US20110311965A1 - Methods of enhancing translocation of charged analytes through transmembrane protein pores - Google Patents

Methods of enhancing translocation of charged analytes through transmembrane protein pores Download PDF

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US20110311965A1
US20110311965A1 US13/129,278 US200913129278A US2011311965A1 US 20110311965 A1 US20110311965 A1 US 20110311965A1 US 200913129278 A US200913129278 A US 200913129278A US 2011311965 A1 US2011311965 A1 US 2011311965A1
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pore
analyte
translocation
dna
amino acids
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Giovanni Maglia
John Hagan Pryce Bayley
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Oxford University Innovation Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention relates to enhancing translocation of a charged analyte through a transmembrane protein pore. Translocation is enhanced by increasing the net opposing charge of the barrel or channel and/or entrance of the pore.
  • the invention also relates to a pore enhanced in accordance with 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 ⁇ -hemolysin ( ⁇ -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).
  • analytes When analytes translocate through a transmembrane protein pore, they are capable of affecting the current flowing through the pore in a manner specific for that analyte. This allows the analyte to be detected using stochastic sensing.
  • the analyte usually affects the current flowing through a pore by interacting with the pore. A distinctive current flows through the pore whenever the analyte interacts with the pore.
  • the inventors have surprisingly demonstrated that increasing the net opposing charge of the barrel or channel and/or entrance of a pore enhances the translocation of charged analytes through the pore. For instance, increasing the net positive charge of the barrel or channel and/or entrance of a pore enhances the translocation of negatively charged analytes through the pore. Increasing the net opposing charge of the barrel or channel and/or entrance increases the frequency of translocation of the analyte through the pore. It also decreases the threshold voltage for translocation of the analyte through the pore. It also decreases the translocation speed of the analyte through the pore. Further, events in which the analyte enters the pore, but is not translocated, are almost eliminated. Pores enhanced in this way are useful tools for stochastic sensing of charged analytes, especially for sequencing nucleic acids.
  • the invention therefore provides a method of enhancing translocation of a charged analyte through a transmembrane protein pore, comprising:
  • the invention also provides:
  • FIG. 1 shows sections through the ⁇ -hemolysin ( ⁇ -HL) nanopore (PDB: 7AHL).
  • ⁇ -HL ⁇ -hemolysin
  • PDB 7AHL
  • FIG. 2 shows DNA translocation through the ⁇ -HL pore at +120 mV.
  • Type A event a simple low-amplitude event (interpreted as the passage of DNA straight through the pore); type B: a simple mid-amplitude event (DNA is captured in the vestibule and exits the pore from the side of entry, the cis side); type C: a mid-amplitude signal, followed by a low-amplitude signal within the same event (DNA is detained in the vestibule before it passes through the ⁇ barrel to exit at the trans side); type D: a low-amplitude signal, followed by a mid-amplitude signal within the same event (DNA enters the ⁇ barrel directly and then retracts into the vestibule to exit the pore from the cis side); type E: a mid-amplitude signal, followed by a low-amplitude signal, followed by a second mid-amplitude signal within the same event (DNA explores the vestibule, threads
  • FIG. 3 shows the effects of charged side-chains, introduced or removed by mutagenesis, on the frequency of DNA translocation at +120 mV.
  • M113R-WT (0.46 ⁇ M DNA, cis)
  • E111N-WT (0.47 ⁇ M)
  • WT (0.53 ⁇ M)
  • M113R-RL2 (0.74 ⁇ M)
  • RL2 (0.60 ⁇ M)
  • M113D-RL2 2.0 ⁇ M.
  • M113D-RL2 showed no current blockades at up to +300 mV. Additional conditions are described in the legend to FIG. 2 .
  • FIG. 4 shows the effects of arginine substitution in the ⁇ barrel of ⁇ -HL on the frequency of DNA translocation through the pore +120 mV.
  • the frequency of DNA translocation is normalized to 1 ⁇ M DNA.
  • the line is a single exponential fit of the frequency of DNA translocation versus the distance of the arginine substitution from the constriction as represented by residue number.
  • the mutants are in the RL2 background. Additional conditions are described in the legend to FIG. 2 .
  • FIG. 5 shows the voltage dependence of the DNA capture rates.
  • the DNA translocation frequency is plotted on a logarithmic scale and normalized to 1 ⁇ M DNA. Only the translocation events (i.e. type A and C) are included in the analysis.
  • M113R-WT red circles
  • E111N-WT blue diamonds
  • M113R-RL2 black squares
  • WT green triangles down
  • RL2 blue triangles up
  • the lines show fits of the data with equation 1.
  • FIG. 6 shows the model for the translocation of DNA through the ⁇ -HL pore.
  • DNA To pass through the pore (V), DNA must first collide with the cis entrance of the pore (II), be transported through the vestibule (III) and enter the ⁇ barrel (IV). The DNA can prolong its visit within the vestibule and produce mid-amplitude and low-amplitude events corresponding to VI and VII.
  • the processes that can be observed experimentally are labelled as events and are shown with slow inter-event rates. All steps are reversible with exception of translocation.
  • type A events DNA collides with the pore, enters the vestibule and then directly translocates through the ⁇ barrel to exit on the trans side of the pore.
  • Type B events DNA enters the pore but instead of penetrating the ⁇ barrel it has a prolonged interaction with the vestibule to produce the mid-amplitude event (I ⁇ II ⁇ III ⁇ VI). In this case, DNA exits the pore from the same cis entrance from which it entered (VI ⁇ III ⁇ II ⁇ I).
  • type C events first show a mid-amplitude current (I ⁇ II ⁇ III ⁇ VI), but then one end of the DNA enters the ⁇ barrel (VII) and a translocation event occurs (VII ⁇ V ⁇ I).
  • type D events the DNA most likely enters the ⁇ barrel (I ⁇ II ⁇ III ⁇ IV) but then stops translocating (VII), retracts back into the vestibule (VI) and exits the pore from the cis side (VI ⁇ III ⁇ II ⁇ I).
  • type E events the DNA begins to translocate through the ⁇ barrel as in type C events (I ⁇ II ⁇ III ⁇ VI ⁇ VII ⁇ V), but then retracts into the vestibule (V ⁇ VII ⁇ VI) and exits the pore from the cis side as in type D events (VI ⁇ III ⁇ II ⁇ I).
  • Intermediates expected to give mid-amplitude events are numbered in yellow, although II and III would be too short for observation.
  • Intermediates expected to give low-amplitude events are numbered in red, although IV would be too short for observation.
  • FIG. 7 shows DNA blockades for the homoheptameric mutant ⁇ -HL pores E111N-WT and M113R-WT.
  • the DNA was added to the cis chamber at ⁇ 0.5 ⁇ M; the exact concentration was determined after each experiment. For further conditions, see FIG. 2 , legend.
  • FIG. 8 shows amplitude histograms of mean event currents for DNA blockades of various ⁇ -HL pores.
  • the main peak represents the type A events (DNA translocation).
  • Each plot comprises 1000 events. The conditions are described in the legend to FIG. 2 .
  • FIG. 9 shows current blockades after DNA addition (1.0 ⁇ M) to the trans chamber for: (a) M113R-RL2 and (b) N121R-RL2.
  • the applied potential was ⁇ 120 mV.
  • the currents through the open pores are much noisier at negative potentials than at positive potentials. Further conditions are described in the legend to FIG. 2 .
  • FIG. 10 shows rare ( ⁇ 1%) current blockade events for DNA translocation through WT- ⁇ -HL nanopores at +120 mV showing multiple transitions between the mid-amplitude and low-amplitude states. The conditions are described in the legend to FIG. 2 .
  • FIG. 11 shows: a) Section through a 7R nanopore.
  • the amino acids at position 113, 115, 117, 119, 121, 123 and 125 were replaced by arginine using PyMOL (DeLano Scientific LLC, v 1.0) software from the WT nanopore (PDB:7AHL). Negative charged residues are coloured in red and positively charged residues in blue.
  • FIG. 12 shows DNA translocation through 7R nanopores.
  • FIG. 13 shows residual current through WT and modified ⁇ -HL nanopores during ssDNA translocation at +120 mV. Errors express the standard deviation. All nanopores, except from WT, and 2N are in RL2 background. The fourth to last columns from the left concern the nanopores with additional positive charges.
  • FIG. 14 shows the dependence of the DNA translocation speed on the internal charge of 7R heteromers.
