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Abstract

A modified portal protein of a bacteriophage DNA packaging motor, wherein the modified portal protein is capable of direct insertion into a membrane, and wherein the portal protein is modified such that one or more amino acid residues on the outer surface of the portal protein are substituted with one or more other amino acid residues compared to a wild-type portal protein, and / or wherein one or more amino acid residues are inserted on the outer surface of the portal protein to alter the outer surface hydrophobicity of the modified portal protein compared to a wild-type portal protein.

Description

Technical Field

[0001] The present disclosure relates to modified portal proteins, membranes comprising modified portal proteins, and methods of characterizing analytes using membranes comprising modified portal proteins. Background Art

[0002] Nanopore sensing is a method for detecting and characterizing analytes that relies on the observation of individual binding or interaction events between analyte molecules and ion-conducting channels. Nanopore sensors can be created by placing a single nanometer-sized pore in an electrically insulating membrane and measuring the voltage-driven ionic current through the pore in the presence of analyte molecules. The presence of an analyte inside or near the nanopore alters the ion flux through the pore, causing a change in the ion or current measured across the channel. The identity of the analyte is revealed by its unique current signature, particularly the duration and extent of current spikes and changes in current level during interaction with the pore. Analytes can be organic and inorganic small molecules as well as various biological or synthetic macromolecules and polymers, including polynucleotides, polypeptides, and polysaccharides. Nanopore sensing can reveal the identity of the sensed analyte and perform single-molecule counting. It can also provide information about the analyte composition, such as nucleotide, amino acid, or glycan sequence, and the presence of base, amino acid, or glycan modifications, such as methylation and acylation, phosphorylation, hydroxylation, oxidation, reduction, glycosylation, decarboxylation, and deamination. Nanopore sensing has the potential to allow rapid and inexpensive polynucleotide sequencing, providing single-molecule sequence reads of polynucleotides that are tens to tens of thousands of bases in length.

[0003] Bioderived nanopores are based on naturally occurring membrane proteins and can be inserted into copolymer membranes by contacting the membrane with purified proteins and applying a voltage potential to the membrane.

[0004] The phi29 bacteriophage gp10 portal protein is assembled from the 12 subunits of gp10 into a propeller-like structure. The portal protein has an outer diameter of 14.6 nm and a height of 7.5 nm. At its narrowest point, the wild-type channel is 3.6 nm. Each of the 12 subunits has an elongated shape containing a central α-helical domain composed of a three-helical bundle, an α-β motif, and six heavy-chain SH3-like domains at the wider C-terminus. The portal protein is not a natural membrane protein or ion channel, but has been proposed as a nanopore for the characterization of analytes. To be inserted into the membrane, the bacteriophage phi29 portal protein must first be inserted into liposomes, which then fuse with a planar lipid bilayer. Summary of the Invention

[0005] Disclosed herein are modified bacteriophage portal proteins that spontaneously insert into membranes. The inserted portal proteins can function as nanopores.

[0006] In one aspect, a modified portal protein of a bacteriophage DNA-packaging motor capable of direct membrane insertion is provided, wherein one or more amino acid residues on the outer surface of the portal protein are substituted with one or more other amino acid residues, and / or one or more amino acid residues are inserted into the outer surface of the portal protein to alter the outer surface hydrophobicity of the portal protein compared to the wild-type portal protein. The introduction of one or more amino acid residues by substitution and / or insertion can increase or decrease the outer surface hydrophobicity compared to the wild-type portal protein. The outer surface hydrophobicity of a specific region of the protein can be increased or decreased.

[0007] Because portal protein channels are assembled from 12 subunits, changing one or more residues in one monomer will trigger effects throughout the entire channel, provided the mutations reside in the same plane of the molecule. Generally speaking, portal proteins are composed of two domains: the wing domain and the stalk domain. The stalk domain comprises the hydrophobic band beneath the portal protein's wings.

[0008] In one embodiment, at least one of the one or more introduced amino acid residues is located in the central hydrophobic zone of the portal protein. The one or more introduced amino acid residues can be introduced, for example, by substitution and / or insertion. The residues in the hydrophobic zone of the portal protein of the Phi29 DNA packaging motor include F24, I25, L28, F60, F128, P129, and P132. In one embodiment, the amino acid within one or two residues of any one or more of these positions, or at one or more corresponding positions in a similar portal protein, can be substituted with one or more amino acids that are more hydrophobic than the naturally occurring amino acid at the substituted position. In one embodiment, a hydrophobic amino acid can be inserted within one or two residues of any one or more of these positions, or at one or more corresponding positions in a similar portal protein. The N-terminal residue of each subunit of the portal protein is located in the hydrophobic zone. In one embodiment, at least one of the one or more amino acid residues is located within 30 amino acids of the N-terminus of the portal protein. For example, at least one of the one or more amino acid residues is at a position corresponding to R10, E14, R17, Q18 and / or R22 of the portal protein of the Phi29 DNA packaging motor, or at a corresponding position in a similar portal protein.

[0009] In one embodiment, at least one of the one or more introduced amino acid residues is located in the hydrophilic cis-layer and / or trans-layer of the portal protein. Examples of amino acid residues in the cis-layer of the portal protein include positions corresponding to Q32, Y36, F52, K55, Q59, F60, Y62, N77, G78, A79, L80, S81, R84, R94, A96, S97, P98, and Q101 in the wing domain of the portal protein of the Phi29 DNA packaging motor. Examples of amino acid residues in the trans-layer of the portal protein include positions corresponding to P129, T131, E135, and Q168 in the stem domain of the portal protein of the Phi29 DNA packaging motor.

[0010] In specific embodiments, at least one of the one or more introduced amino acid residues in the cis-layer or trans-layer of the portal protein at a position corresponding to A79, E135 and / or Q168 of the portal protein of the Phi29 DNA-packaging motor is modified.

[0011] In one aspect, a modified portal protein of a bacteriophage DNA-packaging motor capable of direct membrane insertion is provided, wherein one or more amino acid residues are introduced onto the outer surface of the portal protein to introduce a binding site for a molecule on the outer side of the wing domain or in the stem domain that alters the hydrophobicity of the outer surface of the portal protein compared to the wild-type portal protein. The binding site can be introduced by replacing a residue present on the surface of the portal protein with another amino acid residue or by inserting one or more amino acid residues.

[0012] In one embodiment, at least one of the one or more amino acid residues introduced into the portal protein to introduce a binding site on the outside of the wing domain or in the stalk domain is cysteine ​​and / or an unnatural amino acid.

[0013] In one embodiment, at least one of the one or more amino acid residues introduced into the portal protein to introduce a binding site is located in the hydrophilic cis-layer and / or trans-layer of the portal protein. Examples of amino acid residues in the cis-layer of the portal protein include those located at positions corresponding to one or more of Q32, Y36, F52, K55, Q59, F60, Y62, N77, G78, A79, L80, S81, R84, R94, A96, S97, P98, or Q101 in the wing domain of the portal protein of the Phi29 DNA packaging motor. Examples of amino acid residues in the trans-layer of the portal protein include those located at positions corresponding to P129, T131, E135, or Q168 in the stem domain of the portal protein of the Phi29 DNA packaging motor.

[0014] In specific embodiments, at least one amino acid residue among the one or more amino acid residues in the cis-layer or trans-layer of the portal protein at a position corresponding to A79, E135 and / or Q168 of the portal protein of the Phi29 DNA packaging motor is modified to introduce a binding site.

[0015] In one embodiment, the molecule that alters the hydrophobicity of the outer surface of the portal protein compared to the wild-type portal protein is a hydrophobic molecule. Exemplary hydrophobic molecules are those comprising porphyrin, tetraphenylporphyrin, protoporphyrin IX, octaethylporphyrin, cholesterol, heme, or biliverdin.

[0016] In some embodiments, the modified portal protein is modified by adding and / or deleting one or more amino acid residues at the N-terminus of the portal protein.

[0017] In certain embodiments, the modified portal protein is a modified portal protein from a DNA packaging motor of a bacteriophage selected from the group consisting of phi29, T3, T4, T5, T7, SPP1, HK97, λ, G20c, P2, P3, and P22.

[0018] In one embodiment, the modified portal protein is composed of identical subunits.

[0019] In other aspects, the following is provided:

[0020] - a subunit of a modified portal protein as disclosed herein;

[0021] - a membrane comprising a modified portal protein as disclosed herein;

[0022] - an array comprising two or more membranes comprising a modified portal protein as disclosed herein;

[0023] - a device comprising an array comprising two or more membranes, each membrane comprising a modified portal protein as disclosed herein, means for applying an electrical potential across the membranes and means for detecting the charge across the membranes; and

[0024] A method of characterizing a target analyte, the method comprising: contacting a membrane comprising a modified portal protein as disclosed herein with the target analyte and applying a potential across the membrane such that the target analyte moves relative to a nanopore, and making one or more measurements as the target analyte moves relative to the pore, thereby determining the presence, absence, or one or more characteristics of the analyte.

[0025] In one embodiment, the membrane is a lipid membrane or a copolymer membrane, such as a diblock or triblock copolymer membrane.

[0026] In one embodiment, the array is adapted to be inserted into a sensor device.

[0027] In one embodiment, the device further comprises a fluidic system configured to supply a sample to the membrane.

[0028] In one embodiment, the method comprises performing electrical and / or optical measurements. In one embodiment of the method, multiple target analytes are characterized. In one embodiment of the method, the target analytes are polynucleotides, proteins, peptides, carbohydrates, metabolites, or other chemicals. In one embodiment of the method, the target analytes are associated with a medical condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1The structure of the phi29 gp10 portal protein is shown from different angles. The structures of the subunits (monomer units) of the pore are also shown. Representative positioning for generating conjugation sites for binding hydrophobic groups on the surface of the phi29 gp10 portal protein is shown in one monomer of the protein. A. Side view of the wild-type phi29 gp10 pore. Regions of interest requiring protein engineering for direct membrane insertion are boxed. Residues are present in either the wing domain or the stem domain, as shown in B. Two different domains of the pore are shown as monomer units. B. The monomer unit (one of 12 assembly subunits) that assembles the pore is shown. The wing domain is shown in black (residues 1-125), and the stem domain is shown in gray (residues 120-309). The boxed region of interest in Figure A encompasses portions of both protein domains. C. Representative positioning of conjugation sites on the WT phi29 gp10 protein for generating conjugation sites for binding a hydrophobic membrane anchoring module. Specific mutation points in the wing domain include residues Q32, Y36, F52, K55, Q59, F60, Y62, N77, G78, A79, L80, S81, R84, R94, A96, S97, P98, and Q101; specific mutation points in the stem domain include P129, T131, E135, and Q168. D. The hydrophobicity scale of 20 natural amino acids is shown. Typically, I, VL, or F is introduced at the target mutation point to enhance the relative hydrophobicity of the region of interest.

