pore

Abstract

To provide: a modified portal protein; a membrane containing the modified portal protein; and a method for characterizing an analyte using the membrane containing the modified portal protein.SOLUTION: Provided is a modified portal protein of a bacteriophage DNA packaging motor. The modified portal protein is capable of direct insertion into a membrane. The portal protein is modified compared to a wild type portal protein such that one or more amino acid residues on an outer surface of the portal protein is substituted by one or more other amino acid residue, and / or one or more amino acid residue is inserted on the outer surface of the portal protein so as to alter outer surface hydrophobicity of the modified portal protein compared to the wild type portal protein.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present disclosure relates to modified portal proteins, membranes comprising modified portal proteins, and a method for determining a characteristic of an analyte using a membrane containing the modified portal protein. Regarding. [Background technology]

[0002] Nanopore sensing involves the detection of individual bonds or interactions between analyte molecules and ion-conducting channels. Nanoparticles are an approach to the detection and characterization of analytes that relies on the observation of interaction events. The pore sensor places a single pore of nanometer dimensions in an insulating film and detects the pore in the presence of an analyte molecule. The internal structure of the nanopore can be fabricated by measuring the voltage-driven ionic current through the pore. In the presence of an analyte nearby, the ionic flow through the pore changes and is measured across the channel. This results in a change in ionic current or electrical current. The identity of the analyte is determined by its unique current signature. Specifically, the duration and extent of current block, as well as the current level during the interaction time with the pore. Analytes are revealed through the variation of the analyte chain. A variety of biological or synthetic polymers and polysaccharides, including leucocytes, polypeptides, and polysaccharides. Nanopore sensing can reveal the identity of the sensed analyte and provide a simple Single molecule counting can be performed, but not nucleotides, amino acids, or glycans. The composition of the analyte, such as the sequence, as well as methylation, acylation, phosphorylation, hydroxylation , oxidation, reduction, glycosylation, decarboxylation, deamination, etc. of bases, amino acids, or glycans Nanopore sensing can also provide information about the presence of amine modifications. This may enable the efficient determination of polynucleotide sequences with lengths of several tens to several tens of thousands of bases. The present invention provides single molecule sequence reads of leutions.

[0003] Biological nanopores are based on natural membrane proteins and are characterized by the interaction of purified proteins with membrane proteins. and applying a potential across the membrane.

[0004] The phi29 bacteriophage gp10 portal protein binds the 12 subunits of gp10. The propeller-like structure is made up of subunits with an outer diameter of 14.6 nm. At the narrowest constriction, the wild-type channel is 3.6 nm. Each of the 12 subunits contains a three-helical bundle, an α-β motif, and a central α-helical domain, composed of a broader C-terminal six-stranded SH3-like domain Portal proteins are naturally occurring membrane proteins or ion channels. Although not a channel, it has been proposed as a nanopore for characterizing analytes. To achieve this, the bacteriophage phi29 portal protein was first introduced into the liposome. The endonuclease must then be inserted into a membrane, which then fuses with the planar lipid bilayer. Summary of the Invention

[0005] Disclosed herein are modified bacteriophage polypeptides that spontaneously insert into membranes. The inserted portal protein functions as a nanopore. This can be done.

[0006] In one embodiment, a bacteriophage DNA package capable of directly inserting into a membrane is provided. A modified portal protein of the ginking motor is provided, and the outer surface of the portal protein is one or more of the above amino acid residues are replaced by one or more other amino acid residues, and and / or one or more amino acid residues are inserted on the exterior surface of the portal protein to produce a wild-type Altering the exterior hydrophobicity of the portal protein relative to the portal protein. One or more The introduction of amino acid residues by substitution and / or insertion of The hydrophobicity of the outer surface of a protein can be increased or decreased compared to the inner surface. The exterior hydrophobicity of the region can be increased or decreased.

[0007] The portal protein channel is assembled from 12 subunits, By changing one or more residues in one monomer, mutations are introduced in the same plane of the molecule. In general, the portal tank will have a large number of channels, which will cause effects throughout the channel. Proteins consist of two domains: the wing domain and the stalk domain. The stalk domain contains a hydrophobic belt region beneath the wings of the portal protein.

[0008] In one embodiment, at least one of the one or more introduced amino acid residues is a porcine amino acid. The amino acid residues introduced are located in the central hydrophobic belt region of the tal protein. For example, it can be introduced by substitution and / or insertion. Residues in the hydrophobic belt region of the portal protein of the ligation motor include F24, I25, and L In one embodiment, these include F28, F60, F128, P129, and P132. or a similar portal junction within one or two residues at any one or more of these positions. The amino acid at one or more of the corresponding positions in the protein is a naturally occurring amino acid at the substituted position. In one embodiment, the hydrophobic amino acid may be substituted with one or more amino acids that are more hydrophobic than the acid. The acid may be present in one or two residues or in a group of residues at any one or more of these positions. The protein may be inserted into one or more corresponding positions in a similar portal protein. The N-terminal residue of each subunit of is in the hydrophobic belt region. At least one of the amino acid residues is located in the 30 amino acid residues at the N-terminus of the portal protein. For example, at least one of the one or more amino acid residues is Phi29 Portal proteins R10, E14, R17, and Q1 of the DNA packaging motor 8, and / or positions corresponding to R22, or their counterparts in similar portal proteins It is in a position to do so.

[0009] In one embodiment, at least one of the one or more introduced amino acid residues is a porcine amino acid. The hydrophilic cis and / or trans layers of the portal protein. Examples of amino acid residues in the cis layer include the portal junction of the Phi29 DNA packaging motor. Q32, Y36, F52, K55, Q59, F60, and Y in the wing domain of the protein 62, N77, G78, A79, L80, S81, R84, R94, A96, S97, P 98, and Q101. An example of an amino acid residue is the nucleotide sequence of the portal protein of the Phi29 DNA packaging motor. These are located at positions corresponding to P129, T131, E135, and Q168 in the stalk domain.

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

[0011] In one embodiment, a bacteriophage DNA package capable of directly inserting into a membrane is provided. The modified portal protein of the sing motor is provided, wherein one or more amino acid residues are , which are introduced onto the outer surface of the portal protein to enhance the portal fold change relative to the wild-type portal protein. On the outside of the wing domain or on the strand, a molecule that changes the hydrophobicity of the outer surface of the tal protein A binding site is introduced into the portal domain. The binding site is located on the surface of the portal protein. by substituting an existing residue with another amino acid residue, or by substituting one or more amino acid residues It can be introduced by inserting:

[0012] In one embodiment, binding sites are introduced on the outside of the wing domain or within the stalk domain. At least one of the one or more amino acid residues introduced into the portal protein to The other is cysteine ​​and / or an unnatural amino acid.

[0013] In one embodiment, one or more amino acids are introduced into the portal protein to introduce a binding site. At least one of the amino acid residues of Examples of amino acid residues in the cis layer of portal proteins include Phi 29 Q32 in the wing domain of the portal protein of the DNA packaging motor , Y36, F52, K55, Q59, F60, Y62, N77, G78, A79, L80 , S81, R84, R94, A96, S97, P98, or Q101 Examples of amino acid residues in the translaminar layer of portal proteins include those at positions corresponding to The stalk domain of the portal protein of the Phi29 DNA packaging motor Includes positions corresponding to P129, T131, E135, or Q168 in .

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

[0015] In one embodiment, the outer surface of the portal protein is sparser than that of a wild-type portal protein. Molecules that alter the aqueous state are hydrophobic molecules. Exemplary hydrophobic molecules are porphyrins, tetrahydrofurans, and the like. Traphenylporphyrin, protoporphyrin IX, octaethylporphyrin, cholesteryl A molecule containing sterol, heme, or biliverdin.

[0016] In some embodiments, the modified portal protein is the N It is modified at the termini by the addition and / or deletion of one or more amino acid residues.

[0017] In certain embodiments, the modified portal protein is phi29, T3, T4, T 5, T7, SPP1, HK97, lambda, G20c, P2, P3, and P22 A modified DNA packaging motor from a bacteriophage selected from the group It is a portal protein.

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

[0019] In other aspects, the following is provided: a modified portal protein subunit as disclosed herein, - a membrane comprising a modified portal protein as disclosed herein, - an array comprising two or more membranes comprising a modified portal protein as disclosed herein; - an array comprising two or more membranes each containing a modified portal protein as disclosed herein; a means for applying a potential across the membrane; and a means for detecting a charge across the membrane. Devices that include - A method for determining a characteristic of a target analyte, comprising the steps of: contacting the membrane containing the protein with a target analyte; and causing the target analyte to migrate relative to the nanopore. A potential is applied across the membrane to induce a potential gradient across the pore, and a potential gradient is applied across the pore as the target analyte moves toward the pore. The above measurements are then taken to determine the presence, absence, or one or more characteristics of the analyte. A method comprising:

[0020] In one embodiment, the membrane is a lipid membrane, or a diblock or triblock copolymer membrane. and the like.

[0021] In one embodiment, the array is adapted for insertion into a sensor device.

[0022] In one embodiment, the device further comprises a fluidics system configured to deliver the sample to the membrane. Prepare for.