  • the data are fitted to a single-exponential curve (upper panel).
  • Heteromers nanopores were obtained by mixing monomers of RL2 (open circles) with monomer of 7R (purple circles).
  • FIG. 15 shows the potential uses of 7R nanopores.
  • a) Single strands are sequenced by reading the tunneling current.
  • the 7R channel is incorporated into solid state nanopore functionalized with tunneling probes. Transverse tunneling currents through a ssDNA that is translocation through the pore are measured for recognition of single bases. 7R will control the time that each nucleotide remains between the tunneling probes.
  • a ⁇ -barrel containing intra-positive charges is attached to the CIS entrance of the ⁇ -HL nanopore for controlling the speed of ssDNA. Differences between single nucleotides are measured by changes in conductance of the nanopore generated by the translocation of DNA.
  • SEQ ID NO: 1 shows the polynucleotide sequence that encodes one subunit of wild-type (WT) ⁇ -hemolysin.
  • SEQ ID NO: 2 shows the amino acid sequence of one subunit of WT ⁇ -hemolysin.
  • Amino acids 2 to 6, 73 to 75, 207 to 209, 214 to 216 and 219 to 222 form ⁇ -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 ⁇ -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 that encodes one subunit of ⁇ -hemolysin RL2.
  • RL2 is the product of a semisynthetic gene that was devised to permit cassette mutagenesis of the sequence encoding the transmembrane ⁇ barrel (Cheley, S., Braha, O., Lu, X., Conlan, S., and Bayley, H. (1999) Protein Sci. 8, 1257-1267). It contains six silent restriction sites and five altered amino acids in the encoded polypeptide sequence (K8A, V124L, G130S, N139Q and I142L). D8RL2 is RL2 with an octa-aspartate tail.
  • SEQ ID NO: 4 shows the amino acid sequence of one subunit of ⁇ -hemolysin RL2.
  • the same amino acids discussed above for SEQ ID NO: 2 form ⁇ -helices, ⁇ -strands, loop regions and terminal amino acids.
  • SEQ ID NO: 5 shows the polynucleotide sequence that encodes one subunit of ⁇ -hemolysin M113R-WT.
  • SEQ ID NO: 6 shows the amino acid sequence of one subunit of ⁇ -hemolysin M113R-WT.
  • SEQ ID NO: 7 shows the polynucleotide sequence that encodes one subunit of ⁇ -hemolysin E111N-WT.
  • SEQ ID NO: 8 shows the amino acid sequence of one subunit of ⁇ -hemolysin E111N-WT.
  • SEQ ID NO: 9 shows the polynucleotide sequence that encodes one subunit of ⁇ -hemolysin A8R-RL2 used in the Example.
  • SEQ ID NO: 10 shows the amino acid sequence of one subunit of ⁇ -hemolysin A8R-RL2 used in the Example.
  • SEQ ID NO: 11 shows the polynucleotide sequence that encodes one subunit of ⁇ -hemolysin M113K-RL2 used in the Example.
  • SEQ ID NO: 12 shows the amino acid sequence of one subunit of ⁇ -hemolysin M113K-RL2 used in the Example.
  • SEQ ID NO: 13 shows the polynucleotide sequence that encodes one subunit of ⁇ -hemolysin M113R-RL2 used in the Example.
  • SEQ ID NO: 14 shows the amino acid sequence of one subunit of ⁇ -hemolysin M113R-RL2 used in the Example.
  • SEQ ID NO: 15 shows the DNA sequence used in the Example.
  • the present invention provides a method of enhancing translocation of a charged analyte through a transmembrane protein pore.
  • the transmembrane protein pore is typically for use in detecting the analyte via stochastic sensing. As the analyte translocates through the pore, it typically interacts with the pore and affects the current flowing through the pore in a manner specific for that analyte. A distinctive current flows through the pore whenever the analyte interacts with the pore. This allows the analyte to be detected using stochastic sensing.
  • the method may enhance the translocation of the analyte in any way.
  • the method preferably (1) increases the frequency of translocation of the analyte through the pore, (2) decreases the threshold voltage for translocation of the analyte through the pore, (3) decreases the speed of translocation of the analyte through the pore or (4) decreases the number of non-translocation interactions between the analyte and the pore.
  • Non-translocation interactions are events where the analyte enters the pore and interacts with the pore, but does not translocate through the pore. In some instance, the non-translocation interactions are eliminated.
  • the method more preferably increases (1) and (2); (1) and (3); (1) and (4); (2) and (3); (2) and (4); (3) and (4); (1), (2) and (3); (1), (3) and (4); (1), (2) and (4); or (2), (3) and (4).
  • the method most preferably increases (1), (2), (3) and (4). In other words, the method most preferably increases the frequency of translocation of the analyte through the pore, decreases the threshold voltage for translocation of the analyte through the pore, decreases the speed of translocation of the analyte through the pore and decreases the number of non-translocation interactions between the analyte and the pore.
  • the analyte may be contacted with a non-enhanced or wild-type pore and the same type of pore in which the net opposing charge of its barrel or channel and/or entrance has been increased and any difference in translocation of the analyte between the two pores may be determined.
  • Analyte translocation may be measured using any method known in the art. One method is described in the Examples.
  • the method of the invention provides an improved pore for stochastic sensing of the analyte.
  • An enhanced translocation through the pore results in a more sensitive system that: (1) allows the detection of the analyte at both high and low concentrations; (2) allows the detection of the analyte even amongst impurities; (3) allows more rapid detection of the analyte; (4) reduces the background noise produced by non-translocation interactions and (5) allows the detection of the analyte at a lower voltage.
  • an enhanced translocation, and in particular an enhanced frequency of translocation results in a reduced deadtime between reads of the consecutive nucleotides in the sequence. This allows nucleic acids to be sequenced more quickly.
  • a decreased translocation speed through the pore increases the read time of the consecutive nucleotides in the nucleic acid sequence and so allows the sequence of the nucleic acid to be determined more accurately.
  • the analyte can be any charged substance. An analyte is charged if it has a net charge. The analyte may be negatively charged or positively charged. An analyte is negatively charged if it has a net negative charge. An analyte is positively charged if it has a net positive charge.
  • Suitable analytes include, but are not limited to, metal ions, inorganic salts, polymers, such as a polymeric acids or bases, dyes, bleaches, pharmaceuticals, diagnostic agents, recreational drugs, explosives and environmental pollutants.
  • the analyte can be an analyte that is secreted from cells.
  • the analyte can be an analyte that is present inside cells such that the analyte must be extracted from cells.
  • the analyte is preferably a polymer.
  • the polymer is preferably a nucleic acid sequence. Nucleic acids are negatively charged.
  • a nucleic acid is a macromolecule comprising two or more nucleotides.
  • the nucleic acid handled by the enzyme may comprise any combination of any nucleotides.
  • the nucleotides can be naturally occurring or artificial.
  • 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.
  • Suitable 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), deoxyaden
  • the nucleic acid is preferably double stranded, such as DNA.
  • the nucleic acid may be single stranded, such as cDNA or RNA.
  • the polymer can be a peptide, polypeptide or a protein.
  • the peptide, polypeptide or protein can be naturally-occurring or non-naturally-occurring.
  • the polypeptide or protein can include within it synthetic or modified amino acids. A number of different types of modification to amino acids are well known in the art. For the purposes of the invention, it is to be understood that the analyte can be modified by any method available in the art.
  • the protein can be an enzyme, antibody, hormone, growth factor or growth regulatory protein, such as a cytokine.
  • the cytokine may be selected from an interleukin, preferably IFN-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 or IL-13, an interferon, preferably IL- ⁇ or other cytokines such as TNF- ⁇ .
  • the protein may be a bacterial protein, fungal protein, virus protein or parasite-derived protein. Before it is contacted with the pore, the protein may be unfolded to form a polypeptide chain and thereby allow it to enter the barrel or channel of the pore and interact with the pore.
  • the analyte may be an amino acid.
  • Charged amino acids are well known in the art and are discussed in more detail below.
  • the amino acid can be naturally-occurring or non-naturally-occurring.
  • the amino acid may be synthetic or modified. A number of different types of modification to amino acids are well known in the art.
  • the analyte may be an individual nucleotide or a single nucleotide.
  • An individual nucleotide is one which is not bound to another polynucleotide by a nucleotide bond.
  • a nucleotide 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 by a nucleotide bond to another polynucleotide 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.