[0030] Figure 2 Representative locations for generating cysteine ​​mutations on the surface of the phi29 gp10 portal protein to bind hydrophobic groups are shown. These locations include A79C in the wing domain and E135C and Q168C in the stem domain. Other locations not shown in this figure include Q32, Y36, F52, K55, Q59, F60, Y62, N77, G78, L80, S81, R84, R94, A96, S97, P98, Q101 in the wing domain; and P129 and T131 in the stem domain. Because the protein assembles as a dodecamer, each mutation creates a loop. Cysteine ​​residues can be attached to hydrophobic modules using standard sulfhydryl chemistry. In addition to cysteine, any unnatural amino acid, such as those with alkyne side chains, can also be incorporated for chemical conjugation mediated by "click" chemistry.

[0031] Figure 3Representative locations used to create hydrophobic mutations on the surface of the phi29 gp10 portal protein to facilitate insertion into polymer membranes are shown. These locations include R10, E14, R17, Q18, and R22 in the wing domain, near the N-terminus of the subunit. These residues are typically mutated with hydrophobic residues I, V, L, or F. Because the protein assembles as a dodecamer, each mutation creates a ring of hydrophobic residues along the plane. The positioning of the hydrophobic amino acids or hydrophobic anchoring module relative to the membrane core determines the location of the pore in the membrane.

[0032] Figure 4 Figure 1 is an SDS-PAGE gel showing the expression and purification of a representative phi29 gp10 portal protein mutant A79C. The mutant A79C gene was cloned into an expression vector and then transformed into E. coli. Successfully transformed bacteria were cultured overnight in LB medium. Protein expression was induced by adding IPTG. After induction, the bacteria were collected and then lysed. The protein and other components were separated by centrifugation. Ni-NTA His-binding resin with a His tag was applied to purify the mutant protein. As shown in the figure, the protein was eluted using elution buffer containing increasing concentrations of imidazole. The eluate was collected and concentrated, and then subjected to FPLC purification. An SDS-PAGE gel was run to examine the protein samples.

[0033] Figure 5 Figure 1 is an SDS-PAGE gel showing the expression and purification of a representative phi29 gp10 portal protein mutant, E135C. The mutant E135C gene was cloned into an expression vector and then transformed into E. coli. Successfully transformed bacteria were cultured overnight in LB medium. Protein expression was induced by adding IPTG. After induction, the bacteria were collected and then lysed. The protein and other components were separated by centrifugation. Ni-NTAHis-bound resin with a His tag was applied to purify the mutant protein. As shown in the figure, the protein was eluted using elution buffer containing increasing concentrations of imidazole. The eluate was collected and concentrated, and then subjected to FPLC purification. An SDS-PAGE gel was run to examine the protein samples.

[0034] Figure 6Figure 1 is an SDS-PAGE gel showing that the representative phi29 gp10 portal protein mutant Q168C was expressed and purified. The mutant Q168C gene was cloned into an expression vector and then transformed into E. coli. Successfully transformed bacteria were cultured overnight in LB medium. Protein expression was induced by adding IPTG. After induction, the bacteria were collected and then lysed. The protein and other components were separated by centrifugation. Ni-NTA His-binding resin with a His tag was applied to purify the mutant protein. As shown in the figure, the protein was eluted using elution buffer containing increasing concentrations of imidazole. The eluate was collected and concentrated, and then subjected to FPLC purification. An SDS-PAGE gel was run to examine the protein samples.

[0035] Figure 7 Figure 1 is an SDS-PAGE gel showing representative phi29 gp10 portal protein mutants R10L, E14V, R17L and N-7Δ (mutant-b) (left side of the marker in the figure) and R10L, E14V, R17L, Q18L, R22I and N-7Δ (mutant-c) (right side of the marker in the figure) being expressed and purified. The mutant genes were cloned into expression vectors and then transformed into E. coli. Successfully transformed bacteria were cultured overnight in LB medium. Protein expression was induced by adding IPTG. After induction, the bacteria were collected and then lysed. The protein and other components were differentiated by centrifugation. Ni-NTA His-binding resin with a His tag was applied to purify the mutant protein. As shown in the figure, the protein was eluted using an elution buffer containing increasing concentrations of imidazole. The eluate was collected and concentrated, and then FPLC purification was performed. An SDS-PAGE gel was run to examine the protein samples.

[0036] Figure 8 Figure 1 is an SDS-PAGE gel showing the expression and purification of the representative phi29 gp10 portal protein mutant NIL (mutant-d) (left side of the marker in the figure) and N-terminal-7Δ (mutant-e) with R10L, E14V, R17L, and IL added to the N-terminus (right side of the marker in the figure). The mutant gene was cloned into an expression vector and then transformed into E. coli. The successfully transformed bacteria were cultured overnight in LB medium. Protein expression was induced by adding IPTG. After induction, the bacteria were collected and then lysed. The protein and other components were differentiated by centrifugation. Ni-NTA His-binding resin with a His tag was applied to purify the mutant protein. As shown in the figure, the protein was eluted using an elution buffer containing increasing concentrations of imidazole. The eluate was collected and concentrated, and then FPLC purification was performed. An SDS-PAGE gel was run to examine the protein samples.

[0037] Figure 9Data obtained using engineered mutants of the phi29 gp10 portal protein in an Oxford Nanopore Technologies MinION device are shown. A. No direct insertion of the WT phi29 gp10 pore into the ONT membrane was observed. BF. Direct insertion of engineered phi29 gp10 pores into the ONT membrane: B. Mutants (R10L, E14V); C. Mutant A79C with a conjugated porphyrin; D. Mutants (R10L, E14V, R17L, 7aa deletion, and N-terminal addition of an IL tag); E. Mutants (R10L, E14V, R17L, 7aa deletion of the N-terminus); F. Mutant (Q168C) with conjugated cholesterol. In order to insert the engineered protein channel into the ONT membrane, the protein at a concentration of 1 mg / ml was diluted 1000 times in C13 buffer (25 mM potassium phosphate, 150 mM potassium ferrocyanide, 150 mM potassium ferricyanide, pH 8). 200 ul of diluted protein sample was added through the perfusion port of the MinION flow cell. A ramp voltage of +50 to +350 mV (5 mV increments; held for 20 seconds) was then applied to help insert the protein channel. The flow cell was then rinsed with 2 mL of C13 buffer. IV curves were then typically run, ±50, ±100, ±150, ±200 mV, with variable holding times (held for 2 to 10 minutes at each voltage) to observe the pore behavior over time. Analytes such as DNA or peptides (1 pM concentration) were suspended in C13 buffer and added to the flow cell to check pore function. In AF, the applied voltage was 100 mV. Conductive buffer: C13 (ONT reagent). The analyte, a TAT peptide that elicits a unique current-blocking event, indicates a functional pore. G. Relative insertion rates for WT and engineered mutants. The rates are relative to WT. There was variation among the different mutants within each of the mentioned categories, but the overall trends are shown. DETAILED DESCRIPTION

[0038] The present invention will be described with respect to specific embodiments and with reference to certain drawings, but the invention is not limited thereto but only by the claims. Any reference signs in the claims should not be construed as limiting the scope. Of course, it should be understood that not all aspects or advantages may be achieved according to any particular embodiment of the present invention. Thus, for example, those skilled in the art will recognize that the present invention may be embodied or performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein.

[0039] The present invention (both with respect to organization and method of operation) and its features and advantages can be best understood by reference to the following detailed description when read in conjunction with the accompanying drawings. Various aspects and advantages of the present invention will become apparent from one or more embodiments described below and will be explained with reference to the embodiments. Reference to "one embodiment" or "embodiment" throughout this specification means that the specific features, structures or characteristics described in conjunction with the embodiment are included in at least one embodiment of the present invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout this specification do not necessarily refer to the same embodiment, but may refer to the same embodiment. Similarly, it should be understood that in the description of exemplary embodiments of the present invention, for the purpose of simplifying the disclosure and helping to understand one or more of the various inventive aspects, the various features of the present invention are sometimes grouped together in a single embodiment, figure or description thereof. However, the method of the present disclosure should not be interpreted as reflecting the intention that the claimed invention requires more features than those explicitly stated in each claim. On the contrary, as reflected in the following claims, the inventive aspects are less than all the features of a single aforementioned disclosed embodiment.

[0040] In addition, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes two or more polynucleotides; reference to a "polynucleotide binding protein" includes two or more such proteins; reference to a "helicase" includes two or more helicases; reference to a "monomer" refers to two or more monomers; reference to a "pore" includes two or more pores, etc.

[0041] In all discussions herein, standard single-letter codes for amino acids are used. These codes are as follows: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). Standard substitution notation is also used, i.e., Q42R means that the Q at position 42 is replaced by R.

[0042] portal protein

[0043] Nanopores are modified portal proteins of viral DNA-packaging motors, such as those of bacteriophages.

[0044] The portal protein of the bacteriophage DNA packaging motor has a truncated conical structure. The protein has a central channel formed by twelve portal protein subunits (also known as connexin subunits). An exemplary unmodified viral DNA packaging motor portal protein from bacteriophage phi29 has been purified, and its three-dimensional structure has been crystallographically characterized (e.g., Guasch et al., 1998 FEBS Lett. 430:283; Marais et al., 2008 Structure 16:1267). The phi29 channel has a narrow end of 3.6 nm and a wide end of 6 nm, which is larger than most membrane protein channels. Thus, various embodiments as described herein refer to the phi29 DNA packaging gp10 motor portal protein (e.g., GenBank Accession No. ACE96033 UniProt ID: P04332; Gene ID: 6446518; SEQ ID NO: 1) and / or its polypeptide subunits, including fragments, variants and derivatives capable of forming a channel (e.g., Accession Nos. gi 29565762, gi31072023, gi 66395194, gi 29565739, gi 157738604).