[0023] In one embodiment, the method includes taking electrical and / or optical measurements. In one embodiment of the method, a plurality of target analytes are characterized. The analyte may be a polynucleotide, a protein, a peptide, a carbohydrate, a metabolite, or other In one embodiment of the method, the target analyte is associated with a disease state. [Brief description of the drawings]

[0024] [Figure 1]The structure of the phi29 gp10 portal protein is shown from different angles. The structure of the subunits (monomer units) of the pore is also shown. Representative locations for creating conjugation sites on the phi29 gp10 portal protein surface for incorporating hydrophobic groups are shown in one monomer of the protein. A. Side view of the wild type phi29 gp10 pore. Regions of interest that require protein engineering for direct membrane insertion are boxed. Residues are in either the wing domain or the stalk domain as shown in B. The two separate domains of the pore are shown in monomer units. B. A monomer unit (one of 12 assembled subunits) of the assembled pore is shown. The wing domain is shown in black (residues 1-125) and the stalk domain in grey (residues 120-309). The region of interest boxed in panel A encompasses parts of both protein domains. C. Representative locations in the WT phi29 gp10 protein for generating conjugation sites to incorporate hydrophobic membrane anchoring modules. 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, and specific mutation points in the stalk domain include P129, T131, E135 and Q168. D. Chart showing the hydrophobicity scale of the 20 natural amino acids. Typically, I, VL or F are introduced at the target points of mutation to improve the relative hydrophobicity of the region of interest. [Diagram 2]Representative positions for generating cysteine ​​mutations on the phi29 gp10 portal protein surface to incorporate hydrophobic groups are shown. These positions include A79C in the wing domain, and E135C and Q168C in the stalk domain. Other positions 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, T131 in the stalk domain. Since the protein is assembled as a dodecamer, each mutation generates a ring. The cysteine ​​residues allow for linkage of hydrophobic modules via standard sulfhydryl chemistry. Any unnatural amino acid, such as an amino acid with an alkyne side chain, may also be incorporated in place of cysteine ​​for "click" chemistry-mediated chemical conjugation. [Diagram 3] Representative locations are shown for generating hydrophobic mutations on the phi29 gp10 portal protein surface to facilitate insertion into polymeric membranes. 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 is assembled as a dodecamer, each mutation generates a ring of hydrophobic residues along a plane. The location of the hydrophobic amino acids or hydrophobic anchoring modules relative to the membrane core determines where the pore resides in the membrane. [Figure 4]Figure 1 shows an SDS-PAGE gel showing that a representative phi29 gp10 portal protein mutant A79C was expressed and purified. The mutant A79C gene was cloned into an expression vector and then transformed into E. coli. Successfully transformed bacteria were grown overnight in LB medium. Protein expression was induced by adding IPTG. After induction, bacteria were collected and then lysed. Protein and other components were separated by centrifugation. His-tagged Ni-NTA His-binding resin was applied to purify the mutant protein. Protein was eluted using elution buffer containing increasing concentrations of imidazole as indicated. The eluate was collected and concentrated, and then purified by FPLC. An SDS-PAGE gel was run to check the protein sample. [Diagram 5] Figure 1 shows 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 grown overnight in LB medium. Protein expression was induced by adding IPTG. After induction, bacteria were collected and then lysed. Protein and other components were separated by centrifugation. His-tagged Ni-NTA His-binding resin was applied to purify the mutant protein. Protein was eluted using elution buffer containing increasing concentrations of imidazole as indicated. The eluate was collected and concentrated, and then purified by FPLC. An SDS-PAGE gel was run to check the protein sample. [Figure 6]Figure 1 shows an SDS-PAGE gel of a representative phi29 gp10 portal protein mutant Q168C expressed and purified. The mutant Q168C gene was cloned into an expression vector and then transformed into E. coli. Successfully transformed bacteria were grown overnight in LB medium. Protein expression was induced by adding IPTG. After induction, bacteria were collected and then lysed. Protein and other components were separated by centrifugation. His-tagged Ni-NTA His-binding resin was applied to purify the mutant protein. Protein was eluted using elution buffer containing increasing concentrations of imidazole as indicated. The eluate was collected and concentrated, and then purified by FPLC. An SDS-PAGE gel was run to check the protein sample. [Figure 7] SDS-PAGE gel showing that representative phi29 gp10 portal protein mutants R10L, E14V, R17L, and N-7Δ (mutant-b) (left of the figure marker) and R10L, E14V, R17L, Q18L, R22I, and N-7Δ (mutant-c) (right of the figure marker) were expressed and purified. The mutant genes were cloned into an expression vector and then transformed into E. coli. Successfully transformed bacteria were grown overnight in LB medium. Protein expression was induced by adding IPTG. After induction, bacteria were collected and then lysed. Proteins and other components were separated by centrifugation. His-tagged Ni-NTA His-binding resin was applied to purify the mutant proteins. Proteins were eluted using elution buffer containing increasing concentrations of imidazole as indicated in the figure. The eluate was collected and concentrated, and then purified by FPLC. SDS-PAGE gel was run to check the protein samples. [Figure 8]SDS-PAGE gel showing that representative phi29 gp10 portal protein mutants NIL (mutant-d) (left of the figure marker) and N-ter-7Δ (mutant-e) with R10L, E14V, R17L, and N-terminal IL additions (right of the figure marker) were expressed and purified. The mutant genes were cloned into an expression vector and then transformed into E. coli. Successfully transformed bacteria were grown overnight in LB medium. Protein expression was induced by adding IPTG. After induction, bacteria were collected and then lysed. Proteins and other components were separated by centrifugation. His-tagged Ni-NTA His-binding resin was applied to purify the mutant proteins. Proteins were eluted using elution buffer containing increasing concentrations of imidazole as indicated in the figure. The eluate was collected and concentrated, and then purified by FPLC. SDS-PAGE gel was run to check the protein samples. [Figure 9-1]Figure 1 shows data obtained using engineered mutants of the phi29 gp10 portal protein on an Oxford Nanopore Technologies MinION device. A. Direct insertion into the ONT membrane was not observed with the WT phi29 gp10 pore. B-F. Direct insertion of the engineered phi29 gp10 pore into the ONT membrane. B. Mutant (R10L, E14V), C. Porphyrin-conjugated mutant A79C, D. Mutant (R10L, E14V, R17L, 7aa deleted and IL-tagged N-terminus), E. Mutant (R10L, E14V, R17L, 7a.a deleted N-terminus), F. Cholesterol-conjugated mutant (Q168C). To insert engineered protein channels into the ONT membrane, proteins with a 1 mg / ml concentration were diluted 1000-fold in C13 buffer (25 mM potassium phosphate, 150 mM potassium ferrocyanide, 150 mM potassium ferrocyanide, pH 8). 200 ul of diluted protein sample was added through the priming port of the MinION flow cell. Voltage was then applied while increasing from +50 to +350 mV (5 mV increments, 20 s hold) to assist in the insertion of protein channels. The flow cell was then flushed with 2 mL of C13 buffer. IV curves were then run at ±50, ±100, ±150, ±200 mV with varying hold times (2 min to 10 min hold 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 the pore function. In A-F, a voltage of -100 mV was applied. Conductivity buffer: C13 (ONT Reagent). Analyte - TAT peptide elevated at characteristic current block event, indicative of a functional pore. G. Relative insertion rate of WT and engineered mutants. Rates are relative to WT. There is variation for different mutants within each category described, but the overall trends are as shown in the chart. [Figure 9-2] Shown are data obtained using engineered mutants of the phi29 gp10 portal protein on the Oxford Nanopore Technologies MinION device (continued from Figure 9-1). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention will now be described with respect to particular embodiments and with reference to certain drawings. The disclosure is not limited thereto, but is limited only by the scope of the claims. Of course, any reference signs should not be construed as limiting the scope. It is to be understood that not all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. It is to be understood that, for example, those skilled in the art may be able to make other modifications as may be taught or suggested herein. It is understood that one advantage or benefit taught herein may be achieved without necessarily achieving all of the aspects or advantages. The invention may be embodied or practiced in any manner that achieves or optimizes a group of points. will recognize that.

[0026] The present invention, both as to organization and method of operation, together with its features and advantages, is described in the accompanying The present invention can be best understood by reference to the following detailed description taken in conjunction with the drawings in which: Aspects and advantages will be apparent from and elucidated with reference to the embodiments described hereinafter. Throughout this specification, references to "one embodiment" or "an embodiment" refer to an embodiment. It is understood that a particular feature, structure, or characteristic described herein is included in at least one embodiment of the present invention. Thus, the term "in one embodiment" is used in various places throughout this specification. Appearances of the phrases "in" or "in an embodiment" do not necessarily all refer to the same embodiment. Similarly, in describing exemplary embodiments of the present invention, Various features of the invention may be grouped together in a single embodiment, figure, or description thereof to simplify the disclosure. may be intended to simplify the description and aid in the understanding of one or more of the various inventive aspects. It should be understood, however, that this method of disclosure does not necessarily require that the claimed invention be reflects an intention to require more features than are explicitly enumerated in the paragraph. Rather, as the following claims reflect, the present invention should not be construed as being inventive. An aspect may include less than all features of a single foregoing disclosed embodiment.

[0027] Additionally, as used in this specification and the appended claims, the singular forms "a," "a The words "one", "n" and "the" include plural referents unless the context clearly indicates 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. A reference to "a helicase" includes two or more helicases; a reference to "a monomer" includes two or more and reference to "a pore" includes two or more pores, etc.

[0028] In all discussions herein, the standard one-letter codes for amino acids are used. They are: alanine (A), arginine (R), and 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). Onine (T), Tryptophan (W), Tyrosine (Y), and Valine (V). Substitution notation is also used, i.e., Q42R indicates that Q at position 42 is replaced by R. This means:

[0029] Portal Proteins Nanopores are used to transport viral DNA, such as the bacteriophage DNA packaging motor. A modified portal protein of the packaging motor.

[0030] The portal protein of the bacteriophage DNA packaging motor has a cone-shaped structure. The protein has a structure consisting of 12 subunits, also called connector protein subunits. It has a central channel formed by the portal protein subunits. An exemplary unmodified viral DNA packaging motor domain from phage phi29. The tal protein has been purified and its three-dimensional structure characterized crystallographically ( For example, Guasch et al., 1998 FEBS Lett. 430:283 , Marais et al.,2008 Structure 16:1267). p The hi29 channel has a narrow section of 3.6 nm and an end that is 6 nm wide, which is This is larger than most membrane protein channels. Embodiments include phi29 DNA packaging gp10 motor portal protein (e.g. For example, Genbank accession number ACE96033 UniProt ID: P04332 , gene ID: 6446518, SEQ ID NO: 1), and / or forming a channel and polypeptide subunits thereof, including fragments, variants, and derivatives thereof, which are capable of (e.g., accession numbers GI29565762, GI31072023, GI66395 194, gi29565739, gi157738604).

[0031] The viral portal proteins share little sequence homology and differ in molecular weight. There are striking underlying structural similarities between the DNA packages of other dsDNA viruses, in particular Zygotic motor connector proteins (e.g., T4, lambda, P22, P2, T3, T 5, and T7) share little sequence homology with the phi29 connector and have molecular weights Despite their differences, they exhibit striking underlying structural similarities (e.g., Bazinet et al.,1985 Ann Rev.Microbiol.39:109-29 ).