  • the individual nucleotide has been digested from a target polynucleotide sequence, such as a DNA or RNA strand. Nucleotides are negatively charged.
  • the nucleotide may be any of those discussed above.
  • the nucleotide may be derived from the digestion of a nucleic acid sequence such as ribonucleic acid (RNA) or deoxyribonucleic acid.
  • Nucleic acid sequences can be digested using any method known in the art. Suitable methods include, but are not limited to, those using enzymes or catalysts. Catalytic digestion of nucleic acids is disclosed in Deck et al., Inorg. Chem., 2002; 41: 669-677.
  • nucleotides from a single nucleic acid sequence may be contacted with the pore in a sequential manner in order to sequence the whole or part of the nucleic acid. Sequencing nucleic acids in accordance with the second embodiment of the invention is discussed in more detail below.
  • the nucleotide is typically unmodified, such as when the nucleotide is derived from the digestion of a nucleic acid sequence. Alternatively, the nucleotide may be modified or damaged. The nucleotide is typically methylated or oxidised.
  • the nucleotide may be labelled with a revealing label.
  • the revealing label may be any suitable label which allows the nucleotide to be detected. Suitable labels include fluorescent molecules, radioisotopes, e.g. 125 I, 35 S, and linkers such as biotin.
  • the nucleotide is typically present in any suitable biological sample. Suitable biological samples are discussed below.
  • a transmembrane protein pore is a polypeptide that permits ions to flow from one side of the membrane to the other along an applied potential.
  • the pore preferably permits the analyte to flow from one side of the membrane to the other along the applied potential.
  • the pore is typically an oligomer.
  • the pore is preferably made up of several repeating subunits, such as 6, 7 or 8 subunits.
  • the pore is more preferably heptameric.
  • 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 ⁇ barrel or channel or a transmembrane ⁇ -helix bundle or channel.
  • Pores for use in accordance with the invention can be ⁇ -barrel pores or ⁇ -helix bundle pores.
  • ⁇ -barrel pores comprise a barrel or channel that is formed from ⁇ -sheets.
  • Suitable ⁇ -barrel pores include, but are not limited to, ⁇ -toxins, such as ⁇ -hemolysin 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).
  • ⁇ -helix bundle pores comprise a barrel or channel that is formed from ⁇ -helices.
  • Suitable ⁇ -helix bundle pores include, but are not limited to, inner membrane proteins and ⁇ outer membrane proteins, such as WZA.
  • the most preferred pore for use in the invention is ⁇ -hemolysin or a variant thereof.
  • the ⁇ -hemolysin pore is formed of seven identical subunits (i.e. it is heptameric).
  • the sequence of one subunit of wild-type ⁇ -hemolysin is shown in SEQ ID NO: 2.
  • the sequence of one subunit of ⁇ -hemolysin RL2 is shown in SEQ ID NO: 4.
  • a variant is a heptameric pore in which one or more of the seven subunits has an amino acid sequence which varies from that of SEQ ID NO: 2 or 4 and which retains pore activity.
  • a variant may include modifications that facilitate interaction with the analyte.
  • 1, 2, 3, 4, 5, 6 or 7 of the subunits in a variant ⁇ -hemolysin may have an amino acid sequence that varies from that of SEQ ID NO: 2 or 4. All seven subunits within a variant pore are typically identical but may be different, particularly if one or more of the subunits has been modified to facilitate interaction with the analyte.
  • the variant may be a naturally-occurring variant which is expressed by an organism, for instance by a Staphylococcus bacterium.
  • Variants also include non-naturally occurring variants produced by recombinant technology.
  • a subunit of a variant will preferably be at least 50% homologous to that sequence based on amino acid identity. More preferably, the subunit 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.
  • Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 2 or 4, 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.
  • One or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 or 4 may alternatively or additionally be deleted. Up to 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.
  • Variants may include subunits made of 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. Such fragments may be used to produce chimeric pores. A fragment preferably comprises the pore forming domain of SEQ ID NO: 2 or 4.
  • Variants include chimeric protein pores comprising fragments or portions of SEQ ID NO: 2 or 4.
  • Chimeric protein pores are formed from subunits each comprising fragments or portions of SEQ ID NO: 2 or 4.
  • the pore or channel part of a chimeric protein pore is typically formed by the fragments or portions 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 N-terminus or C-terminus of the amino acid sequence of SEQ ID NO: 2 or 4 or polypeptide variant or fragment thereof.
  • the extension may be quite short, for example from 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example up to 50 or 100 amino acids.
  • a carrier protein may be fused to an amino acid sequence according to the invention.
  • 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 ⁇ -HL, which contains a ⁇ -barrel, is provided by ⁇ -sheets in each subunit.
  • a variant of SEQ ID NO: 2 or 4 typically comprises the regions in SEQ ID NO: 2 or 4 that form ⁇ -sheets.
  • One or more modifications can be made to the regions of SEQ ID NO: 2 or 4 that form ⁇ -sheets as long as the resulting variant retains its ability to form a pore. Specific modifications that can be made to the ⁇ -sheet regions of SEQ ID NO: 2 or 4 in accordance with the invention are discussed below.
  • a variant of SEQ ID NO: 2 or 4 preferably includes one or more modifications, such as substitutions, additions or deletions, within its ⁇ -helices and/or loop regions.
  • Amino acids 2 to 6, 73 to 75, 207 to 209, 214 to 216 and 219 to 222 of SEQ ID NO: 2 and 4 form ⁇ -helices.
  • Amino acids 1 and 294 are terminal amino acids. Specific modifications that can be made to the loop regions of SEQ ID NO: 2 or 4 in accordance with the invention are discussed below.
  • Standard methods in the art may be used to determine homology.
  • 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, p 387-395).
  • 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.
  • HSPs high scoring sequence pair
  • T some positive-valued threshold score
  • 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 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.
  • P(N) the smallest sum probability
  • 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.
  • a transmembrane protein pore may be modified for example by the addition of histidine or aspartic acid residues to assist their 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.
  • a pore 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. 125 I, 35 S, enzymes, antibodies, antigens, polynucleotides and ligands such as biotin.
  • the pore may be derived from a pore producing organism, such as Staphylococcus aureus, or made synthetically or by recombinant means.
  • the pore may be synthesized by in vitro transcription and translation (IVTT).
  • IVTT in vitro transcription and translation
  • the amino acid sequence of the pore may be modified to include non-naturally occurring amino acids, for instance to increase the stability of the pore.
  • such amino acids may be introduced during production.
  • the pore may also be produced using D-amino acids.
  • the pore 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 pore can also be altered following either synthetic or recombinant production.
  • the pore may contain other non-specific modifications as long as they do not interfere with translocation of the analyte.
  • a number of non-specific side chain modifications are well known in the art and may be made to the side chains of the pore. Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH 4 , amidination with methylacetimidate and thiol-specific modifications using ethyleneamine.
  • the pore can be produced using standard methods known in the art. Polynucleotide sequences encoding a pore or a pore subunit may be derived and replicated using standard methods in the art. Such sequences are discussed in more detail below. Polynucleotide sequences encoding a pore or a pore subunit may be expressed in a bacterial host cell using standard techniques in the art. The pore or pore subunit 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 pore subunit 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.
  • the naturally occurring or recombinantly-produced pore or pore subunit may then be inserted into a naturally occurring or artificial membrane for use in accordance with the invention. Methods for inserting pore into membranes are discussed below.
  • the transmembrane pore may comprise a molecular adaptor, such as a cyclodextrin, a cyclic peptide or a cucurbituril, that facilitates the interaction between the pore and the analyte by improving the host-guest chemistry of the pore and the analyte.
  • a molecular adaptor such as a cyclodextrin, a cyclic peptide or a cucurbituril
  • Suitable adaptors are well known in the art.
  • the molecular adaptor may be covalently attached to the pore.
  • the method of the invention comprises increasing the net opposing charge of the barrel or channel and/or entrance of the pore.
  • the opposing charge is the opposite charge to the charge of the analyte. If the analyte is negatively charged, the method involves increasing the net positive charge of the barrel or channel and/or entrance of the pore. If the analyte is positively charged, the method comprises increasing the net negative charge of the barrel or channel and/or entrance of the pore.
  • the net opposing charge may be increased by any means known in the art.
  • the net opposing charge is increased in a manner that does not interfere with translocation of the analyte through the pore.