[0045] Although viral portal proteins share little sequence homology and differ in molecular weight, there is significant underlying structural similarity. Specifically, the DNA packaging motor connector proteins of other dsDNA viruses (e.g., T4, λ, P22, P2, T3, T5, and T7), although sharing little sequence homology with the phi29 connector and differing in molecular weight from the connector, exhibit significant underlying structural similarity (e.g., Bazinet et al., 1985 Ann Rev. Microbiol. 39:109-29).

[0046] In certain embodiments, isolated viral DNA packaging motor portal proteins from other dsDNA viruses are contemplated for use, including but not limited to isolated viral DNA packaging motor portal proteins from any of bacteriophages lambda, P2, P3, P22, T3, T4, T5, SPP1, HK97, and T7, such as isolated dsDNA viral DNA packaging motor portal proteins (e.g., T4 (Accession No. NP-049782) (Driedonks et al., 1981 J Mol Biol 152:641), lambda (Accession Nos. gi549295, gi 6723246, gi 15837315, gi 16764273) (Kochan et al., 1984 J Mol Biol 174:433), SPP1 (Accession No. P54309), P22 (Accession No. AAA72961) (Cingolani et al., 2002 J Struct Biol 139:46), G20c (accession number KX987127.1), P2 (accession number NP-046757), P3 (Nutter et al., 1972 J. Viral. 10(3):560-2), T3 (accession number CAA35152) (Carazo et al., 1986 J. Ultrastruct Mol Struct Res 94:105), T5 (accession numbers AAX12078, YP-006980; AAS77191; AAU05287), T7 (accession number NP-041995) (Cerritelli et al., 1996 J. Mol. Biol. 285:299; Agirrezabala et al., 2005 J. Mol. Biol. 347:895)). In some embodiments, the connexin comprises bacteriophage T3 connexin gp8. In some embodiments, the connexin comprises bacteriophage T7 connexin gp8. In some embodiments, the connexin comprises bacteriophage T4 connexin gp20. In some embodiments, the connexin comprises bacteriophage T5 connexin gp7. In some embodiments, the connexin comprises bacteriophage SPP1 connexin gp6. In some embodiments, the connexin comprises bacteriophage HK97 connexin gp3.

[0047] As with the phi29 DNA packaging motor portal protein exemplified herein, these and other dsDNA viral packaging motor portal proteins that have been substantially structurally characterized can be modified such that the portal proteins are incorporated into membrane layers to form pores through which electrical conduction can occur when an electrical potential is applied across the membrane in the same manner as the portal protein of the phi29 DNA packaging motor. Thus, the disclosure herein regarding the phi29 portal protein is intended to illustrate related embodiments contemplated using any of these other isolated dsDNA viral DNA packaging motor portal proteins.

[0048] The portal protein of the phi29 DNA packaging motor, or the portal protein from another bacteriophage DNA packaging motor, can be modified according to the teachings discovered herein.

[0049] According to the present disclosure, isolated DNA packaging motor portal proteins that have been artificially engineered to have membrane-binding properties (e.g., stable transmembrane integration in a membrane layer) can be used as conductive biosensors for cancer biomarkers. Portal proteins can also be artificially engineered to affect the conductive properties of the transmembrane channel formed by the portal protein.

[0050] Modified isolated double-stranded DNA viral DNA packaging motor protein connectors, such as the phi29 connector, can be engineered to have the desired structure for the presently disclosed embodiments, for which protein crystallographic structural data are readily available. Large-scale production and purification procedures for the phi29 connector have been developed (Guo et al., 2005; Ibanez et al., Nucleic Acids Res. 12, 2351-2365 (1984); Robinson et al., Nucleic Acids Res. 34, 2698-2709 (2006); Xiao et al., ACS Nano 3, 100-107 (2009).

[0051] In one embodiment, a modified bacteriophage phi29 viral DNA packaging motor portal protein (e.g., SEQ ID NO: 1) is at least 80%, 90% or 95% identical to the wild-type protein or a portion derived from the wild-type phi29 viral DNA packaging motor connecting protein, the portion containing at least 150, 175, 200, 225, 250, 275, including at least 240, 260, 280, 285, 290, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330 or more amino acids.

[0052] In other embodiments, the modified portal protein is a modified double-stranded DNA portal protein from another bacteriophage, such as bacteriophage T4, lambda phage (accession numbers gi549295, gi6723246, gi15837315, gi16764273), bacteriophage SPP1 (P54309), bacteriophage P22 (accession number AAA72961), bacteriophage P2 (accession number NP_046757), bacteriophage P3 (Nutter et al., 1972 Journal of Virology 10(3):560-2), bacteriophage T3 (accession number CAA35152), bacteriophage T5 (accession numbers AAX12078, YP006980; AAS77191; AAU05287), bacteriophage T7 (accession number NP041995), and bacteriophage HK97 (accession number NP_037699). For example, the modified portal protein can be a mutant of any of these phage virus DNA packaging motor portal proteins and can, for example, be at least 80%, 90% or 95% identical to the polypeptides disclosed herein and to fragments of such polypeptides. "Fragments" of mutant portal protein subunits typically contain at least 150, 175, 200, 225, 250, 275 amino acids, including at least 240, 260, 280, 285, 290, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330 or more amino acids.

[0053] As used herein, the term "amino acid identity" refers to the degree to which a sequence is identical on an amino acid to amino acid basis over a comparison window. Therefore, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appear in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to produce percentage of sequence identity.

[0054] The portal protein can be a modified analog of any of the above-mentioned phage portal proteins. "Analog" when referring to the viral DNA packaging motor portal protein means a naturally occurring homolog or variant of the viral DNA packaging motor portal protein. Portal proteins are typically composed of subunits that can self-assemble into oligomeric (e.g., homododecamer) channels.

[0055] The portal protein may be (i) a portal protein in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and such substituted amino acid residues may or may not be amino acid residues encoded by the genetic code, or (ii) a portal protein in which one or more amino acid residues comprise a substitution, or (iii) a portal protein in which additional amino acids are genetically fused to one or more portal protein subunits, said additional amino acids comprising amino acids for detection or specific functional alteration of the mutant portal protein.

[0056] Isolating the modified portal protein. The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it occurs naturally). For example, a naturally occurring protein present in an intact naturally occurring virus is not isolated, but the same protein separated from some or all of the coexisting materials in the natural system is isolated. Such a protein can be part of a composition and still be isolated because such a vector or composition is not part of its natural environment. Methods of isolating portal proteins of bacteriophage motor proteins are known in the art. The portal proteins of bacteriophage motor proteins used in the methods and compositions described herein can be produced by recombinant methods well known in the art.

[0057] In one embodiment, the modified protein is a truncated version of the portal protein, and / or the modified protein may include additional amino acids at one or both ends of one or more of the subunits of the portal protein, and / or may include one or more amino acid substitutions, deletions, or additions within the amino acid sequence of the portal protein.

[0058] The truncated portal protein can be truncated at the N-terminus and / or the C-terminus. For example, the N-terminus can be deleted up to about 30 amino acids, such as the N-terminus can be deleted up to about 20, 10, 9, 8 or 7 amino acids. Alternatively or additionally, the C-terminus can be deleted up to about 30 amino acids, such as the C-terminus can be deleted up to about 20, 10, 9, 8 or 7 amino acids. One or more, such as 2 to about 30 amino acids, such as 3 to about 20, 4 to about 10, 5 to 9 or 6, 7 or 8 amino acids, can be added to the N-terminus and / or to the C-terminus, or to the truncated N-terminus and / or the truncated C-terminus.

[0059] The modified portal protein comprises a channel. In one embodiment, the portal protein is modified to alter one or more properties of the channel of the nanopore. In one embodiment, this is achieved by modifying one or more amino acid residues at the channel lining and / or the channel entrance.

[0060] In some embodiments, the nanopore comprises only full-length subunits of the portal protein.

[0061] In some embodiments, the nanopore is a multimeric protein formed by six or more portal protein subunits (such as 7, 8, 9, 10, 11 or 12 subunits). For example, the nanopore can be a dodecameric protein. One or more of the subunits can be modified, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In one embodiment, one or more of the subunits can be modified at the C-terminus and / or N-terminus, for example to increase the hydrophilicity at one or both ends of the nanopore. For example, one or more of the subunits can be modified by adding a flexible linker and / or a peptide tag at the C-terminus and / or N-terminus. In some embodiments, the nanopore is composed of identical subunits. Any suitable linker can be used, for example, a linker comprising 3 to 12 amino acids, such as 4 or 5 to 10, preferably 6 to 8 amino acids. The amino acids in the linker can be selected from lysine, serine, arginine, proline, glycine, alanine aspartic acid, tyrosine, isoleucine and / or threonine. Examples of suitable linkers include, but are not limited to, the following: GGGS, PGGS, PGGG, RPPPPP, RPPPP, VGG, RPPG, PPPP, RPPG, PPPPPPP, RPPG, GGG, GGGG, GGGGG, GGGGGG, and DYDIPTT.

[0062] The modified portal protein can include a tag, for example, to facilitate its purification. Any suitable peptide tag can be used to facilitate the purification of the portal protein. For example, in one embodiment, the tag can be a Streptococcus tag. In one embodiment, the Streptococcus tag has a length of 8 to 11 amino acids and / or the Streptococcus tag amino acid sequence contains the motif HPQ. The Streptococcus tag can, for example, include or consist of the amino acid sequence WSHPQSEK, WSHPQFEK, NWSHPQFEK, PWSHPQFEK or GGSHPQFEG. As long as the core "HPQ" motif is maintained, this sequence can be varied by adding, deleting or replacing one or more, such as 2, 3, 4 or 5 amino acids. The variant sequence is typically 8 to 11 amino acids NWSHPQFEK, PWSHPQFEK, and GGSHPQFEG. In another embodiment, the tag can be a His tag (typically His6 (HHHHHH)).