[0032] In certain embodiments, viral DNA packages isolated from other dsDNA viruses are The use of dsDNA motor portal proteins is contemplated, including isolated dsDNA A viral DNA packaging motor portal protein (e.g., T4 (accession no. NP-049782)(Driedonks et al.,1981 J Mol B iol 152:641), lambda (accession nos. gi549295, gi6723246, gi15837315, gi16764273)(Kochan et al.,198 4 J Mol Biol 174:433), SPP1 (accession number P54309), P 22 (Accession No. AAA72961) (Cingolani et al., 2002 J Struct Biol 139:46), G20c (Accession No. KX987127.1) , P2 (accession number NP-046757, P3 (Nutter et al., 1972J .Viral.10(3):560-2), T3 (accession number CAA35152) (Car azo et al.,1986Jl,.Ultrastruct Mol Struc t Res 94:105), T5 (accession number AAX12078, YP-006980; AAS77191;AAU05287), T7(Acc.No.NP-041995)( Cerritelli et al.,1996 J Mol Biol 285:29 9, Agirrezabala et al.,2005 J Mol Biol 34 7:895)), phages such as lambda, P2, P3, P22, T3, T4, T5, and SPP 1, viral DNA packaging isolated from either HK97 or T7 In some embodiments, the motor portal protein may be a protein that is involved in the induction of a cellular signaling pathway. Connector proteins include the bacteriophage T3 connector protein gp8. In some embodiments, the connector protein is a bacteriophage T7 connector protein. In some embodiments, the connector protein comprises the bacterium gp8. In some embodiments, the T4 connector protein gp20 is Nectar proteins include the bacteriophage T5 connector protein gp7. In some embodiments, the connector protein is a bacteriophage SPP1 connector. In some embodiments, the connector protein comprises the bacterium gp6. Contains the phage HK97 connector protein gp3.

[0033] Exemplary phi29 DNA packaging motor portal proteins herein These and other dsDNA virus patterns have been extensively structurally characterized. The packaging motor portal protein, phi29, is integrated into the membrane layer. A potential is applied across the membrane in the same way as the portal protein of the DNA packaging motor. When the ion exchange membrane is exposed, it can be modified to form an opening through which conductance can occur. The disclosure herein regarding the phi29 portal protein is intended to provide a basis for the identification of such other isolated Any of the dsDNA viral DNA packaging motor portal proteins These are intended to illustrate relevant embodiments in which use of

[0034] The portal protein of the phi29 DNA packaging motor, or another bacterium, The portal protein of the lyophile DNA packaging motor is shown herein. The invention can be modified according to the teachings of the present invention.

[0035] The present disclosure provides a method for the preparation of a medicament having membrane-integrating properties (e.g., stable transmembrane integration into a membrane layer). The isolated DNA packaging motor portal protein has been engineered to The protein can be used as a conductive biosensor for cancer biomarkers Portal proteins also mediate the formation of transmembrane channels. It can also be artificially engineered to affect its conductive properties.

[0036] Modified isolated double-stranded DNA viral DNA packages, including the phi29 connector. The aging motor protein connector is a protein for which crystallographic structure data is readily available. If possible, manipulate the material to have the desired structure for use in the embodiments of the present disclosure. Procedures for large-scale production and purification of the phi29 connector have been developed. (Guo et al., 2005, Ibanez et al., Nucleic Acids Res. 12, 2351-2365 (1984), Robinson e t al.,Nucleic Acids Res.34,2698-2709(200 6), Xiao et al., ACS Nano 3, 100-107 (2009).

[0037] In one embodiment, the modified bacteriophage phi29 viral DNA package The motor portal protein (e.g., SEQ ID NO: 1) is a wild-type protein or a wild-type The live phi29 virus DNA packaging motor connector protein having at least 80%, 90%, or 95% identity to a portion thereof, , at least 240, 260, 280, 285, 290, 295, 296, 297, 29 8, 299, 300, 301, 302, 303, 304, 305, 306, 307, 30 8, 309, 310, 311, 312, 313, 314, 315, 316, 317, 31 8, 319, 320, 321, 322, 323, 324, 325, 326, 327, 32 At least 150, 175, 200, 225, 250, including 8, 329, 330 and above It contains 275 amino acids.

[0038] In other embodiments, the modified portal protein is a phage T4, lambda phage (Accession numbers gi549295, gi6723246, gi15837315, and gi167 64273), phage SPP1 (accession no. P54309), and phage P22 (accession no. AAA72961), phage P2 (accession number NP-046757), phage P3 (N utter et al., 1972 J.Virol.10(3):560-2), Phage T3 (accession number CAA35152), phage T5 (accession number AAX12078, YP006980;AAS77191;AAU05287), phage T7 (accession no. N P041995), and other phage such as HK97 (accession no. NP_037699). A modified double-stranded DNA portal protein from a bacteriophage. For example, The modified portal protein mediates the DNA packaging of these bacteriophage viruses. The protein may be a mutant of any of the ginseng motor portal proteins, for example, At least 80% of the polypeptides disclosed herein and fragments of such polypeptides. , 90%, or 95% identity. A "fragment" generally has at least 240, 260, 280, 285, 290, 295, 2 96, 297, 298, 299, 300, 301, 302, 303, 304, 305, 3 06, 307, 308, 309, 310, 311, 312, 313, 314, 315, 3 16, 317, 318, 319, 320, 321, 322, 323, 324, 325, 3 At least 150, 175, 200, including 26, 327, 328, 329, 330 and above , 225, 250, and 275 amino acids.

[0039] As used herein, the term "amino acid identity" refers to a sequence that is a function of the amino acid sequence over a window of comparison. "Percent sequence identity" refers to the degree to which sequences are identical amino acid by amino acid. The "page" compares two optimally aligned sequences over a window of comparison and finds identical amino acids. Residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, P he, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, C Determine the number of positions where α, β, γ, and Met occur in both sequences and calculate the number of matching positions. The number of matched positions is calculated by multiplying the total number of positions in the comparison window (i.e., the window size). The percentage of sequence identity is calculated by dividing the result by the number of residues (number of residues) and multiplying the result by 100.

[0040] The portal protein is one of the bacteriophage portal proteins listed above. The viral DNA packaging motor portal protein may be a modified analogue of The "analog" when referring to proteins is the viral DNA packaging motor portal tract Portal proteins are typically naturally occurring homologs or variants of a given protein. The subunits are capable of self-assembling into oligomers, e.g., homododecameric channels. It is composed of

[0041] Portal proteins are characterized by (i) one or more of the amino acid residues being conserved or non-conserved amino acids; and such substituted amino acid residues are replaced with amino acid residues (preferably conserved amino acid residues). The acid residue may or may not be encoded by the genetic code; or (ii) one or more of the amino acid residues includes a substituent; or (iii) an additional The additional amino acids are used to detect mutant portal proteins or to identify functional changes. Genetically fused to one or more portal protein subunits, including amino acids It is possible that.

[0042] The modified portal protein is isolated. The term "isolated" refers to the material It means removed from its original environment (e.g., the natural environment if naturally occurring). For example, The native proteins present in the intact native virus have not been isolated, but they coexist in the natural system. The same protein separated from some or all of the materials that Such proteins may be part of a composition, and such vectors or compositions may be However, it is still isolated in that it is not part of its natural environment. Methods for isolating the portal protein of a dimotor protein are known in the art. Bacteriophage vectors for use in the methods and compositions described herein are disclosed. Portal proteins of target proteins are used in recombinant DNA technology well known in the art. It can be produced by any method known in the art.

[0043] In one embodiment, the modified protein is a truncated version of the portal protein. The modified and / or modified proteins are the subunits of the portal protein. and / or a portal protein. It may involve the substitution, deletion, or addition of one or more amino acids within the amino acid sequence of the protein.

[0044] A truncated portal protein may be truncated at the N-terminus and / or C-terminus. Up to about 30 amino acids, such as about 20, can be deleted from the N-terminus, including 10, 9, 8, or Alternatively, or in addition, up to about 20 amino acids may be deleted from the N-terminus. Up to about 30 amino acids may be deleted from the C-terminus, and 10, 9, 8, or 7 amino acids may be deleted. 3 to about 20, 4 to about 10, 5 to 9, or 6, 7, or One or more amino acids, such as 2 to about 30, such as about 8 amino acids, are attached to the N-terminus and / or or C-terminus, or a truncated N-terminus and / or a truncated C-terminus obtain.

[0045] The modified portal protein comprises a channel. In one embodiment, the portal protein The protein is modified to change one or more properties of the channel of the nanopore. In the present state, this is due to the presence of amino acid residues inside and / or at the entrance to the channel. This is achieved by modifying one or more of the groups.

[0046] In some embodiments, the nanopore comprises only a full-length subunit of the portal protein. Includes.

[0047] In some embodiments, the nanopore has 7, 8, 9, 10, 11, or 12 subpores. A multimeric protein formed from six or more portal protein subunits, such as the For example, the nanopore may be a dodecamer protein. One or more of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, etc., are qualified. In one embodiment, one or more of the subunits may be, for example, aligned to one end of the nanopore. or modified at the C-terminus and / or N-terminus to increase hydrophilicity at both ends. For example, one or more of the subunits may have flexible attachment to the C-terminus and / or N-terminus. In some embodiments, the nucleic acid sequence may be modified by the addition of a suitable linker and / or peptide tag. In this embodiment, the nanopore is composed of identical subunits, e.g., 4 or 5 to 10, preferably Any suitable linker, such as a linker containing 3 to 12 amino acids, preferably 6 to 8 amino acids. Any suitable linker may be used. The amino acids of the linker may be lysine, serine, arginine, proline, or ribozyme. oleic acid, glycine, alanine aspartic acid, tyrosine, isoleucine, and / or Examples of suitable linkers include the following: GGGS, PGGS, P GGG, RPPPPP, RPPPP, VGG, RPPG, PPPP, RPPG, PPPP PPPPP, RPPG, GGG, GGGG, GGGGG, GGGGGG, and DYDI These include, but are not limited to, PTT.