  • the net opposing charge is increased in a manner that does not interfere with the interaction between the analyte and the pore.
  • the analyte is negatively charged and the method involves increasing the net positive charge of the barrel or channel and/or entrance of the pore.
  • the net positive charge is preferably increased by introducing one or more positively charged amino acids into the barrel or channel and/or entrance of the pore.
  • the one or more positively charged amino acids may be introduced by addition.
  • the one or more positively charged amino acids are preferably introduced by substitution.
  • a positively charged amino acid is an amino acid with a net positive charge.
  • the positively charged amino acid(s) can be naturally-occurring or non-naturally-occurring.
  • the positively charged amino acids may be synthetic or modified.
  • modified amino acids with a net positive charge may be specifically designed for use in the invention.
  • a number of different types of modification to amino acids are well known in the art.
  • Preferred naturally-occurring positively charged amino acids include, but are not limited to, histidine (H), lysine (K) and arginine (R). Any number and combination of H, K and/or R may be introduced into the barrel or channel and/or entrance of the pore.
  • methionine (M) may be substituted with arginine (R) by replacing the codon for methionine (ATG) with a codon for arginine (AGA) at the relevant position in a polynucleotide encoding the pore.
  • AGA codon for arginine
  • non-naturally-occurring amino acids may be introduced by including synthetic aminoacyl-tRNAs in the IVTT system used to express the pore.
  • they may be introduced by expressing the pore in E. coli that are auxotrophic for specific amino acids in the presence of synthetic (i.e. non-naturally-occurring) analogues of those specific amino acids. They may also be produced by naked ligation if the pore is produced using partial peptide synthesis.
  • Any amino acid may be substituted with a positively charged amino acid.
  • One or more uncharged amino acids, non-polar amino acids and/or aromatic amino acids may be substituted with one or more positively charged amino acids.
  • Uncharged amino acids have no net charge. Suitable uncharged amino acids include, but are not limited to, cysteine (C), serine (S), threonine (T), methionine (M), asparagine (N) and glutamine (Q).
  • Non-polar amino acids have non-polar side chains. Suitable non-polar amino acids include, but are not limited to, glycine (G), alanine (A), proline (P), isoleucine (I), leucine (L) and valine (V).
  • Aromatic amino acids have an aromatic side chain.
  • Suitable aromatic amino acids include, but are not limited to, histidine (H), phenylalanine (F), tryptophan (W) and tyrosine (Y).
  • one or more negatively charged amino acids are substituted with one or more positively charged amino acids.
  • Suitable negatively charged amino acids include, but are not limited to, aspartic acid (D) and glutamic acid (E).
  • Preferred introductions include, but are not limited to, substitution of M with R, substitution of M with H, substitution of M with K, substitution of D with R, substitution of D with H, substitution of D with K, substitution of E with R, substitution of E with H, substitution of E with K, substitution of N with R, substitution of T with R and substitution of G with R.
  • any number of positively charged amino acids may be introduced. For instance, 1, 2, 5, 10, 15, 20, 25 or more positively charged amino acids may be introduced. If the pore is oligomeric, the one or more positively charged amino acids may be introduced into one, some or all of the subunits of the pore. In the case of ⁇ -HL (i.e. SEQ ID NO: 2 and 4 and variants thereof discussed above), the one or more positively charged amino acids may be introduced into 1, 2, 3, 4, 5, 6 or 7 of the subunits in the pore. In each of the seven subunits, the one or more positively charged amino acids may be introduced at the same or different positions. Preferably, the pore is a homoheptamer and one or more positive amino acids are introduced at the same position(s) in each subunit.
  • the net positive charge may also be increased by replacing by substitution one or more negatively charged amino acids with one or more uncharged amino acids, non-polar amino acids and/or aromatic amino acids.
  • the removal of negative charge from the barrel or channel and/or entrance increases the net positive charge.
  • the uncharged amino acids, non-polar amino acids and/or aromatic amino acids can be naturally-occurring or non-naturally-occurring. They may be synthetic or modified. Suitable uncharged amino acids, non-polar amino acids and aromatic amino acids are discussed above.
  • Preferred substitutions include, but are not limited to, substitution of E with N, substitution of D with N, substitution of E with T, substitution of D with T, substitution of E with G and substitution of D with G.
  • any number and combination of uncharged amino acids, non-polar amino acids and/or aromatic amino acids may substituted into the barrel or channel and/or entrance.
  • 1, 2, 5, 10, 15, 20, 25 or more uncharged amino acids, non-polar amino acids and/or aromatic amino acids may be may substituted into the barrel or channel and/or entrance.
  • the pore is oligomeric, the uncharged amino acids, non-polar amino acids and/or aromatic amino acids may be substituted into one, some or all of the subunits of the pore.
  • ⁇ -HL i.e.
  • the uncharged amino acids, non-polar amino acids and/or aromatic amino acids may be substituted into 1, 2, 3, 4, 5, 6 or 7 of the subunits in the pore.
  • the one or more uncharged amino acids, non-polar amino acids and/or aromatic amino acids may be substituted into the same or different positions.
  • the pore is a homoheptamer and uncharged amino acids, non-polar amino acids and/or aromatic amino acids are substituted into the same position(s) in each subunit.
  • Negatively charged amino acids may be substituted with (1) uncharged amino acids; (2) non-polar amino acids; (3) aromatic amino acids; (4) uncharged amino acids and non-polar amino acids; (5) uncharged amino acids and aromatic amino acids; and (5) non-polar amino acids and aromatic amino acids; or (6) uncharged amino acids, non-polar amino acids and aromatic amino acids.
  • the analyte is positively charged and the method involves increasing the net negative charge of the barrel or channel and/or entrance of the pore.
  • the net negative charge is preferably increased by introducing one or more negatively charged amino acids into the barrel or channel and/or entrance of the pore.
  • the one or more negatively charged amino acids may be introduced by addition.
  • the one or more negatively charged amino acids are preferably introduced by substitution. Methods for adding and substituting amino acids are well known in the art.
  • Suitable negatively charged amino acids are discussed above.
  • the negatively charged amino acid(s) can be naturally-occurring or non-naturally-occurring.
  • the negatively charged amino acids may be synthetic or modified.
  • Any amino acid may be substituted with a negatively charged amino acid.
  • One or more uncharged amino acids, non-polar amino acids and/or aromatic amino acids may be substituted with one or more negatively charged amino acids.
  • one or more positively charged amino acids are substituted with one or more negatively charged amino acids. Any number of negatively charged amino acids may be introduced as discussed above.
  • the net negative charge may also be increased by replacing by substitution one or more positively charged amino acids with one or more uncharged amino acids, non-polar amino acids and/or aromatic amino acids.
  • a preferred substitution is substitution of K with N.
  • the removal of positive charge from the barrel or channel and/or entrance increases the net negative charge.
  • the uncharged amino acids, non-polar amino acids and/or aromatic amino acids can be naturally-occurring or non-naturally-occurring. They may be synthetic or modified.
  • Any number and combination of uncharged amino acids, non-polar amino acids and/or aromatic amino acids may substituted into the barrel or channel and/or entrance as discussed above.
  • the net opposing charge of the barrel or channel of the pore is increased.
  • the barrel or the channel of the pore is the portion of the pore through which ions travel through the pore across the membrane.
  • the analyte also translocates through the barrel or the channel.
  • the barrel or channel may be formed from ⁇ -helices or ⁇ -strands depending on the type of pore.
  • the net opposing charge of the lumen of the barrel or channel is increased.
  • Amino acid(s) may be introduced or substituted at any position in the barrel or channel as long as translocation of the analyte is enhanced. Amino acid(s) are preferably introduced or substituted at position(s) that form part of the lumen of the barrel or channel. In other words, it is preferred that amino acid(s) are not introduced or substituted at positions in the barrel or channel that are buried in the pore or hidden from the lumen.
  • Amino acid(s) are preferably introduced or substituted towards the end of the barrel or channel through which the analyte enters.
  • the end of the pore through which the analyte enters may be the cis end or the trans end.
  • the end is preferably the cis end.
  • Amino acid(s) are preferably introduced or substituted near a constriction of the barrel or channel.
  • Amino acid(s) are more preferably introduced or substituted near a constriction of the lumen of the barrel or channel.
  • the net opposing charge of the entrance of the pore is increased.