[0063] The portal protein may comprise a cleavage site to allow removal of the tag and / or linker from the subunit before or after pore assembly. Any suitable cleavage site may be used. An example is the TEV (tobacco etch virus) clearance site (ENLYFQG; where cleavage occurs between the Q and G residues).

[0064] Modification by introducing amino acids to facilitate insertion

[0065] In one aspect, the portal protein is modified by introducing one or more amino acids to alter the hydrophobicity of the pore surface to facilitate its direct insertion into the membrane. The one or more amino acids can be introduced by substitution and / or insertion. The inserted amino acid can be inserted at one or both ends of the amino acid chain of the portal protein subunit and / or between two amino acids in the chain. When the subunits fold and assemble into the pore, the introduced amino acid is present on the outer surface of the pore.

[0066] At least one amino acid is introduced into one or more of the subunits in the pore. Each subunit in the pore can independently include one or more introduced amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. The pore can contain identical modified subunits, but one or more of the subunits in the pore can be different from the other subunits. For example, the pore can be composed of two or more different subunits, such as from 3 or more different subunits. For example, 4, 5, 6, 7, 8, 9, 10 or 11 different modified subunits can be present in the pore. In one embodiment, all subunits can be different from each other. Typically, at least one amino acid is introduced into each subunit in the pore. For example, in the case of a pore comprising 12 subunits, the pore includes 12 or more introduced or substituted amino acids to change the hydrophobicity of the pore surface.

[0067] The modification is usually made in a central hydrophobic zone surrounding the outside of the pore. The positioning of the central hydrophobic zone region is as follows Figure 1 The central zone region of the pore is typically modified to increase its hydrophobicity. For example, increased hydrophobicity can be achieved by replacing hydrophilic, neutral, or relatively less hydrophobic amino acids (e.g., alanine and / or methionine) with more hydrophobic residues (e.g., leucine, valine, and / or isoleucine). Figure 1 D shows the relative hydrophobicity of the amino acids. Substitutions to increase hydrophobicity can be used e.g. Figure 1 Any amino acid having a more positive number on the hydrophobicity scale shown in D than the amino acid being replaced replaces one or more of the amino acids present in the pore.

[0068] The increase in hydrophobicity can be achieved by inserting one or more hydrophobic amino acids into and / or at the termini of the amino acid chains of the portal protein subunits. Figure 1 Shown in D.

[0069] The positioning of the introduced hydrophobic amino acids can determine where the pore is located in the membrane. The position of the pore relative to the membrane can be shifted upward or downward (e.g., by up to 0.5 nm in either direction). Thus, the position of the pore in the membrane can be controlled to improve the stability of the pore in the membrane. The intrinsic electrophysiology of the pore is generally not altered by changes in the amino acids on the outer surface of the pore.

[0070] In one embodiment, the hydrophobicity is altered by introducing one or more amino acids in the band below the wing domain. The target locations include Phe24, Ile25, Leu28, Phe60, Phe128, Pro129 and Pro132 in the phi29 portal protein subunit, and corresponding positions in similar subunits. Additional hydrophobic residues can be substituted or inserted within one or two amino acids before and / or after any one or more of these target locations (such as 2, 3, 4, 5, 6, 7 or 8 of these target locations). For example, the hydrophobicity of the amino acid residues at positions corresponding to positions 22, 23, 26, 27, 29, 30, 58, 59, 61, 62, 126, 127, 130, 131, 133 and / or 134 in SEQ ID NO: 1 can be increased by substitution or insertion of amino acid residues. Alterations can be made at any one or more of these positions, such as any two, three, four, five, six, seven, eight, nine, or ten or more positions. Other exemplary mutation positions include those corresponding to Arg10, Glu14, Arg17, Gln18, and Arg22 in SEQ ID NO: 1. Any one or more of these amino acids can be substituted with a more hydrophobic residue to increase the hydrophobicity of the surface of the central region of the pore.

[0071] In one embodiment, the exposed charged residues in the stem region (such as the residues corresponding to Arg17, Arg22 and / or Lys172 of SEQ ID NO: 1) and the Asn / Gln residues concentrated in the two distal parts of the protein stem (such as the residues corresponding to Asn166, Asn167, Gln168, Gln173, Asn176 and / or Gln177 in SEQ ID NO: 1) can be altered to change the hydrophilicity.

[0072] Modification by introduction of amino acids to facilitate conjugation of molecules with altered hydrophobicity

[0073] In one aspect, the modified portal protein of the phage DNA packaging motor that has been modified so that it can be directly inserted into the membrane is a portal protein in which one or more amino acid residues on the outer surface of the portal protein are replaced with another amino acid residue and / or one or more amino acid residues introduced on the outer surface of the portal protein to introduce a binding site for a molecule that changes the hydrophobicity of the outer surface of the portal protein compared to the wild-type portal protein. The binding site is introduced on the outer side of the wing domain or in the stem domain. The binding site serves as an attachment site for the molecule. The molecule is typically a hydrophobic molecule that increases the hydrophobicity of the portal protein surface.

[0074] In one embodiment, the introduced binding site can be a cysteine ​​residue. In another embodiment, the binding site can be an unnatural amino acid.

[0075] Non-natural amino acids are amino acids that are not naturally present in proteins. Non-natural amino acids are preferably not histidine, alanine, isoleucine, arginine, leucine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, serine or tyrosine. Non-natural amino acids are more preferably not any one of the twenty amino acids in the previous sentence or selenocysteine.

[0076] Preferred unnatural amino acids for use in the present invention include, but are not limited to, 4-azido-L-phenylalanine (Faz), 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine, 4-acetoacetyl-L-phenylalanine, O-allyl-L-tyrosine, 3-(phenylseleno)-L-alanine, O-2-propyn-1-yl-L-tyrosine, 4-(dihydroxyboryl)-L-phenylalanine, 4-[(ethylsulfanyl)carbonyl]-L-phenylalanine, (2S)-2-amino-3-{4-[(prop-2-ylsulfanyl)carbonyl]phenyl}propanoic acid, (2S)-2-amino-3-{4-[(2-amino-3-sulfanylpropionyl)amino]phenyl}propanoic acid, O-methyl-L-tyrosine, 4-amino-L -phenylalanine, 4-cyano-L-phenylalanine, 3-cyano-L-phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-L-phenylalanine, 4-bromo-L-phenylalanine, O-(trifluoromethyl)tyrosine, 4-nitro-L-phenylalanine, 3-hydroxy-L-tyrosine, 3-amino-L-tyrosine, 3-iodo-L-tyrosine, 4-isopropyl-L-phenylalanine, 3-(2-naphthyl)-L-alanine, 4-phenyl-L-phenylalanine, (2S)-2-amino-3-(naphthylacetamido-2-ylamino)propionic acid, 6-(methylsulfanyl)norleucine, 6-oxo-L-lysine, D-tyrosine, (2R)-2-hydroxy-3-(4-hydroxyphenyl)propionic acid, (2R)-2-octanamide 3-(2,2'-dipyridin-5-yl)-D-alanine, 2-amino-3-(8-hydroxy-3-quinolinol)propionic acid, 4-benzoyl-L-phenylalanine, S-(2-nitrobenzyl)cysteine, (2R)-2-amino-3-[(2-nitrobenzyl)sulfanyl]propionic acid, (2S)-2-amino-3-[(2-nitrobenzyl)oxy]propionic acid, O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine, (2S)-2-amino-6-({[(2-nitrobenzyl)oxy]carbonyl}amino)hexanoic acid, O-(2-nitrobenzyl)-L-tyrosine, 2-nitrophenylalanine, 4-[(E)-phenyldiazenyl]-L-phenylalanine, 4-[3-(trifluoromethyl) -3H-diaziridin-3-yl]-D-phenylalanine, 2-amino-3-[[5-(dimethylamino)-1-naphthyl]sulfonylamino]propionic acid, (2S)-2-amino-4-(7-hydroxy-2-oxo-2H-chromen-4-yl)butanoic acid, (2S)-3-[(6-acetylnaphthylacetamido-2-yl)amino]-2-aminopropionic acid, 4-(carboxymethyl)phenylalanine, 3-nitro-L-tyrosine, O-sulfo-L-tyrosine, (2R)-6-acetamido-2-aminohexanoic acid, 1-methylhistidine, 2-aminononanoic acid, 2-aminodecanoic acid, L-homocysteine, 5-sulfonylnorvaline, 6-sulfonyl-L-norleucine, 5-(methylsulfonyl)-L-pentanoic acid, N6 -{[(2R,3R)-3-methyl-3,4-dihydro-2H-pyrrol-2-yl]carbonyl}-L-lysine, N 6 -[(Benzyloxy)carbonyl]lysine, (2S)-2-amino-6-[(cyclopentylcarbonyl)amino]hexanoic acid, N 6 -[(cyclopentyloxy)carbonyl]-L-lysine, (2S)-2-amino-6-{[(2R)-tetrahydrofuran-2-ylcarbonyl]amino}hexanoic acid, (2S)-2-amino-8-[(2R,3S)-3-ethynyltetrahydrofuran-2-yl]-8-oxooctanoic acid, N 6 -(tert-Butoxycarbonyl)-L-lysine, (2S)-2-hydroxy-6-({[(2-methyl-2-propanyl)oxy]carbonyl}amino)hexanoic acid, N 6 -[(allyloxy)carbonyl]lysine, (2S)-2-amino-6-({[(2-azidobenzyl)oxy]carbonyl}amino)hexanoic acid, N 6 -L-prolyl-L-lysine, (2S)-2-amino-6-{[(prop-2-yn-1-yloxy)carbonyl]amino}hexanoic acid and N 6 -[(2-azidoethoxy)carbonyl]-L-lysine. The most preferred unnatural amino acid is 4-azido-L-phenylalanine (Faz).