[0048] The modified portal protein may, for example, contain a tag to facilitate its purification. Any suitable peptide tag can be used to facilitate purification of the portal protein. For example, in one embodiment, the tag may be a strep tag. the strep tag has a length of 8 to 11 amino acids, and / or The tag amino acid sequence contains the motif HPQ. A strep tag can be, for example, Arrays WSHPQSEK, WSHPQFEK, NWSHPQFEK, PWSHPQFEK, or GGSHPQFEG. This sequence may comprise or consist of the core "HP 2, 3, 4, or 5 of the amino acids, provided that the "Q" motif is maintained Variants can be varied by one or more additions, deletions, or substitutions such as The sequence is typically 8-11 amino acids NWSHPQFEK, PWSHPQFEK, and GGSHPQFEG. In another embodiment, the tag is a His tag (typically , His6(HHHHHH)).

[0049] The portal protein can be modified with tags and / or linkers either before or after pore assembly. The nucleic acid may include a cleavage site to allow removal of the from the subunit. A cleavage site can be used. One example is TEV (Tobacco Etch Virus). us) cleavage site (ENLYFQG, where cleavage occurs between the Q and G residues).

[0050] Modification by introduction of amino acids to facilitate insertion In one embodiment, the portal protein introduces one or more amino acids to enhance the hydrophobicity of the surface of the pore. The modified cleavage site is modified to facilitate direct insertion into the membrane by altering its molecular weight. The amino acids may be introduced by substitution and / or insertion. The inserted amino acids are At one or both ends of the amino acid chain of the portal protein subunit and / or at the chain The subunits fold and assemble into a pore. When introduced, the introduced amino acid is present on the exterior surface of the pore.

[0051] The introduction is performed on at least one amino acid of one or more of the subunits of the pore. Each subunit of the pore can independently have 2, 3, 4, 5, 6, 7, 8, 9, 10 or more The pore may contain one or more introduced amino acids, such as the amino acid introductions listed above. The pore may be composed of separate subunits, but one or more of the subunits of the pore may be separate from the others. The pore may be composed of two or more different subunits, e.g., from three or more different subunits. For example, 4, 5, 6, 7, 8, 9, 10, or 11 In one embodiment, all of the subunits may be present in the pore. The units may be different from each other. Typically, at least one amino acid is present in each site of the pore. For example, if the pore contains 12 subunits, the pore is Contains 12 or more introduced or substituted amino acids to alter the hydrophobicity of the pore surface. nothing.

[0052] The modification is typically performed in a central hydrophobic belt around the outside of the pore. The location of the pore belt region is shown in Figure 1. The central belt region of the pores typically has a The increased hydrophobicity can be achieved by, for example, modifying the hydrophilic, neutral, or relatively hydrophobic nature of the molecule. Replace amino acids with low hydrophobicity (e.g., alanine and / or methionine) with more hydrophobic residues. (e.g., leucine, valine, and / or isoleucine, etc.) This can be achieved by increasing the relative hydrophobicity of amino acids. Substitutions for the amino acid that is more hydrophobic than the amino acid that it replaces are more hydrophobic on the hydrophobicity scale shown in Figure 1D. By substituting any amino acid with a positive number for one or more amino acids present in the pore, could be.

[0053] The increase in hydrophobicity occurs in the middle and / or at the end of the amino acid chain of the portal protein subunit. This can be achieved by inserting one or more hydrophobic amino acids at the ends. , as shown in Figure 1D.

[0054] The location of the introduced hydrophobic amino acid may determine the location of the pore in the membrane. The position of the pore relative to the membrane shifts up or down (e.g., by up to 0.5 nm in either direction). Therefore, the location of the pores in the membrane can be controlled to improve the stability of the pores in the membrane. The intrinsic electrophysiology of the pore is typically determined by the presence of a mesh on the outer surface of the pore. It is not altered by changes in acid.

[0055] In one embodiment, one or more amino acids are introduced into the belt region below the wing domain. The target site is the phi29 portal protein subunit. Units Phe24, Ile25, Leu28, Phe60, Phe128, Pro1 29, and Pro132, as well as the corresponding positions in similar subunits. Additional hydrophobic residues may be located at 2, 3, 4, 5, 6, 7, or 8 of these target positions. 1, either before and / or after any one or more of these target locations. Substitutions or insertions may be made within one or two amino acids. For example, at position 22 of SEQ ID NO:1, 23, 26, 27, 29, 30, 58, 59, 61, 62, 126, 127, 130, 1 The hydrophobicity of the amino acid residues at positions corresponding to 31, 133, and / or 134 is The modification can be increased by the substitution or insertion of an acid residue at these positions. Any one or more of the following: 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more Other exemplary positions for mutation include Arg10, Glu These positions correspond to Arg14, Arg17, Gln18, and Arg22. by replacing any one or more of the amino acids in the central region of the pore with more hydrophobic residues. The hydrophobicity of the surface can be increased.

[0056] In one embodiment, the two distal portions of the protein stalk are The exposed charged residues in the stalk region (Arg17, Arg22 in SEQ ID NO:1) were enriched in , and / or residues corresponding to Lys172), as well as Asn / Gln residues (sequence Row number 1: Asn166, Asn167, Gln168, Gln173, Asn176, and / or such residue corresponding to Gln177) may be altered.

[0057] By introducing amino acids to facilitate conjugation of molecules that alter hydrophobicity qualification In one embodiment, a bacteriophage modified to be capable of directly inserting into a membrane The modified portal protein of the DNA packaging motor is the portal protein and / or one or more amino acid residues on the outer surface of Alternatively, one or more amino acid residues may be introduced onto the exterior surface of the portal protein to produce a wild-type portal protein. Molecular binding sites that alter the hydrophobicity of the exterior surface of the portal protein compared to the tal protein The binding site is located on the outside of the wing domain or on the stalk domain. The binding site serves as the site of attachment of the molecule. The molecule is typically It is a hydrophobic molecule that increases the hydrophobicity of the surface of the portal protein.

[0058] The introduced binding site may in one embodiment be a cysteine ​​residue. Alternatively, the binding site may be a non-natural amino acid.

[0059] Unnatural amino acids are amino acids that are not naturally found in proteins. , preferably histidine, alanine, isoleucine, arginine, leucine, asparagus Gin, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamine acid, threonine, glutamine, tryptophan, glycine, valine, proline, serine The unnatural amino acid is more preferably one of the 20 amino acids in the previous sentence. Neither selenocysteine ​​nor selenocysteine.

[0060] Preferred unnatural amino acids for use in the present invention include 4-azido-L-phenylalanine. 4-Acetyl-L-phenylalanine (Faz), 4-Acetyl-L-phenylalanine, 3-Acetyl-L-phenylalanine Lanine, 4-acetoacetyl-L-phenylalanine, O-allyl-L-tyrosine, 3- (Phenylselanyl)-L-alanine, O-2-propyn-1-yl-L-tyrosine, 4 -(Dihydroxyboryl)-L-phenylalanine, 4-[(ethylsulfanyl)carbamoyl] (2S)-2-amino-3-{4-[(propane-2 -ylsulfanyl)carbonylphenyl}propanoic acid, (2S)-2-amino-3-{ 4-[(2-amino-3-sulfanylpropanoyl)amino]phenyl}propanoic acid, O-Methyl-L-tyrosine, 4-amino-L-phenylalanine, 4-cyano-L-phenyl 3-Cyano-L-phenylalanine, 4-Fluoro-L-phenylalanine 4-iodo-L-phenylalanine, 4-bromo-L-phenylalanine, O-(triphenylamine) (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-(naphthalene-2-ylamino)propane Acid, 6-(methylsulfanyl)norleucine, 6-oxo-L-lysine, D-tyrosine , (2R)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid, (2R)- 2-Ammoniooctanoate 3-(2,2'-bipyridin-5-yl)-D-alanine , 2-amino-3-(8-hydroxy-3-quinolyl)propanoic acid, 4-benzoyl-L -Phenylalanine, S-(2-nitrobenzyl)cysteine, (2R)-2-amino- 3-[(2-nitrobenzyl)sulfanyl]propanoic acid, (2S)-2-amino-3- [(2-nitrobenzyl)oxy]propanoic acid, O-(4,5-dimethoxy-2-nitro benzyl)-L-serine, (2S)-2-amino-6-({[(2-nitrobenzyl)oxy) O-(2-nitrobenzyl)-L-tyrosine, 2-Nitrophenylalanine, 4-[(E)-phenyldiazenyl]-L-phenylalanine Nine, 4-[3-(trifluoromethyl)-3H-diaziren-3-yl]-D-phenyl Luaranine, 2-amino-3-[[5-(dimethylamino)-1-naphthyl]sulfonyl Amino]propanoic acid, (2S)-2-amino-4-(7-hydroxy-2-oxo-2H -chromen-4-yl)butanoic acid, (2S)-3-[(6-acetylnaphthalene-2-yl) 4-(carboxymethyl)phenylalanine, 3-aminopropanoic acid, -Nitro-L-tyrosine, O-sulfo-L-tyrosine, (2R)-6-acetamido-2 -Ammoniohexanoate, 1-methylhistidine, 2-aminononanoic acid, 2-amino Decanoic acid, L-homocysteine, 5-sulfanylnorvaline, 6-sulfanyl-L- Norleucine, 5-(methylsulfanyl)-L-norvaline, N 6-{[(2R,3R )-3-Methyl-3,4-dihydro-2H-pyrrol-2-yl]carbonyl}-L- Jin, 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-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-azido benzyl)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).

[0061] Examples of suitable hydrophobic molecules that can be conjugated to the portal protein include porphyrin, tetraphenylporphyrin, protoporphyrin IX, octaethylporphyrin These and other Hydrophobic molecules are attached to portal proteins to enable their membrane anchoring. It can be worn.

[0062] Controlling the precise location of binding sites for hydrophobic molecules determines where pores exist within the membrane Hydrophobic molecules can be used to shift the position of the pores relative to the membrane. For example, pores can be formed by hydrophobic molecules moving up and down the membrane (e.g., up to 100 nm in either direction). The stability of the pores in the membrane can be controlled by shifting the pore size by 0.5 nm. Placing hydrophobic molecules on the exterior surface of the pore does not alter the intrinsic electrophysiological properties of the pore. It will not be changed.