  • An entrance of the pore is the portion of the pore through which the analyte enters into the barrel or channel. Entrances are typically formed primarily from loop regions in the pore.
  • amino acid(s) may be introduced or substituted at any position in the entrance as long as translocation of the analyte is enhanced.
  • amino acid(s) are introduced or substituted at position(s) that form part of the lumen of the entrance. In other words, it is preferred that amino acid(s) are not introduced or substituted at positions in the entrance that are buried in the pore or hidden from the lumen.
  • Amino acid(s) may be introduced or substituted in one or both entrances. Amino acid(s) are typically introduced or substituted near the entrance through which the analyte enters the pore. Amino acid(s) may be introduced or substituted in the barrel or channel and in one or both entrance(s).
  • amino acid(s) may be introduced or substituted at any of positions 1 to 22, 35 to 41, 54 to 59, 100 to 155 and 218 to 229 to increase the net opposing charge. All of these positions form part of the lumen of the barrel or channel or part of the lumen of an entrance of SEQ ID NOs: 2 and 4. Positions 111 to 147 form part of the lumen of the barrel or channel. Positions 1 to 22, 35 to 41, 54 to 59, 100 to 110, 148 to 155 and 218 to 229 form part of the lumen of an entrance. Amino acid(s) are preferably introduced or substituted at positions of SEQ ID NO: 2 or 4 located near the constriction of the barrel or channel. Such positions include, but are not limited to, positions 111, 113, 115, 117, 119, 121, 123, 125, 127, 145 and 147. A preferred position in the entrance of SEQ ID NO: 2 is position 8.
  • Preferred substitutions located near the constriction of the barrel or channel of SEQ ID NO: 2 or 4 include, but are not limited to, M113R, E111N, M113K, T115R, T117R, G119R, N121R, N123R, M113D, K147N, D127R, T125R and T145R.
  • Preferred substitutions located in the entrance of SEQ ID NO: 4 include, but are not limited to, A8R and A8K.
  • the present invention also provides a transmembrane protein pore enhanced in accordance with the invention.
  • a transmembrane protein pore enhanced in accordance with the invention.
  • Such a pore will have an increased net opposing charge in its barrel or channel and/or entrance.
  • An increased net positive charge enhances translocation of a negatively charged analyte through the pore.
  • An increased net negative charge enhances translocation of a positively charged analyte through the pore.
  • the pore may be enhanced using any method described above.
  • 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.
  • 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.
  • a pore of the invention may be present in a lipid bilayer.
  • a pore of the invention may be present as an individual or single pore.
  • a pore of the invention may be present in a homologous or heterologous population of two or more pores.
  • the transmembrane pore preferably comprises at least one subunit comprising SEQ ID NO: 6, 8, 10 or a variant thereof.
  • the transmembrane pore is more preferably a homoheptamer comprising seven subunits comprising SEQ ID NO: 6, 8 or 10 or a variant thereof.
  • Variants of SEQ ID NO: 6, 8 and 10 may differ from SEQ ID NO: 6, 8 and 10 to the same extent discussed above for variants of SEQ ID NOs: 2 and 4.
  • Variants of SEQ ID NO: 6 must comprise arginine at position 113 (M113R).
  • the variant of SEQ ID NO: 6 is preferably not M113R-RL2 (SEQ ID NO: 14).
  • Variants of SEQ ID NO: 8 must comprise asparagine at position 111 (E111N).
  • Variants of SEQ ID NO: 10 must comprise arginine at position 8 (A8R-RL2).
  • the present invention also provides a polynucleotide sequence which encodes a transmembrane protein pore of the invention.
  • the polynucleotide will encode a pore having an increased net opposing charge in its barrel or channel and/or entrance.
  • An increased net positive charge enhances translocation of a negatively charged analyte through the pore.
  • An increased net negative charge enhances translocation of a positively charged analyte through the pore.
  • the pore may be enhanced using any method described above.
  • the present invention also provides a polynucleotide sequence encoding a transmembrane protein pore subunit having the sequence of SEQ ID NO: 6, 8 or 10 or a variant thereof.
  • the polynucleotide sequence preferably comprises the sequence shown in SEQ ID NO: 5, 7 or 9 or a variant thereof.
  • a variant of SEQ ID NO: 5, 7 or 9 is a sequence at least 50%, 60%, 70%, 80%, 90% or 95% homologous based on nucleotide identity to sequence of SEQ ID NO: 5, 7 or 9 over the entire sequence.
  • nucleotide identity may be calculated as described above.
  • the variant may comprise a sequence that differs from SEQ ID NO: 5, 7 or 9 on the basis of the degeneracy of the genetic code.
  • a variant of SEQ ID NO: 5 must encode a pore subunit comprising an arginine at position 113 (M113R) of SEQ ID NO: 6 or at the position equivalent to position 113 (M113R) of SEQ ID NO: 6.
  • a variant of SEQ ID NO: 5 preferably does not encode M113R-RL2 (SEQ ID NO: 14).
  • a variant of SEQ ID NO: 7 must encode a pore subunit comprising asparagine at position 111 (E111N) of SEQ ID NO: 8 or at the position equivalent to position 111 (E111N) of SEQ ID NO: 8.
  • a variant of SEQ ID NO: 9 must encode a pore subunit comprising arginine at the position 8 (A8R) of SEQ ID NO: 10 or at the position equivalent to position 8 (A8R) of SEQ ID NO: 10.
  • Polynucleotide sequences may be derived and replicated using standard methods in the art. Chromosomal DNA may be extracted from a pore producing organism, such as Staphylococcus aureus. The gene encoding the pore subunit may be amplified using PCR involving specific primers. The amplified sequence 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.
  • polynucleotide sequences encoding a pore subunit may be made by introducing a polynucleotide encoding a pore subunit 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 encoding a pore subunit are well known in the art and described in more detail below.
  • the polynucleotide sequence encoding a pore subunit may be cloned into suitable expression vector.
  • the polynucleotide sequence encoding a pore subunit 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 pore subunit.
  • 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 pore subunit sequences may be introduced into the vector.
  • a pore subunit can be produced by inserting a polynucleotide sequence encoding a pore subunit 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 encoding the pore subunit.
  • the recombinantly-expressed pore subunit may self-assemble into a pore in the host cell membrane. Alternatively, the recombinant pore produced in this manner may be removed from the host cell and inserted into another membrane.
  • the 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 a tetracycline 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 ⁇ L promoter is typically used.
  • the host cell typically expresses the pore subunit at a high level. Host cells transformed with a polynucleotide sequence encoding a pore subunit will be chosen to be compatible with the expression vector used to transform the cell.
  • the host cell is typically bacterial and preferably Escherichia coli. Any cell with a ⁇ 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 present invention also relates to a method of determining the presence or absence of an analyte in a sample.
  • the analyte is charged.
  • the analyte may be any of those discussed above.
  • the method comprises (1) contacting the sample with a transmembrane protein pore of the invention under conditions that allow the analyte, if present, to translocate through and interact with the pore and (2) measuring the current passing through the pore during the interaction and thereby determining the presence or absence of the analyte.
  • Any transmembrane protein pore of the invention can be used.
  • the benefits associated with using a transmembrane protein pore of the invention to detect an analyte are discussed above.
  • the method further comprises enhancing translocation of the analyte through the pore as described above. The enhancement is of course carried out before the analyte is detected.
  • the analyte is present if the current flows through the pore in a manner specific for the analyte (i.e. if a distinctive current associated with the analyte is detected flowing through the pore).
  • the analyte is absent if the current does not flow through the pore in a manner specific for the analyte.
  • the invention therefore involves stochastic sensing of an analyte.
  • the invention can be used to differentiate analytes of similar structure on the basis of the different effects they have on the current passing through a transmembrane protein pore.
  • the invention can also be used to measure the concentration of a particular analyte in a sample.
  • the invention may also be used in a sensor that uses many or thousands of pores of the invention in bulk sensing applications.
  • the method may be carried out using any suitable membrane/pore system in which a transmembrane protein pore of the invention is inserted into a membrane.
  • the method is typically carried out using (i) an artificial membrane comprising a transmembrane protein pore of the invention, (ii) an isolated, naturally-occurring membrane comprising a transmembrane protein pore of the invention, or (iii) a cell expressing a transmembrane protein pore that has been modified in accordance with the invention.
  • the method is 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 transmembrane protein pore of the invention.
  • the method of the invention is typically carried out in vitro.
  • 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.
  • the method 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.