[0077] Examples of suitable hydrophobic molecules that can be conjugated to portal proteins include porphyrin, tetraphenylporphyrin, protoporphyrin IX, octaethylporphyrin, cholesterol, heme, and biliverdin. These and other hydrophobic molecules can be attached to portal proteins to achieve membrane anchoring of portal proteins.

[0078] The exact location of the binding site of the hydrophobic molecule can be controlled to determine the position of the hole in the membrane. The hydrophobic molecule can be used to change the position of the hole relative to the membrane. For example, the hydrophobic molecule can cause the hole to shift upward or downward in the membrane (for example, in either direction, up to 0.5nm). Thus, the stability of the hole in the membrane can be controlled. The positioning of the hydrophobic molecule on the outer surface of the hole will not change the inherent electrophysiological properties of the hole.

[0079] Examples of locations where binding (or conjugation) sites can be introduced into the Phi29 Gp10 portal protein include Q32, Y36, F52, K55, Q59, F60, Y62, N77, G78, A79, L80, S81, R84, R94, A96, S97, P98, Q101, P129, T131, E135, and Q168. Any one or more of these positions in the Phi29 Gp10 portal protein or residues at corresponding positions in other portal proteins can be substituted, for example, with cysteine ​​or a non-natural amino acid to introduce a binding side for a hydrophobic molecule. Cysteine ​​residues or non-natural amino acid residues can alternatively be inserted within one or both residues at these positions.

[0080] In one embodiment, hydrophobicity is adjusted by adding one or more natural or unnatural amino acids to one or both of the termini of the subunit molecule to facilitate insertion of the portal protein into the membrane. For example, a hydrophilic or hydrophobic tag can be added to one or both of the termini. Typically, a hydrophobic tag is added to the N-terminus present in the central band region of the molecule and / or a hydrophilic tag can be added to the C-terminal domain. The hydrophilic and / or hydrophobic tags can be attached to the portal protein via a linker. Suitable linkers are described above.

[0081] The tag can include, for example, two to twelve amino acids, such as 3 or 4 to 10, such as 5, 6, 7, 8 or 9 amino acids. In one embodiment, all amino acids in the tag are hydrophilic amino acids. Hydrophilic amino acids are amino acids with negative numbers on the hydrophobicity scale (e.g. Figure 1 D). One or more of the residues in the hydrophilic tag can be the residue at position 0 on the hydrophobicity scale. In one embodiment, the hydrophilic tag can be generally hydrophilic but include one or more hydrophobic residues with positive numbers on the hydrophobicity scale.

[0082] In embodiments using a hydrophobic tag, the hydrophobic tag may, for example, contain only residues with positive numbers on the hydrophobicity scale. Alternatively, the hydrophobic tag may contain one or more residues with a hydrophobicity of 0. As long as the tag is generally hydrophobic, the tag may contain one or more polar or charged amino acids with negative numbers on the hydrophobicity scale.

[0083] Mutations used to facilitate use as nanopore sensors

[0084] The modified portal protein may include one or more additional modifications that alter other properties of the pore. Such changes generally facilitate the use of the pore as a nanopore sensor. Examples of such modifications include the following:

[0085] The overall electronegativity of the channel interior can be altered by changing the ring of negatively charged Arg / Lys or Asp / Glu residues. The Arg / Gly residues can, for example, be replaced by positively charged or neutral amino acids. One or more Asp / Glu residues can be replaced by positively charged, negatively charged, or neutral amino acids. The acidic residues at the inner channel entrance at the narrow end, such as Glu189, Asp19, and Asp194 in SEQ ID NO: 1, can be used to alter the hydrophilicity.

[0086] Several amino acids (any natural or unnatural) are added at the termini, with the goal of using these amino acids as anchor points to add functionality or to alter the electrophysiological properties of the pore.

[0087] Alterations (deletions, truncations, mutations) of internal flexible loops, such as residues 229-244 in the phi29 Gp10 portal protein, can alter the electrophysiological properties and / or detection capabilities of the pore.

[0088] Mutant or modified protein, monomer or peptide can also be chemically modified in any way and at any site.Preferably by attaching the molecule to one or more cysteines (cysteine ​​connection), attaching the molecule to one or more lysines, attaching the molecule to one or more non-natural amino acids, enzyme modification of epi-positions or modification of the ends to the mutant or modified monomer. Suitable methods for carrying out such modifications are well known in the art. The mutant of modified protein, monomer or peptide can be chemically modified by the attachment of any molecule. For example, the mutant of modified protein, monomer or peptide can be chemically modified by the attachment of a dye or fluorophore.

[0089] membrane

[0090] Any suitable membrane can be used in the system. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed by amphiphilic molecules such as phospholipids, which have both hydrophilic and lipophilic properties. Amphiphilic molecules can be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles that form a monolayer are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymeric materials in which two or more monomer subunits are polymerized together to produce a single polymer chain. Block copolymers generally have properties contributed by each monomer subunit. However, block copolymers may have unique properties that polymers formed by individual subunits do not have. Block copolymers can be engineered so that one of the monomer subunits is hydrophobic (i.e., lipophilic) in an aqueous medium, while the other subunits are hydrophilic. In this case, the block copolymer may have amphiphilic properties and may form a structure that simulates a biological membrane. Block copolymers can be diblock (composed of two monomer subunits), but can also be constructed from more than two monomer subunits to form more complex arrangements that behave as amphiphiles. The copolymers can be triblock, tetrablock, or pentablock copolymers. The membrane is preferably a triblock copolymer membrane.

[0091] Archaeal bipolar tetraether lipids are naturally occurring lipids that are constructed to allow lipids to form monolayer membranes. These lipids are generally found in extremophiles, thermophiles, halophiles, and acidophiles that survive in harsh biological environments. It is believed that their stability is due to the fusogenic properties of the final bilayer. A straightforward approach is to construct block copolymer materials that mimic these biological entities by producing triblock polymers with the general motif hydrophilicity-hydrophobicity-hydrophilicity. Such materials can form monomeric membranes that behave similarly to lipid bilayers and encompass a series of staged behaviors from vesicles to lamellar membranes. Membranes formed from these triblock copolymers maintain several advantages over biological lipid membranes. Because the triblock copolymers are synthetic, the exact construction can be carefully controlled to provide the correct chain length and properties required to form the membrane and interact with pores and other proteins.

[0092] Block copolymers can also be constructed from subunits that are not classified as lipid submaterials; for example, hydrophobic polymers can be made from siloxanes or other non-hydrocarbon-based monomers. The hydrophilic subsegments of the block copolymers can also possess low protein binding properties, which allows for the creation of membranes that are highly resistant when exposed to raw biological samples. This head group unit can also be derived from non-classical lipid head groups.

[0093] Compared to biological lipid membranes, triblock copolymer membranes also have increased mechanical and environmental stability, such as much higher operating temperature or pH ranges.The synthetic nature of block copolymers provides a platform for tailoring polymer-based membranes for a wide range of applications.

[0094] The membrane is most preferably one of the membranes disclosed in International Application No. WO2014 / 064443 or WO2014 / 064444.

[0095] The amphiphilic molecules can be chemically modified or functionalized to facilitate coupling to polynucleotides. The amphiphilic layer can be a single layer or a double layer. The amphiphilic layer is typically planar. The amphiphilic layer can be curved. The amphiphilic layer can be supported.

[0096] Amphiphilic membranes are usually naturally mobile, essentially at a rate of about 10 -8 cm s -1 The lipid diffusion rate is 2.5 Å, which acts as a two-dimensional liquid. This means that the pore and the coupled polynucleotide can generally move within the amphiphilic membrane.

[0097] The membrane can be a lipid bilayer. The lipid bilayer is a model of the cell membrane and serves as an excellent platform for a range of experimental studies. For example, the lipid bilayer can be used for in vitro studies of membrane proteins by single-channel recording. Alternatively, the lipid bilayer can be used as a biosensor to detect the presence of a range of substances. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers, or liposomes. The lipid bilayer is preferably a flat lipid bilayer. Suitable lipid bilayers are disclosed in WO 2008 / 102121, WO 2009 / 077734, and WO 2006 / 100484.

[0098] Methods for forming lipid bilayers are known in the art. Lipid bilayers are typically formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA, 1972; 69: 3561-3566), in which a lipid monolayer is carried on an aqueous solution / air interface passing through an opening perpendicular to the interface. The lipids are typically added to the surface of an aqueous electrolyte solution by first dissolving the lipids in an organic solvent and then evaporating a drop of solvent on the surface of the aqueous solution on either side of the opening. Once the organic solvent has evaporated, the solution / air interface on either side of the opening is physically moved back and forth through the opening until a bilayer is formed. A flat lipid bilayer can be formed across an opening in a membrane or across an opening in a groove.

[0099] The method of Montal and Mueller is commonly used because it is a cost-effective and relatively straightforward method for forming good quality lipid bilayers suitable for protein pore insertion. Other common methods for bilayer formation include tip immersion, bilayer painting, and patch clamping of liposomal bilayers.

[0100] Tip-immersion bilayer formation requires contacting the surface of the pore (e.g., pipette tip) with the surface of the test solution carrying the lipid monolayer. Similarly, a lipid monolayer is first generated at the solution / air interface by evaporating a drop of lipid dissolved in an organic solvent at the solution surface. The bilayer is then formed by the Langmuir-Schaefer process, and mechanical automation is required to move the pore relative to the solution surface.

[0101] For a brushed bilayer, a drop of lipid dissolved in an organic solvent is applied directly to the aperture, which is then immersed in an aqueous test solution. Using a brush or equivalent, the lipid solution is thinly diffused within the aperture. This thinning of the solvent allows for the formation of a lipid bilayer. However, it is very difficult to completely remove the solvent from the bilayer, and the bilayers formed by this method are therefore less stable and more prone to noise during electrochemical measurements.

[0102] Patch clamping is commonly used in biological cell membrane research. The cell membrane is clamped to the end of the pipette by pumping, and the membrane patch becomes attached to the perforation. The method is suitable for producing lipid bilayers by clamping and then bursting liposomes to leave the lipid bilayer sealed in the perforation of the pipette. The method requires stable, large and unilamellar liposomes and the production of small perforations in materials with glass surfaces.