[0063] The binding (or conjugation) site is the Phi29 Gp10 portal protein Examples of positions that can be introduced include Q32, Y36, F52, K55, Q59, F60, Y6 2, N77, G78, A79, L80, S81, R84, R94, A96, S97, P9 8, Q101, P129, T131, E135, and Q168. These positions in the p10 portal protein or corresponding positions in other portal proteins any one or more of the residues in Alternatively, a cysteine ​​residue or a non-naturally occurring Amino acid residues may be inserted within one or two residues of these positions.

[0064] In one embodiment, the hydrophobicity is provided by one or more phosphate groups at one or both ends of the subunit molecule. Facilitate the insertion of portal proteins into membranes by adding natural or unnatural amino acids. For example, hydrophilic or hydrophobic tags can be added to one or both termini. Typically, the hydrophobic tag is added to the N-terminus of the central belt region of the molecule. A hydrophilic tag may be added to the C-terminal domain. and / or the hydrophobic tag may be attached to the portal protein via a linker. Suitable linkers are described above.

[0065] For example, the tag may be 3 or 4 to 10, e.g., 5, 6, 7, 8, or 9 amino acids. In one embodiment, all amino acids of the tag may comprise from 2 to 12 amino acids, such as Hydrophilic amino acids are negative on the hydrophobicity scale (shown in FIG. 1D). One or more of the residues of the hydrophilic tag have a position on the hydrophobicity scale. In one embodiment, the hydrophilic tag may be an overall hydrophilic residue, , may contain one or more hydrophobic residues having a positive number on the hydrophobicity scale.

[0066] In embodiments that use hydrophobic tags, the hydrophobic tag may be, for example, a positive number on the hydrophobicity scale. Alternatively, the hydrophobic tag may comprise only residues having a hydrophobicity of 0. The tag may comprise residues ranging from 0 to 1 on the hydrophobicity scale, provided that the tag is overall hydrophobic. It may contain one or more polar or charged amino acids having a negative number.

[0067] Mutations to facilitate use as nanopore sensors The modified portal protein may then be modified with one or more additional molecules to alter other properties of the pore. Such alterations may include modifications of the pore. Such alterations typically affect the use of the pore as a nanopore sensor. Examples of such modifications include:

[0068] By altering the ring of negatively charged Arg / Lys or Asp / Glu residues By doing so, they change the overall electronegative character of the channel interior. Arg / Gly residues, e.g. For example, one or more Asp / Glu residues may be substituted with positively charged or neutral amino acids. The groups can be substituted with positively charged, negatively charged, or neutral amino acids. To modify the sequence, narrow amino acids such as Glu189, Asp19, and Asp194 of SEQ ID NO:1 are used. The acidic residues at the inner channel entrance at one end are changed with any amino acid.

[0069] These are used to add functionality or to alter the electrophysiological properties of the pore. The goal is to use amino acids as anchoring points, with some amino acids (optional) at the ends. The addition of (natural or non-natural)

[0070] Flexible internal loops to modify the pore's electrophysiological properties and / or sensing capabilities For example, residues 229 to 244 of the phi29 Gp10 portal protein were changed (deleted) , shortening, mutating).

[0071] The mutant or modified proteins, monomers, or peptides may also be synthesized by any method and It may also be chemically modified at any site. The bond is preferably a bond of the molecule to one or more cysteines (cysteine ​​bond), one or more conjugation of the molecule to one or more non-naturally occurring amino acids; To carry out such modifications, Suitable methods for the synthesis of modified proteins, monomers, or peptides are well known in the art. The mutants of the proteins can be chemically modified by the attachment of any molecule. For example, modified proteins Proteins, monomers, or peptide variants are labeled with dyes or fluorophores. The peptide may be chemically modified.

[0072] film Any suitable membrane can be used in the system. The membrane is preferably an amphiphilic layer. The amphiphilic layer is made up of amphiphilic molecules, such as phospholipids, that have both hydrophilic and lipophilic properties. The amphiphilic molecules can be synthetic or natural. Amphiphilic materials and monolayer-forming amphiphiles are known in the art, e.g., Block copolymers (Gonzalez-Perez et al., Langmuir ,2009,25,10447-10450). Block copolymers are A polymeric material in which the monomer subunits are polymerized together to create a single polymer chain. Block copolymers typically consist of a number of monomeric subunits each of which contributes However, block copolymers are made up of individual subunits. Block copolymers can have unique properties that are not possessed by polymers that have been synthesized using monomers. One of the subunits is hydrophobic (i.e., lipophilic), while the other subunit is It can be engineered to be hydrophilic in aqueous media. In this case, the block copolymer can possess amphiphilic properties and can form structures that mimic biological membranes. can be a diblock (consisting of two monomer subunits), but can be composed of three or more monoblocks. There are also more complex arrangements built from mer subunits that behave as amphiphiles. The copolymer may be a triblock, tetrablock, or pentablock copolymer. The membrane is preferably a triblock copolymer membrane.

[0073] Archaeal bipolar tetraether lipids are naturally organized structures in which lipids are organized to form monolayer membranes. These lipids are generally found in extremophilic bacteria and microbial bacteria that survive in harsh biological environments. They are found in thermophiles, halophiles, and eosinophils. Their stability is determined by the fusion of the final bilayer. It is believed that this is due to the synthetic nature of the molecules. We mimic these biological entities by creating block copolymers. It is easy to construct polymers. This material is a monolayer that behaves similarly to lipid bilayers. These tomographic membranes can form a wide range of phase behaviors, from vesicles to lamellar membranes. Membranes formed from triblock copolymers have several advantages over biological lipid membranes. Because triblock copolymers are synthetic, their exact structure can be carefully controlled to create membranes. The correct chain length and structure required to form the pore and interact with other proteins and properties can be provided.

[0074] Block copolymers are also constructed from subunits that are not classified as lipid submaterials. For example, the hydrophobic polymer may be made from siloxane or other non-hydrocarbon monomers. The hydrophilic subsection of the block copolymer may also be made of low protein binding. The membrane may possess synthetic properties that allow for the creation of membranes that are highly resistant when exposed to live biological samples. The head group unit may also be derived from a non-classified lipid head group.

[0075] Triblock copolymer membranes also exhibit increased mechanical and environmental resistance compared to biological lipid membranes. Stability, e.g., a much higher operating temperature or pH range. The synthetic nature provides a platform for customizing polymer-based membranes for a wide range of applications.

[0076] The membrane is most preferably a membrane according to the International Application No. WO2014 / 064443 or WO20 This is one of the membranes disclosed in 14 / 064444.

[0077] The amphiphilic molecule is chemically modified to facilitate coupling to a polynucleotide. or functionalized. The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is typically planar. The amphiphilic layer may be curved. It may be possible.

[0078] Amphiphilic membranes are typically mobile in nature, with -8 cm s -1 Lipid diffusion The pores and the bound polynuclear This means that the leutide is typically capable of moving within an amphiphilic membrane.

[0079] The membrane may be a lipid bilayer. Lipid bilayers are a model for cell membranes and have been used extensively in experiments. For example, lipid bilayers are ideal for single channel recordings. Alternatively, lipid bilayers can be used for in vitro investigation of membrane proteins. The molecular layer can be used as a biosensor to detect the presence of a wide range of substances. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include planar lipid bilayers. Examples of suitable membrane bilayers include, but are not limited to, a lipid bilayer, a supported bilayer, or a liposome. The lipid bilayer is preferably a planar lipid bilayer. A suitable lipid bilayer is WO20 08 / 102121, WO2009 / 077734, and WO2006 / 100484 is disclosed in.

[0080] Methods for forming lipid bilayers are known in the art. The layers are based on Montal and Mueller (Proc.Natl.Acad.Sci .USA., 1972;69:3561-3566), and The monolayer is located on either side of an opening perpendicular to the aqueous / air interface. The lipid is usually first dissolved in an organic solvent and then loaded onto the By allowing evaporation on the interface of the aqueous solutions on either side of the opening, the electrolyte When the organic solvent evaporates, the solution / air interface on either side of the opening becomes The planar lipid bilayer is then physically moved up and down through the opening until a molecular layer is formed. , may be formed across an opening in the membrane or across an opening to a recess.

[0081] The Montal & Mueller method produces high-quality lipid bilayers suitable for protein pore insertion. It is popular because it is a cost-effective and relatively easy way to form a child layer. Other common methods of molecular layer formation include tip-dipping, bilayer deposition, Painting bilayers and patch cladding of liposome bilayers Includes pump.

[0082] The tip-dipping bilayer formation involves depositing an open surface ( The lipid monolayer is again contacted with an organic solvent. First, a small amount of lipid dissolved in the solvent is evaporated at the solution surface, resulting in a The bilayer is then formed by the Langmuir-Schäfer method, and the solution Mechanical automation is required to move the opening relative to the surface.

[0083] For bilayer coating, a small amount of lipid dissolved in an organic solvent is immersed in the test aqueous solution. The lipid solution is applied directly to the opening that is currently being used. The lipid solution is applied to the opening using a paint brush or equivalent. The thinning of the solvent results in the formation of a lipid bilayer. However, it is difficult to completely remove the solvent from the bilayer, and as a result, this method The bilayer formed by is less stable and prone to noise during electrochemical measurements.

[0084] Patch clamping is commonly used to study biological cell membranes. The membrane is then clamped onto the end of the pipette so that the patch of membrane adheres over the opening. The liposomes are then clamped and ruptured to seal the lipid bilayer over the opening of the pipette. This method has been adapted to produce lipid bilayers by the addition of glycerol to the cells. To fabricate small openings in materials with large and monolayer liposomes and glass surfaces. It is necessary to

[0085] Liposomes can be formed by sonication, extrusion, or the Mozafari method. (Colas et al. (2007) Micron 38:841-847) .

[0086] In a preferred embodiment, the lipid bilayer is a lipid bilayer as described in International Application No. WO2009 / 077734. Advantageously, in this method, the lipid bilayer is formed from dry lipids. In a most preferred embodiment, the lipid bilayer is The opening is formed across the opening so that the

[0087] A lipid bilayer is formed from two opposing layers of lipids. The two lipid layers are separated by their loosely packed surfaces. The hydrophilic tails are arranged facing each other to form a hydrophobic interior. The head groups face outward toward the aqueous environment on either side of the bilayer. Disordered phase (fluid lamellar), liquid ordered phase, solid ordered phase (lamellar gel phase, comb-like gel phase), and Planar bilayer crystals (lamellar subgel phase, lamellar crystalline phase), including but not limited to: It may be present in several lipid phases.

