may be non-native residues
introduced by mutagenesis. Alternatively, the one or more accessible residues may be native
residues remaining once the other native accessible cysteine residues are removed. The
invention therefore provides a construct comprising a nucleic acid binding protein and a
surface, wherein at least one native accessible cysteine residue is removed from the binding
protein, wherein the binding protein is attached to the surface via one or more accessible
cysteine residues and wherein the binding protein retains its ability to bind nucleic acids.
The nucleic acid binding protein is preferably a nucleic acid handling enzyme. The surface is
preferably a pore or a pore subunit.
The inventors have also surprisingly demonstrated that the constructs of the invention
can be used to generate transmembrane pores that are capable of both binding a nucleic acid
and sequencing the nucleic acid via stochastic sensing. The fixed nature and close proximity
of the nucleic acid binding protein to the pore means that a proportion of the nucleotides in a
target nucleic acid will interact with the pore and affect the current flowing through the pore in
a distinctive manner. As a result, transmembrane pores comprising such constructs are useful
tools for stochastic sensing and especially for sequencing nucleic acids.

Accordingly, the invention also provides:
a modified pore for use in sequencing nucleic acids, comprising at least one construct
of the invention;
a kit for producing a modified pore for use in sequencing nucleic acids, comprising:
(a) at least one construct of the invention in which the surface is a pore subunit;
and
(b) the remaining subunits needed to form a pore;
a method of producing a construct of the invention, comprising:
(a) attaching a nucleic acid binding protein comprising one or more accessible
cysteine residues to a surface via the cysteine residues; and
(b) determining whether or not the resulting construct is capable of binding
nucleic acids;
a method of producing a modified pore of the invention, comprising:
(a) allowing at least one construct of the invention in which the surface is a
pore subunit to form a pore with other suitable subunits; and
(b) determining whether or not the resulting pore is capable of binding nucleic
acids and detecting nucleotides;
a method of sequencing a target nucleic acid sequence, comprising:
(a) contacting the target sequence with a pore of the invention, which comprises
an exonuclease, such that the exonuclease digests an individual nucleotide from
one end of the target sequence;
(b) contacting the nucleotide with the pore so that the nucleotide interacts with
the adaptor;
(c) measuring the current passing through the pore during the interaction and
thereby determining the identity of the nucleotide; and
(d) repeating steps (a) to (c) at the same end of the target sequence and thereby
determining the sequence of the target sequence;
a method of sequencing a target nucleic acid sequence, comprising:
(a) contacting the target sequence with a pore of the invention comprising a
nucleic acid handling enzyme so that the enzyme pushes or pulls the target
sequence through the pore and a proportion of the nucleotides in the target
sequence interacts with the pore; and
(b) measuring the current passing through the pore during each interaction and
thereby determining the sequence of the target sequence;

an exonuclease enzyme comprising the sequence shown in any one of SEQ ID NOs: 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46,48 and 50 or a variant
thereof; and
a polynucleotide sequence which encodes an exonuclease enzyme of the invention.
Description of the Figures
Figure 1 shows a cartoon of the structure of EcoExo I showing the position and residue
number of naturally occurring cysteines (obtained from crystal structure).
Figure 2 shows a surface diagram of EcoExo I showing the position of accessible
cysteines (obtained from crystal structure).
Figure 3 shows the relative activity of Exo I mutants compared to the wild type.
Figure 4 shows a diagram of EcoExo I showing the positions at which a single cysteine
could be introduced for coupling to a protein nanopore
Figures 5a and 5b show the relative activity of Exo I mutants compared to the wild
type.
Figure 6 shows a schematic representation of the Exonuclease I assay.
Figure 7 shows an example of data from the Exonuclease I assay.
Figure 8 shows that the attachment of a linker to Exo-ATTTT-A83C does not affect
enzyme activity. The Y-axis is relative activity. The left-hand pair of columns is ONLP1403
and the right-hand pair of columns is ONLP1405. In each pair, the left-hand (darker) column
is the enzyme with linker and the right-hand (lighter column) is enzyme without linker.
Figure 9 shows that the attachment of PNA to Exo-ATTTT-A83C does not
affect enzyme activity. The Y-axis is relative activity. The left-hand column is ONLP1498
and the right-hand column is ONLP1499. Figure 10 shows that the attachment of a PEG
linker to Exo-ATTTT or Exo-ATTTT-M184C does not affect enzyme activity. The Y-axis is
relative activity. The left-hand pair of columns is Exo-ATTTT and the right-hand pair of
columns is Exo-ATTTT-M184C. In each pair, the left-hand (darker) column is the enzyme
with linker and the right-hand (lighter column) is enzyme without linker.
Description of the Sequence Listing
SEQ ID NO: 1 shows the polynucleotide sequence encoding one subunit of wild type
α-hemolysin (α-HL).
SEQ ID NO: 2 shows the amino acid sequence of one subunit of wild type α-HL.
Amino acids 2 to 6, 73 to 75, 207 to 209, 214 to 216 and 219 to 222 form α-helices. Amino

acids 22 to 30, 35 to 44, 52 to 62, 67 to 71, 76 to 91, 98 to 103, 112 to 123, 137 to 148, 154 to
159, 165 to 172, 229 to 235, 243 to 261, 266 to 271, 285 to 286 and 291 to 293 form β-strands.
All the other non-terminal amino acids, namely 7 to 21, 31 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 and 287 to 290 form loop regions. Amino acids 1 and
294 are terminal amino acids.
SEQ ID NO: 3 shows the polynucleotide sequence encoding one subunit of α-HL
L135C/N139Q(HL-CQ).
SEQ ID NO: 4 shows the amino acid sequence of one subunit of α-HL L135C/N139Q
(HL-CQ). The same amino acids that form α-helices, β-strands and loop regions in wild type
α-HL form the corresponding regions in this subunit.
SEQ ID NO: 5 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: 6 shows the amino acid sequence of exonuclease I enzyme (EcoExo I)
from E. coli. This enzyme performs processive digestion of 5' monophosphate nucleosides
from single stranded DNA (ssDNA) in a 5' to 3' direction. Amino acids 60 to 68, 70 to 78, 80
to 93, 107 to 119, 124 to 128,137 to 148,165 to 172, 182 to 211, 213 to 221, 234 to 241, 268
to 286, 313 to 324. 326 to 352, 362 to 370, 373 to 391, 401 to 454 and 457 to 475 form ot-
helices. Amino acids 10 to 18, 28 to 26, 47 to 50, 97 to 101, 133 to 136, 229 to 232, 243 to
251. 258 to 263,298 to 302 and 308 to 311 form (3-strands. All the other non-terminal amino
acids, 19 to 27, 37 to 46, 51 to 59, 69, 79, 94 to 96102 to 106,120 to 123, 129 to 132,149 to
164,173 to 181,212, 222 to 228 233,242, 252 to 257, 264 to 267, 287 to 297, 303 to 307,
312, 325, 353 to 361, 371, 372, 392 to 400, 455 and 456, form loops. Amino acids 1 to 9 are
terminal amino acids. The overall fold of the enzyme is such that three regions combine to
form a molecule with the appearance of the letter C, although residues 355 - 358, disordered
in the crystal structure, effectively convert this C into an O-like shape. The amino terminus
(1 -206) forms the exonuclease domain and has homology to the DnaQ superfamily, the
following residues (202-354) form an SH3-like domain and the carboxyl domain (359-475)
extends the exonuclease domain to form the C-like shape of the molecule. Four acidic residues
of EcoExo I are conserved with the active site residues of the DnaQ superfamily
(corresponding to D15, E17, D108 and D186). It is suggested a single metal ion is bound by
residues D15 and 108. Hydrolysis of DNA is likely catalyzed by attack of the scissile
phosphate with an activated water molecule, with H181 being the catalytic residue and
aligning the nucleotide substrate.

SEQ ID NO: 7 shows the codon optimised polynucleotide sequence encoding EcoExo
I C98S/C306S/C330T/C51A (ONLD0393).
SEQ ID NO: 8 shows the amino acid sequence of EcoExo I C98S/C306S/C330T/C51A
(ONLD0393).
SEQ ID NO: 9 shows the codon optimised polynucleotide sequence encoding EcoExo
I C98S/C306S/C330T/C144M (ONLD0403).
SEQ ID NO: 10 shows the amino acid sequence of EcoExo I
C98S/C306S/C330T/C144M (ONLD0403).
SEQ ID NO: 11 shows the codon optimised polynucleotide sequence encoding EcoExo
I C98S/C306S/C330T/C144T (ONLD0404).
SEQ ID NO: 12 shows the amino acid sequence of EcoExo I
C98S/C306S/C330T/C144T(ONLD0404).
SEQ ID NO: 13 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98S/C144M/C306S/C330T/V42C (ONLD0415).
SEQ ID NO: 14 shows the amino acid sequence of EcoExo I
C51A/C98S/C144M/C306S/C330T/V42C (ONLD0415).
SEQ ID NO: 15 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98S/C144T/C306S/C330T/V42C (ONLD0416).
SEQ ID NO: 16 shows the amino acid sequence of EcoExo I
C51A/C98S/C144T/C306S/C330T/V42C (ONLD0416).
SEQ ID NO: 17 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98S/C144M/C306S/C330T/M184C (ONLD0417).
SEQ ID NO: 18 shows the amino acid sequence of EcoExo I
C51A/C98S/C144M/C306S/C330T/M184C (ONLD0417).
SEQ ID NO: 19 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98S/C144T/C306S/C330T/M184C (ONLD0418).
SEQ ID NO: 20 shows the amino acid sequence of EcoExo I
C51A/C98S/C144T/C306S/C330T/M184C (ONLD0418).
SEQ ID NO: 21 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98S/C144T/C306S/C330T (ONLD0411).
SEQ ID NO: 22 shows the amino acid sequence of EcoExo I
C51A/C98S/C144T/C306S/C330T(ONLD0411).
SEQ ID NO: 23 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98T/C144T/C306S/C330T (ONLD0432).

SEQ ID NO: 24 shows the amino acid sequence of EcoExo I
C51A/C98T/C144T/C306S/C330T(ONLD0432).
SEQ ID NO: 25 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98T/C144T/C306T/C330T (ONLD0433).
SEQ ID NO: 26 shows the amino acid sequence of EcoExo I
C51A/C98T/C144T/C306T/C330T(ONLD0433).
SEQ ID NO: 27 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98T/C144M/C306S/C330T (ONLD0451).
SEQ ID NO: 28 shows the amino acid sequence of EcoExo I
C51A/C98T/C144M/C306S/C330T(ONLD0451).
SEQ ID NO: 29 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98T/C144M/C306T/C330T (ONLD0452).
SEQ ID NO: 30 shows the amino acid sequence of EcoExo I
C51A/C98T/C144M/C306T/C330T (ONLD0452).
SEQ ID NO: 31 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98G/C144T/C306S/C330T (ONLD0453).
SEQ ID NO: 32 shows the amino acid sequence of EcoExo I
C51A/C98G/C144T/C306S/C330T(ONLD0453).
SEQ ID NO: 33 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98D/C144T/C306S/C330T (ONLD0491).
SEQ ID NO: 34 shows the amino acid sequence of EcoExo I
C51A/C98D/C144T/C306S/C330T(ONLD0491).
SEQ ID NO: 35 shows the codon optimised polynucleotide sequence encoding EcoExo
1 C51A/C98K/C144T/C306S/C330T (ONLD0454).
SEQ ID NO: 36 shows the amino acid sequence of EcoExo I
C51A/C98K/C144T/C306S/C330T (ONLD0454).
SEQ ID NO: 37 shows the codon optimised polynucleotide sequence encoding EcoExo
1 C51A/C98L/C144T/C306S/C330T (ONLD0455).
SEQ ID NO: 38 shows the amino acid sequence of EcoExo I
C51A/C98L/C144T/C306S/C330T(ONLD0455).
SEQ ID NO: 39 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98V/C144T/C306S/C330T (ONLD0456).
SEQ ID NO: 40 shows the amino acid sequence of EcoExo I
C51A/C98V/C144T/C306S/C330T(ONLD0456).

SEQ ID NO: 41 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98V/C144T/C306T/C330T (ONLD0476).
SEQ ID NO: 42 shows the amino acid sequence of EcoExo I
C51A/C98V/C144T/C306T/C330T(ONLD0476).
SEQ ID NO: 43 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98T/C144T/C306M/C330T (ONLD0477).
SEQ ID NO: 44 shows the amino acid sequence of EcoExo I
C51A/C98T/C144T/C306M/C330T(ONLD0477).
SEQ ID NO: 45 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98T/C144T/C306N/C330T (ONLD0478).
SEQ ID NO: 46 shows the amino acid sequence of EcoExo I
C51A/C98T/C144T/C306N/C330T (ONLD0478).
SEQ ID NO: 47 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98T/C144T/C306D/C330T (ONLD0479).
SEQ ID NO: 48 shows the amino acid sequence of EcoExo I
C51A/C98T/C144T/C306D/C330T(ONLD0479).
SEQ ID NO: 49 shows the codon optimised polynucleotide sequence encoding EcoExo
I C51A/C98T/C144T/C306A/C330T (ONLD0480).
SEQ ID NO: 50 shows the amino acid sequence of EcoExo I
C51A/C98T/C144T/C306A/C330T(ONLD0480).
In all of the mutants described in SEQ ID NOs: 7 to 50, the same amino acids that form
α-helices, B-strands and loop regions in wild type EcoExo I form the corresponding regions in
this mutant.
SEQ ID NO: 51 shows the codon optimised polynucleotide sequence derived from the
xthA gene from E. coli. It encodes the exonuclease III enzyme from E. coli.
SEQ ID NO: 52 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. Amino acids 11 to 13, 15 to 25, 39 to
41, 44 to 49, 85 to 89, 121 to 139, 158 to 160, 165 to 174, 181 to 194, 198 to 202, 219 to 222,
235 to 240 and 248 to 252 form α-helices. Amino acids 2 to 7, 29 to 33, 53 to 57, 65 to 70, 75
to 78, 91 to 98, 101 to 109, 146 to 151, 195 to 197, 229 to 234 and 241 to 246 form β-strands.
All the other non-terminal amino acids, 8 to 10, 26 to 28, 34 to 38, 42, 43, 50 to 52, 58 to 64,
71 to 74, 79 to 84, 90, 99, 100, 110 to 120, 140 to 145, 152 to 157, 161 to 164, 175 to 180, 203