  • the pore 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.
  • the pore 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 method of the invention is typically carried out in vitro.
  • the analyte may be contacted with the pore on either side of the membrane.
  • the analyte may be introduced to the pore on either side of the membrane.
  • the analyte may be contacted with the side of the membrane that allows the analyte to pass through the pore to the other side of the membrane.
  • the analyte is contacted with an end of the pore, which in its native environment allows the entry of ions or small molecules, such as analytes, into the barrel or channel of the pore such that the analyte may pass through the pore.
  • the analyte interacts with the pore as it passes across the membrane through the barrel or channel of the pore.
  • the analyte may be contacted with the side of the membrane that allows the analyte to interact with the pore, dissociate from the pore and remain on the same side of the membrane.
  • the analyte may interact with the pore in any manner and at any site.
  • the analyte may reversibly bind to the pore.
  • the analyte affects the current flowing through the pore in a manner specific for that analyte. For example, a particular analyte 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 analyte.
  • Control experiments may be carried out to determine the effect a particular analyte 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 analyte in the sample or determine whether a particular analyte is present in the sample.
  • the frequency at which the current flowing through the pore is affected in a manner indicative of a particular analyte can be used to determine the concentration of that analyte in the sample.
  • the method may be carried out using any apparatus that is suitable for investigating a membrane/pore system in which a transmembrane protein pore is inserted into a membrane.
  • the method may be carried out using any apparatus that is suitable for stochastic sensing.
  • 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 analyte may be contacted with the pore by introducing the analyte into the chamber.
  • the analyte may be introduced into either of the two sections of the chamber.
  • the method may be carried out using the apparatus described in International Application No. PCT/GB08/000562.
  • the method of the invention involves measuring the current passing through the pore during interaction with the analyte. Therefore the apparatus also comprises an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane and pore.
  • the method may be carried out using a patch clamp or a voltage clamp.
  • the method preferably involves the use of a voltage clamp.
  • the Example discloses one way to carry out a voltage clamp method.
  • the analyte is present in any suitable sample.
  • the invention is typically carried out on a sample that is known to contain or suspected to contain the analyte.
  • the invention may be carried out on a sample that contains one or more analytes whose identity is unknown.
  • the invention may be carried out on a sample to confirm the identity of one or more analytes whose presence in the sample is known or expected.
  • the sample may be a biological sample.
  • the invention may be carried out in vitro on a sample obtained from or extracted from any organism or microorganism.
  • the organism or microorganism is typically prokaryotic or eukaryotic and typically belongs to one the five kingdoms: plantae, animalia, fungi, monera and protista.
  • the invention may be carried out in vitro on a sample obtained from or extracted from any virus.
  • the sample is preferably a fluid sample.
  • the sample typically comprises a body fluid of the patient.
  • the sample may be urine, lymph, saliva, mucus or amniotic fluid but is preferably blood, plasma or serum.
  • the sample is human in origin, but alternatively it may be from another mammal animal such as from commercially farmed animals such as horses, cattle, sheep or pigs or may alternatively be pets such as cats or dogs.
  • the sample may be a non-biological sample.
  • the non-biological sample is preferably a fluid sample.
  • Examples of a non-biological sample include surgical fluids, water such as drinking water, sea water or river water, and reagents for laboratory tests.
  • the sample is typically processed prior to being assayed, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells.
  • the sample may be measured immediately upon being taken.
  • the sample may also be typically stored prior to assay, preferably below ⁇ 70° C.
  • the method of the invention involves the measuring of a current passing through the pore during interaction with the analyte. Suitable conditions for measuring ionic currents through transmembrane protein pores are well known in the art and disclosed in the Example. The method is carried out with a voltage applied across the membrane and pore. The voltage used is typically from ⁇ 400 mV to +400 mV.
  • the voltage used is preferably in a range having a lower limit selected from ⁇ 400 mV, ⁇ 300 mV, ⁇ 200 mV, ⁇ 150 mV, ⁇ 100 mV, ⁇ 50 mV, ⁇ 20 mV 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 120 mV to 170 mV. Enhancing translocation of the analyte through the pore in accordance with the invention allows lower voltages to be used.
  • the method is carried out in the presence of any alkali metal chloride salt.
  • the salt is present in the aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl) or caesium chloride (CsCl) is typically used. KCl 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.
  • the salt concentration is preferably from 150 to 500 mM. High salt concentrations provide a high signal to noise ratio and allow for currents indicative of the presence of a analyte to be identified against the background of normal current fluctuations.
  • the salt concentration is preferably from 150 to 500 mM. Good nucleotide discrimination at these low salt concentrations can be achieved by carrying out the method at temperatures above room temperature, such as from 30° C. to 40° C.
  • the method is typically carried out in the presence of a buffer.
  • the buffer is present in the aqueous solution in the chamber. Any buffer may be used in the method of the invention.
  • One suitable buffer is Tris-HCl buffer.
  • the method is 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 method is 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 method may be carried out at room temperature.
  • the method is preferably carried out at a temperature that supports enzyme function, such as about 37° C. Good analyte discrimination can be achieved at low salt concentrations if the temperature is increased.
  • 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 KCl and rely on an additional charge carrying species that is less disruptive to enzyme activity.
  • the present invention also relates to a method of sequencing a target nucleic acid sequence.
  • the method comprises (a) pushing or pulling the target sequence through a transmembrane pore of the invention so that 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.
  • 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.
  • any pore of the invention may be used.
  • a pore of the invention in which the net positive charge of the barrel or channel and/or entrance has been increased is particularly suited to this method.
  • the enhanced translocation of the negatively charged nucleic acid through the pore reduces the non-translocation events and deadtime between reads of each nucleotide in the sequence and/or increases the read time for each nucleotide in the sequence.
  • the method further comprises enhancing translocation of the analyte through the pore as described above. The enhancement is of course carried out before the nucleic acid is sequenced.
  • 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.
  • 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 is preferably DNA or RNA.
  • the nucleic acid sequence can be naturally occurring or artificial.
  • the method may be used to verify the sequence of a manufactured oligonucleotide. The method is typically carried out in vitro.
  • the method may be carried out using any of the samples, membrane/pore systems and conditions discussed above.
  • the nucleic acid may be contacted with the pore on either side of the membrane.
  • the 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 an enzyme is attached to the pore. This allows the enzyme to handle the nucleic acid during the method as discussed below.
  • a proportion of the nucleotides of the target nucleic acid sequence interact with the pore as it passes across the membrane through the barrel or channel of the pore.
  • 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.
  • 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.
  • Strand sequencing in accordance with the invention involves the controlled and stepwise translocation of nucleic acid polymers through a pore.
  • the nucleic acid may be pushed or pulled through the pore in any manner known to a person skilled in the art.
  • the nucleic acid could be pushed or pulled through the pore using an electroosmotic effect.
  • the nucleic acid is preferably pushed or pulled through the pore using a DNA handling enzyme.
  • nucleic acid handling enzymes are suitable for use in this application provided they hydrolyse, polymerise or process single stranded nucleic acids, such as DNA or RNA.
  • Preferred enzymes that are capable of pushing or pulling the target nucleic acid sequence through the pore include polymerases, exonucleases, helicases and topoisomerases, such as gyrases.
  • 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 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 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.
  • a helicase that unwinds the double stranded DNA can also be used in a similar manner.
  • 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.
  • 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.
  • the present invention also relates to a kit that may be used to carry out the method of sequencing a target nucleic acid sequence.
  • the kit is therefore suitable for sequencing nucleic acids.
  • the kit comprises a transmembrane pore of the invention and a nucleic acid handling enzyme.
  • the pore and the enzyme may be any of those discussed above.
  • the kit may additionally comprise one or more other reagents or instruments which enable any of the embodiments of the method mentioned above to be carried out.
  • 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 nucleic acid 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.
  • ⁇ -HL was produced as described elsewhere (Cheley S, Braha O, Lu X, Conlan S, Bayley H: A functional protein pore with a “retro” transmembrane domain. Protein Sci. 1999, 8:1257-1267).
  • the sequence of one subunit of ⁇ -HL is shown in SEQ ID NO: 2.
  • the protein was expressed in the presence of [ 35 S]methionine in an E. coli in vitro transcription and translation (IVTT) system ( E. coli T7 S30 Extract System for Circular DNA, Cat. #L1130, Promega, Madison, Wis.).