[0088] Any lipid composition that forms a lipid bilayer may be used. The properties that allow the membrane to function are determined by, for example, surface charge, ability to support membrane proteins, packing density, or mechanical properties. The lipid composition is selected to form a lipid bilayer having one or more different lipids. For example, the lipid composition may include up to 100 lipids. The lipid composition preferably comprises 1 to 10 lipids. It may also include artificial lipids.

[0089] Lipids typically consist of a head group, an interfacial portion, and two hydrophobic groups, which may be the same or different. Suitable head groups include diacylglyceride (DG) and ceramide ( Neutral head groups such as phosphatidylcholine (PC) and phosphatidylethanolamine (PHA). Zwitterionic head groups such as amines (PE) and sphingomyelins (SM), phosphatase Negatively charged head groups such as glycerol (PG), phosphatidylserine (PS ), phosphatidylinositol (PI), phosphatidic acid (PA) and cardiolipin cations, such as cations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 3 Suitable interface moieties include, but are not limited to, naturally occurring interface moieties. Examples of suitable glycerol-based or ceramide-based moieties include, but are not limited to, glycerol-based or ceramide-based moieties. Suitable hydrophobic tail groups include saturated hydrocarbon chains, such as lauric acid (n-dodecanol acid (n -Dodecanolic acid), myristic acid (n-tetradecononinoic acid (n -Tetradecononic acid), Palmitic acid (n-hexadecanoic acid) , stearic acid (n-octadecanoic acid), and arachidic acid (n-eicosanoic acid), Saturated hydrocarbon chains, such as oleic acid (cis-9-octadecanoic acid), as well as branched hydrocarbons The length of the chain and the amount of the non-functional chain are also included, but are not limited to, phytanoyl. The position and number of double bonds in saturated hydrocarbon chains can vary. The length of the chain in branched hydrocarbon chains The position and number of branches, such as methyl groups, can be varied. The lipids can be attached to the interface as ethers or esters. It's fine.

[0090] Lipids can also be chemically modified. The head or tail groups of the lipids can be chemically modified. Suitable lipids whose head groups have been chemically modified include PEG-modified lipids, For example, 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methacrylamide (polyethylene glycol)-2000]; functionalized PEG lipids, e.g., 1,2-distearate Aroyl-sn-glycero-3 phosphoethanolamine-N-[biotinyl(polyethylene ethylene glycol) 2000]; as well as lipids modified for conjugation, e.g. For example, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(sucrose) cinyl) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine -N-(biotinyl) whose tail group is chemically modified. Suitable lipids that are attached include polymerizable lipids, such as 1,2-bis(10,12-tricosapentaerythritol) and 1,2-bis(10,12-tricosapentaerythritol). Trichosadioyl-sn-glycero-3-phosphocholine; Fluorinated lipids, such as 1-palmitoyl-2-(16-fluoropalmitoyl)-sn-glycosyl Lysero-3-phosphocholine; deuterated lipids, e.g. 1,2-dipalmitoyl-D62-s n-glycero-3-phosphocholine; and ether-linked lipids, such as 1,2-di-O-phosphocholine. These include, but are not limited to, isopropyl-sn-glycero-3-phosphocholine. The material is chemically modified or functionalized to facilitate coupling of polynucleotides. This is also fine.

[0091] The amphiphilic layer, e.g., lipid composition, typically contains one or more additives that affect the properties of the layer. Suitable additives include fatty acids such as palmitic acid, myristic acid, and oleic acid; Fatty alcohols such as palmitic alcohol, myristic alcohol and oleic alcohol Cholesterol, cholesterol, ergosterol, lanosterol, sitosterol and sucrose Sterols such as stigmasterol, 1-acyl-2-hydroxy-sn-glycero-3 -Lysophospholipids such as phosphocholine, and ceramides, but are not limited to these. stomach.

[0092] In another preferred embodiment, the membrane comprises a solid layer. The solid layer is made of a material selected from the group consisting of fine electronic materials, insulating materials, and the like. , e.g., Si3N4, A12O3, and SiO, organic and inorganic polymers, e.g., poly amides, plastics such as Teflon® or elastomers such as 2 Organic and inorganic, including but not limited to component-additive cured silicone rubbers, and glass The solid layer may be made of graphene. Graphene layers are disclosed in WO2009 / 035647. A pore is typically a hole, well, gap, channel, groove, etc., in a solid layer. or in an amphiphilic membrane or layer contained within the gap. It is possible to prepare amphiphilic / amphiphilic hybrid systems. Suitable systems are described in WO2009 / 02 0682 and WO2012 / 005857. Either a film or a layer can be used.

[0093] The method typically comprises: (i) an artificial amphiphilic layer containing pores; (ii) an isolation layer containing pores; (iii) using cells with pores inserted inside This method typically involves the application of an artificial amphiphilic polymer such as an artificial triblock copolymer layer. This is carried out using a layer, which in addition to the pores contains other transmembrane proteins and / or membrane The method may include the steps of: The methods of the invention are typically carried out in vitro.

[0094] Methods for inserting modified pores into membranes Disclosed herein are modifications of bacteriophage DNA packaging motors. and a method for inserting the portal protein into a membrane for use as a nanopore. be.

[0095] The modified polysaccharide was synthesized by contacting the purified protein with the membrane and applying a potential to the membrane. Such a method can insert extracellular proteins into the copolymer membrane. It has been used in the art to insert into membranes.

[0096] One exemplary method involves contacting a membrane with a modified portal protein and increasing the voltage. By applying a voltage to the membrane, the portal protein is inserted into the membrane to form a channel. Those skilled in the art can readily determine suitable portal protein concentrations and voltages. For example, the voltage could be increased in 5 mV increments, with each voltage held for about 20 seconds. While applying the voltage, for example, an increased voltage of +50 to +350 mV can be applied. Excess portal protein may be washed away before use as a surface active agent.

[0097] array The present disclosure provides an array of membranes containing nanopores, the nanopores being formed by modifying portal tandems. In a preferred embodiment, each membrane of the array contains one nanopore. Due to the manner in which the array is formed, for example, the array may include one or more nanopores that do not include nanopores. The array may include one or more membranes containing two or more nanopores. About 2 to about 1000, for example, about 10 to about 800, about 20 to about 600, or about 30 to about 5 00 membrane.

[0098] In one embodiment, the array of membranes containing modified portal protein nanopores is The nucleic acid sequence may be present in a device suitable for high throughput sequencing.

[0099] Sensor Device The present disclosure provides a method for detecting and determining whether a target protein is present, comprising: For example, the device may include a chamber containing an aqueous solution and a chamber divided into two compartments. and a barrier separating the two compartments. The barrier is typically formed of a membrane containing nanopores. Alternatively, the barrier may form a membrane in which pores exist.

[0100] Thus, the device may include a first chamber and a second chamber, and a second chamber separated by a membrane containing a modified portal protein nanopore. When used to determine the characteristics of a target polynucleotide, the device The method may further comprise the step of: detecting a target polynucleotide by a portal protein; The first channel is temporarily located within the channel formed by the first reaction, and one end of the target polynucleotide is One end of the target polynucleotide is located in a first chamber and one end of the target polynucleotide is located in a second chamber.

[0101] In one embodiment, the device is capable of supporting a plurality of nanopores and membranes; The nanopore and membrane are operable to perform characterization of an analyte. In one embodiment, the device includes a small amount of material for delivering the material to perform the characterization. In one embodiment, the device includes at least one port for performing the characterization. In one embodiment, the device includes at least one reservoir for holding the material. The sensor is adapted to controllably supply material from at least one reservoir to the sensor device. and one or more containers for receiving respective samples. a fluidics system for selectively delivering sample from one or more reservoirs to the sensor device; The device also applies a potential and generates an electrical signal across the membrane and the complex. The device may include an electrical circuit that is capable of measuring the temperature.

[0102] The device is disclosed in WO2008 / 102120, WO2009 / 077734, and WO201 No. 0 / 122293, WO2011 / 067559, or WO00 / 28312 It can be any of the following:

[0103] In one embodiment, the device forms part of a system for determining a characteristic of an analyte. In one embodiment, the system includes a conductive solution in contact with the nanopore and a potential applied across the membrane. and a measurement system for measuring the current through the nanopore. In an embodiment, the voltage applied to the membrane and pore complex is between +5V and -5V, for example -600 The voltage used is more Preferably, in the range of 100 mV to 240 mV, more preferably, 120 mV to 220 mV By using increased applied potentials, different nuclei can be obtained for each pore. It is possible to increase discrimination between nucleotides. Any suitable conductive solution can be used. For example, the solution may contain metal salts, such as alkali metal salts, halide salts, e.g. The charge carrier may include any charge carrier, such as a chloride salt, for example an alkali metal chloride salt. is an ionic liquid or an organic salt, for example, tetramethylammonium chloride, trimethylammonium chloride, phenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3 In an exemplary system, the salt may include methylimidazolium chloride. It exists in aqueous solutions of potassium chloride (KCl), sodium chloride (NaCl), Cesium (CsCl), or a mixture of potassium ferrocyanide and potassium ferricyanide KCl, NaCl, and potassium ferrocyanide and ferricyanide are typically used. A mixture with potassium cyanide is preferred.The charge carriers may be asymmetric across the membrane. For example, the type and / or concentration of charge carriers may be different on each side of the membrane, e.g., in each chamber. It could be.

[0104] The salt concentration can be saturated. The salt concentration is 3M or less, typically 0.1 to 2.5 M, 0.3~1.9M, 0.5~1.8M, 0.7~1.7M, 0.9~1.6M, and The salt concentration may be 1 M to 1.4 M. The salt concentration is preferably 150 mM to 1 M. is preferably at least 0.3M, for example at least 0.4M, at least 0.5 M, at least 0.6M, at least 0.8M, at least 1.0M, at least 1.5 Use a salt concentration of at least 2.0M, at least 2.5M, or at least 3.0M. The high salt concentration provides a high signal-to-noise ratio and reduces the normal current fluctuations. It allows identification of a current indicative of the presence of a nucleotide against background.