to 218, 223 to 228, 247 and 253 to 261, form loops. Amino acids 1, 267 and 268 are terminal
amino acids. The enzyme active site is formed by loop regions connecting β1 - α1, β3 - β4, β5
- β6, βIII- α1, βIV - αII and βv - βIV (consisting of amino acids 8 - 10, 58 - 64, 90, 110 - 120,
152 - 164, 175 - 180, 223 - 228 and 253 - 261 respectively). A single divalent metal ion is
bound at residue E34 and aids nucleophihc attack on the phosphodiester bond by the D229 and
H259 histidine-aspartate catalytic pair.
SEQ ID NO: 53 shows the codon optimised polynucleotide sequence derived from the
recJ gene from T. thermophilus. It encodes the RecJ enzyme from T. thermophilus (TthRecJ-
cd).
SEQ ID NO: 54 shows the amino acid sequence of the RecJ enzyme from T.
thermophilus (TthRecJ-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. Amino acids 19 to 33, 44 to 61, 80 to 89, 103 to 111, 136 to 140, 148 to 163,
169 to 183, 189 to 202, 207 to 217, 223 to 240, 242 to 252, 254 to 287, 302 to 318, 338 to 350
and 365 to 382 form α-helices. Amino acids 36 to 40, 64 to 68, 93 to 96, 116 to 120, 133 to
135, 294 to 297, 321 to 325, 328 to 332, 352 to 355 and 359 to 363 form p-strands. All the
other non-terminal amino acids, 34, 35, 41 to 43, 62, 63, 69 to 79, 90 to 92, 97 to 102, 112 to
115,121 to 132, 141 to 147, 164 to 168, 184 to 188203 to 206, 218 to 222, 241, 253, 288 to
293. 298 to 301, 319, 320, 326, 327, 333 to 337, 351 to 358 and 364, form loops. Amino acids
1 to 18 and 383 to 425 are terminal amino acids. The crystal structure has only been resolved
for the core domain of RecJ from Thermus thermophilus (residues 40 - 463). To ensure
initiation of translation and in vivo expression of the RecJ core domain a methionine residue
was added at its amino terminus, this is absent from the crystal structure information. The
resolved structure shows two domains, an amino (2-253) and a carboxyl (288-463) region,
connected by a long a-helix (254-287). The catalytic residues (D46, D98, H122, and D183)
co-ordinate a single divalent metal ion for nucleophihc attack on the phosphodiester bond.
D46 and H120 proposed to be the catalytic pair; however, mutation of any of these conserved
residues in the E. coli RecJ was shown to abolish activity.
SEQ ID NO: 55 shows the codon optimised polynucleotide sequence derived from the
bacteriphage lambda exo (redX) gene. It encodes the bacteriophage lambda exonuclease.
SEQ ID NO: 56 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. Amino acids 3 to 10, 14 to 16, 22 to 26, 34 to 40, 52 to 67, 75 to 95,135 to
149, 152 to 165 and 193 to 216 form α-helices. Amino acids 100 to 101, 106 to 107, 114 to
116, 120 to 122, 127 to 131, 169 to 175 and 184 to 190 form β-strands. All the other non-
terminal amino acids, 11 to 13, 17 to 21, 27 to 33, 41 to 51, 68 to 74, 96 to 99, 102 to 105, 108
to 113, 117 to 119, 123 to 126, 132 to 134, 150 to 151, 166 to 168, 176 to 183, 191 to 192,217
to 222, form loops. Amino acids 1, 2 and 226 are terminal amino acids. Lambda exonuclease
is a homo-trimer that forms a toroid with a tapered channel through the middle, apparently
large enough for dsDNA to enter at one end and only ssDNA to exit at the other. The catalytic
residues are undetermined but a single divalent metal ion appears bound at each subunit by
residues D119, E129 and L130.
SEQ ID NO: 57 shows the nucleic sequence from which preferred nucleic acid linkers
can be generated.
SEQ ID NO: 58 shows a preferred nucleic acid linker. MAL is maleimide. This linker
is used in combination with SEQ ID NO: 61.
SEQ ID NO: 59 shows a preferred nucleic acid linker. MAL is maleimide. This linker
is used in combination with SEQ ID NO: 62.
SEQ ID NO: 60 shows a preferred nucleic acid linker. MAL is maleimide. This linker
is used in combination with SEQ ID NO: 63.
SEQ ID NO: 61 shows a preferred nucleic acid linker. MAL is maleimide. This linker
is complementary to and used in combination with SEQ ID NO: 58.
SEQ ID NO: 62 shows a preferred nucleic acid linker. MAL is maleimide. This linker
is complementary to and used in combination with SEQ ID NO: 59.
SEQ ID NO: 63 shows a preferred nucleic acid linker. MAL is maleimide. This linker
is complementary to and used in combination with SEQ ID NO: 60.
SEQ ID NO: 64 shows a preferred nucleic acid linker. This linker is used in
combination with SEQ ID NO: 65.
SEQ ID NO: 65 shows a preferred nucleic acid linker. This linker is used in
combination with SEQ ID NO: 64.
SEQ ID NO: 66 shows a preferred nucleic acid linker. This linker is used in
combination with SEQ ID NO: 68.
SEQ ID NO: 67 shows a preferred nucleic acid linker. This linker is used in
combination with SEQ ID NO: 69.

SEQ ID NO: 68 shows a preferred nucleic acid linker. This linker is complementary to
and used in combination with SEQ ID NO: 66.
SEQ ID NO: 69 shows a preferred nucleic acid linker. This linker is complementary to
and used in combination with SEQ ID NO: 67.
SEQ ID NO: 70 shows a preferred nucleic acid linker. This linker is complementary to
and used in combination with SEQ ID NO: 73.
SEQ ID NO: 71 shows a preferred nucleic acid linker. This linker is complementary to
and used in combination with SEQ ID NO: 74.
SEQ ID NO: 72 shows a preferred nucleic acid linker. This linker is used in
combination with SEQ ID NO: 75.
SEQ ID NO: 73 shows a preferred nucleic acid linker. This linker is used in
combination with SEQ ID NO: 70.
SEQ ID NO: 74 shows a preferred nucleic acid linker. This linker is used in
combination with SEQ ID NO: 71.
SEQ ID NO: 75 shows a preferred nucleic acid linker. This linker is complementary to
and used in combination with SEQ ID NO: 72.
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 "a construct" includes "constructs", reference to "a
transmembrane protein pore" includes two or more such pores, reference to "a molecular
adaptor" includes two or more such adaptors, and the like.
All publications, patents and patent applications cited herein, whether supra or infra,
are hereby incorporated by reference in their entirety.
Constructs
The invention provides constructs that are useful for binding nucleic acids. In
particular, the invention provides constructs that are useful for handling nucleic acids, such as
when sequencing nucleic acids. The constructs comprise a nucleic acid binding protein and a

surface. At least one native accessible cysteine residue is removed from the nucleic acid
binding protein. However, the nucleic acid binding protein comprises one or more accessible
cysteine residues and is attached to the surface via those residues. The presence of a limited
number of accessible cysteine residues allows controlled attachment to the surface.
The ionisable side chain of cysteine residues is a potent nucleophile for engaging in
addition reactions and so is a popular choice for use in bioconjugation techniques. However, a
common barrier to the effective use of these techniques is the native cysteine residues present
in wild type proteins. Modification of one or more of the native cysteine residues can cause
both ambiguous enzyme activity and uncontrolled linkage.
The invention concerns site directed mutagenesis of native cysteine residues from
nucleic acid binding protein. All but one or more of the accessible native cysteines can be
removed. Alternatively, all the accessible cysteine residues can be removed and one or more
cysteine residues can be introduced to the protein. The presence of specific accessible
cysteine residues facilitates the attachment of the binding protein to a surface, such as another
protein, a solid support or a chemical reagent.
Removal of native cysteine residues and targeted addition of single or multiple
cysteine residue improves on previous methodologies as it conveys the ability to control
attachment, while also minimising the possible interaction of the nucleic acid binding proteins
with one another to form dimers, trimers and the like via undesired surface thiols. If the
protein contains only a single cysteine residue at a designed position in its structure, then the
thiol group of that residue can be used to form either a direct disulphide bond with other
sulfhydryl groups or coupling can be mediated by a reactive linker. These targeted cysteine
residues ensure only the desired number of enzyme molecules cross-link to the desired number
of target molecules, whilst also conferring a degree of favourable conformation, so that for
instance active sites can be optimally orientated.
The native cysteine residues present in the wild type enzyme do not readily permit the
use of specific linkers containing thiol reactive groups, such as maleimide, iodoactemide or
ortho-pyridyl disulphide (OPSS). The potential for reaction with any structurally or
catalytically important cysteine residues could impact upon the binding protein's activity,
which is of particular importance for use in single molecule applications. The use of linkers
for bioconjugation is preferred to ensure some spatial separation so that binding protein
activity is not affected by being in close proximity to another protein or a surface, such as
solid support.

The surface is preferably a pore or a pore subunit. The constructs of the invention are
therefore useful tools for forming pores that are capable of sequencing nucleic acids by
stochastic sensing. The constructs of the invention are particularly useful for generating
transmembrane pores that can both bind a target nucleic acid sequence and discriminate
between the different nucleotides in the target sequence. As described in more detail below,
the nucleic acid binding protein preferably handles a target nucleic acid in such a way that the
pore can identify nucleotides in the target sequence and thereby sequence the target sequence.
In particular, the constructs of the invention not only allow co-localisation of a nucleic acid
binding protein, such as a nucleic acid handling enzyme, to a nanopore, but also the enzyme
active site to be orientated to optimise base translocation once catalysis has occurred.
The surface does not have to be a pore or pore subunit. Other sequencing techniques
commonly rely on nucleic acids being immobilised onto a solid support, such as a bead, before
an enzyme binds from solution and metal ions are added to trigger catalytic activity.
Liberated bases can then be detected downstream in a number of manners. However, one
barrier to this method is the time consuming and expensive amplification and subsequent
chemical modification required in order to couple the template nucleic acid to the bead
surface. Having a nucleic acid binding protein pre-attached to a bead minimises the need for
any nucleic acid modification. Existing methods of enzyme attachment would not be suitable
as they rely on the undirected bulk attachment of nucleic binding proteins and so are highly
variable in terms of the amount of attached protein and the activity of the attached protein due
to the unknown orientation or spatial separation from the surface. The constructs of the
invention allow controlled addition of a single nucleic acid binding protein to a surface and
therefore vastly improve on this sequencing method.
One important class of nucleic acid binding proteins is the exonucleases. The common
use for exonucleases in molecular biology is the removal of the amplification primers from
PCR reactions to allow the direct use of the product for sequencing by addition of a specific
sequencing primer. There is still a requirement however for the exonuclease to be either
denatured or removed in order to prevent degradation of the sequencing primer. A method for
the rapid and easy removal of the exonuclease at this stage is therefore desirable both to aid in
the automation of this process, as well as reduce costly DNA purification procedures. A
construct of the invention comprising an exonuclease attached to a surface would be ideal for
such applications. The problems associated with the existing methods of attaching
exonucleases to a surface, such as a glass slide, the bottom of a well or a bead, have already
been outlined above. The covalent coupling of an exonuclease to a surface, such as an agarose

bead, through a single cysteine residue would vastly improve upon known methods, as well as
help any potential enzyme solubility problems. In addition, as can be seen from the Example,
removal of native cysteine residues is capable of producing proteins with an altered activity
and stability. An exonuclease with a high activity, but lowered stability may also aid in the
removal of the enzyme activity from the PCR reaction. Similar potential applications for
many other commercially important enzymes should also be considered.
The constructs of the invention may be isolated, substantially isolated, purified or
substantially purified. A construct is isolated or purified if it is completely free of any other
components, such as lipids or other pore monomers. A construct is substantially isolated if it
is mixed with carriers or diluents which will not interfere with its intended use. For instance, a
construct is substantially isolated or substantially purified if it present in a form that comprises
less than 10%, less than 5%, less than 2% or less than 1% of other components, such as lipids
or other pore monomers. A construct of the invention may be present in a lipid bilayer.
Mutagenesis of the cysteine residues
Constructs of the invention comprise a nucleic acid binding protein in which at least
one accessible cysteine residue is removed. The protein also has one or more accessible
cysteine residues. Accessible cysteine residues are residues that are available for reaction with
a thiol specific group. Methods for determining whether cysteine residues are available for
reaction with a thiol specific group are well known in the art. Accessible cysteine residues are
typically present on the accessible surface of the protein. Accessible cysteine residues are
cysteine residues that are not buried within the binding protein and/or do not form an
intramolecular disulphide bond. Cysteine residues that are buried within the binding protein
and/or form intramolecular disulphide bonds do not need to be removed from the binding
protein because they will not interfere with attachment to the surface. Cysteine residues can
of course form disulphide bonds with other cysteine residues in the protein or with other
chemical conjugates containing a reactive thiol group.
Accessible cysteine residues can be mapped using sequence information and molecular
modeling. Methods for such modeling are known in the art. One method is also described in
the Example. Once the accessible cysteine residues have been mapped, at least one of them
can be removed as described below.
Any number of native accessible cysteine residues, such as two, five, ten or more, can
be removed from the binding protein. In some embodiments, all the native accessible cysteine

residues are removed from the nucleic acid binding protein. This is discussed in more detail
below.
Native accessible cysteine residues can be removed using any method known in the art.
Native residues are residues that are present in the native or wild type protein. Native cysteine
residues can be removed by deletion. Native cysteine residues are preferably removed by
substitution. Native cysteine residues can be substituted with naturally occurring residues or
non-natural ly occurring residues. Native cysteine residues are typically substituted with
residues lacking a reactive thiol group. Native cysteine residues are preferably substituted
with residues having a similar structure. Suitable residues for replacing cysteine residues
include, but are not limited to, alanine, serine, threonine, methionine and valine. Cysteine
residues are most preferably substituted with threonine. The number of accessible cysteines
removed from the nucleic acid binding protein will of course depend on the number present in
the native protein.
In one embodiment, all the native accessible cysteine residues are removed from the
binding protein and one or more one, such as two, three, four, five or more, non-native
accessible cysteine residue are introduced into the binding protein. Non-native residues are
residues that are not present in the native or wild type protein. Non-native cysteine residues
can be introduced by addition. Non-native cysteine residues are preferably introduced by
substitution. Non-native cysteine residues are typically introduced in place of residues lacking
a reactive thiol group. Any residue, such as methionine, valine, serine or alanine, can be
replaced with cysteine.
In another embodiment, all but one or more of the native accessible cysteine residues
are removed from the binding protein. In this embodiment, the nucleic acid binding protein
comprises one or more, such as two, three, four, five or more, native accessible cysteine
residues. Preferably, all but one or all but two of the native accessible cysteine residues are
removed from the binding protein. The remaining native accessible cysteine residues are used
to attach the binding protein to the surface in a specific manner. In this embodiment, the
nucleic acid binding protein may further comprise one or more, such as two, three, four, five
or more, non-native accessible cysteine residues introduced as described above. The nucleic
acid binding protein may be attached to the surface by the native residues, the non-native
residues or both the native and non-native residues.
In preferred embodiments, the nucleic acid binding protein comprises only one or only
two accessible cysteine residues. The cysteine residues may be native, non-native or a
combination of the two as described above. The nucleic acid protein is attached to the surface

via these cysteine residues. The presence of only one or only two accessible cysteine residues
can be determined using any method known in the art. For instance, the nucleic acid binding
protein can be reacted with conjugates containing a reactive thiol group and the number of
conjugates attached to the protein can be determined. Methods for carrying out such reactions
are discussed in more detail below. A nucleic acid binding protein comprises only one
accessible cysteine residue if only one conjugate is attached to the protein following such a
reaction. A nucleic acid binding protein comprises only two accessible cysteine residues if
only two conjugates are attached to the protein following such a reaction.
The nucleic acid binding protein retains its ability to bind nucleic acids. This allows the
construct to bind nucleic acids and preferably sequence nucleic acids as described below. The
ability of a construct to bind nucleic acids can be assayed using any method known in the art.
For instance, constructs or pores formed from the constructs can be tested for their ability to
bind specific sequences of nucleic acids. The ability of a construct or a pore to bind nucleic
acids is typically assayed as described in the Example.
The nucleic acid binding protein also retains its ability to be expressed using standard
techniques. An inability to express the protein once one or more native cysteine residues are
removed of course means that the protein cannot be attached to the surface. It is
straightforward to determine whether a particular mutant can be expressed or not. The ability
of a nucleic acid binding protein to be expressed is typically assayed as described in the
Example.
Nucleic acid binding protein
The constructs of the invention comprise a nucleic acid binding protein. Examples of
such proteins include, but are not limited to, nucleic acid handling enzymes, such as nucleases,
polymerases, topoisomerases, ligases and helicases, and non-catalytic binding proteins such as
those classified by SCOP (Structural Classification of Proteins) under the Nucleic acid-binding
protein superfamily (50249). The nucleic acid binding protein is modified to remove and/or
replace cysteine residues as described above.
A nucleic acid is a macromolecule comprising two or more nucleotides. The nucleic
acid bound by the protein may comprise any combination of any nucleotides. The nucleotides
can be naturally occurring or artificial. The nucleotide can be oxidized or methylated. A
nucleotide typically contains a nucleobase, a sugar and at least one phosphate group. The
nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and
pyrimidines and more specifically adenine, guanine, thymine, uracil and cytosine. The sugar

is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and
deoxyribose. The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The
nucleotide typically contains a monophosphate, diphosphate or triphosphate. Phosphates may
be attached on the 5' or 3' side of a nucleotide.
Nucleotides include, but are not limited to, adenosine monophosphate (AMP),
adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate
(GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine
monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine
monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine
monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic
adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP),
deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP),
deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP),
deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine
monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate
(dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP),
deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine
diphosphate (dCDP) and deoxycytidine triphosphate (dCTP). The nucleotides are preferably
selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP.
The nucleic acid can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The
nucleic acid may be any synthetic nucleic acid known in the art, such as peptide nucleic acid
(PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA)
or other synthetic polymers with nucleotide side chains. The nucleic acid bound by the
protein is preferably single stranded, such as cDNA, RNA, GNA, TNA or LNA. The nucleic
acid bound by the protein is preferably double stranded, such as DNA. Proteins that bind
single stranded nucleic acids may be used to sequence double stranded DNA as long as the
double stranded DNA is dissociated into a single strand before it is bound by the protein.
It is preferred that the tertiary structure of the nucleic acid binding protein is known.
Knowledge of the three dimensional structure of the binding protein allows modifications to
be made to the protein to facilitate its function in the construct or pore of the invention.
The protein may be any size and have any structure. For instance, the protein may be
an oligomer, such as a dimer or trimer. The protein is preferably a small, globular polypeptide
formed from one monomer. Such proteins are easy to handle and are less likely to interfere