[0103] Liposomes can be formed by sonication, extrusion, or the Mozafari method (Colas et al. (2007) Micron 38:841-847).

[0104] In a preferred embodiment, the lipid bilayer is formed as described in International Application No. WO 2009 / 077734. Advantageously, the lipid bilayer is formed by drying the lipids. In a most preferred embodiment, the lipid bilayer is formed across an opening as described in WO 2009 / 077734.

[0105] The lipid bilayer is formed by two relative layers of lipid. Two lipid layers are arranged so that their hydrophobic tail groups face each other, forming hydrophobic inside. The hydrophilic head group of lipid faces outwards towards the aqueous environment on bilayer every side. Bilayer can be present in multiple lipid stages, and the stage includes but is not limited to liquid disordered stage (liquid lamella), liquid ordered stage, solid ordered stage (lamella gel stage, interlaced gel stage) and flat double layer crystal (lamella subgel stage, lamella crystallization stage).

[0106] Any lipid composition that forms lipid bilayer can be used. Select lipid composition so that lipid bilayer has required characteristic, for example surface charge, the ability of supporting membrane protein, packing density or the mechanical property formed. Lipid composition can comprise one or more different lipids. For example, lipid composition can contain up to 100 kinds of lipids. Lipid composition preferably contains 1 to 10 kinds of lipids. Lipid composition can comprise naturally occurring lipid and / or artificial lipid.

[0107] Lipids typically include a head group, an interfacial moiety, and two hydrophobic tail groups that may be the same or different. Suitable head groups include, but are not limited to, neutral head groups such as diacylglycerides (DG) and ceramide (CM); zwitterionic head groups such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM); negatively charged head groups such as phosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), and cardiolipin (CA); and positively charged head groups such as trimethylammoniumpropane (TAP). Suitable interfacial moieties include, but are not limited to, naturally occurring interfacial moieties, such as glycerol-based or ceramide-based moieties. Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains such as lauric acid (n-dodecanoic acid), myristic acid (n-tetradecanoic acid), palmitic acid (n-hexadecanoic acid), stearic acid (n-octadecanoic acid), and arachidic acid (n-eicosanoic acid); unsaturated hydrocarbon chains such as oleic acid (cis-9-octadecanoic acid); and branched hydrocarbon chains such as phytanyl. The chain length and the position and number of double bonds in the unsaturated hydrocarbon chain can vary. The chain length and the position and number of branches (e.g., methyl groups) in the branched hydrocarbon chain can vary. The hydrophobic tail group can be attached to the interfacial portion as an ether or ester. The lipid can be a mycolic acid.

[0108] The lipids can also be chemically modified. The head group or tail group of the lipid can be chemically modified. Suitable lipids whose head groups have been chemically modified include, but are not limited to: PEG-modified lipids, such as 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; functionalized PEG lipids, such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)2000]; and lipids modified for conjugation, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl). Suitable lipids with chemically modified tail groups include, but are not limited to, polymerizable lipids such as 1,2-bis(10,12-tricosadiynyl)-sn-glycero-3-phosphocholine; fluorinated lipids such as 1-palmitoyl-2-(16-fluoropalmitoyl)-sn-glycero-3-phosphocholine; deuterated lipids such as 1,2-dipalmitoyl-D62-sn-glycero-3-phosphocholine; and ether-linked lipids such as 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine. The lipids can be chemically modified or functionalized to facilitate coupling to polynucleotides.

[0109] An amphiphilic layer, such as a lipid composition, typically includes one or more additives that will affect the properties of the layer. Suitable additives include, but are not limited to: fatty acids, such as palmitic acid, myristic acid, and oleic acid; fatty alcohols, such as palmityl, myristyl, and oleyl alcohol; sterols, such as cholesterol, ergosterol, lanosterol, sitosterol, and stigmasterol; lysophospholipids, such as 1-acyl-2-hydroxy-sn-glycero-3-phosphocholine; and ceramides.

[0110] In another preferred embodiment, the membrane comprises a solid layer. The solid layer can be formed from both organic and inorganic materials, including but not limited to: microelectronic materials, insulating materials (such as Si3N4, Al2O3 and SiO), organic and inorganic polymers (such as polyamide), plastics (such as ) or elastomers (such as two-component addition-cured silicone rubber) and glass. The solid-state layer can be formed from graphene. Suitable graphene layers are disclosed in WO 2009 / 035647. If the membrane includes a solid-state layer, the pores are typically present in the amphiphilic membrane or layer, and the amphiphilic membrane or layer is contained within the solid-state layer, for example, within holes, pores, gaps, channels, grooves or gaps within the solid-state layer. A skilled person can prepare suitable solid-state / amphiphilic hybrid systems. Suitable systems are disclosed in WO 2009 / 020682 and WO 2012 / 005857. Any of the amphiphilic membranes or layers discussed above can be used.

[0111] The methods are typically performed using: (i) an artificial amphiphilic layer comprising a pore, (ii) an isolated naturally occurring lipid bilayer comprising a pore, or (iii) a cell into which a pore is inserted. The methods are typically performed using an artificial amphiphilic layer (e.g., an artificial triblock copolymer layer). The layer may include other transmembrane and / or intramembrane proteins and other molecules in addition to the pore. Suitable equipment and conditions are discussed below. The methods of the present invention are typically performed in vitro.

[0112] Methods for inserting modified pores into membranes

[0113] Disclosed herein are methods for inserting modified portal proteins of bacteriophage DNA-packaging motors into membranes for use as nanopores.

[0114] The modified portal protein can be inserted into the copolymer membrane by contacting the membrane with the purified protein and applying a voltage potential to the membrane.Such methods are used in the art to insert nanopores into membranes.

[0115] One exemplary method involves contacting a membrane with a modified portal protein and applying a voltage ramp to facilitate insertion of the portal protein into the membrane to form a channel. A skilled artisan will readily be able to determine the appropriate portal protein concentration and voltage. For example, a voltage ramp from +50 to +350 mV can be applied, with the voltage increasing in 5 mV increments, for example, for about 20 seconds at each voltage. Excess portal protein can be washed away prior to use as a sensor.

[0116] Array

[0117] The present disclosure provides arrays of membranes comprising nanopores, wherein the nanopores comprise modified portal proteins. In preferred embodiments, each membrane in the array comprises one nanopore. Due to the manner in which the array is formed, for example, the array may comprise one or more membranes that do not comprise a nanopore, and / or one or more membranes that comprise two or more nanopores. The array may comprise from about 2 to about 1000, e.g., from about 10 to about 800, from about 20 to about 600, or from about 30 to about 500 membranes.

[0118] In one embodiment, a membrane array containing modified portal protein nanopores can be present in a device suitable for high-throughput sequencing.

[0119] sensor device

[0120] The present disclosure provides a device comprising a membrane array containing modified portal protein nanopores. For example, the device may include a chamber containing an aqueous solution and a barrier dividing the chamber into two sections. The barrier typically has an orifice, into which the membrane containing the nanopores is formed. Alternatively, the barrier may form a membrane having pores therein.

[0121] Thus, the device may comprise a first chamber and a second chamber, wherein the first chamber and the second chamber are separated by a membrane comprising a modified portal protein nanopore. When used to characterize a target polynucleotide, the device may further comprise a target polynucleotide, wherein the target polynucleotide is transiently localized within a channel formed by the portal protein, and wherein one end of the target polynucleotide is localized in the first chamber and one end of the target polynucleotide is localized in the second chamber.

[0122] In one embodiment, the device is capable of supporting a plurality of nanopores and membranes and is operable to perform analyte characterization using the nanopores and membranes. In one embodiment, the device includes at least one port for delivering a material for performing the characterization. In one embodiment, the device includes at least one reservoir for holding a material for performing the characterization. In one embodiment, the device includes a fluidic system configured to controllably supply material from at least one reservoir to the sensor device; and one or more containers for receiving corresponding samples, the fluidic system configured to selectively supply samples from the one or more containers to the sensor device. The device may also include circuitry capable of applying a potential and measuring an electrical signal across the membrane and pore complex.

[0123] The apparatus may be any of those described in WO 2008 / 102120, WO 2009 / 077734, WO 2010 / 122293, WO 2011 / 067559 or WO 00 / 28312.

[0124] In one embodiment, the device forms part of a system for characterizing an analyte. In one embodiment, the system may further include a conductive solution in contact with the nanopore, electrodes for providing a voltage potential across the membrane, and a measurement system for measuring the current through the nanopore. In one embodiment, the voltage applied across the membrane and pore complex is between +5V and -5V, such as -600mV to +600mV or -400mV to +400mV. The voltage used is preferably in the range of 100mV to 240mV, and more preferably in the range of 120mV to 220mV. By using an increased applied potential, the discrimination between different nucleotides can be increased through the pore. Any suitable conductive solution can be used. For example, the solution can include a charge carrier, such as a metal salt, such as an alkali metal salt; a halide salt, such as a chloride salt, such as an alkali metal chloride salt. The charge carrier can comprise an ionic liquid or an organic salt, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3-methylimidazole chloride. In an exemplary system, the salt is present in an aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl), cesium chloride (CsCl), or a mixture of potassium ferrocyanide and potassium ferrocyanide is commonly used. KCl, NaCl, and a mixture of potassium ferrocyanide and potassium ferrocyanide are preferred. The charge carriers can be asymmetric across the membrane. For example, the type and / or concentration of charge carriers on each side of the membrane (e.g., in each chamber) can be different.

[0125] The salt concentration can be at saturation. The salt concentration can be 3M or lower, and is typically 0.1 to 2.5M, 0.3 to 1.9M, 0.5 to 1.8M, 0.7 to 1.7M, 0.9 to 1.6M, or 1M to 1.4M. The salt concentration is preferably 150mM to 1M. The method is preferably performed using a salt concentration of at least 0.3M, such as at least 0.4M, at least 0.5M, at least 0.6M, at least 0.8M, at least 1.0M, at least 1.5M, at least 2.0M, at least 2.5M, or at least 3.0M. High salt concentrations provide a high signal-to-noise ratio and allow identification of current indicating the presence of nucleotides relative to a background of normal current fluctuations.