  • IVTT reactions 100 ⁇ l
  • ⁇ -HL monomers were incubated with rabbit red blood cell membranes for 1 h at 37° C.
  • the solution was spun at 25,000 ⁇ g and the pellet containing ⁇ -HL heptamers was loaded onto a 5% SDS-polyacrylamide gel.
  • the gel was run for 4 h at 100 V and vacuum dried for 3 to 4 h without heating onto Whatman 3M filter paper.
  • the dried gel was exposed to photographic film for 2 h and the developed film was used to locate the position of the heptameric protein in the gel. This region of the gel was cut out, rehydrated and crushed in 400 ⁇ L of 10 mM Tris.HCl, pH 8.0, containing 100 ⁇ M EDTA.
  • the polyacrylamide was removed by centrifuging the suspension at 25,000 ⁇ g for 7 min at room temperature through a cellulose micro spin column (Microfilterfuge tubes, Cat. #7016-024, Rainin, Oakland, Calif.). Aliquots of the purified protein were stored at ⁇ 80° C.
  • Mutants ⁇ -HL genes were prepared either by using a site-directed mutagenesis kit (QuickChange® II XL, Cat. #200522-5, Stratagene, Cedar Creek, Tex.) or by cassette mutagenesis (Cheley S, Gu L-Q, Bayley H: Stochastic sensing of nanomolar inositol 1,4,5-trisphosphate with an engineered pore. Chem. Biol. 2002, 9:829-838).
  • the RL2 gene encodes four amino acid replacements in the barrel domain (Val-124 ⁇ Leu, Gly-130 ⁇ Ser, Asn-139 ⁇ Gln, Ile-142 ⁇ Leu) and one in the amino latch region of the pore (Lys-8 ⁇ Ala).
  • the sequence of one subunit of ⁇ -HL-RL2 is shown in SEQ ID NO: 4. The DNA sequence of each new gene was verified.
  • a bilayer of 1,2-diphytanoyl-sn-glycero-3-phosphocholine was formed on an aperture ( ⁇ 100 ⁇ m in diameter) in a 25- ⁇ m thick polytetrafluoroethylene film (Goodfellow Cambridge Limited, Cat. no. FP301200/10, Huntingdon, UK) that divided a chamber into cis and trans compartments. Both compartments contained 0.4 mL of 1 M KCl, 25 mM Tris.HCl, pH 8, containing 100 ⁇ M EDTA. Planar bilayer current recordings were performed with a patch clamp amplifier (Axopatch 200B, Axon Instruments, Foster City, Calif.).
  • the protein and subsequently the DNA were added to the cis compartment, which was connected to ground.
  • the DNA was prepared and PAGE-purified by Sigma Genosys, UK.
  • the concentration of the DNA was determined by measuring the absorbance at 260 nm of the solution in the cis chamber at the end of each experiment.
  • the DNA sequence was: 5′-AAAAAAAAAAAAAAAAAAAAATTCCCCCCCCCCCCCCCCCTTAAAAAAAAAATT CCCCCCCCTTAAAAAAAAAATTCCCCCCCCCC-3′ (SEQ ID NO: 15).
  • the amplified signal arising from the ionic current passing through the ⁇ -HL pore was low-pass filtered at 50 kHz and sampled at 250 kHz with a computer equipped with a Digidata 1440A digitizer (Molecular Devices, Sunnyvale, Calif.).
  • the signal was filtered at 40 kHz, while at +60 mV and +40 mV the signal was filtered at 30 kHz and 20 kHz, respectively.
  • the sampling rate was at least five times the filtering rate.
  • the data used in Table 2 were analyzed with pClamp 10.1 software (Axon Instruments), by using three different current levels. Level 0 corresponded to the open channel current, level 1 to the mid-amplitude event current and level 2 to the low-amplitude event current. The assignment to each level was checked manually.
  • the data used in FIG. 2 b were analyzed and prepared for presentation by using only two levels: level 0 was set to the open channel current, and level 1 was placed between the mid-amplitude and low-amplitude event currents.
  • the frequency of DNA translocations was calculated by fitting the interevent intervals to a single exponential (logarithmic binning, 10 bin/decade) and normalizing the mean value of the duration to a DNA concentration of 1 ⁇ M.
  • the DNA translocation time was calculated from the peak of a histogram of the distribution of the translocation times (Meller A, Nivon L, Brandin E, Golovchenko J, Branton D: Rapid nanopore discrimination between single polynucleotide molecules. Proc. Natl. Acad. Sci. USA 2000, 97:1079-1084).
  • f max , b and V 0 were determined by fitting the voltage dependence of the frequency of DNA translocation for the WT and mutated ⁇ -HL nanopores to equation 1 ( FIG. 5 ) by using Origin data analysis software (OriginLab, Northampton, Mass.). The errors in these values are expressed as standard errors. The curve fittings were adjusted for the experimental error of each point by using the instrumental weight.
  • f max b V 0 V 1 a M113R-WT 7,300 ⁇ 2,200 1200 ⁇ 100 ⁇ 90 ⁇ 7 45 E111N-WT 1,600 ⁇ 200 780 ⁇ 40 ⁇ 56 ⁇ 3 50 M113R-RL2 990 ⁇ 300 990 ⁇ 90 ⁇ 67 ⁇ 7 77 WT 300 ⁇ 60 420 ⁇ 40 25 ⁇ 6 100 RL2 300 ⁇ 150 980 ⁇ 130 20 ⁇ 17 151
  • the values were derived by fitting the data to equation 1 (see Methods). Additional conditions are described in the legend to FIG. 2.
  • the peak in the events histogram at a fractional current of 0.58 comprises type B current blockades, which show a mid-state current level only ( FIG. 2 b ).
  • Type C, D and E events contain both mid-amplitude and low-amplitude current levels.
  • Type C events corresponded to 10% of the total number of events, type D events 2% of the total and type E events, which displayed a mid-low-mid pattern, were only 0.6% of the total.
  • the type C, D and E events are scattered between the two main peaks in the histogram, reflecting their wide distribution of mean currents.
  • the pores examined were homoheptamers, so the mutations appear in all seven subunits, e.g. M113R has a ring of seven positively charged side chains at position 113.
  • the mutants were made by using the WT or RL2 genes as templates.
  • the RL2 gene encodes four amino acid replacements in the barrel domain (Val-124 ⁇ Leu, Gly-130 ⁇ Ser, Asn-139 ⁇ Gln, Ile-142 ⁇ Leu) and one in the cis entrance of the pore (Lys-8 ⁇ Ala).
  • the properties of the mutants differed significantly from the WT pore with respect to the open-pore current and the frequency of occurrence of DNA translocation events (Table 4).
  • the translocation times of DNA through the nanopores were not changed considerably by the point mutations.
  • the mutant with the slowest mean translocation time was N117R-RL2 at 0.35 ⁇ 0.04 ms, which is only twice the mean translocation time observed for the WT pore (Table 4).
  • the various mutants showed different extents of current block during the translocation of DNA through the pore (as determined from the type A events or the lowest level of type C events).
  • WT, RL2, A8K-RL2 and E111N-WT showed fractional residual currents of 0.10 of the open channel current, while the introduction of positive charges in the ⁇ barrel produced higher extents of block (Table 4).
  • Lys-8 is the charged residue in the closest proximity to entering DNA.
  • the substitution of Lys-8 with Ala decreased the number of translocation events per unit time by almost an order of magnitude, while substitution with Arg (in A8R-RL2 i.e. seven subunits of SEQ ID NO: 10) produced a three-fold increase in the translocation rate (Table 4).
  • the additional mutations in RL2 did not affect DNA translocation, as confirmed by reintroducing Lys-8 into the RL2 background (A8K-RL2, Table 4 and FIG. 8 ).
  • Amino acid substitutions within the ⁇ barrel of ⁇ -HL also affected the frequency of DNA translocation. Individual replacements of the amino acids at positions 111, 113, 115, 117, 119, 121 and 123 with Arg (in the RL2 background) produced ⁇ -HL pores that showed a progressive decrease in the translocation frequency as the Arg residue became located further from the cis entrance ( FIG. 4 and Table 4).
  • Arg in the RL2 background
  • ‘f max ’ is the rate of DNA translocation at a hypothetical infinite applied potential
  • ‘V 0 ’ is the threshold for DNA translocation
  • a larger value of ‘b’ gives a faster approach to ‘f max ’ ( FIG. 5 ).