with the surface. For instance, such proteins are less likely to interfere with the pore forming
ability of a pore subunit.
It is also preferred that the location and function of the active site of the protein is
known. This prevents modifications being made to the active site that abolish the activity of
the protein. It also allows the protein to be attached to the surface so that the protein binds the
target nucleic acid sequence in a particular way. For instance, if the protein is being attached
to a transmembrane protein pore for sequencing purposes, it allows a proportion of the
nucleotides in a target sequence to interact with the pore as described below. In such
embodiments, it is beneficial to position the active site of the protein as close as possible to the
opening of the barrel of channel of the pore, without the protein itself presenting a block to the
flow of current. Knowledge of the way in which a protein may orient nucleic acids also
allows an effective construct to be designed.
As discussed in more detail below, it may be necessary to purify the construct of the
invention. It is preferred that the nucleic acid binding protein is capable of withstanding the
conditions used to purify the construct.
The surface in a construct of the invention may comprise a pore. Such pores may be
used to sequence nucleic acids. In order that most of the nucleotides in the target nucleic acid
are correctly identified by stochastic sensing, the protein preferably binds the nucleic acid in a
buffer background which is compatible with discrimination of the nucleotides. The protein
preferably has at least residual activity in a salt concentration well above the normal
physiological level, such as from 100 mM to 2000 mM. The protein is more preferably
modified to increase its activity at high salt concentrations. The protein may also be modified
to improve its processivity, stability and shelf life.
Suitable modifications can be determined from the characterisation of nucleic acid
handling enzymes from extremphiles such as halophilic, moderately halophilic bacteria,
thermophilic and moderately thermophilic organisms, as well as directed evolution approaches
to altering the salt tolerance, stability and temperature dependence of mesophilic or
thermophilic exonucleases.
The enzyme also preferably retains at least partial activity at temperatures from 10 °C
to 60 °C, such as at room temperature. This allows the construct to sequence nucleic acids at
a variety of temperatures, including room temperature.
The nucleic acid binding protein is preferably a nucleic acid handling enzyme. A
nucleic acid handling enzyme is a polypeptide that is capable of interacting with and
modifying at least one property of a nucleic acid. The enzyme may modify the nucleic acid by

cleaving it to form individual nucleotides or shorter chains of nucleotides, such as di- or
trinucleotides. The enzyme may modify the nucleic acid by orienting it or moving it to a
specific position.
The nucleic acid handling enzyme is preferably derived from a nucleolytic enzyme.
The nucleic acid handling enzyme used in the construct of the enzyme is more preferably
derived from a member of any of the Enzyme Classification (EC) groups 3.1.11, 3.1.13,
3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. The nucleic
acid handling enzyme is more preferably based on any one of the following enzymes:
• 3.1.11.- Exodeoxyribonucleases producing 5'-phosphomonoesters.
o 3.1.11.1 Exodeoxyribonuclease I.
o 3.1.11.2 Exodeoxyribonuclease III.
o 3.1.11.3 Exodeoxyribonuclease (lambda-induced).
o 3.1.11.4 Exodeoxyribonuclease (phage SP3-induced).
o 3.1.11.5 Exodeoxyribonuclease V.
o 3.1.11.6 Exodeoxyribonuclease VII.
• 3.1.13.- Exoribonucleases producing 5'-phosphomonoesters.
o 3.1.13.1 Exoribonuclease II.
o 3.1.13.2 Exoribonuclease H.
o 3.1.13.3 Oligonucleotidase.
o 3.1.13.4 Poly( A)-specific ribonuclease.
o 3.1.13.5 Ribonuclease D.
• 3.1.14.- Exoribonucleases producing 3'-phosphomonoesters.
o 3.1.14.1 Yeast ribonuclease.
• 3.1.15.- Exonucleases active with either ribo- or deoxyribonucleic acid
producing 5' phosphomonoesters
c 3.1.15.1 Venom exonuclease.
• 3. 1.16.- Exonucleases active with either ribo-or deoxyribonucleic acid
producing 3' phosphomonoesters
o 3.1.16.1 Spleen exonuclease.

• 3.1.21.- Endodeoxyribonucleases producing 5'-phosphomonoesters.
o 3.1.21.1 Deoxyribonuclease I.
o 3.1.21.2 Deoxyribonuclease IV (phage-T(4)-induced).
o 3.1.21.3 Type I site-specific deoxyribonuclease.
o 3.1.21.4 Type II site-specific deoxyribonuclease.
o 3.1.21.5 Type III site-specific deoxyribonuclease.
o 3.1.21.6 CC-preferring endodeoxyribonuclease.
o 3.1.21.7 Deoxyribonuclease V.
• 3.1.22.- Endodeoxyribonucleases producing other than 5'-phosphomonoesters.
o 3.1.22.1 Deoxyribonuclease II.
o 3.1.22.2 Aspergillus deoxyribonuclease K(l).
o 3.1.22.3 Transferred entry: 3.1.21.7.
o 3.1.22.4 Crossover junction endodeoxyribonuclease.
o 3.1.22.5 Deoxyribonuclease X.
• 3.1.25.- Site-specific endodeoxyribonucleases specific for altered bases.
o 3.1.25.1 Deoxyribonuclease (pyrimidine dimer).
o 3.1.25.2 Transferred entry: 4.2.99.18.
• 3. 1.26.- Endoribonucleases producing S'-phosphomonoesters.
o 3.1.26.1 Physarum polycephalum ribonuclease.
o 3.1.26.2 Ribonuclease alpha.
o 3.1.26.3 Ribonuclease III.
o 3.1.26.4 Ribonuclease H.
o 3.1.26.5 Ribonuclease P.
o 3.1.26.6 Ribonuclease IV.
o 3.1.26.7 Ribonuclease P4.
o 3.1.26.8 Ribonuclease M5.
o 3.1.26.9 Ribonuclease (poly-(U)-specific).
o 3.1.26.10 Ribonuclease IX.
o 3.1.26.11 Ribonuclease Z.

• 3.1.27.- Endoribonucleases producing other than 5'-phosphomonoesters.
o 3.1.27.1 Ribonuclease T(2).
o 3.1.27.2 Bacillus subtilis ribonuclease.
o 3.1.27.3 Ribonuclease T(l).
o 3.1.27.4 Ribonuclease U(2).
o 3.1.27.5 Pancreatic ribonuclease.
o 3.1.27.6 Enterobacter ribonuclease.
o 3.1.27.7 Ribonuclease F.
o 3.1.27.8 Ribonuclease V.
o 3.1.27.9 tRNA-intron endonuclease.
o 3.1.27.10 rRNA endonuclease.
• 3. 1.30.- Endoribonucleases active with either ribo- or deoxyribonucleic
producing 5' phosphomonoesters
o 3.1.30.1 Aspergillus nuclease S(l).
o 3.1.30.2 Serratia marcescens nuclease.
• 3. 1.31.- Endoribonucleases active with either ribo- or deoxyribonucleic
producing 3' phosphomonoesters
3.1.31.1 Micrococcal nuclease.
The enzyme is most preferably derived from an exonuclease, such as a
deoxyribonuclease, which cleaves nucleic acids to form individual nucleotides. The
advantages of exodeoxyribonucleases are that they are active on both single stranded and
double stranded DNA and hydrolyse bases either in the 5' - 3' or 3' - 5' direction.
An individual nucleotide is a single nucleotide. An individual nucleotide is one which
is not bound to another nucleotide or nucleic acid by any bond, such as a phosphodiester bond.
A phosphodiester bond involves one of the phosphate groups of a nucleotide being bound to
the sugar group of another nucleotide. An individual nucleotide is typically one which is not
bound in any manner to another nucleic acid sequence of at least 5, at least 10, at least 20, at
least 50, at least 100, at least 200, at least 500, at least 1000 or at least 5000 nucleotides.
Preferred enzymes for use in the invention include exonuclease I from E. coli (SEQ ID
NO: 6), exonuclease III enzyme from E. coli (SEQ ID NO: 52), RecJ from T. thermophilus
(SEQ ID NO: 54) and bacteriophage lambda exonuclease (SEQ ID NO: 56) and variants

thereof. Three identical subunits of SEQ ID NO: 56 interact to form a trimer exonuclease.
The enzyme is most preferably based on exonuclease I from E. coli (SEQ ID NO: 6).
The nucleic acid handling enzyme is preferably derived from an exonuclease enzyme
comprising any of the sequences shown in SEQ ID NOs: 6, 52, 54 and 56 or a variant thereof.
In other words, the enzyme preferably comprises any of the sequences shown in SEQ ID NOs:
6. 52. 54 and 56 or a variant thereof before its cysteine residues are modified as described
above.
A variant of SEQ ID NO: 6, 52, 54 or 56 is an enzyme that has an amino acid sequence
which varies from that of SEQ ID NO: 6, 52, 54 or 56 and which retains nucleic acid handling
ability. The ability of a variant to handle nucleic acids can be assayed using any method
known in the art. For instance, the ability of a variant to handle nucleic acids can be assayed
as described in the Example. The variant must also retain its ability to be expressed as
described in the Example.
The variant may include modifications that facilitate handling of the nucleic acid
and/or facilitate its activity at high salt concentrations and/or room temperature.
The enzyme, may be a naturally occurring variant which is expressed by an organism,
for instance by an E. coli bacterium. Variants also include non-naturally occurring variants
produced by recombinant technology. Over the entire length of the amino acid sequence of
SEQ ID NO: 6, 52, 54 or 56, 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: 6, 52, 54 or 56 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 200 or more, for example 230, 250, 270 or 280 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/). This algorithm
involves first identifying high scoring sequence pair (HSPs) by identifying short words of
length W in the query sequence that either match or satisfy some positive-valued threshold
score T when aligned with a word of the same length in a database sequence. T is referred to
as the neighbourhood word score threshold (Altschul et al, supra). These initial
neighbourhood word hits act as seeds for initiating searches to find HSP's containing them.
The word hits are extended in both directions along each sequence for as far as the cumulative
alignment score can be increased. Extensions for the word hits in each direction are halted
when: the cumulative alignment score falls off by the quantity X from its maximum achieved
value; the cumulative score goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The
BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix
(see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments
(B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.
The BLAST algorithm performs a statistical analysis of the similarity between two
sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787.
One measure of similarity provided by the BLAST algorithm is the smallest sum probability
(P(N)), which provides an indication of the probability by which a match between two amino
acid sequences would occur by chance. For example, a sequence is considered similar to
another sequence if the smallest sum probability in comparison of the first sequence to the
second sequence is less than about 1, preferably less than about 0.1, more preferably less than
about 0.01, and most preferably less than about 0.001.
Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 6,
52, 54 or 56 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, for example, according to Table 1
below.
Table 1 - Conservative substitutions
Amino acids in the same block in the second column and preferably in the same line in the
third column may be substituted for each other.


One or more amino acid residues of the amino acid sequence of SEQ ID NO: 6, 52, 54
or 56 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: 6, 52, 54 or 56. Such fragments retain
nucleic acid handling activity. Fragments may be at least 50, 100, 200 or 250 amino acids in
length. A fragment preferably comprises the nucleic acid handling domain of SEQ ID NO: 6,
52, 54 or 56.
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: 6, 52, 54 or 56 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.
As discussed above, a variant of SEQ ID NO: 6, 52, 54 or 56 is a protein that has an
amino acid sequence which varies from that of SEQ ID NO: 6,52, 54 or 56 and which retains
its ability to handle nucleic acids. A variant typically contains the regions of SEQ ID NO: 6,
52, 54 or 56 that are responsible for handling nucleic acids. The catalytic domains of SEQ ID
NOs: 6, 52, 54 and 56 are discussed above in the description of the sequence listing. A variant
of SEQ ID NO: 6. 52, 54 or 56 preferably comprises the relevant catalytic domain. A variant
SEQ ID NO: 6, 52, 54 or 56 typically includes one or more modifications, such as
substitutions, additions or deletions, outside the relevant catalytic domain. Specific variants of
SEQ ID NO: 6 are discussed in more detail below.
The variant may be modified for example by the addition of histidine or aspartic acid
residues to assist its identification or purification 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.
Preferred enzymes that are capable of pushing or pulling the target nucleic acid
sequence through the pore include polymerases, nucleases, helicases and topoisomerases, such
as gyrases. The nucleic acid handling enzyme can be derived from any of these types of
enzymes. The polymerase is preferably a member of any of the Enzyme Classification (EC)
groups 2.7.7.6, 2.7.7.7,2.7.7.19, 2.7.7.48 and 2.7.7.49. The polymerase is preferably a DNA-
dependent DNA polymerase, an RNA-dependent DNA polymerase, a DNA-dependent RNA
polymerase or an RNA-dependent RNA polymerase. The helicase is preferably based on a
member of any of the Enzyme Classification (EC) groups 3.6.1.- and 2.7.7.-. The helicase is
preferably an ATP-dependent DNA helicase (EC group 3.6.1.8), an ATP-dependent RNA
helicase (EC group 3.6.1.8) or an ATP-independent RNA helicase. The topoisomerase is
preferably a member of any of the Enzyme Classification (EC) groups 5.99.1.2 and 5.99.1.3.
The nucleic acid binding protein 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. 1251,35S, 14C, enzymes,
antibodies, antigens, polynucleotides and ligands such as biotin.
The nucleic acid binding protein may be isolated from a binding protein producing
organism, such as E. coli, T. thermophilus or bacteriophage, or made synthetically or by
recombinant means. For example, the nucleic acid binding protein may be synthesised by in
vitro translation and transcription. The amino acid sequence of the nucleic acid binding
protein may be modified to include non-naturally occurring amino acids or to increase the
stability of the protein. When the nucleic acid binding protein is produced by synthetic means,
such amino acids may be introduced during production. The nucleic acid binding protein may
also be altered following either synthetic or recombinant production.
The nucleic acid binding protein may also be produced using D-amino acids. For
instance, the nucleic acid binding proteins may comprise a mixture of L-amino acids and D-
amino acids. This is conventional in the art for producing such proteins or peptides.
The nucleic acid binding protein may also contain other non-specific chemical
modifications as long as they do not interfere with its ability to handle nucleic acids or attach
to the surface. A number of non-specific side chain modifications are known in the art and
may be made to the side chains of the pores. 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

modifications to the nucleic acid binding protein can be made after expression of the nucleic
acid binding protein or after the nucleic acid binding protein has been used to form a construct
of the invention.
The nucleic acid binding protein can be produced using standard methods known in the
art. Polynucleotide sequences encoding a nucleic acid binding protein may be isolated and
replicated using standard methods in the art. Such sequences are discussed in more detail
below. Polynucleotide sequences encoding a nucleic acid binding protein may be expressed in
a bacterial host cell using standard techniques in the art. The nucleic acid binding protein may
be produced in a cell by in situ expression of the polypeptide from a recombinant expression
vector. The expression vector optionally carries an inducible promoter to control the
expression of the polypeptide.
A nucleic acid binding protein may be produced in large scale following purification
by any protein liquid chromatography system from pore producing organisms or after
recombinant expression as described below. Typical protein liquid chromatography systems
include FPLC, AKTA systems, the Bio-Cad system, the Bio-Rad BioLogic system and the
Gilson HPLC system.
Preferred nucleic acid handling enzymes
The nucleic acid handling enzyme is preferably derived from an exonuclease enzyme
comprising the sequence shown in SEQ ID NO: 6 or a variant thereof. In other words, the
enzyme preferably comprises the sequence shown in SEQ ID NO: 6 or a variant thereof before
its cysteine residues are modified as described above. Variants of SEQ ID NO: 6 are
discussed above.
SEQ ID NO: 6 has five native cysteine residues at positions 51,98,144,306 and 330.
All five cysteine residues at these positions are accessible. A variant of SEQ ID NO: 6
preferably comprises all five of these residues before it is modified in accordance with the
invention.
In one embodiment, all five of the cysteines at positions 51,98,144, 306 and 330 are
removed from SEQ ID NO: 6 and one or more non-native cysteine residues are introduced.
In another embodiment, all but one or more of the five cysteine residues at positions
51, 98, 144, 306 and 330 are removed from SEQ ID NO: 6. Any combination of one or more
of the five cysteine residues at positions 51,98,144, 306 and 330 can remain in an enzyme
derived from SEQ ID NO: 6 after mutagenesis. Preferred combinations include, but are not