[0105] A buffer solution may be present in the conductive solution. Typically, the buffer solution is a phosphate buffer. The preferred buffers for the conductive solution are HEPES and Tris-HCl buffers. 4.0~12.0, 4.5~10.0, 5.0~9.0, 5.5~8.8, 6.0~ The pH used may be 8.7, or 7.0 to 8.8, or 7.5 to 8.5. Preferably, it is about 7.5.

[0106] The device may be compatible with high throughput equipment. For example, the device may be Sm developed by Ord Nanopore Technologies Ltd. It can be an idgION, MinION, GridION, or PromethION device. These devices are different types of flow cytometers in which nanopores are embedded in a copolymer membrane. The copolymer membrane can be attached to a cell. The device can be a flow cell. , stable for at least several months and can withstand higher voltages. Each channel has its own It contains a pair of body electrodes so that the electrical signals between the channels are separated.

[0107] Methods for characterizing an analyte In a further embodiment, a method for determining the presence, absence, or one or more characteristics of a target analyte comprises The method includes contacting a target analyte with a membrane comprising a pore complex, the method comprising: This allows the target analyte to be detected in relation to the successive channels, e.g., in or between the successive channels. The nanopore and auxiliary proteins or peptides are transported through the membrane to the pore complex, respectively. and contacting the analyte with at least two of the components provided by the channel. One or more measurements are taken while the analyte is moving relative to the sample, thereby determining the presence, absence, or or determining one or more characteristics of the analyte. In an alternative embodiment, the analyte can pass through a pore complex in the membrane. Depending on the orientation of the body, it can pass through the auxiliary protein constriction and subsequently the nanopore constriction.

[0108] In one embodiment, the method determines the presence, absence, or one or more characteristics of a target analyte. The method is for determining the presence, absence, or one or more The method may be for determining the presence, absence, or characteristic of two or more analytes. or one or more characteristics of the analyte. For example, the presence of 2, 5, 10, 15, 20, 30, 40, 50, 100 or more analytes; The method may include determining the absence, presence, or one or more characteristics of one or more analytes. Numeric features may be determined, for example 1, 2, 3, 4, 5, 10 or more features.

[0109] The binding of molecules within the channels of the pore complex or near any opening of the channels This affects the open channel ion flow through the pore, which in turn affects the “molecular cell” of the pore channel. This is the essence of "sequencing." In a similar way to nucleic acid sequencing applications, The variation in the cell ion current can be measured using a suitable measurement technique by changing the electrical current. (See, for example, WO2000 / 28312 and D. Stoddart et al., P roc.Natl.Acad.Sci.,2010,106,7702-7 or WO2 The degree of reduction in ion flow, as measured by the reduction in current, is , which is related to the size of the disorder in or near the pore. Binding of a molecule of interest, also referred to as an "analyte," to the target molecule provides a detectable and measurable event. Thus, they form the basis of "biological sensors". Molecules include nucleic acids, proteins, peptides, polysaccharides, and small molecules (herein referred to as low molecular weight ( <900 Da or <500 Da), e.g., organic or inorganic compounds Detecting the presence of biological molecules, including drugs, toxins, cytokines, and pollutants is focused on personalized drug development, medical diagnostics, life science research, environmental monitoring, and in the security and / or defense industries.

[0110] The target analytes may be metal ions, inorganic salts, polymers, amino acids, peptides, polypeptides, Proteins, nucleotides, oligonucleotides, polynucleotides, polysaccharides, dyes, bleach Whitening agents, pharmaceuticals, diagnostic agents, recreational drugs, explosives, toxic compounds, or environmental pollutants The method can be used to detect two or more analytes of the same type, e.g., two or more proteins. The presence, absence, or presence of one or more proteins, two or more nucleotides, or two or more drugs Alternatively, the method may involve determining the characteristics of two or more different types analytes, e.g., one or more proteins, one or more nucleotides, and one or more therapeutic It may relate to determining the presence, absence, or one or more characteristics of a drug.

[0111] The target analyte may be secreted from the cell. Alternatively, the target analyte may be present inside the cell. The analyte may be an analyte that does not need to be extracted from the cell before the method can be performed. It must be.

[0112] In one embodiment, the analyte is an amino acid, a peptide, a polypeptide, or a protein. An amino acid, peptide, polypeptide, or protein may be naturally occurring or non-naturally occurring. Polypeptides or proteins may contain synthetic or modified amino acids within them. Several different types of modifications to amino acids are known in the art. Suitable amino acids and modifications thereof are described above. The target analyte is It is understood that the modification may be by any method available.

[0113] In a preferred embodiment, the analyte is a polynucleotide, such as a nucleic acid. A nucleotide is defined as a polymer containing two or more nucleotides. Naturally occurring nucleobases in nucleic acids may be distinguished by their physical size. As molecules or individual bases pass through the nanopore channel, the size differences between the bases cause There is a direct correlation between the ion flow through the channel and the reduction in the ion flow. The fluctuations in the ion flow can be recorded. Suitable electrical measurement techniques for recording the fluctuations in ion current are described, for example, in WO2000 / 2 8312 and D. Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106, pp7702-7 (single channel recording device), as well as examples For example, see WO2009 / 077734 (Multi-channel recording techniques). Through precise calibration, the characteristic reduction in ion current can be used to identify specific nucleosides passing through the channel. The nucleic acid sequence and associated bases can be identified in real time. In sequencing, the nucleotide of interest is blocked due to partial blocking of the channel by the nucleotide. While the individual nucleotides of the sequence pass sequentially through the nanopore channel, The current of ions in the nozzle is reduced. It is this ion current that is measured using the preferred recording technique described above. The reduction in ion flux is measured for a known nucleotide through the channel. This allows the ion current reduction to be calibrated, so that it is possible to determine which nucleotides are chalcogenides. This provides a means for determining whether the nanoparticles pass through the nanotube, if performed sequentially. A method for determining the nucleotide sequence of a nucleic acid that passes through a pore is provided. To accurately determine , typically, a reduction in ion flow through a channel is indicative of constriction (or This directly correlates with the size of the individual nucleotides passing through the DNA (the "reading head"). Sequencing is necessary, for example, through the action of associated polymerases or helicases. It will be understood that this can be done on intact nucleic acid polymers that are "threaded" into pores. Alternatively, the sequence may be a sequence of nucleotides successively removed from the target nucleic acid adjacent to the pore. This can be determined by the passage of the triphosphate group (see, for example, WO2014 / 187924). (I want to be).

[0114] A polynucleotide or nucleic acid can contain any combination of any nucleotides. The nucleotides may be natural or artificial. The nucleotides may be oxidized or methylated. The nucleotide may be damaged. For example, the polynucleotide may be a pyrimidine dimer. Such dimers are typically associated with UV damage and may be involved in the formation of dermal One or more nucleotides in a polynucleotide may be a target nucleic acid. The polynucleotide may be modified with a recognition sequence or tag, suitable examples of which are known to those skilled in the art. A nucleotide may contain one or more spacers. A nucleotide typically consists of a nucleobase, a sugar, and at least one phosphate group. The nucleobase and sugar form a nucleoside. Nucleobases are typically heterocyclic. Nucleobases include purines and pyrimidines. More specifically, adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). The sugars are typically pentoses. Nucleotide sugars include ribose and deoxyribose. The sugar is preferably deoxyribose. The polynucleotide is preferably or the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG), and deoxycytidine (dT). Nucleotides are typically ribonucleotides or deoxyribonucleic acids. Nucleotides are typically monophosphate, diphosphate, or triphosphate. A nucleotide may contain more than three phosphates, for example, four or five phosphates. The phosphate can be attached to the 5' or 3' side of the nucleotide. The nucleotides can be linked together in any manner. Nucleotides are typically the same as nucleic acids. Nucleotides are linked by their sugar and phosphate groups in the same way. Polynucleotides can be linked through their nucleobases, as can mers. At least a portion of the polynucleotide is preferably double-stranded. The polynucleotide is most preferably a ribonucleic acid (RNA) or deoxyribonucleic acid (DTA) chain. DNA. In particular, polynucleotides are used as alternative analytes. The method includes determining whether the polynucleotide is a polynucleotide having a length greater than or equal to 100; (i) the sequence of the polynucleotide; (iv) the secondary structure of the polynucleotide; and (v) the structure of the polynucleotide. determining one or more characteristics selected from whether the nucleotide is modified or not .

[0115] A polynucleotide can be of any length (i). For example, a polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 20 0, at least 250, at least 300, at least 400, or at least 500 A polynucleotide can be any length of nucleotide or nucleotide pair. Nucleotides or nucleotide pairs above, 5000 or more nucleotides or nucleotide pairs above nucleotide pairs or lengths of 100,000 or more nucleotides or nucleotide pairs Any number of polynucleotides can be interrogated. For example, the method can include , 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides When two or more polynucleotides are characterized, the are different polynucleotides or are two instances of the same polynucleotide The polynucleotides may be natural or artificial. For example, This method allows the sequence of the oligonucleotides produced to be confirmed. is carried out in vitro.

[0116] The nucleotide may have identity (ii) and may be adenosine monophosphate (AMP), guanosine monophosphate (GUA), Glycine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP ), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine Cyclic adenosine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (CAMP) cGMP, deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine deoxycytidine monophosphate (dUMP), deoxycytidine monophosphate (dCMP), and deoxymethionine monophosphate (dCMP) Nucleotides may include, but are not limited to, cytidine monophosphate. For example, AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, d The nucleotide is selected from abasic (i.e., nucleobase A nucleotide may also lack a nucleobase and a sugar (i.e., The nucleotide sequence (iii) is The consecutive identity of the following nucleotides linked together in the 5' to 3' direction of the strand: is determined by.

[0117] The following examples illustrate the invention.