[0126] A buffer may be present in the conductive solution. Typically, the buffer is a phosphate buffer. Other suitable buffers are HEPES and Tris-HCl buffer. The pH of the conductive solution may be 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, or 7.0 to 8.8, or 7.5 to 8.5. The pH used is preferably about 7.5.

[0127] The device can be compatible with high-throughput equipment. For example, the device can be a SmidgION, MinION, GridION, or PromethION instrument developed by Oxford Nanopore Technologies Ltd. These instruments can be equipped with different types of flow cells, in which the nanopores are embedded in the copolymer membrane. The device can be a flow cell. The copolymer membrane is stable for at least several months and can also withstand higher voltages. Each channel contains its own pair of electrodes, thereby separating the electrical signal between the channels.

[0128] Methods for characterizing analytes

[0129] In another aspect, a method for determining the presence, absence or one or more characteristics of a target analyte is disclosed. The method involves contacting the target analyte with a membrane comprising a pore complex so that the target analyte moves relative to (such as into or through) a continuous channel, the continuous channel comprising at least two structures provided by a nanopore and an auxiliary protein or peptide in the pore complex, respectively, and performing one or more measurements as the analyte moves relative to the channel to determine the presence, absence or one or more characteristics of the analyte. The analyte can pass through the nanopore constriction and then through the auxiliary protein constriction. In an alternative embodiment, depending on the orientation of the pore complex in the membrane, the analyte can pass through the auxiliary protein constriction and then through the nanopore constriction.

[0130] In one embodiment, the method is used to determine the presence, absence, or one or more characteristics of a target analyte. The method can be used to determine the presence, absence, or one or more characteristics of at least one analyte. The method can involve determining the presence, absence, or one or more characteristics of two or more analytes. The method can include determining the presence, absence, or one or more characteristics of any number of analytes (such as 2, 5, 10, 15, 20, 30, 40, 50, 100, or more analytes). Any number of characteristics of one or more analytes can be determined, such as 1, 2, 3, 4, 5, 10, or more characteristics.

[0131] The binding of molecules in the channel of the pore complex or near any opening of the channel will affect the open channel ion flow through the pore, which is the essence of "molecular sensing" of the pore channel. In a manner similar to nucleic acid sequencing applications, changes in open channel ion flow can be measured by changes in current using suitable measurement techniques (e.g., WO 2000 / 28312 and D. Stoddart et al., Proceedings of the National Academy of Sciences of the United States of America, 2010, 106, 7702-7 or WO 2009 / 077734). The degree of reduction in ion flow, measured by the reduction in current, is related to the size of the obstruction in or near the pore. Therefore, the binding of a molecule of interest (also called an "analyte") in or near the pore provides a detectable and measurable event, thus forming the basis of a "biosensor". Suitable molecules for nanopore sensing include nucleic acids; proteins; peptides; polysaccharides and small molecules (here refers to low molecular weight (e.g., <900 Da or <500 Da) organic or inorganic compounds) such as drugs, toxins, cytokines and pollutants. Detecting the presence of biomolecules has applications in personalized drug development, medicine, diagnostics, life science research, environmental monitoring, and the security and / or defense industries.

[0132] The target analyte can be a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a polysaccharide, a dye, a bleach, a drug, a diagnostic agent, a recreational drug, an explosive, a toxic compound, or an environmental pollutant. The method can involve determining the presence, absence, or one or more characteristics of two or more analytes of the same type (e.g., two or more proteins, two or more nucleotides, or two or more drugs). Alternatively, the method can involve determining the presence, absence, or one or more characteristics of two or more different types of analytes (e.g., one or more proteins, one or more nucleotides, and one or more drugs).

[0133] The analyte of interest may be secreted from the cell. Alternatively, the analyte of interest may be an analyte present inside the cell, such that the analyte must be extracted from the cell prior to performing the method.

[0134] In one embodiment, the analyte is an amino acid, peptide, polypeptide, or protein. The amino acid, peptide, polypeptide, or protein may be naturally occurring or non-naturally occurring. The polypeptide or protein may be contained within a synthetic or modified amino acid. Several different types of amino acid modifications are known in the art. Suitable amino acids and their modifications are described above. It should be understood that the target analyte may be modified by any method available in the art.

[0135] In a preferred embodiment, the analyte is a polynucleotide, such as a nucleic acid. A polynucleotide is defined as a macromolecule comprising two or more nucleotides. Naturally occurring nucleic acid bases in DNA and RNA can be distinguished by their physical size. When a nucleic acid molecule or a single base passes through the channel of a nanopore, the size difference between the bases causes the ion flow through the channel to be directly related to a reduction. Changes in ion flow can be recorded. Suitable electrical measurement techniques for recording changes in ion flow are described in, for example, WO 2000 / 28312 and D. Stoddart et al., Proceedings of the National Academy of Sciences of the United States of America, 2010, 106, pp. 7702-7 (single-channel recording equipment); and, for example, WO 2009 / 077734 (multi-channel recording technology). Through appropriate calibration, the characteristic reduction of ion flow can be used to identify specific nucleotides and related bases passing through the channel in real time. In typical nanopore nucleic acid sequencing, because the channel is partially blocked by nucleotides, when the single nucleotides of the nucleic acid sequence of interest pass through the channel of the nanopore in order, the open channel ion flow decreases. It is this reduction in ion flow that is measured using the above-mentioned suitable recording technology. The reduction in ion flow can be calibrated to the reduction in measured ion flow of known nucleotides through the channel, thereby generating a means for determining which nucleotide is passing through the channel, and therefore, when performed sequentially, a way to determine the nucleotide sequence of the nucleic acid passing through the nanopore. In order to accurately determine individual nucleotides, it is generally necessary to directly correlate the reduction in ion flow through the channel with the size of the individual nucleotides passing through the constriction (or "reading head"). It will be understood that, for example, sequencing can be performed on complete nucleic acid polymers that are "passed through" the hole, for example, by the action of an associated polymerase or helicase. Alternatively, the sequence can be determined by passage of nucleotide triphosphate bases that have been sequentially removed from a target nucleic acid adjacent to the hole (see, for example, WO 2014 / 187924).

[0136] A polynucleotide or nucleic acid can include any combination of nucleotides. Nucleotides can be naturally occurring or artificial. One or more nucleotides in a polynucleotide can be oxidized or methylated. One or more nucleotides in a polynucleotide can be damaged. For example, a polynucleotide can include a pyrimidine dimer. Such dimers are often associated with UV damage and are a major cause of skin melanoma. One or more nucleotides in a polynucleotide can be modified, for example, with a marker or label, suitable examples of which are known to those skilled in the art. A polynucleotide can include one or more spacers. Nucleotides typically contain a nucleobase, a sugar, and at least one phosphate group. The nucleobase and sugar form a nucleoside. Nucleobases are typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, and more specifically include adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). Sugars are typically pentoses. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably deoxyribose. Polynucleotides preferably include the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC). Nucleotides are generally ribonucleotides or deoxyribonucleotides. Nucleotides generally contain monophosphate, diphosphate or triphosphate. Nucleotides can include more than three phosphates, such as 4 or 5 phosphates. The phosphate can be attached to the 5' or 3' side of the nucleotide. The nucleotides in the polynucleotide can be attached to each other in any way. Nucleotides are generally attached through their sugar and phosphate groups, as in nucleic acids. Nucleotides can be connected through their core bases, as in pyrimidine dimers. Polynucleotides can be single-stranded or double-stranded. At least a portion of the polynucleotide is preferably double-stranded. The polynucleotide is most preferably ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Specifically, the method using a polynucleotide as an analyte may alternatively comprise determining one or more properties selected from the group consisting of: (i) the length of the polynucleotide; (ii) the identity of the polynucleotide; (iii) the sequence of the polynucleotide; (iv) the secondary structure of the polynucleotide; and (v) whether the polynucleotide is modified.

[0137] Polynucleotide can be any length (i). For example, the length of the polynucleotide 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 or nucleotide pairs. The length of the polynucleotide can be 1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotides or nucleotide pairs or a length of 100000 or more nucleotides or nucleotide pairs. Any number of polynucleotides can be studied. For example, the method can involve characterizing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides. If characterizing two or more polynucleotides, it can be two examples of different polynucleotides or the same polynucleotide. Polynucleotide can be naturally occurring or artificial. For example, the method can be used to verify the sequence of manufactured oligonucleotides. The method is usually carried out in vitro.

[0138] Nucleotides may have any identity (ii) and include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP), and deoxymethylcytidine monophosphate. Nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP, and dUMP. Nucleotides may be abasic (i.e., lack a nucleobase). Nucleotides may also lack a nucleobase and a sugar (i.e., be a C3 spacer). The sequence of nucleotides (iii) is determined by the consecutive identity of the following nucleotides attached to each other throughout the polynucleotide strain in the 5' to 3' direction of the chain.

[0139] The following examples illustrate the invention.

[0140] Example 1: Protein expression and purification

[0141] The engineered gene for the phi29 portal protein channel was cloned into an expression vector. The newly constructed clone was transformed into BL21 (DE3) Escherichia coli bacteria. The successfully transformed bacteria were cultured overnight at 37°C in 10 mL of Luria-Bertani (LB) medium. These cultured bacteria were transferred to 500 mL of fresh LB medium. When the OD600 reached 0.5-0.6, 0.5 mM IPTG was added to the medium to induce protein expression. The bacteria were collected after 3 hours of induction after centrifugation. The bacterial wall was lysed using a French press, and the protein and other components were differentiated by centrifugation. Ni-NTA His-binding resin with a His tag was applied to purify the mutant protein. Briefly, 2 ml of regenerated His resin was loaded into the column. The supernatant differentiated by centrifugation was loaded into the column. The column was then washed with wash buffer to remove any contaminant proteins. The protein was eluted using an elution buffer containing 500 mM imidazole. The eluate was collected and concentrated to 5 mL. The eluate was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was then aspirated and injected into an AKTA FPLC using a syringe. Prior to injection, the sample loop was washed with 10 mL of lysis buffer. The protein was collected after passing through a size exclusion column. An SDS-PAGE gel was run to examine the protein samples. All wild-type and mutant proteins were expressed and purified in this manner. Typically, the protein was stored at -20°C in aliquots in multiple tubes to avoid repeated freeze-thaw cycles.