limited, to 144 and 330. In a preferred embodiment, only the native cysteine residue at
position 144 or 330 of SEQ ID NO: 6 remains.
One or more of the five cysteine residues in SEQ ID NO: 6 are preferably substituted
with alanine, serine, methionine or threonine. The cysteine residue at position 51 in SEQ ID
NO: 6 is more preferably substituted with alanine. The cysteine residue at position 98 in SEQ
ID NO: 6 is more preferably substituted with serine or threonine. The cysteine residue at
position 144 in SEQ ID NO: 6 is more preferably substituted with methionine or threonine.
The cysteine residue at position 306 in SEQ ID NO: 6 is more preferably substituted with
serine or threonine. The cysteine residue at position 330 in SEQ ID NO: 6 is more preferably
substituted with threonine. In the most preferred embodiment, the cysteine residue at position
51 in SEQ ID NO: 6 is substituted with alanine, the cysteine residue at position 98 in SEQ ID
NO: 6 is substituted with threonine, the cysteine residue at position 144 in SEQ ID NO: 6 is
substituted with threonine, the cysteine residue at position 306 in SEQ ID NO: 6 is substituted
with threonine and the cysteine residue at position 330 in SEQ ID NO: 6 is substituted with
threonine.
The constructs of the invention most preferably comprise a nucleic acid handling
enzyme comprising the sequence shown in any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20,
22, 24. 26, 28. 30, 32, 34, 36, 38. 40, 42, 44, 46, 48 and 50 or a variant thereof. A variant of
SEQ ID NO: 8,10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38,40,42,44, 46,48 and
50 is a protein that has an amino acid sequence which varies from that of SEQ ID NO: 8, 10,
12,14,16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38,40,42,44,46,48 and 50 and which
retains its ability to handle nucleic acids. Variants may differ from SEQ ID NO: 8, 10,12,14,
16, 18. 20, 22. 24, 26, 28, 3.0, 32, 34, 36, 38,40,42,44, 46, 48 or 50 to the same extent as
variants of SEQ ID NO: 6 differ from SEQ ID NO: 6 as discussed above. However, variants
of SEQ ID NOs: 8, 10. 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48
and 50 must comprise the changes made compared with the wild type enzyme. For instance,
variants of SEQ ID NOs: 8,10 and 12 must comprise the same residues as SEQ ID NOs: 8,10
and 12 at positions 98, 144, 306 and 330. Variants of SEQ ID NOs: 14 and 16 must comprise
the same residues as SEQ ID NOs: 14 and 16 at positions 42, 51, 98, 144, 306 and 330.
Variants of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50
must comprise the same residues as SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48 and 50 at positions 51, 98, 144, 184, 306 and 330.
The nucleic acid handling enzyme is typically attached to the surface via one or more
of its surface accessible residues having side chains of a preferred orientation. The following

residues in SEQ ID NO: 6 should not be used as attachment points because they are buried and
inaccessible to solvent or attachment to a linker will result is severe disruption of the protein
global structure: 13, 101, 104, 111, 114, 115, 117, 190, 194, 197,208,232,233,236,246,247,
248, 258, 259, 261, 262, 283, 298, 309, 310, 347, 406, and 267. The following residues in
SEQ ID NO: 6 are close to DNA binding groove and would not be a good location to attach a
linker even though they are surface accessible: 21, 22, 103,105,106,107, 109,110, 114, 115,
117. 118, 231, 242, 252, 256,257, 285, 287, 288, 289, 300, 301, 302, 304, 305, 307, 355, 356,
357. 358, 359, 360 368, 369 and 371. All the remaining residues in SEQ ID NO: 6 could
potentially be used as attachment points.
The following residues in SEQ ID NO: 6 are preferred attachment points because they
provide a side chain, are exposed and are solvent accessible and mutation of the side chain
would be predicted to lead to little disruption of the overall protein structure: 8, 9, 37, 38, 39,
41. 43. 44, 45, 47, 76, 77, 96, 150,151,153,156,159,161,171, 173, 176, 178, 179, 184,195,
198, 199, 200, 203, 209, 218, 222. 225,227,256, 273, 275,277, 278, 280, 281, 282,285, 292,
293, 311. 313, 316, 318, 321, 326, 327, 328, 332, 335, 338, 339, 340, 342, 345, 353, 374, 381,
385, 387. 389. 390, 395, 397, 401,417, 420, 423, 424, 429, 432, 437, 438, 441, 445, 448, 452,
456,458,459, 465, 458, 459,462, 466 and 467. The following residues are most preferred as
attachment points: 184, 83,42, 94, 90,188,184,185,186,187,188, 189,190, 191,192,193,
194, 195, 196, 197, 198, 199, 215, 216, 217, 218, 219, 220, 221, 222 and 223. Any of the
preferred attachment residues may be modified by substitution. Preferably one or more of the
preferred attachment residues is substituted with cysteine. Preferred substitutions in SEQ ID
NO: 6 or a variant thereof include, but are not limited to, M184C, A83C, V42C, V94C, S90C,
Y188C, Y188C, A219C, A219C, M218C and M218C. As discussed in more detail below, the
enzyme may be attached to the surface via more than one residue.
Surface
The constructs of the invention comprise a surface. Any surface that can be attached to
the nucleic acid binding protein via the one or more accessible cysteine residues can be used in
accordance with the invention. Suitable surfaces include, but are not limited to beads (such as
agarose or magnetic beads), column supports, immobilised metal affinity matrices (such as
NiNTA, Streptavidin-tag or cobalt), glutathione sepharose, dextrin sepharose, affinity matrix,
IgG sepharose. activated thiol-sepharose, metal films (such as gold solid supports),
nanoparticles, glass, treated glass, plastics, resin surfaces, solid state nanopores, sensor
surfaces, single molecular detectors and transmembrane protein pores.

Suitable solid state pores include, but are not limited to, silicon nitride pores, silicon
dioxide pores and graphene pores. Other suitable solid state pores and methods of producing
them are discussed in US Patent No. 6,464,842, WO 03/003446, WO 2005/061373, US Patent
No. 7.258.838. US Patent No. 7,466,069. US Patent No. 7,468,271 and US Patent No.
7,253,434.
The surface is preferably derived from a transmembrane protein pore. A
transmembrane protein pore is a polypeptide or a collection of polypeptides that permits ions
driven by an applied potential to flow from one side of a membrane to the other side of the
membrane. The pore preferably permits nucleotides to flow from one side of a membrane to
the other along the applied potential. The pore preferably allows a nucleic acid, such as DNA
or RNA. to be pushed or pulled through the pore.
The 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 pore.
The 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 P barrel or channel or a transmembrane ot-helix bundle or channel.
The barrel or channel of the pore typically comprises amino acids that facilitate
interaction with nucleotides or nucleic acids. These amino acids are preferably located near a
constriction of the barrel or channel. The pore typically comprises one or more positively
charged amino acids, such as arginine, lysine or histidine. These amino acids typically
facilitate the interaction between the pore and nucleotides or nucleic. The nucleotide detection
can be facilitated with an adaptor. This is discussed in more detail below.
Pores for use in accordance with the invention can be P-barrel pores, a-helix bundle
pores or solid state pores. P-barrel pores comprise a barrel or channel that is formed from P-
strands. Suitable p-barrel pores include, but are not limited to, p-toxins, such as a-hemolysin,
anthrax toxin and leukocidins, and outer membrane proteins/porins of bacteria, such as
Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF), outer
membrane porin G (OmpG), outer membrane phospholipase A and Neisseria autotransporter
lipoprotein (NalP). α-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.
The surface may be a pore itself. Alternatively, if the pore is an oligomer, the surface
may be a pore subunit. The constructs of the invention may be part of a pore. Alternatively,

the construct may be isolated, substantially isolated, purified or substantially purified as
described above.
The pore or subunit is preferably derived from α-hemolysin (α-HL). The wild type a-
HL pore is formed of seven identical monomers or subunits (i.e. it is heptameric). The
sequence of one wild type monomer or subunit of α-hemolysin is shown in SEQ ID NO: 2.
The surface in the constructs of the invention preferably comprises the sequence shown in
SEQ ID NO: 2 or a variant thereof. Amino acids 1, 7 to 21, 31 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: 2 form loop
regions. Residues 113 and 147 of SEQ ID NO: 2 form part of a constriction of the barrel or
channel of α-HL. The nucleic acid binding protein is preferably attached to one or more of
amino acids 8, 9, 17, 18, 19, 44, 45, 50 and 51 of SEQ ID NO: 2.
A variant of SEQ ID NO: 2 is a subunit that has an amino acid sequence which varies
from that of SEQ ID NO: 2 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 membrane 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).
The variant may include modifications that facilitate covalent attachment to or
interaction with the 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: 2.
Preferred variants comprise a substitution of the residue at position 8, 9, 17, 237, 239 and 287
of SEQ ID NO: 2 with cysteine (K8C, T9C, N17C, K237C, S239C or E287C).
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 has a glutamine at position 139 of SEQ ID NO: 2.
The variant preferably has a cysteine at position 119, 121 or 135 of SEQ ID NO: 2. SEQ ID
NO: 4 shows the sequence of SEQ ID NO: 2 except that it has an cysteine at position 135
(LI 35C) and a glutamine at position 139 (N139Q). SEQ ID NO: 4 or a variant thereof may be
used to form a pore in accordance with the invention. The variant may have an arginine at
position 113 of SEQ ID NO:2.
The variant may be a naturally occurring variant which is expressed naturally by an
organism, for instance by a Staphylococcus bacterium, or expressed 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: 2 or 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: 2 or 4 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 200 or
more, for example 230, 250, 270 or 280 or more, contiguous amino acids ("hard homology").
Homology can be measured as described above.
Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 2 or
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, for example, according to Table 1
above.
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 fragments of SEQ ID NO: 2 or 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: 2 or 4. Fragments typically
include residues 119, 121, 135. 113 and 139 of SEQ ID NO: 2 or 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: 2 or 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: 2 or 4 is a subunit that has an amino acid
sequence which varies from that of SEQ ID NO: 2 or 4 and which retains its ability to form a
pore. A variant typically contains the regions of SEQ ID NO: 2 or 4 that are responsible for
pore formation. The pore forming ability of α-HL, which contains a P-barrel, is provided by
P-strands in each subunit. A variant of SEQ ID NO: 2 or 4 typically comprises the regions in
SEQ ID NO: 2 that form p-strands. The amino acids of SEQ ID NO: 2 or 4 that form p-
strands are discussed above. One or more modifications can be made to the regions of SEQ
ID NO: 2 or 4 that form p-strands as long as the resulting variant retains its ability to form a
pore. Specific modifications that can be made to the P-strand regions of SEQ ID NO: 2 or 4
are discussed above.
A variant of SEQ ID NO: 2 or 4 preferably includes one or more modifications, such as
substitutions, additions or deletions, within its α-helices and/or loop regions. Amino acids that
form α-helices and loops are discussed above.
The variant may be modified for example by the addition of histidine or aspartic acid
residues to assist its identification or purification 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.
Variants may also comprise any of the non-specific modifications discussed above for
the nucleic acid binding protein. Subunits or pores can be made as discussed above.
Attachment
The nucleic acid binding protein is attached to the surface via one or more accessible
cysteine residues. The nucleic acid binding protein may be attached to the surface at more
than one, such as two or three, points. Attaching the nucleic acid binding protein to the
surface at more than one point can be used to constrain the mobility of the protein. For
instance, multiple attachments may be used to constrain the freedom of the protein to rotate or
its ability to move away from the surface.
If the surface is the subunit of an oligomeric pore, the subunit may be in a monomeric
form when it is attached to the nucleic acid binding protein (post expression modification).
Alternatively, the subunit may be part of an oligomeric pore when it is attached to the nucleic
acid binding protein (post oligomerisation modification).

The nucleic acid binding protein can be attached to the surface using any method
known in the art. The nucleic acid binding protein and surface may be produced separately
and then attached together. If the surface is itself a protein, such as a pore subunit, the two
components may be attached in any configuration. For instance, they may be attached via
their terminal (i.e. amino or carboxy terminal) amino acids. Suitable configurations include,
but are not limited to, the amino terminus of the nucleic acid binding protein being attached to
the carboxy terminus of the surface and vice versa. Alternatively, the two components may be
attached via amino acids within their sequences. For instance, the nucleic acid binding
protein may be attached to one or more amino acids in a loop region of the surface. In a
preferred embodiment, terminal amino acids of the nucleic acid binding protein are attached to
one or more amino acids in the loop region of the surface. Terminal amino acids and loop
regions are discussed above.
The nucleic acid binding protein is preferably chemically fused to the surface. A
nucleic acid binding protein is chemically fused to a surface if the two parts are chemically
attached, for instance via a linker molecule. Any method of chemical fusion or attachment can
be used. Suitable methods include, but are not limited to, histidine tag binding to a metal
affinity matrix, Ni-NTA, biotin binding to streptavidin, antibody binding to an antigen,
primary amine coupling, GST tags binding to glutathione, MBP tags binding to dextrin,
Protein A binding to IgG, reaction between thiols, nucleic acid hybridization linkers and
cysteine linkage. DNA hybridization linkers and cysteine linkage are discussed in more detail
below. The nucleic acid binding protein is preferably covalently attached to the surface.
If the surface is a protein, the nucleic acid binding protein may be genetically fused to
the surface. A nucleic acid binding protein is genetically fused to a protein surface if the
whole construct is expressed from a single polynucleotide sequence. The coding sequences of
the nucleic acid binding protein and surface may be combined in any way to form a single
polynucleotide sequence encoding the construct.
The nucleic acid binding protein and surface may be genetically fused in any
configuration, such as via their terminal amino acids. The amino acid sequence of the nucleic
acid binding protein is typically added in frame into the amino acid sequence of the surface.
In a preferred embodiment, the nucleic acid binding protein is inserted into a loop region of a
transmembrane protein pore or pore subunit. In an especially preferred embodiment, the
nucleic acid binding protein is inserted between amino acids, 18 and 19,44 and 45 or 50 and
51 of SEQ ID NO:2.

The nucleic acid binding protein retains its ability to bind nucleic acids. This ability is
typically provided by its secondary structural elements (α-helices and β-strands) and tertiary
structural elements. In order to avoid adversely affecting the nucleic acid binding ability of
the protein, it is preferably attached to the surface in a manner that does not affect its
secondary or tertiary structure.
If the surface is a pore or pore subunit, the pore or pore subunit retains it ability to form
pores. The pore forming ability of subunits is typically provided by their α-helices and β-
strands. β-barrel pores comprise a barrel or channel that is formed from β-strands, whereas α-
helix bundle pores comprise a barrel or channel that is formed from α-helices. The α-helices
and β-strands are typically connected by loop regions. In order to avoid affecting the pore
forming ability of the subunit, the nucleic acid binding protein is preferably attached to a loop
region of the subunit. The loop regions of specific subunits are discussed in more detail
above.
The nucleic acid binding protein may be attached directly to the surface. For instance,
native and/or non-native accessible cysteine residues can be attached directly to activated
thiol-sepharose.
The nucleic acid binding protein can be attached to the surface at one or more
positions, such as at one, two, three or four positions. The nucleic acid binding protein is
preferably attached to the surface at one or two positions. After removal of native cysteine
residues from the nucleic acid binding protein, one or more cysteine residuess can be
incorporated into the protein at specific positions for the attachments. Attachments can be
done either by direct cross linking of the cytseine residues in the nucleic acid binding protein
to cysteines in the surface (i.e. via a disulphide bond) or by using cross linkers. Attachment at
two positions can reduce the flexibility of the complex and can fix the nucleic acid binding
protein on the surface in a chosen specific orientation.
In a preferred embodiment, the construct comprises (1) exonuclease I from E. coli
(SEQ ID NO: 6) or a variant thereof with the substitutions M184C and V94C and (2) α-HL
(SEQ ID NO: 2) or a variant thereof having the substitutions N17C and E287C. The M184C
position in SEQ ID NO: 6 or a variant thereof is cross linked to the N17C position in SEQ ID
NO: 2 or a variant thereof either by direct cysteine cross linking or by short linkers. The V94C
position in SEQ ID NO: 6 or a variant therof is cross linked to the E287C position in SEQ ID
NO: 2 or a variant thereof by a different linker. In this example, the nucleic acid binding
protein is attached to the surface via at two distant positions.