  • More positive charge produced pores with a greatly reduced threshold and a steeper approach to f max (Table 3).
  • pores with no additional charge at the constriction had lower values of f max (300 ⁇ 60 s ⁇ 1 ⁇ M ⁇ 1 and 300 ⁇ 150 s ⁇ 1 ⁇ M ⁇ 1 for WT and RL2, respectively), while additional positive charge at both the constriction and the cis entrance gave high values of f max (7,300 ⁇ 2,200 s ⁇ 1 ⁇ M ⁇ 1 and 1,600 ⁇ 200 s ⁇ 1 ⁇ M ⁇ 1 for M113R-WT and E111N-WT, respectively).
  • a pore formed with a ring of additional charge at the constriction, but no charge at the cis entrance displayed an intermediate value of f max (990 ⁇ 300 s ⁇ 1 ⁇ M ⁇ 1 ).
  • Dwell-time/amplitude distributions of the events for the M113R-WT and E111N-WT homoheptamers yielded two distinct clusters of current blockades at 100 to 200 mV ( FIG. 7 ).
  • the relative intensities of the two peaks in the distributions were voltage dependent, with the longer events increasing with the applied potential ( FIG. 7 c ).
  • the first peak comprised blockades with a fast translocation time and a fractional residual current at +120 mV of 0.96 for M113R-WT and 0.89 for E111N-WT.
  • the second cluster of current blockades included events with a longer dwell time and almost full current block.
  • DNA immobilized inside the ⁇ barrel as part of a DNA- ⁇ -HL rotaxane structure causes a 85% block of the pore current (Sánchez-Quesada J, Saghatelian A, Cheley S, Bayley H, Ghadiri M R: Single molecule DNA rotaxanes of a transmembrane pore protein. Angew. Chem. Int. Ed. Engl. 2004, 43:3063-3067).
  • Occupancy of the vestibule is associated with a reduced conductance (but not to the extent caused by occupancy of the barrel).
  • PEG molecules Movileanu L, Cheley S, Howorka S, Braha O, Bayley H: Location of a constriction in the lumen of a transmembrane pore by targeted covalent attachment of polymer molecules. J. Gen. Physiol. 2001, 117:239-251
  • PAMAM dendrimers with a hydrodynamic diameter of 4.1 nm (Martin H, Kinns H, Mitchell N, Astier Y, Madathil R, Howorka S: Nanoscale protein pores modified with PAMAM dendrimers.
  • FIG. 6 A scheme for DNA capture and translocation that incorporates reversible sampling of the lumen is depicted ( FIG. 6 ).
  • a DNA molecule Prior to a typical translocation event (via intermediate V, FIG. 6 ), a DNA molecule first collides with the pore (II). Upon visiting the vestibule (III), the DNA is transported by electrophoresis into the narrow ⁇ barrel (IV). Because the pores used here are anion selective, electroosmotic solvent flow will occur in the same direction (cis to trans) and assist DNA transport. At this point, the effect of the applied field on the DNA molecule is strongest and the polymer is pulled through the pore into the trans compartment (V ⁇ I), the only irreversible step in our scheme.
  • the translocation intermediate (V) was observed experimentally by the abrupt reduction of the current to 90% of the open pore value (during type A and C events), an assignment that is supported by several observations in the literature. For example, Kasianowicz and co-workers showed that the cis addition of ssDNA produces short-lived current blockades of 85-100% of the open pore current.
  • PCR analysis of the trans chamber revealed that DNA had passed through the ⁇ HL pore (Kasianowicz, J. J., Brandin, E., Branton, D., & Deamer, D. W. (1996) Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl. Acad. Sci. USA 93, 13770-13773).
  • DNA molecules captured in the mid-amplitude configuration can either exit the pore from the cis compartment (type B event, FIG. 2 a ), or proceed into the ⁇ barrel producing a low-amplitude signal (intermediate VII). From this configuration, the DNA can either complete translocation through the pore and exit on the trans side of the bilayer (type C event, FIG. 2 a , via intermediate V, FIG. 6 ) or retract back into the vestibule and exit on the cis side (type E event, FIG. 2 a ).
  • a ring of seven positive charges introduced by site-directed mutagenesis near the internal constriction increased the frequency of translocation of DNA molecules ( FIG. 3 ), and reduced the fraction of events with mid-amplitude components ( FIG. 2 ab ).
  • the DNA is within a Debye length of the residues forming the constriction only as intermediate IV, V or VII ( FIG. 6 ) and cannot sense these charged groups from bulk solution. Therefore, to explain the effect of charge at the constriction, we propose that a DNA molecule enters and quickly exits the top end of the barrel (sampling, III IV, FIG. 6 ) many times before either retracting back into the cis compartment or proceeding to intermediate V, VI or VII.
  • step IV The depth to which DNA samples the ⁇ barrel (step IV) was investigated by introducing a ring of arginines at various positions.
  • DNA translocation frequency decreased roughly exponentially as the ring was moved from the constriction (M113R-RL2)towards the trans entrance (N123R-RL2) of the pore ( FIG. 4 ), suggesting that DNA molecules can sample the ⁇ barrel deeply.
  • the DNA When presented from the cis side, the DNA must occasionally come close to residue 119, which is 2.3 nm away from the constriction. Accordingly, when the DNA was added on the trans side of the pore, more translocation events were observed (at ⁇ 120 mV) when the ring of arginines was closer to the trans entrance ( FIG.
  • P + /P ⁇ 0.38, in 1 M NaCl cis and 0.2 M NaCl trans
  • the new engineered pores or derivatives of them will be useful for enhancing the sensitivity of ⁇ -HL as a biosensor of nucleic acids.
  • An additional potential application is in nanopore sequencing, where the in a single DNA strand are read off one by one during translocation.
  • An increase in the frequency of ssDNA translocation through ⁇ -HL pores, as demonstrated here, might, for example, be used to reduce the deadtime between reads.
  • engineered pores might also be used to increase the capture efficiency of individual bases during exonuclease sequencing, which will be vital for accurate reads.
  • Example 1 The same Materials and Methods used in Example 1 were also used in this Example.
  • ⁇ G is the most likely DNA translocation speed
  • g is the unitary conductance of the nanopores
  • I RES is the residual current percent during DNA translocation. All mutants, except for WT and 2N, are in RL2 background (SEQ ID NOs: 3 and 4). Errors are shown in standard deviation. *data from Example 1 and Maglia, Proc. Natl. Acad. Sci. USA , 2008, supra
  • the current rectification ratios calculated as the value of ionic current at +50 divided by the current at ⁇ 50, also varied depending on the position of the charges.
  • Nanopores with additional positive charges introduced near the trans exit showed current rectification lower than 1 (e.g. they show higher current at negative applied potentials than at positive applied potentials), while mutants with positive charges introduced close to the cis side displayed similar rectifying behaviour to WT- ⁇ -HL (higher than 1).
  • nanopores with no charges at the central constriction displayed almost non-rectifying behaviour (R ⁇ 1 in 2N)).
  • ssDNA 92 bases nucleotide in this work
  • the most likely translocation time, ⁇ G is defined as the peak of the Gaussian fit the histogram of events duration ( FIG. 11 a ).
  • the residual current during the translocation of DNA molecules through ⁇ -HL nanopores is defined as the blocked pore current (I B ) divided by the open pore current (I O ) expressed in percent. WT nanopores showed a fractional residual current of 11%.
  • I RES is important because it is the most likely mean to which single bases will be discriminated as they are translocated through the pore.
  • arginine residues allow the control of DNA translocation through ⁇ -HL nanopores
  • the residual current while DNA is translocating is greatly reduced (Table 5).
  • Single and double arginines substitutions in the lumen of the pore reduced the residual current to 4% and 2%, respectively (Maglia, Proc. Natl.
  • Hetero-heptamers ⁇ -HL pores were prepared by mixing 7R monomers containing a D8 tail at the C termini with RL2 monomers on rabbit red blood cells, and mono-derivatized heptamers were separated by SDS-PAGE (SI).
  • the modified pores contained, therefore, lines of single arginine residues along the barrel of the pore stretching from the constriction to the trans exit of the pore, forming a slide to guide DNA translocation through the ⁇ -HL pore. It was hoped that the arginine residues would reduce DNA translocation by interacting strongly with the phosphate groups of DNA, while allowing K+ ions still to pass through the pore, thus allowing base recognition.
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