[0118] Example 1: Protein Expression and Purification The engineered gene for the phi29 portal protein channel was cloned into an expression vector. The newly constructed clones were transformed into BL21(DE3) E. coli bacteria. The successfully transformed bacteria were transferred to 10 mL of Luria-Bertani (LB) medium. The bacteria were cultured overnight at 37°C in 500 mL of fresh LB medium. When the OD600 reached 0.5-0.6, add 0.5 mM IPTG to the culture medium. Protein expression was induced by adding 100 mM NaCl. Bacteria were harvested 3 hours after induction by centrifugation. The bacterial walls were lysed using an anti-press, and proteins and other components were separated by centrifugation. Ni-NTA His-binding resin with His-tag was applied to identify mutant proteins. Briefly, 2 ml of regenerated His resin was packed into a column. The supernatant separated by this procedure was loaded onto the column. The column was then washed with a washing buffer to remove contaminants. All proteins were removed. An elution buffer containing 500 mM imidazole was used. The protein was eluted by rotating at 12000 rpm. The eluate was collected and concentrated to 5 mL. Centrifuge the eluate for 10 min, then absorb the supernatant with a syringe and load it onto an AKTA FPLC. Prior to injection, the sample loop was washed with 10 mL of lysis buffer. Size Exclusion Column After passing the protein through the ELISA kit, the protein was collected. SDS-PAGE gel was run to determine the protein The samples were checked. In this way, all wild-type and mutant proteins were expressed and Typically, the protein is stored at -20°C and eluted by repeated freeze-thaw cycles. The solution was aliquoted into multiple tubes to avoid contamination.

[0119] The sequence of the phi29 portal protein is known and available from Genbank. k Accession No. ACE96033). The hi29 gp10 portal protein was generated: -A79C, -E135C, -Q168C, -R10L, E14V, R17L, and N-7Δ (mutant-b), -R10L, E14V, R17L, Q18L, R22I, and N-ter-7Δ Variant-c), -IL added to the N-terminus (variant-d), and -R10L, E14V, R17L, N-ter-7Δ (mutation Body-e)

[0120] Example 2: Pore insertion into MinION device To insert the engineered protein channels into the ONT membrane, The protein was dissolved in C13 buffer (25 mM potassium phosphate, 150 mM potassium ferrocyanide). The solution was diluted 1000-fold with 150 mM potassium ferrocyanide, pH 8. 200 μl of diluted protein sample was injected through the priming port of the ON flow cell. Then, to support the insertion of the protein channel, a voltage of +50 to +350 mV was added. A voltage was applied while increasing the voltage by 5 mV (5 mV increments, held for 20 seconds). The flow cell was then 1 mL of C13 buffer. Then, typically, the retention time was varied (at each voltage hold for 2-10 minutes at ±50, ±100, ±150, ±200mV, run IV curves Analytes such as DNA or peptides (1 pM concentration) were added to the C 13 buffer and added to the flow cell to check the pore function.

[0121] Unless otherwise defined, all technical and scientific terms used herein are defined by the It has the same meaning as commonly understood by a person skilled in the art to which the invention pertains. Publications cited herein and the material for which they are cited are expressly incorporated by reference. do.

[0122] Those skilled in the art will recognize many equivalents to the specific embodiments of the invention described herein. or could be verified using no more than routine experimentation. Such equivalents are intended to be encompassed by the scope of the following claims.

[0123] Array List SEQ ID NO:1 - Amino acid sequence of wild-type phi29gp-10 1 MARKRSNTYR SINEIQRQKR NRWFIHYLNY LQSL AYQLFE WENLPPTINP 51 SFLEKSIHQF GYVGFYKDPV ISYIACNGAL SGQ RDVYNQA TVFRAASPVY 101 QKEFKLYNYR DMKEEDMGVV IYNNDMAFPT TP TLELFAAE LAELKEIISV 151 NQNAQKTPVL IRANDNNQLS LKQVYNQYEG NA PVIFAHEA LDSDSIEVFK 201 TDAPYVVDKL NAQKNAVWNE MMTFLGIKNA NL EKKERMVT DEVSSNDEQI 251 ESSGTVFLKS REEACEKINE LYGLNVKVKF RY DIVEQMRR ELQQIENVSR 301 GTSDGETNE

Claims

1. Modified portal protein of the bacteriophage DNA packaging motor wherein the modified portal protein is capable of directly inserting into a membrane and One or more amino acid residues on the exterior surface of the portal protein are linked to one or more other amino acid residues. The portal protein is modified relative to a wild-type portal protein such that the and / or has a modified amino acid sequence similar to that of the wild-type portal protein. The portal protein may be modified with one or more amino acid residues to alter the hydrophobicity of the outer surface of the modified portal protein. a modified portal protein having a modified amino acid sequence inserted on the outer surface of the portal protein; Stuffy.

2. At least one of the one or more amino acid residues is in the portal protein. The modified portal protein of claim 1 , which is in the central hydrophobic belt region.

3. The introduction of one or more amino acid residues results in a priori, as compared to the wild-type portal protein.

3. The modified portal protein of claim 1 or 2, which has increased outer surface hydrophobicity.

4. At least one of the one or more amino acid residues is involved in the Phi29 DNA packaging F24, I25, L28, F60, and F128 of the portal protein of the ging motor , P129, and P132. The modified portal protein of any one of claims 1 to 3, which is in an intracellular position. 。

5. At least one of the one or more amino acid residues is N of the portal protein. The modified polypeptide according to any one of claims 1 to 3, which is within about 30 amino acids of the terminal end. Total protein.

6. At least one of the one or more amino acid residues is The portal proteins R10, E14, R17, Q18, and and R22. protein.

7. At least one of the one or more amino acid residues is a parent of the portal protein. The modified aqueous phase according to any one of the preceding claims, in the aqueous cis and / or trans phase. Portal proteins.

8. At least one of said one or more amino acid residues of said cis layer of said portal protein The other is the portal protein of the Phi29 DNA packaging motor. Q32, Y36, F52, K55, Q59, F60, Y62, N77, G78, A79, Corresponding to L80, S81, R84, R94, A96, S97, P98, and Q101 and / or at the trans layer of the portal protein, At least one of the amino acid residues is a member of the Phi29 DNA packaging motor. - Positions corresponding to P129, T131, E135, and Q168 of the portal protein The modified portal protein of claim 7,

9. the one or more amino acid residues of the cis layer or the trans layer of the portal protein At least one of the poles of the phi29 DNA packaging motor a cDNA clone located at a position corresponding to A79, E135, and / or Q168 of the tal protein; 9. The modified portal protein of claim 8.

10. Modified portal protein of the bacteriophage DNA packaging motor wherein the modified portal protein is capable of directly inserting into a membrane, At least one amino acid residue is introduced onto the outer surface of the portal protein to form a wild-type portal protein. a molecule that alters the hydrophobicity of the exterior surface of the portal protein compared to the portal protein; One or more binding sites are introduced on the outside of the wing domain or within the stalk domain. A modified portal protein.

11. At least one of the one or more amino acid residues introduced into the portal protein The modified portal ligase of claim 10, wherein one of the amino acids is cysteine ​​or an unnatural amino acid. le protein.

12. At least one of the one or more amino acid residues is a parent of the portal protein. The modified glycerol according to claim 10 or 11, which is in the aqueous cis and / or trans phase. Portal proteins.

13. At least one of said one or more amino acid residues of said cis layer of said portal protein The other is Q3 of the portal protein of the Phi29 DNA packaging motor. 2, Y36, F52, K55, Q59, F60, Y62, N77, G78, A79, L8 Positions corresponding to 0, S81, R84, R94, A96, S97, P98, and Q101 and / or the one or more amines of the trans layer of the portal protein At least one of the amino acid residues of the Phi29 DNA packaging motor At positions corresponding to P129, T131, E135, and Q168 of the portal protein The modified portal protein of claim 12.

14. the one or more amino acid residues of the cis layer or the trans layer of the portal protein At least one of the poles of the phi29 DNA packaging motor a cDNA clone located at a position corresponding to A79, E135, and / or Q168 of the tal protein; 14. The modified portal protein of claim 13.

15. The at least one amino acid is introduced by substitution and / or insertion.

2. A modified portal protein according to any one of claims 1 to 11.

16. The portal protein comprises one or more amino acid residues at the N-terminus of the portal protein. The modified form of any one of the preceding claims, modified by the addition and / or deletion of groups. Decorated portal proteins.

17. phi29, T3, T4, T5, T7, SPP1, HK97, Lambda, G20c, P2 A DNA package from a bacteriophage selected from the group consisting of P1, P2, and P3. A modified portal protein of a binding motor according to any one of the preceding claims. The modified portal protein described herein.

18. A modified form according to any one of the preceding claims, which is composed of identical subunits. Portal proteins.

19. the outer surface of the portal protein compared to the wild-type portal protein; 19. The method according to claim 10, wherein the molecule that changes hydrophobicity is a hydrophobic molecule. The modified portal protein described herein.

20. The hydrophobic molecule is porphyrin, tetraphenylporphyrin, protoporphyrin IX, octaethylporphyrin, cholesterol, heme, or biliverdin; 10. A modified portal protein according to any one of the preceding claims.

21. A modified portal protein subunit according to any one of claims 1 to 20. 。

22. A membrane comprising a modified portal protein according to any one of claims 1 to 20.

23. 23. The membrane of claim 22 which is a lipid membrane or a copolymer membrane.

24. 23. The copolymer membrane of claim 22, wherein the copolymer membrane is a diblock or triblock copolymer membrane. The membrane according to claim 1.

25. An array comprising two or more membranes according to any one of claims 22 to 24.

26. 26. The array of claim 25 adapted for insertion into a sensor device.

27. 27. An array according to claim 25 or 26, means for applying an electrical potential across the membrane, and and means for detecting the charge across the membrane.

28. 28. The method of claim 27, further comprising a fluidics system configured to supply a sample to the membrane. Devices listed.

29. A method for determining a characteristic of a target analyte, comprising the steps of: Contacting the membrane with the target analyte and allowing the target analyte to translocate relative to the nanopore. A potential is applied across the membrane as the target analyte moves relative to the pore. and performing one or more measurements on the analyte to determine the presence, absence, or one or more characteristics of the analyte. and determining.

30. 30. The method of claim 29, wherein the measurement is an electrical measurement and / or an optical measurement.

31. 31. The method of claim 29 or 30, wherein a plurality of target analytes are characterized.

32. The target analyte may be a polynucleotide, a protein, a peptide, a carbohydrate, a metabolite, or other chemical substance.

33. The method of any one of claims 29 to 32, wherein the target analyte is associated with a disease state. Law.

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