The nucleic acid binding protein is preferably attached to the surface using one or
more, such as two or three, linkers. The one or more linkers may be designed to constrain the
mobility of the nucleic acid binding protein. The linkers are typically attached to the one or
more accessible cysteine residues in the nucleic acid binding protein. The linkers may be
attached to one or more reactive groups, such as cysteine residues, reactive lysine residues or
non-natural amino acids, in the surface. Suitable linkers are well known in the art. Suitable
linkers include, but are not limited to, chemical crosslinkers and peptide linkers. Preferred
chemical crosslinkers are nucleic acid hybridization linkers. The length, flexibility and
hydrophilicity of the nucleic acid hybridization linkers are typically designed such that they do
not to disturb the functions of the nucleic acid binding protein and surface. An advantage of
using hybridization linkers is that the formation of unwanted dimers (surface-surface or
protein-protein) is minimized. The nucleic acid hybridization linkers can comprise any of the
nucleic acids discussed above. For instance, they may comprise deoxyribonucleic acid
(DNA). ribonucleic acid (RNA) or any synthetic nucleic acid known in the art, such as peptide
nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic
acid (LNA) or other synthetic polymers with nucleotide side chains. The linkers can also be
modified such they react with one another once they have hybridised. Alternatively, agents
may be used to crosslink the linkers once they have hybridised to one another.
Preferred nucleic acid hybridization linkers correspond to the first 15, 25 or 35
nucleotides from the 5' end of SEQ ID NO: 57. The linker preferably also has TT at the 3'
end to provide extra flexibility. At the 3' end, the linkers have a group, such as maleimide or
Thiol, that allows the linker to be attached to the nucleic acid binding protein or surface.
Maleimide or Thiol modified oliognucleotides can be obtained commercially, for instance

from ATDBio. More preferred linkers are shown in SEQ ID NOs: 58, 59 and 60.
Complementary linkers are shown in SEQ ID NOs: 61, 62 and 63. SEQ ID NO: 58, 59 or 60
may be attached to one of the nucleic acid binding protein and surface and the complementary
linker (SEQ ID NO: 61, 62 or 63 respectively) is attached to the other of the nucleic acid
binding protein and surface. The nucleic acid binding protein and surface can then be attached
together by hybridizing the linkers.
The stability of the hybridization depends on the melting temperature of the
hybridizing linkers. Depending on the application and the required stability, this can be
optimized by changing the sequences of the linkers (e.g. changing the linkers to more GC rich
will increase their melting temperature and hence the stability), the length of the linkers (i.e.
increasing their length will increase the stability) or the reaction conditions (e.g. increasing
their concentration will increase the stability).
For maximum stability of hybridization, it is desirable to have long hybridizing linkers
with high melting temperatures, for example linkers more than 15 nucleotides in length,
particularly 15 to 45 nucleotides in length, such as 15,20,25, 30,35,40 or 45 nucleotides in
length. However, the use of long linkers increases the distance between the moieties. This
may be disadvantageous because interaction between the moieties is disrupted or because
proximity is required for the surface to detect a substrate which has been released from the
nucleic acid binding protein. Increased distance may be advantageous as it may prevent
aggregation or electrostatic interactions and may permit flexing. The disadvantages of the
increased distance can be overcome by changing the orientation of the nucleic acid
attachment. Most preferably, the linkers comprise a nucleic acid that is from 6 to 15
nucleotides in lenght, such as 6, 8 or 10 nucleotides long.
The hybridization linkers preferably have an affinity of from 1 fM to 1 uM at
concentrations of from 1 pM to 1 mM. The linkers more preferably have an affinity of from 1
fM to 1 OnM at concnetrations of from 1 pM to 1 uM. The linkers most preferably have an
affinity of from 1 pM to 100 pM at concentrations of from 100 pM to 10 nM.
A preferred linker is shown in the SEQ ID NO: 64. The 3' end of this linker can be
attached to a cysteine residue on the surface. The linker preferably also has TTTTT at the 3'
end to provide extra flexibility. The 5' end of SEQ ID NO: 65 can then be attached to the
nucleic acid binding protein. In this example, the 3' end of the SEQ ID NO 64 is
complementary to a stretch of sequence at the 5' end of SEQ ID NO: 65.
Once the linkers are hybridized to each other, they melt (fall apart) under certain
conditions (for example, at high temperatures or lower salt conditions) unless there is a

permanent bond between the two linkers. To form a permanent bond, the linkers are
preferably modified such they react with one another once they have hybridized. Each linker
preferably contains a group capable of forming a covalent bond with a group in another linker.
A pair of linkers can be linked by one or more covalent bonds, for example one, two or three,
covalent bonds.
Typically the bond will be a simple disulfide bond between the two linkers. The
linkers can also be modified to incorporate thiol groups at one or more, such as two, positions.
Depending on the application and preferences, thiols groups can be internal or terminal.
Linkers can also be modified either internally or terminally to include one or more,
such as two. iodoacetamide groups. A hybridizing linker with one or more iodoactamide
groups can be covalently linked to thiols on the complementary hybridizing linker.
Linkers can also be modified with alkene groups. One or more internal or terminal
alkene groups in preferred positions can be subjected to olefin metathesis to make a covalent
bond between the alkenes in the hybridization linkers.
If necessary, a small linker can be added between the linker and the reactive groups,
such as thiol groups, iodoacetamide groups and alkene groups, to obtain the proper distances
necessary to make an efficient covalent bond between the linkers.
In a preferred embodiment, the covalent bond between the linkers can be made using
the click chemistry. Click chemistry is a term first introduced by Kolb et ah in 2001 to
describe an expanding set of powerful, selective, and modular building blocks that work
reliably in both small- and large-scale applications (Kolb HC, Finn, MG, Sharpless KB, Click
chemistry: diverse chemical function from a few good reactions, Angew. Chem. Int. Ed. 40
(2001) 2004-2021). They have defined the set of stringent criteria for click chemistry as
follows: 'The reaction must be modular, wide in scope, give very high yields, generate only
inoffensive byproducts that can be removed by nonchromatographic methods, and be
stereospecific (but not necessarily enantioselective). The required process characteristics
include simple reaction conditions (ideally, the process should be insensitive to oxygen and
water), readily available starting materials and reagents, the use of no solvent or a solvent that
is benign (such as water) or easily removed, and simple product isolation. Purification if
required must be by nonchromatographic methods, such as crystallization or distillation, and
the product must be stable under physiological conditions".
Suitable example of click chemistry include, but are not limited to, the following:
(a) copper-free variant of the 1,3 dipolar cycloaddition reaction, where an azide reacts
with an alkyne under strain, for example in a cyclooctane ring;

(b) the reaction of an oxygen nucleophile on one linker with an epoxide or aziridine
reactive moiety on the other; and
(c) the Staudinger ligation, where the alkyne moiety can be replaced by an aryl phosphine,
resulting in a specific reaction with the azide to give an amide bond.
Preferably the click chemistry reaction is the Cu (I) catalysed 1,3 dipolar cycloaddition
reaction between an alkyne and an azide. Nucleic acid bases have already been synthesized
incorporating azide and alkyne groups in preferred positions (for example Kocalka P, El-
Sagheer AH, Brown T, Rapid and efficient DNA strand cross-linking by click chemistry,
Chembiochem. 2008. 9(8): 1280-5).
If nucleotides within the linkers' nucleic acid acid regions are modified to include
groups that can form covalent bonds, the modified nucleotides are preferably offset from one
another by one nucleotide in order to achieve the link. This follows the published work of Tom
Brown (Kocalka et al. (2008) ChemBiochem 9 8 1280-1285).
In a preferred embodiment, a single azide group (SEQ ID NO: 66) or more such as two
(SEQ ID NO: 67) can be incorporated into uracil bases at specific places in a 15 base
deoxyribonucleic acid sequence. The cysteine residues on the surface can then be modified
with these azide hybridization linkers using the thiol group at the 5'end (SEQ ID NOs: 66 and
67). Alkyne groups can also be incorporated into uracil bases at preferred positions in
sequences complementary to the SEQ ID NOs: 66 and 67 (SEQ ID NOs: 68 and 69
respectively). These sequences can be used to modify the cysteines on the nucleic acid
binding protein. Using DNA hybridization followed by 'click chemistry' between the azide
and alkyne groups, hybridization linkers can be covalently cross linked.
The distance between the surface and the nucleic acid binding protein can be
modulated by changing the length of the hybridization linkers. The position of the azide and
alkyne modified bases then needs to be changed accordingly.
In a preferred embodiment 6 mer (SEQ ID NO 70), 8 mer (SEQ ID NO 71) or 10 mer
(SEQ ID NO 72) DNA in which two uracil bases are modified with azide groups can be attach
to the cysteines of the moiety. Complementary sequences of 6 mer (SEQ ID NO 73), 8 mer
(SEQ ID NO 74) or 10 mer (SEQ ID NO 75) DNA in which two uracil bases are modified
with alkyne groups can be attached to the cysteines of a moiety such as a DNA binding
protein. Covalent cross linking between these hybridization linkers will bring the moieties
closer to each other than with the hybridization linkers (SEQ ID NO 67 and 69). Incorporation
of azide and alkyne groups into uracil base units of DNA has been developed by ATDBio.
Other preferred chemical crosslinkers are shown in the following Table 3.

Linkers may be attached to the nucleic acid binding protein first and then the surface,
the surface first and then the nucleic acid binding protein or the surface and nucleic acid
binding protein at the same time. When the linker is attached to a pore subunit (as the
surface), it may be a monomeric subunit, part of an oligomer of two or more monomers or part
of complete oligomeric pore. It is preferred that the linker is reacted before any purification
step to remove any unbound linker.
The preferred method of attaching the nucleic acid binding protein to the surface is via
cysteine linkage. This can be mediated by a bi-functional chemical linker or by a polypeptide
linker with a terminal presented cysteine residue. α-HL (SEQ ID NO: 2) lacks native cysteine
residues so the introduction of a cysteine into the sequence of SEQ ID NO: 2 enables the
controlled covalent attachment of the nucleic acid binding protein to the subunit. Cysteines
can be introduced at various positions, such as position K8, T9, N17 or E287 of SEQ ID NO: 2
or at the carboxy terminus of SEQ ID NO: 2. The length, reactivity, specificity, rigidity and
solubility of any bi-functional linker may be designed to ensure that the enzyme is positioned
correctly in relation to the subunit and the function of both the subunit and enzyme is retained.
Suitable linkers include those described above.
Cross-linkage of subunits or enzymes to themselves may be prevented by keeping the
concentration of linker in a vast excess of the nucleic acid binding protein and/or the surface.
Alternatively, a ''lock and key" arrangement may be used in which two linkers are used. For
instance, click chemistry, such as azide alkyne Huisgen cycloaddition, may be used to ensure
that the nucleic acid binding protein only binds to the surface and not to itself and vice versa.
In a preferred embodiment, the azide-PEG-maleimide and alkyne-PEG-maleimide linkers
shown in Table 3 above are used. One is attached to the nucleic acid binding protein and the
other is attached to the surface. This ensures that binding only occurs between the nucleic acid
binding protein and the surface.
Only one end of each linker may react together to form a longer linker and the other
ends of the linker each react with a different part of the surface (i.e. subunit or monomer).

In a preferred embodiment, the site of covalent attachment is selected such that, when
the construct is used to form a pore, the nucleic acid binding protein handles a target nucleic
acid sequence in such a way that a proportion of the nucleotides in the target sequence
interacts with the pore. Nucleotides are then distinguished on the basis of the different ways in
which they affect the current flowing through the pore during the interaction.
There are a number of ways that pores can be used to sequence nucleic acid molecules.
One way involves the use of an exonuclease enzyme, such as a deoxyribonuclease. In this
approach, the exonuclease enzyme is used to sequentially detach the nucleotides from a target
nucleic strand. The nucleotides are then detected and discriminated by the pore in order of
their release, thus reading the sequence of the original strand. For such an embodiment, the
exonuclease enzyme is preferably attached to a pore subunit such that a proportion of the
nucleotides released from the target nucleic acid is capable of entering and interacting with the
barrel or channel of a pore comprising the subunit. The exonuclease is preferably attached to
the subunit at a site in close proximity to the part of the subunit that forms the opening of the
barrel of channel of the pore. The exonuclease enzyme is more preferably attached to the
subunit such that its nucleotide exit trajectory site is orientated towards the part of the subunit
that forms part of the opening of the pore.
Another way of sequencing nucleic acids involves the use of an enzyme that pushes or
pulls the target nucleic acid strand through the pore in combination with an applied potential.
In this approach, the ionic current fluctuates as a nucleotide in the target strand passes through
the pore. The fluctuations in the current are indicative of the sequence of the strand. For such
an embodiment, the enzyme is preferably attached to a pore subunit such that it is capable of
pushing or pulling the target nucleic acid through the barrel or channel of a pore comprising
the subunit and does not interfere with the flow of ionic current through the pore. The enzyme
is preferably attached to the subunit at a site in close proximity to the part of the subunit that
forms part of the opening of the barrel of channel of the pore. The enzyme is more preferably
attached to the subunit such that its active site is orientated towards the part of the subunit that
forms part of the opening of the pore.
A third way of sequencing a nucleic acid strand is to detect the byproducts of a
polymerase in close proximity to a pore detector. In this approach, nucleoside phosphates
(nucleotides) are labelled so that a phosphate labelled species is released upon the addition of a
polymerase to the nucleotide strand and the phosphate labelled species is detected by the pore.
The phosphate species contains a specific label for each nucleotide. As nucleotides are
sequentially added to the nucleic acid strand, the bi-products of the base addition are detected.

The order that the phosphate labelled species are detected can be used to determine the
sequence of the nucleic acid strand.
The nucleic acid binding protein is preferably attached to the part of a pore or a subunit
thereof that forms part of the cis side of a pore. In electrophysiology, the cis side is the
grounded side by convention. If a hemolysin pore is inserted correctly into an
electrophysiology apparatus, the Cap region is on the cis side. It is well known that, under a
positive potential, nucleotides will migrate from the cis to the trans side of pores used for
stochastic sensing. Positioning the nucleic acid binding protein at the cis side of a pore allows
it to handle the target nucleic acid such that a proportion of the nucleotides in the sequence
enters the barrel or channel of the pore and interacts with it. Preferably, at least 20%, at least
40%, at least 50%, at least 80% or at least 90% of the nucleotides in the sequence enters the
barrel or channel of the pore and interacts with it.
The site and method of covalent attachment is preferably selected such that mobility of
the nucleic acid binding protein is constrained. This helps to ensure that the protein handles
the target nucleic acid sequence in such a way that a proportion of the nucleotides in the target
sequence interacts with the pore. For instance, constraining the ability of nucleic acid binding
protein to move means that its active site can be permanently orientated towards the part of the
subunit that forms part of the opening of the barrel of channel of the pore. The mobility of the
nucleic acid binding protein may be constrained by increasing the number of points at which
the protein is attached to the surface and/or the use of specific linkers.
Preferred conjugates
In a preferred embodiment, the construct comprises exonuclease I from E. coli (SEQ
ID NO: 6) or a variant thereof having at least one native accessible cysteine residue removed
attached to oc-HL (SEQ ID NO: 2) or a variant thereof via one or more accessible cysteine
residues. In a more preferred embodiment, the construct comprises exonuclease I from E. coli
(SEQ ID NO: 6) or a variant thereof having all its native accessible cysteine residue removed
attached to α-HL (SEQ ID NO: 2) or a variant thereof via one or more accessible cysteine
residues that have been substituted into the exonuclease. In a more preferred embodiment, the
construct comprises exonuclease I from E. coli (SEQ ID NO: 6) or a variant thereof having
only the native cysteine residues at position 144 and/or 330 attached to α-HL (SEQ ID NO: 2)
or a variant thereof via one or more of the native cysteine residues. In a more preferred
embodiment, the construct comprises exonuclease I from E. coli (SEQ ID NO: 6) or a variant

thereof having no native cysteine residues and A83C attached to α-HL (SEQ ID NO: 2) or a
variant thereof having E287C via the non-native cysteine residues.
In a preferred embodiment, the construct comprises exonuclease I from E. coli (SEQ
ID NO: 6) or a variant thereof having at least one native accessible cysteine residue removed
attached to α-HL or a variant thereof having L135C and N139Q (e.g. SEQ ID NO: 4) via one
or more accessible cysteine residues. In a more preferred embodiment, the construct
comprises exonuclease I from E. coli (SEQ ID NO: 6) or a variant thereof having all its native
accessible cysteine residue removed attached to α-HL or a variant thereof having L135C and
N139Q (e.g. SEQ ID NO: 4) via one or more accessible cysteine residues that have been
substituted into the exonuclease. In a more preferred embodiment, the construct comprises
exonuclease I from E. coli (SEQ ID NO: 6)or a variant thereof having only the native cysteine
residues at position 144 and/or 330 attached to α-HL or a variant thereof having L135C and
Nl 39Q (e.g. SEQ ID NO: 4) via one or more of the native cysteine residues. In a more
preferred embodiment, the construct comprises exonuclease I from E. coli (SEQ ID NO: 6) or
a variant thereof having no native cysteine residues and A83C attached to α-HL or a variant
therof having L135C, N139Q and E287C via the non-native cysteine residues.
In a preferred embodiment, the construct comprises exonuclease I from E. coli (SEQ
ID NO: 6) or a variant thereof having at least one native accessible cysteine residue removed
attached to α-HL (SEQ ID NO: 2) or a variant thereof via one or more hybridization linkers
attached to accessible cysteine residues in the exonuclease. In a more preferred embodiment,
the construct comprises exonuclease I from E. coli (SEQ ID NO: 6) or a variant thereof having
all its native accessible cysteine residue removed attached to α-HL (SEQ ID NO: 2) or a
variant thereof via one or more hybridization linkers attached to accessible cysteine residues
that have beens substituted into the exonuclease. In a more preferred embodiment, the
construct comprises exonuclease I from E. coli (SEQ ID NO: 6) or a variant thereof having
only the native cysteine residues at position 144 and/or 330 attached to α-HL (SEQ ID NO: 2)
or a variant thereof via hybridization linkers attached to one or more of the native cysteine
residue. In a more preferred embodiment, the construct comprises exonuclease I from E. coli
(SEQ ID NO: 6) or a variant thereof having no native cysteine residues and A83C attached to
α-HL (SEQ ID NO: 2) or a variant thereof having E287C via a hybridization linker attached to
the non-native cysteine residues.
In another preferred embodiment, the construct comprises exonuclease I from E. coli
(SEQ ID NO: 6) or a variant thereof having at least one native accessible cysteine residue
removed attached to α-HL or a variant thereof having L135C and N139Q (e.g. SEQ ID NO: 4)