[0142] The sequence of the phi29 portal protein is known and available in Genbank (Genbank Accession No. ACE96033). A mutant phi29 gp10 portal protein was generated with the following mutations:

[0143] -A79C;

[0144] -E135C;

[0145] -Q168C;

[0146] -R10L, E14V, R17L, and N-7Δ (mutant-b);

[0147] -R10L, E14V, R17L, Q18L, R22I, and N-terminal-7Δ (mutant-c);

[0148] -IL was added to the N-terminus (mutant-d); and

[0149] -R10L, E14V, R17L, N-terminal-7Δ, in which IL is added to the N-terminus (mutant-e)

[0150] Example 2: Well Insertion in the MinION Device

[0151] In order to insert the engineered protein channel into the ONT membrane, the protein at a concentration of 1 mg / ml was diluted 1000 times in C13 buffer (25 mM potassium phosphate, 150 mM potassium ferrocyanide, 150 mM potassium ferricyanide, pH 8). 200 μl of diluted protein sample was added through the perfusion port of the MinION flow cell. A ramp voltage (5 mV increments; 20 seconds) from +50 to +350 mV was then applied to help insert the protein channel. The flow cell was then rinsed with 2 mL of C13 buffer. IV curves were then typically run, ±50, ±100, ±150, ±200 mV, with variable holding times (holding 2 minutes to 10 minutes at each voltage) to observe the pore behavior over time. Analytes such as DNA or peptides (1 pM concentration) were suspended in C13 buffer and added to the flow cell to check pore function.

[0152] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs.Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0153] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0154] Sequence Listing

[0155] SEQ ID NO: 1 - Amino acid sequence of wild-type phi29 gp-10

[0156] 1 MARKRSNTYR SINEIQRQKR NRWFIHYLNY LQSLAYQLFE WENLPPTINP

[0157] 51 SFLEKSIHQF GYVGFYKDPV ISYIACNGAL SGQRDVYNQA TVFRAASPVY

[0158] 101 QKEFKLYNYR DMKEEDMGVV IYNNDMAFPT TPTLELFAAE LAELKEIISV

[0159] 151 NQNAQKTPVL IRANDNNQLS LKQVYNQYEG NAPVIFAHEA LDSDSIEVFK

[0160] 201 TDAPYVVDKL NAQKNAVWNE MMTFLGIKNA NLEKKERMVT DEVSSNDEQI

[0161] 251 ESSGTVFLKS REEACEKINE LYGLNVKVKF RYDIVEQMRR ELQQIENVSR

[0162] 301 GTSDGETNE Sequence Listing <110> Oxford Nanopore Technologies <120> hole <130> N416084WO <150> 62 / 831,671 <151> 2019-04-09 <160> twenty one <170> PatentIn version 3.5 <210> 1 <211> 309 <212> PRT <213> bacteriophage phi‑29 <400> 1 Met Ala Arg Lys Arg Ser Asn Thr Tyr Arg Ser Ile Asn Glu Ile Gln 1 5 10 15 Arg Gln Lys Arg Asn Arg Trp Phe Ile His Tyr Leu Asn Tyr Leu Gln 20 25 30 Ser Leu Ala Tyr Gln Leu Phe Glu Trp Glu Asn Leu Pro Pro Thr Ile 35 40 45 Asn Pro Ser Phe Leu Glu Lys Ser Ile His Gln Phe Gly Tyr Val Gly 50 55 60 Phe Tyr Lys Asp Pro Val Ile Ser Tyr Ile Ala Cys Asn Gly Ala Leu 65 70 75 80 Ser Gly Gln Arg Asp Val Tyr Asn Gln Ala Thr Val Phe Arg Ala Ala 85 90 95 Ser Pro Val Tyr Gln Lys Glu Phe Lys Leu Tyr Asn Tyr Arg Asp Met 100 105 110 Lys Glu Glu Asp Met Gly Val Val Ile Tyr Asn Asn Asp Met Ala Phe 115 120 125 Pro Thr Thr Pro Thr Leu Glu Leu Phe Ala Ala Glu Leu Ala Glu Leu 130 135 140 Lys Glu Ile Ile Ser Val Asn Gln Asn Ala Gln Lys Thr Pro Val Leu 145 150 155 160 Ile Arg Ala Asn Asp Asn Asn Gln Leu Ser Leu Lys Gln Val Tyr Asn 165 170 175 Gln Tyr Glu Gly Asn Ala Pro Val Ile Phe Ala His Glu Ala Leu Asp 180 185 190 Ser Asp Ser Ile Glu Val Phe Lys Thr Asp Ala Pro Tyr Val Val Asp 195 200 205 Lys Leu Asn Ala Gln Lys Asn Ala Val Trp Asn Glu Met Met Thr Phe 210 215 220 Leu Gly Ile Lys Asn Ala Asn Leu Glu Lys Lys Glu Arg Met Val Thr 225 230 235 240 Asp Glu Val Ser Ser Asn Asp Glu Gln Ile Glu Ser Ser Gly Thr Val 245 250 255 Phe Leu Lys Ser Arg Glu Glu Ala Cys Glu Lys Ile Asn Glu Leu Tyr 260 265 270 Gly Leu Asn Val Lys Val Lys Phe Arg Tyr Asp Ile Val Glu Gln Met 275 280 285 Arg Arg Glu Leu Gln Gln Ile Glu Asn Val Ser Arg Gly Thr Ser Asp 290 295 300 Gly Glu Thr Asn Glu 305 <210> 2 <211> 4 <212> PRT <213> Artificial sequence <220> <223> connector <400> 2 Gly Gly Gly Ser 1 <210> 3 <211> 4 <212> PRT <213> Artificial sequence <220> <223> connector <400> 3 Pro Gly Gly Ser 1 <210> 4 <211> 4 <212> PRT <213> Artificial sequence <220> <223> connector <400> 4 Pro Gly Gly Gly 1 <210> 5 <211> 6 <212> PRT <213> Artificial sequence <220> <223> connector <400> 5 Arg Pro Pro Pro Pro Pro 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial sequence <220> <223> connector <400> 6 Arg Pro Pro Pro Pro 1 5 <210> 7 <211> 4 <212> PRT <213> Artificial sequence <220> <223> connector <400> 7 Arg Pro Pro Gly 1 <210> 8 <211> 4 <212> PRT <213> Artificial sequence <220> <223> connector <400> 8 Pro Pro Pro Pro 1 <210> 9 <211> 4 <212> PRT <213> Artificial sequence <220> <223> connector <400> 9 Arg Pro Pro Gly 1 <210> 10 <211> 9 <212> PRT <213> Artificial sequence <220> <223> connector <400> 10 Pro Pro Pro Pro Pro Pro Pro Pro Pro 1 5 <210> 11 <211> 4 <212> PRT <213> Artificial sequence <220> <223> connector <400> 11 Gly Gly Gly Gly 1 <210> 12 <211> 5 <212> PRT <213> Artificial sequence <220> <223> connector <400> 12 Gly Gly Gly Gly Gly 1 5 <210> 13 <211> 6 <212> PRT <213> Artificial sequence <220> <223> connector <400> 13 Gly Gly Gly Gly Gly Gly 1 5 <210> 14 <211> 7 <212> PRT <213> Artificial sequence <220> <223> connector <400> 14 Asp Tyr Asp Ile Pro Thr Thr 1 5 <210> 15 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Label <400> 15 Trp Ser His Pro Gln Ser Glu Lys 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Label <400> 16 Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Label <400> 17 Asn Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Label <400> 18 Pro Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 19 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Label <400> 19 Gly Gly Ser His Pro Gln Phe Glu Gly 1 5 <210> 20 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Label <400> 20 His His His His His His 1 5 <210> twenty one <211> 7 <212> PRT <213> Artificial sequence <220> <223> Cleavage site <400> twenty one Glu Asn Leu Tyr Phe Gln Gly 1 5

Claims

1. A modified portal protein of a bacteriophage Phi29 DNA-packaging motor, wherein the modified portal protein is capable of directly inserting into a membrane, and wherein the portal protein is modified compared to a wild-type portal protein such that one or more amino acid residues on the outer surface of the portal protein are substituted with one or more other amino acid residues, and / or wherein one or more amino acid residues are inserted onto the outer surface of the portal protein to increase or decrease the hydrophobicity of the outer surface of the modified portal protein compared to the wild-type portal protein, wherein the one or more amino acid residues are at the following positions of the portal protein of the Phi29 DNA-packaging motor, or at positions corresponding to the following positions of the portal protein of the Phi29 DNA-packaging motor: (i) R10L, E14V; (ii) A79C with a conjugated porphyrin; (iii) R10L, E14V, R17L, wherein 7 amino acids are deleted from the N-terminus and an IL tag is added; (iv) R10L, E14V, R17L, wherein 7 amino acids are deleted from the N-terminus; or (v) Q168C with a conjugated cholesterol.

2. The modified portal protein according to claim 1, which is composed of identical subunits.

3. A triblock copolymer membrane comprising the modified portal protein according to claim 1.

4. An array comprising two or more membranes according to claim 3.

5. An array according to claim 4 adapted to be inserted into a sensor device.

6. A device comprising an array according to claim 5, means for applying a voltage potential across the membrane and means for detecting the charge across the membrane. The device of claim 6 , further comprising a fluidic system configured to supply a sample to the membrane.

8. A method for characterizing a target analyte, wherein the target analyte is a polynucleotide, protein, peptide, polysaccharide or small molecule, the method comprising contacting the membrane of claim 3 with the target analyte and applying a voltage potential across the membrane such that the target analyte moves relative to a nanopore, and performing one or more electrical measurements as the target analyte moves relative to the pore to thereby determine the presence, absence or one or more characteristics of the target analyte.

9. The method of claim 8, wherein a plurality of target analytes are characterized.

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