via one or more hybridization linkers attached to accessible cysteine residues in the
exonuclease. In a more preferred embodiment, the construct comprises exonuclease I from E.
coli (SEQ ID NO: 6) or a variant thereof having all its native accessible cysteine residue
removed attached to α-HL or a variant thereof having L135C and N139Q (e.g. SEQ ID NO: 4)
via one or more hybridization linkers attached to accessible cysteine residues that have beens
substituted into the exonuclease. In a more preferred embodiment, the construct comprises
exonuclease I from E. coli (SEQ ID NO: 6) or a variant thereof having only the native cysteine
residues at position 144 and/or 330 attached to α-HL or a variant thereof having L135C and
N139Q (e.g. SEQ ID NO: 4) via hybridization linkers attached to the one or more native
cysteine residue. In a more preferred embodiment, the construct comprises exonuclease I from
E. coli (SEQ ID NO: 6) or a variant thereof having no native cysteine residues and A83C
attached to α-HL or a variant thereof having L135C, N139Q and E287C via a hybridization
linker attached to the non-native cysteine residues.
Modified pores
The invention also provides modified pores for use in sequencing nucleic acids. The
pores comprise at least one construct of the invention in which the surface is a transmembrane
protein pore or a subunit thereof. The pores may comprise more than one, such as 2, 3 or 4,
constructs of the invention.
A pore of the invention may be isolated, substantially isolated, purified or substantially
purified. A pore of the invention is isolated or purified if it is completely free of any other
components, such as lipids or other pores. A pore is substantially isolated if it is mixed with
carriers or diluents which will not interfere with its intended use. For instance, a pore is
substantially isolated or substantially purified if it present in a form that comprises less than
10%, less than 5%, less than 2% or less than 1% of other components, such as lipids or other
pores. Alternatively, a pore of the invention may be present in a lipid bilayer or in a
surfactant micelle.
The nucleic acid binding protein, which is preferably a nucleic acid handling enzyme,
attached to the construct handles a target nucleic acid sequence in such a way that a proportion
of the nucleotide in the target sequence interacts with the pore, preferably the barrel or channel
of the pore. Nucleotides are then distinguished on the basis of the different ways in which
they affect the current flowing through the pore during the interaction.
The fixed nature of the nucleic acid binding protein means that a target nucleic acid
sequence is handled by the pore in a specific manner. For instance, each nucleotide may be

digested from one of the target sequence in a processive manner, or the target sequence may be
pushed or pulled through the pore. This ensures that a proportion of the nucleotides in the
target nucleic acid sequence interacts with the pore and is identified. The lack of any
interruption in the signal is important when sequencing nucleic acids. In addition, the fixed
nature of the enzyme and the pore means they can be stored together, thereby allowing the
production of a ready-to-use sensor.
In a preferred embodiment, an exonuclease enzyme, such as a deoxyribonuclease, is
attached to the pore such that a proportion of the nucleotides is released from the target nucleic
acid and interacts with the barrel or channel of the pore. In another preferred embodiment, an
enzyme that is capable of pushing or pulling the target nucleic acid sequence through the pore
is attached to the pore such that the target nucleic acid sequence is pushed or pulled through
the barrel or channel of the pore and a proportion of the nucleotides in the target sequence
interacts with the barrel or channel. In this embodiment, the nucleotides may interact with the
pore in blocks or groups of more than one, such as 2, 3 or 4. Suitable enzymes include, but are
not limited to, polymerases, nucleases, helicases and topoisomerases, such as gyrases. In each
embodiment, the enzyme is preferably attached to the pore at a site in close proximity to the
opening of the barrel of channel of the pore. The enzyme is more preferably attached to the
pore such that its active site is orientated towards the opening of the barrel of channel of the
pore. This means that a proportion of the nucleotides of the target nucleic acid sequence is fed
in the barrel or channel. The enzyme is preferably attached to the cis side of the pore.
The modified pore may be derived from any of the transmembrane protein pores
discussed above, including the β-barrel pores and α-helix bundle pores.
For constructs comprising the sequence shown in SEQ ID NO: 2 or a variant thereof,
the pore typically comprises an appropriate number of additional subunits comprising the
sequence shown in SEQ ID NO: 2 or a variant thereof. A preferred pore of the invention
comprises one construct comprising the sequence shown in SEQ ID NO: 2 or a variant thereof
and six subunits comprising the sequence shown in SEQ ID NO: 2 or a variant thereof. The
pore may comprise one or more subunits comprising the sequence shown in SEQ ID NO: 4 or
a variant thereof. SEQ ID NO: 4 shows the sequence of SEQ ID NO: 2 except that it has an
cysteine at position 135 (L135C) and a glutamine at position 139 (N139Q). A variant of SEQ
ID NO: 4 may differ from SEQ ID NO: 4 in the same way and to the same extent as discussed
for SEQ ID NO: 2 above. A preferred pore of the invention comprises one construct
comprising the sequence shown in SEQ ID NO: 2 or a variant thereof and six subunits
comprising the sequence shown in SEQ ID NO: 4 or a variant thereof.

The pores may comprise a molecular adaptor that facilitates the interaction between the
pore and the nucleotides or the target nucleic acid sequence. The presence of the adaptor
improves the host-guest chemistry of the pore and nucleotides released from or present in the
target nucleic acid sequence. 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 nucleotides. The adaptor typically alters the charge of the barrel or channel of
the pore or specifically interacts with or binds to nucleotides thereby facilitating their
interaction with the pore.
The adaptor mediates the interaction between nucleotides released from or present in
the target nucleic acid sequence and the pore. The nucleotides preferably reversibly bind to
the pore via or in conjunction with the adaptor. The nucleotides most preferably reversibly
bind to the pore via or in conjunction with the adaptor as they pass through the pore across the
membrane. The nucleotides can also reversibly bind to the barrel or channel of the pore via or
in conjunction with the adaptor as they pass through the pore across the membrane. The
adaptor preferably constricts the barrel or channel so that it may interact with the nucleotides.
The adaptor is typically cyclic. The adaptor preferably has the same symmetry as the
pore. An adaptor having seven-fold symmetry is typically used if the pore is heptameric (e.g.
has seven subunits around a central axis that contribute 14 strands to a transmembrane β
barrel). Likewise, an adaptor having six-fold symmetry is typically used if the pore is
hexameric (e.g. has six subunits around a central axis that contribute 12 strands to a
transmembrane β barrel, or is a 12-stranded β barrel). Any adaptor that facilitates the
interaction between the pore and the nucleotide can be used. Suitable adaptors include, but are
not limited to, cyclodextrins, cyclic peptides and cucurbiturils. The adaptor is preferably a
cyclodextrin or a derivative thereof. The adaptor is more preferably heptakis-6-amino-β-
cyclodextrin (am7-βCD), 6-monodeoxy-6-monoamino-β-cyclodextrin (am1-βCD) or heptakis-
(6-deoxy-6-guanidino)-cyclodextrin (gu7-βCD). Table 4 below shows preferred combinations
of pores and adaptors.


The adaptor is preferably covalently attached to the pore. The adaptor can be
covalently attached to the pore using any method known in the art. The adaptor may be
attached directly to the pore. The adaptor is preferably attached to the pore using a
bifunctional crosslinker. Suitable crosslinkers are well-known in the art. Preferred
crosslinkers include 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propanoate, 2,5-
dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate and 2,5-dioxopyrrolidin-1-yl 8-
(pyridin-2-yldisulfanyl)octananoate. The most preferred crosslinker is succinimidyl 3-(2-
pyridyldithio)propionate (SPDP). Typically, the adaptor is covalently attached to the
bifunctional crosslinker before the adaptor/crosslinker complex is covalently attached to the

pore but it is also possible to covalently attach the bifunctional crosslinker to the pore before
the bifunctional crosslinker/pore complex is attached to the adaptor.
The site of covalent attachment is selected such that the adaptor facilitates interaction
of nucleotides released from or present in the target nucleic acid sequence with the pore and
thereby allows detection of nucleotides. For pores based on α-HL, the correct orientation of
the adaptor within the barrel or channel of the pore and the covalent attachment of adaptor to
the pore can be facilitated using specific modifications to the pore. In particular, every subunit
of the pore, including the construct(s), preferably has a glutamine at position 139 of SEQ ID
NO: 2. One or more of the subunits of the pore, including the construct(s), may have an
arginine at position 113 of SEQ ID NO: 2. One or more of the subunits of the pore, including
the construct(s), may have a cysteine at position 119,121 or 135 of SEQ ID NO: 2 to facilitate
attachment of the molecular adaptor to the pore.
Methods of producing constructs of the invention
The invention also provides methods of producing a construct of the invention. The
methods comprise attaching a nucleic acid binding protein comprising one or more accessible
cysteines to a surface via the cysteine residues. Any of the nucleic acid binding proteins and
surfaces discussed above can be used in the methods. The site of and method of covalent
attachment are selected as discussed above.
As described above, the methods preferably comprise removing all the native accessible
cysteine residues from the binding protein and introducing one or more non-native accessible
cysteine residues into the binding protein. Alternatively, the method comprises removing all
but one or more of the native accessible cysteine residues from the binding protein.
The methods also comprise determining whether or not the construct is capable of
binding nucleic acids. Assays for doing this are described above. If nucleic acids can be
bound, the protein and surface have been attached correctly and a construct of the invention
has been produced. If nucleic acids cannot be bound, a construct of the invention has not been
produced.
Methods of producing modified pores
The invention also provides methods of producing modified pores of the invention. The
modified pore may be formed by allowing at least one construct of the invention in which the
surface is a pore subunit to form a pore with other suitable subunits. Any of the constructs,

binding proteins, surfaces or pores discussed above can be used in the methods. The site of
and method of covalent attachment are selected as discussed above.
The methods also comprise determining whether or not the pore is capable of binding
nucleic acids and detecting nucleotides. The pore may be assessed for its ability to detect
individual nucleotides or short chains of nucleotides, such as di- or trinucleotides. Assays for
doing this are described above and below. If the pore is capable of binding nucleic acids and
detecting nucleotides, the subunit and enzyme have been attached correctly and a pore of the
invention has been produced. If a pore cannot bind nucleic acids and detect nucleotides, a
pore of the invention has not been produced.
The pores can be purified by inserting the construct(s) and remaining subunits into
synthetic lipid vesicles and allowed them to oligomerise. Methods for inserting the
constmct(s) and remaining subunits into synthetic vesicles are well known in the art.
The synthetic vesicles should have similar properties to rabbit cell membranes, but
should lack the rabbit cell membrane proteins. The vesicles may comprise any components
and are typically made of a blend of lipids. Suitable lipids are well-known in the art. The
synthetic vesicles preferably comprise 30 % cholesterol, 30 % phosphatidylcholine (PC), 20 %
phosphatidylethanolamine (PE), 10 % sphingomyelin (SM) and 10 % phosphatidylserine (PS).
The pore is then purified using an anionic surfactants, such as sodium dodecyl sulphate
(SDS), before it is attached to the nucleic acid binding protein.
Methods of sequencing nucleic acids
The invention also provides methods of sequencing a target nucleic acid sequence. In
one embodiment, the method comprises (a) contacting the target sequence with a pore of the
invention, which comprises an exonuclease, such that the exonuclease digests an individual
nucleotide from one end of the target sequence; (b) contacting the nucleotide with the pore so
that the nucleotide interacts with the adaptor; (c) measuring the current passing through the
pore during the interaction and thereby determining the identity of the nucleotide; and (d)
repeating steps (a) to (c) at the same end of the target sequence and thereby determining the
sequence of the target sequence. Hence, the method involves stochastic sensing of a
proportion of the nucleotides in a target nucleic acid sequence in a successive manner in order
to sequence the target sequence. Individual nucleotides are described above.
In another embodiment, the method comprises (a) contacting the target sequence with a
pore of the invention comprising a nucleic acid handling enzyme so that the target sequence is
pushed or pulled through the pore and a proportion of the nucleotides in the target sequence

interacts with the pore and (b) measuring the current passing through the pore during each
interaction and thereby determining the sequence of the target sequence. Hence, the method
involves stochastic sensing of a proportion of the nucleotides in a target nucleic acid sequence
as the nucleotides pass through the barrel or channel in a successive manner in order to
sequence the target sequence.
Pores comprising a construct of the invention are particularly suited to these methods.
In order to effectively sequence the nucleic acid, it is important to ensure that a proportion of
the nucleotides in the nucleic acid is identified in a successive manner. The fixed nature of the
enzyme means that a proportion of the nucleotides in the target sequence affects the current
flowing through the pore.
The whole or only part of the target nucleic acid sequence may be sequenced using this
method. The nucleic acid sequence can be any length. For example, the nucleic acid sequence
can be at least 10. at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at
least 400 or at least 500 nucleotides in length. The nucleic acid sequence can be naturally
occurring or artificial. For instance, the method may be used to verify the sequence of a
manufactured oligonucleotide. The methods are typically carried out in vitro.
The methods may be carried out using any suitable membrane/pore system in which a
pore comprising a construct of the invention is inserted into a membrane. The methods are
typically carried out using (i) an artificial membrane comprising a pore comprising a construct
of the invention, (ii) an isolated, naturally occurring membrane comprising a pore comprising
a construct of the invention, or (iii) a cell expressing a pore comprising a construct of the
invention. The methods are preferably carried out using an artificial membrane. The
membrane may comprise other transmembrane and/or intramembrane proteins as well as other
molecules in addition to the pore of the invention.
The membrane forms a barrier to the flow of ions, nucleotides and nucleic acids. The
membrane is preferably a lipid bilayer. Lipid bilayers suitable for use in accordance with the
invention can be made using methods known in the art. For example, lipid bilayer membranes
can be formed using the method of Montal and Mueller (1972). Lipid bilayers can also be
formed using the method described in International Application No. PCT/GB08/000563 and
PCT/GB07/002856.
The methods of the invention may be carried out using lipid bilayers formed from any
membrane lipid including, but not limited to, phospholipids, glycolipids, cholesterol and
mixtures thereof. Any of the lipids described in International Application No.
PCT/GB08/000563 may be used.

Methods are known in the art for inserting pores into membranes, such as lipid
bi layers. Some of those methods are discussed above.
Interaction between the pore and nucleotides
The nucleotide or nucleic acid may be contacted with the pore on either side of the
membrane. The nucleotide or nucleic acid may be introduced to the pore on either side of the
membrane. The nucleotide or nucleic acid is typically contacted with the side of the
membrane on which the enzyme is attached to the pore. This allows the enzyme to handle the
nucleic acid during the method.
A proportion of the nucleotides of the target nucleic acid sequence interacts with the
pore and/or adaptor as it passes across the membrane through the barrel or channel of the pore.
Alternatively, if the target sequence is digested by an exonuclease, the nucleotide may interact
with the pore via or in conjunction with the adaptor, dissociate from the pore and remain on
the same side of the membrane. The methods may involve the use of pores in which the
orientation of the adaptor is fixed. In such embodiments, the nucleotide is preferably
contacted with the end of the pore towards which the adaptor is oriented. Most preferably, the
nucleotide is contacted with the end of the pore towards which the portion of the adaptor that
interacts with the nucleotide is orientated.
The nucleotides may interact with the pore in any manner and at any site. As discussed
above, the nucleotides preferably reversibly bind to the pore via or in conjunction with the
adaptor. The nucleotides most preferably reversibly bind to the pore via or in conjunction
with the adaptor as they pass through the pore across the membrane. The nucleotides can also
reversibly bind to the barrel or channel of the pore via or in conjunction with the adaptor as
they pass through the pore across the membrane.
During the interaction between a nucleotides and the pore, the nucleotide affects the
current flowing through the pore in a manner specific for that nucleotide. For example, a
particular nucleotide will reduce the current flowing through the pore for a particular mean
time period and to a particular extent. In other words, the current flowing through the pore is
distinctive for a particular nucleotide. Control experiments may be carried out to determine
the effect a particular nucleotide has on the current flowing through the pore. Results from
carrying out the method of the invention on a test sample can then be compared with those
derived from such a control experiment in order to identify a particular nucleotide.

Apparatus
The methods may be carried out using any apparatus that is suitable for investigating a
membrane/pore system in which a pore comprising a construct of the invention is inserted into
a membrane. The methods may be carried out using any apparatus that is suitable for
stochastic sensing. For example, the apparatus comprises a chamber comprising an aqueous
solution and a barrier that separates the chamber into two sections. The barrier has an aperture
in which the membrane containing the pore is formed. The nucleotide or nucleic acid may be
contacted with the pore by introducing the nucleic acid into the chamber. The nucleic acid
may be introduced into either of the two sections of the chamber, but is preferably introduced
into the section of the chamber containing the enzyme.
The methods may be carried out using the apparatus described in International
Application No. PCT/GB08/000562.
The methods involve measuring the current passing through the pore during interaction
with the nucleotides. Therefore the apparatus also comprises an electrical circuit capable of
applying a potential and measuring an electrical signal across the membrane and pore. The
methods may be carried out using a patch clamp or a voltage clamp. The methods preferably
involve the use of a voltage clamp.
Conditions
The methods of the invention involve the measuring of a current passing through the
pore during interaction with nucleotides of a target nucleic acid sequence. Suitable conditions
for measuring ionic currents through transmembrane pores are known in the art and disclosed
in the Examples. The method is carried out with a voltage applied across the membrane and
pore. The voltage used is typically from -400mV to +400mV. The voltage used is preferably
in a range having a lower limit selected from -400 mV, -300mV, -200 mV, -150 mV, -100
mV, -50 mV, -20mV and 0 mV and an upper limit independently selected from +10 mV, + 20
mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used is
more preferably in the range 120mV to 170mV. It is possible to increase discrimination
between different nucleotides by a pore of the invention by varying the applied potential.
The methods are carried out in the presence of any alkali metal chloride salt. In the
exemplary apparatus discussed above, the salt is present in the aqueous solution in the
chamber. Potassium chloride (KC1), sodium chloride (NaCl) or caesium chloride (CsCl) is
typically used. K.C1 is preferred. The salt concentration is typically from 0.1 to 2.5M, from
0.3 to 1.9M, from 0.5 to 1.8M, from 0.7 to 1.7M, from 0.9 to 1.6M or from 1M to 1.4M High

salt concentrations provide a high signal to noise ratio and allow for currents indicative of the
presence of a nucleotide to be identified against the background of normal current fluctuations.
However, lower salt concentrations may have to be used so that the enzyme is capable of
functioning.
The methods are typically carried out in the presence of a buffer. In the exemplary
apparatus discussed above, the buffer is present in the aqueous solution in the chamber. Any
buffer may be used in the methods. One suitable buffer is Tris-HCl buffer. The methods are
typically carried out at a pH of from 4.0 to 10.0, from 4.5 to 9.5, from 5.0 to 9.0, from 5.5 to
8.8. from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pH used is preferably about 7.5.
The methods are typically carried out at from 0°C to 100°C, from 15°C to 95°C, from
16°C to 90°C, from 17°C to 85°C, from 18°C to 80°C, 19°C to 70°C, or from 20°C to 60°C.
The methods may be carried out at room temperature. The methods are preferably carried out
at a temperature that supports enzyme function, such as about 37°C. Good nucleotide
discrimination can be achieved at low salt concentrations if the temperature is increased.
However, lower temperatures, particularly those below room temperature, result in longer
dwell times and can therefore be used to obtain a higher degree of accuracy.
In addition to increasing the solution temperature, there are a number of other
strategies that can be employed to increase the conductance of the solution, while maintaining
conditions that are suitable for enzyme activity. One such strategy is to use the lipid bilayer to
divide two different concentrations of salt solution, a low salt concentration of salt on the
enzyme side and a higher concentration on the opposite side. One example of this approach is
to use 200 mM of KG on the cis side of the membrane and 500 raM KC1 in the trans chamber.
At these conditions, the conductance through the pore is expected to be roughly equivalent to
400 mM KG under normal conditions, and the enzyme only experiences 200 mM if placed on
the cis side. Another possible benefit of using asymmetric salt conditions is the osmotic
gradient induced across the pore. This net flow of water could be used to pull nucleotides into
the pore for detection. A similar effect can be achieved using a neutral osmolyte, such as
sucrose, glycerol or PEG. Another possibility is to use a solution with relatively low levels of
KG and rely on an additional charge carrying species that is less disruptive to enzyme
activity.
Exonuclease-based methods
In one embodiment, the method of sequencing a target nucleic acid sequence involves
contacting the target sequence with a pore having an exonuclease enzyme, such as

deoxyribonuclease, attached thereto. The constructs needed to make such pores are discussed
above. Any of the exonuclease enzymes discussed above may be used in the method. The
exonuclease releases individual nucleotides from one end of the target sequence.
Exonucleases are enzymes that typically latch onto one end of a nucleic acid sequence and
digest the sequence one nucleotide at a time from that end. The exonuclease can digest the
nucleic acid in the 5' to 3' direction or 3' to 5' direction. The end of the nucleic acid to which
the exonuclease binds is typically determined through the choice of enzyme used and/or using
methods known in the art. Hydroxyl groups or cap structures at either end of the nucleic acid
sequence may typically be used to prevent or facilitate the binding of the exonuclease to a
particular end of the nucleic acid sequence.
The method involves contacting the nucleic acid sequence with the exonuclease so that
the nucleotides are digested from the end of the nucleic acid at a rate that allows identification
of a proportion of nucleotides as discussed above. Methods for doing this are well known in
the art. For example, Edman degradation is used to successively digest single amino acids
from the end of polypeptide such that they may be identified using High Performance Liquid
Chromatography (HPLC). A homologous method may be used in the invention.
The rate at which the exonuclease functions can be altered by mutation compared to
the wild type enzyme. A suitable rate of activity of the exonuclease in the method of
sequencing involves digestion of from 0.5 to 1000 nucleotides per second, from 0.6 to 500
nucleotides per second, 0.7 to 200 nucleotides per second, from 0.8 to 100 nucleotides per
second, from 0.9 to 50 nucleotides per second or 1 to 20 or 10 nucleotides per second. The rate
is preferably 1, 10, 100, 500 or 1000 nucleotides per second. A suitable rate of exonuclease
activity can be achieved in various ways. For example, variant exonucleases with a reduced or
improved optimal rate of activity may be used in accordance with the invention.
Pushing or pulling DNA through the pore
Strand sequencing involves the controlled and stepwise translocation of nucleic acid
polymers through a pore. The majority of DNA handling enzymes are suitable for use in this
application provided they hydrolyse, polymerise or process single stranded DNA or RNA.
Preferred enzymes are polymerases, nucleases, helicases and topoisomerases, such as gyrases.
The enzyme moiety is not required to be in as close a proximity to the pore lumen as for
individual nucleotide sequencing as there is no potential for disorder in the series in which
nucleotides reach the sensing moiety of the pore.
The two strategies for single strand DNA sequencing are the translocation of the DNA

through the nanopore, both cis to trans and trans to cis, either with or against an applied
potential. The most advantageous mechanism for strand sequencing is the controlled
translocation of single strand DNA through the nanopore with an applied potential.
Exonucleases that act progressively or processively on double stranded DNA can be used on
the cis side of the pore to feed the remaining single strand through under an applied potential
or the trans side under a reverse potential. Likewise, a helicase that unwinds the double
stranded DNA can also be used in a similar manner. There are also possibilities for
sequencing applications that require strand translocation against an applied potential, but the
DNA must be first "caught" by the enzyme under a reverse or no potential. With the potential
then switched back following binding the strand will pass cis to trans through the pore and be
held in an extended conformation by the current flow. The single strand DNA exonucleases or
single strand DNA dependent polymerases can act as molecular motors to pull the recently
translocated single strand back through the pore in a controlled stepwise manner, trans to cis,
against the applied potential.
Kits
The invention also provides kits for producing a modified pore for use in sequencing
nucleic acids. In one embodiment, the kits comprise at least one construct of the invention in
which the surface is a pore subunit and any remaining subunits need to form a pore. The kits
may comprise enough constructs of the invention to form a complete pore (i.e. a homo-
oligomer). The kits may comprise any of the constructs and subunits discussed above. A
preferred kit comprises (i) a construct comprising a subunit comprising the sequence shown in
SEQ ID NO: 2 or a variant thereof and (ii) six subunits comprising the sequence shown in
SEQ ID NO: 2 or a variant thereof.
The kits of the invention may additionally comprise one or more other reagents or
instruments which enable any of the embodiments mentioned above to be carried out. Such
reagents or instruments include one or more of the following: suitable buffer(s) (aqueous
solutions), means to obtain a sample from a subject (such as a vessel or an instrument
comprising a needle), means to amplify and/or express polynucleotide sequences, a membrane
as defined above or voltage or patch clamp apparatus. Reagents may be present in the kit in a
dry state such that a fluid sample resuspends the reagents. The kit may also, optionally,
comprise instructions to enable the kit to be used in the method of the invention or details
regarding which patients the method may be used for. The kit may, optionally, comprise
nucleotides.

Enzymes and polynucleotide sequences
The invention also provides an exonuclease enzyme comprising the sequence shown in
any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,
46, 48 and 50 or a variant thereof. SEQ ID NOs: 8, 10 and 12 each have all but one native
accessible cysteine residues removed. SEQ ID NOs: 14,16,18, 20, 22, 24, 26, 28, 30, 32, 34,
36, 38,40, 42, 44, 46, 48 and 50 each have all native accessible cysteine residues removed and
one non-native accessible cysteine residue introduced. Variants include any of those discussed
above with reference to the constructs of the invention.
The invention also provides polynucleotide sequences which encode an exonuclease
enzyme of the invention. The polynucleotide sequence preferably comprises SEQ ID NO: 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49 or a variant
thereof. Variants of SEQ ID NO: 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47 or 49 are sequences that are at least 50%, 60%, 70%, 80%, 90% or 95%
homologous based on nucleotide identity to sequence of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19,
21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45,47 or 49 over the entire sequence. There may
be at least 80%, for example at least 85%, 90% or 95% nucleotide identity over a stretch of
600 or more, for example 700, 750, 850 or 900 or more, contiguous nucleotides ("hard
homology"). Homology may be calculated as described above. The polynucleotide sequence
may comprise a sequence that differs from SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,
29. 31. 33. 35, 37, 39, 41. 43. 45, 47 or 49 on the basis of the degeneracy of the genetic code.
Polynucleotide sequences may be isolated and replicated using standard methods in the
art. Chromosomal DNA may be extracted from an enzyme producing organism, such as E.
coli, T. thermophilics or bacteriophage. The gene encoding the enzyme may be amplified
using PCR involving specific primers. The amplified sequences may then be incorporated into
a recombinant replicable vector such as a cloning vector. The vector may be used to replicate
the polynucleotide in a compatible host cell. Thus polynucleotide sequences encoding the
enzyme may be made by introducing a polynucleotide encoding the enzyme into a replicable
vector, introducing the vector into a compatible host cell, and growing the host cell under
conditions which bring about replication of the vector. The vector may be recovered from the
host cell. Suitable host cells for cloning of polynucleotides are known in the art and described
in more detail below.
The polynucleotide sequence may be cloned into suitable expression vector. In an
expression vector, the polynucleotide sequence encoding a construct is typically operably

linked to a control sequence which is capable of providing for the expression of the coding
sequence by the host cell. Such expression vectors can be used to express a construct.
The term "operably linked" refers to a juxtaposition wherein the components described
are in a relationship permitting them to function in their intended manner. A control sequence
"operably linked" to a coding sequence is ligated in such a way that expression of the coding
sequence is achieved under conditions compatible with the control sequences. Multiple copies
of the same or different polynucleotide may be introduced into the vector.
The expression vector may then be introduced into a suitable host cell. Thus, a
construct can be produced by inserting a polynucleotide sequence encoding a construct into an
expression vector, introducing the vector into a compatible bacterial host cell, and growing the
host cell under conditions which bring about expression of the polynucleotide sequence. The
recombinantly-expressed construct may self-assemble into a pore in the host cell membrane.
Alternatively, the recombinant construct produced in this manner may be isolated from the
host cell and inserted into another membrane. When producing an oligomeric pore comprising
a construct of the invention and at least one different subunit, the construct and different
subunits may be expressed separately in different host cells as described above, removed from
the host cells and assembled into a pore in a separate membrane, such as a rabbit cell
membrane.
The vectors may be for example, plasmid, virus or phage vectors provided with an
origin of replication, optionally a promoter for the expression of the said polynucleotide
sequence and optionally a regulator of the promoter. The vectors may contain one or more
selectable marker genes, for example an ampicillin resistance gene. Promoters and other
expression regulation signals may be selected to be compatible with the host cell for which the
expression vector is designed. A T7, trc, lac, ara or ΛL promoter is typically used.
The host cell typically expresses the construct at a high level. Host cells transformed with a
polynucleotide sequence encoding a construct will be chosen to be compatible with the
expression vector used to transform the cell. The host cell is typically bacterial and preferably
E. coll. Any cell with a λ DE3 lysogen, for example C41 (DE3), BL21 (DE3), JM109 (DE3),
B834 (DE3), TUNER, Origami and Origami B. can express a vector comprising the T7
promoter.
The following Example illustrates the invention:

Example
1 Materials and Methods
1.1 Overview
One method of sequencing nucleic acids using stochastic sensing involves the use of an
exonuclease attached to the α-HL pore. This can be done by introducing a chemical linkage
between the two proteins. The chemical linkage joins one cysteine on the exonuclease to one
cysteine on the hemolysin, either directly (a disulphide bond), or via a linker molecule
between the two cysteine residues.
In order to introduce the chemical linkage in a controlled and directed approach it was
necessary to replace 5 naturally occurring cysteine residues found within Exonuclease I with
other residues. An additional mutation would then be made in order to introduce the cysteine
residue attachment point. Computer modeling was used to identify suitable attachment
positions (see below).
1.2 Modelling Exo I CYS Knockout Mutations
Molecular modelling has been used in an attempt to predict which mutations of the 5
wild type cysteine residues of E.coli Exo I are compatible with protein stability. To this end
alchemical free energy perturbation simulations where performed for these mutations,
allowing predictions to be made about their relative stability. These predictions were used to
guide experimental work.
VMD in combination with NAMD was used in conjunction with the CHARMM27
forcefield to perform alchemical free energy perturbation calculations upon wild type
CYS to XXX mutations of the E. coli Exo I protein structure (PDB accession code:
1 fxx [14]) were determined. The resulting lambda vs. deltaG curves were used to determine
the free energy change upon mutation and if the calculations had converged.
1.3 Expressions and Purification
A codon optimised version of the Exonuclease I gene, scsB, from Escherichia coli
(SEQ ID NOs: 5 and 6) was obtained from GenScript Corporation. Site directed mutagenesis
of cysteine residues was performed by an adapted form of in vivo homologous recombination
of PCR products (Jones. D. H. PCR mutagenesis and recombination in vivo. (1994) Genome
Research, 3. ppI41-148). Plasmids containing the gene of interest were transformed into

BL21 (DE3) pLysS (stratagene). Cultures containing 500ml Terrific Broth in 2 litre baffled
shake flasks supplemented with 100µg/ml ampicillin and 20µg/ml chloramphenical were
inoculated with colonies from agar plates and grown to OD600=2 (approx 6hours) before
induction with 0.2mM IPTG at reduced temperature (18°C). Cells were harvested by
centrifugation at 4K followed by lysis with bugbuster (Novagen) and benzonase (Merck). Cell
debris was removed by centrifugation at 10K and the supernatant filtered before loading onto a
HisTrap column (GE Healthcare). Eluted protein was further purified by gel filtration.
1.4 Enzyme Assay
The Exonuclease 1 (Exo I) assay employs an oligodeoxynucleotide (DNA template)
labelled with a fluorescein dye ("Fluor") on a dT at position 15, and a fluorescein quencher
("BHQ1") on dT at position 1. The template initially exists in a quenched state, with the
fluorescein and quencher in close proximity. Upon the introduction of Exo I, the DNA
template is digested in a processive manner from the 3'-end, eventually releasing dT-Fluor into
solution and leading to a subsequent increase in fluorescence as the dye diffuses away from
the quencher (Figure 6).
To conduct the assay, DNA template is transferred into a cuvette and placed in a Cary
Eclipse fluorimeter to equilibrate at a desired temperature. Meanwhile, Exo I is equilibrated
separately using a water bath and then added to the template-containing cuvette. The
subsequent increase in fluorescence signal is monitored over time (see Figure 7 for example
data) with the initial rate of increase extracted. The end-point-fluorescence after the template
has been fully digested is correlated with the initial amount of DNA template in order to
convert the fluorescence-based rate to a molecular one.

It is clear from the tables above that the obvious mutation (Cys to Ser) at each of these
sites is not predicted to be energetically favourable (positive predicted deltaG). The mutations
which are predicted to stabilise the protein vary significantly, as a consequence of their
different local environments.
It should be noted that, although we have both activity and expression data, this does
not directly relate to the delta G values in these tables. The delta G values can be related to
thermodynamic data (such as that obtained from DSC), providing it is assumed that the
mutations do not change the protein folding pathway significantly (which in many applications
of alchemical FEP in the literature, has been found to be applicable). There is no direct
relationship between expression and delta G of mutation or activity.
Comparison of the crystal structures of the Klenow fragment of DNA polymerase I and
EcoExo I [14] suggest indicate that the residues involved in protein catalysis and metal
binding are Aspl5. Glul7, Aspl08, Aspl86 and His181.
Residue types other than Cys can also be chemically modified, including Asp [1,2],
Glu [1,2], Lys[3,4,5], Arg[6,7,8], His[9,10], Tyr[11,12], Trp[11,12] and Met[13]. Therefore
mutating these residues, in order to allow directed chemical modifications of Exo I allows us
to remove competing chemical reactions.
Experimental work (see below) shows that many of these mutations result in proteins
that retain their activity and in some cases appear to enhance activity, either as single
mutations or as combinations of mutants. Comparison of the E. coli Exo I sequence with other
related Exo I protein sequences, and the construction of homology models of these sequences
suggests that many of these Cys residues are conserved both in sequence and in structure.
This suggests that the properties of the Cys mutants of E. coli Exo I may be applicable to
similar mutants of related Exo I enzymes.
A sequence alignment of all the Exo I enzymes in the Swissprot database shows that
C51. C98, C144 and C306 are conserved.
2.2 Identification of 5 Cysteine Residues
Exonuclease I (SEQ ID N06) contains 5 naturally occurring cysteine residues (Figure
1). Some of the cysteine residues are conserved within other homologues of E.coli
Exonuclease I (EcoExo 1) (see below). None of the cysteine residues within Exonuclease I
form internal disulphide bonds.

2.3 Removal of all 5 Cysteines by Mutation to Serine
The serine (Ser) amino acid is the most similar in structure to cysteine (Cys), with the
S atom being replaced by an 0. The most obvious mutation to make is therefore a Cys (C) to
Ser (S). To test the activity of this mutant, all 5 cysteine residues were mutated to serine and
protein expression was attempted.
Expression of ONLD0217 (Exo I C51S/C98S/C144S/C306S/C330S) resulted in
reduced yields, poor expression and unstable protein. The protein was not able to be purified.
This demonstrates that when all the Cys are replaced with Ser, the protein is not stable.
Further investigation of the protein structure was required to engineer a cysteine free EcoExo
I. This was guided in part by computer modeling.
2.4 Replacement of Individual Cysteines by Mutation to Serine
To identify cysteine positions that would tolerate substitution individual cysteine
residues were replaced in turn with a serine residue.
Constructs ONLD0233 (C51S), ONLD0234 (C98S), ONLD0235 (C144S),
ONLD0236 (C306S), ONLD0237 (C330S) were expressed. The mutant C98S expressed to
sufficient levels, after a few attempts sufficient levels of C306S were also achieved. However
expression levels of all the other position were much reduced.
Alternative mutations were therefore considered to produce sufficient expression levels
so that the mutants could be tested for exonuclease activity. The cysteines were grouped into
highly accessible positions (C98, C306, C330; Figure 2) and more buried residues (C51,
CI 44).
2.5 Replacement of C306 and C330 with Alternatives to Serine
Replacement of cysteine residues at positions 306 and 330 were investigated by
examination of the following mutants.
Exo I C306 → D, M, N, S, T
Exo I C330 → D, M, N, T, Y, H, L, M, Q
Expression levels of ONLD0335 (C306T) were highest. Other mutations that were
tolerated in this position were S, D and N.
Expression levels of ONLD0342 (C330T) were highest. Other mutations that were
tolerated in this position were H, Q and M and to a lesser extent D, N, L and Y.

2.6 Combining C98S with C306S and C330T
In case double and triple mutations adversely affected each other the following
combinations were investigated.

The combination ONLD0366 gave highest expression followed by ONLD0368. The
combinations ONLD0365 and ONLD0363 failed to express. Lower yields were obtained from
ONLD0367, ONLD0364.
Activity assays (see Methods) showed that the relative activity of the Exo I mutants
was higher than the wild type (Figure 3).
2.7 Introduction of Cysteine Attachment Points
Attachment points were designed to form a covalent bond from α-HL direct to the
corresponding cysteine of Exonuclease I or via a linker. The attachment positions (V42C,
A83C, S90C, V94C, Ml 84C) predicted via modeling are shown in Figure 4.
Introduction of the above cysteines to the mutant Exo I C98S, C306S, C330T gave the
following constructs:

The activity of the cysteines was assessed by reacting with malemide-PEG which acts
as a gel-shift reagent. The resultant gels showed that each protein had three reactive cysteines.
This data showed that the cysteines at C144 and C51 were more accessible than previously
assumed and were also reacting with the malemide-PEG. Further rounds of mutagenesis were
carried out to replace C144 and C51 to ensure that the protein only react at one position.

2.8 Replacement of Residue C144 with Alternatives to Serine
Possible mutations were assessed from a total of 18 using molecular modeling (A, E,
G, I, L, N, R, T, W, D, F, H, K, M, Q, S, V, Y). The six most stable mutants at residue 144
predicted from the modeling were made:
Exo 1 C98S, C306S, C330S, C144 → A, G, L, T, H, M
From the 6 mutations tested (ONLD0403) Exo I C98S/C306S/C330T/C144M and
(ONLD0404) Exo I C98S/C306S/C330T/C144T expressed well. Expression levels were
reduced in the other mutants.
2.9 Replacement of Residue C51 with Alternatives to Serine
Possible mutations were assessed from a total of 17 using molecular modeling (A, D,
E, F, G, H, I, K, L, M, N, R, S, T, V, W, Y). The eight most stable mutants at residue 51
predicted from the modeling were made:
Exo I C98S, C306S, C330S, C51 → A, G, H, K, L, M, T, W
From the 8 mutations tested only (ONLD0393) Exo I C98S/C306S/C330T/C51A
expressed.
2.10 Combination of 5 Cysteine Replacements and Addition of Attachement Point
The following combinations were assessed. These included all 5 cysteine
replacements and addition of a cysteine attachment point. Attachment could also be made to a
cysteine added to the N or C-termini.


* ONLD0425 did not express well - not enough to measure the concentration but enough to just confirm activity.
The two mutants with N-terminal cysteine residues (OND0413, ONLD0414) failed to
express. All other combinations expressed and were active exonucleases. Combinations
ONLD0415 and ONLD0417 expressed less well than ONLD0416 and ONLD0418. The
activity results are shown in Figure 5.
The following combinations were also assessed.

2.11 DSC analysis of proteins
Differential scanning calorimetry (DSC) is a technique that allows measurement of
thermal transitions within a sample. Its primary use is measuring the thermal stability of a bio-

molecule as a transition mid-point. The transition mid-point is used as a marker of thermal
stability.
The earliest mutant analysed with all 5 cysteines replaced (Exo-ASTST) was found to
be quite unstable with a high loss of activity over time. The Tm was determined to be 35°C,
compared to wildtype at 49°C.
Following further rounds of mutant screening the enzyme Exo-ATTTT was identified
with a Tm of 40°C and greatly improved stability over time. Other combinations eg Exo-
ATTMT and Exo-AVTAT had a similar but not improved Tm of 40°C.
The mutant A VTMT was found to have slightly higher Tm, however was found more
prone to aggregation under some conditions.
Addition of the preferred non-native cysteine (A83C) added for attachment purposes
does not appear to have a detrimental effect on stability.


2.12 Other mutations of interest: C1441 and C330I
C144 is adjacent to both the DNA binding site and the catalytic site of Exo-I. Residue
C144 and C330 have not been mutated as much as other cysteine residues to find the best
combination. Based upon the analysis of protein sequence and previous modelling work,
models of the following 2 proteins (ATTTT and ATITI) were constructed.
Modeling work comparing ATTTT with ATITI suggest that T330 side chain causes
little disruption of the protein secondary structure. T144 however is packed with the
hydrophobic core which appears to be disrupted when the T144 side chain flips, causing
significant disruption to the core. The ATITI model simulation does not show the same degree
of disruption around T144.
2.13 Enzyme activity
The construct examined was Exo-ATTTT-A83C. The linker used in these examples is
a ssDNA linker with the 5'end free and the 3' end attached to the enzyme. The activity of
Exo-ATTTT-A83C was measured with ssDNA linker attached and then the linker was
removed by addition of DTT and activity remeasured. No change in activity was observed
confirming the presence of the linker does not have a detrimental impact on activity (Figure
8). In this examples activity of Exo-ATTTT-A83C was compared of free enzyme and PNA
modified enzyme. No loss of activity was observed by addition of the PNA linker (Figure 9).
Exo ATTTT-M184C (with PEG). Controls were Exo ATTTT in the presence of PEG. No loss
of activity was observed by addition of the PEG linker (Figure 10).
3 Conclusion
We have created a range of mutants of EcoExo I where all the native cysteines have
been removed. In addition, a single cysteine has been introduced to control the attachment
chemistry of the ExoEco I. Somewhat surprisingly, the activity of some of the mutants is
higher than the wild type.

We cliam:
1. A construct comprising a nucleic acid binding protein and a surface,
wherein at least one native accessible cysteine residue is removed from the binding
protein, wherein the binding protein is attached to the surface via one or more
accessible cysteine residues and wherein the binding protein retains its ability to bind
nucleic acids.
2. A construct according to claim 1, wherein all the native accessible cysteine
residues are removed from the binding protein and one or more non-native accessible
cysteine residues are introduced into the binding protein.
3. A construct according to claim 2, wherein the non-native accessible cysteine
residue(s) are introduced by substitution.
4. A construct according to claim 1, wherein all but one or more of the native
accessible cysteine residues are removed from the binding protein.
5. A construct according to any one of claims 2 to 4, wherein the native cysteine
residues are removed by substitution.
6. A construct according to claim 5, wherein the native cysteine residues are
substituted with threonine.
7. A construct according to any one of the preceding claims, wherein the
accessible cysteine(s) are available for reaction with a thiol specific group.
8. A construct according to any one of the preceding claims, wherein the nucleic
acid binding protein is a nucleic acid handling enzyme and retains its ability to
handle nucleic acids.

9. A construct according to claim 8, wherein the nucleic acid handling enzyme
is derived from a nuclease.
10. A construct according to claim 9, wherein the nuclease is member of any of
the Enzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21,
3.1.22.3.1.25. 3.1.26. 3.1.27. 3.1.30 and 3.1.31.
11. A construct according to claim 10, wherein the nucleic acid handling enzyme
is derived from an exonuclease.
12. A construct according to claim 11, wherein the exonuclease comprises the
sequence shown in any one of SEQ ID NOs: 6, 52, 54 and 56 or a variant thereof.
13. A construct according to any one of the preceding claims, wherein the nucleic
acid binding protein comprises the sequence shown in any one of SEQ ID NOs: 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50 or a
variant thereof.
14. A construct according to claim 8, wherein the nucleic acid handling enzyme
is derived from a polymerase, helicase or topoisomerase.
15. A construct according to claim 14, wherein:

(a) the polymerase is member of any of the Enzyme Classification (EC)
groups 2.7.7.6, 2.7.7.7, 2.7.7.19, 2.7.7.48 and 2.7.7.49;
(b) the helicase is member of any of the Enzyme Classification (EC)
groups 3.6.1.- and 2.7.7.-; or
(c) the topoisomerase is member of any of the Enzyme Classification
(EC) groups 5.99.1.2 and 5.99.1.3.
16 A construct according to claim 15, wherein the polymerase is a DNA-
dependent DNA polymerase, an RNA-dependent DNA polymerase, a DNA-
dependent RNA polymerase or an RNA-dependent RNA polymerase.

17. A construct according to claim 15, wherein the helicase an ATP-dependent
DNA helicase. an ATP-dependent RNA helicase or an ATP-independent RNA
helicase.
18. A construct according to claim 15, wherein the topoisomerase is a gyrase.
19. A construct according to any one of the preceding claims, wherein the surface
is a transmembrane protein pore or a subunit thereof.
20. A construct according to claim 19, wherein the transmembrane protein pore is
α-hemolysin (α-HL).
21. A construct according to claim 20, wherein the surface comprises the
sequence shown SEQ ID NO: 2 or a variant thereof.
22. A construct according to any one of the preceding claims, wherein the
enzyme is attached to the surface by one or more linkers.
23. A construct according to claim 22, wherein the one or more linkers are
nucleic acid hybridization linkers.
24. A modified pore for use in sequencing nucleic acids, comprising at least one
construct according to any one of claims 19 to 21.
25. A pore according to claim 24, wherein the pore comprises a construct
according to claim 21 and six subunits comprising the sequence shown SEQ ID NO:
2 or a variant thereof.
26. A pore according to claim 25, wherein all seven subunits have a glutamine at
position 139 of SEQ ID NO: 2 and one of the subunits has a cysteine at position 135
of SEQ ID NO:2.

27. A pore according to claim 26, wherein all seven subunits have an arginine at
position 113 of SEQ ID NO:2.
28. A pore according to any one of claims 24 to 27, wherein the pore comprises a
molecular adaptor that facilitates an interaction between the pore and one or more
nucleotide(s).
29. A pore according to claim 28, wherein the molecular adaptor is a cyclodextrin
or a derivative thereof.
30. A pore according to claim 29, wherein the cyclodextrin is heptakis-6-amino-
β-cyclodextrin (am7-βCD), 6-monodeoxy-6-monoamino-β-cyclodextrin (am1-βCD)
or heptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu7-βCD).
31. A kit for producing a modified pore for use in sequencing nucleic acids,
comprising:

(a) at least one construct according to any one of claims 19 to 21; and
(b) the remaining subunits needed to form a pore.
32. A kit according to claim 31, wherein the kit comprises:
(a) a construct according to claim 20; and
(b) six subunits each comprising the sequence shown in SEQ ID NO: 2 or a
variant thereof.
33. A method of producing a construct according to any one of claims 1 to 23,
comprising:
(a) attaching a nucleic acid binding protein comprising one or more
accessible cysteine residues to a surface via the cysteine residues; and
(b) determining whether or not the resulting construct is capable of binding
nucleic acids.

34. A method according to claim 33, further comprising before step (a):
(i) removing all the native accessible cysteine residues from the binding
protein and introducing one or more non-native accessible cysteine residues into the
binding protein; or
(ii) removing all but one or more of the native accessible cysteine residues
from the binding protein.
35. A method of producing a modified pore according to claim 24, comprising:
(a) allowing at least one construct of any one of claims 19 to 21 to form a
pore with other suitable subunits; and
(b) determining whether or not the resulting pore is capable of binding
nucleic acids and detecting nucleotides.
36. A method of sequencing a target nucleic acid sequence, comprising:
(a) contacting the target sequence with a pore according to claim
24, which comprises an exonuclease, such that the exonuclease digests an individual
nucleotide from one end of the target sequence;
(b) contacting the nucleotide with the pore so that the nucleotide interacts
with the adaptor;
(c) measuring the current passing through the pore during the interaction and
thereby determining the identity of the nucleotide; and
(d) repeating steps (a) to (c) at the same end of the target sequence and
thereby determining the sequence of the target sequence.
37. A method of sequencing a target nucleic acid sequence, comprising:
(a) contacting the target sequence with a pore according to claim 24 so that
the enzyme pushes or pulls the target sequence through the pore and a proportion of
the nucleotides in the target sequence interacts with the pore; and
(b) measuring the current passing through the pore during each interaction
and thereby determining the sequence of the target sequence.

38. An exonuclease enzyme comprising the sequence shown in any one of SEQ
ID NOs: 8. 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48
and 50 or a variant thereof.
39. A polynucleotide sequence which encodes an exonuclease enzyme according
to claim 38.

The invention relates to constructs comprising a nucleic acid binding protein
and a surface. At least one native accessible cysteine residue is removed from the
binding protein. The binding protein is attached to the surface via one or more
accessible cysteine residues. The removal of other accessible cysteine residues from
the protein allows control attachment to the surface. The constructs can be used to
generate transmembrane pores having a nucleic acid binding protein attached thereto.
Such pores are particularly useful for sequencing nucleic acids. The enzyme
handles the nucleic acid in such a way that the pore can detect each of its component
nucleotides by stochastic sensing.