Biological nanopores having tunable pore diameters and uses thereof as analytical tools

Abstract

To provide a strategy that allows for tuning the diameter of Fragaceatoxin C (FraC) nanopores, such that a larger range of peptides sizes can be identified; and to provide applications the nanopores.SOLUTION: Provided is a system comprising oligomeric Fragaceatoxin C (FraC) nanopores comprised in a lipid bilayer, the sum of the nanopore fraction in the heptameric (Type II) state and the nanopore fraction in the hexameric (Type III) state representing at least 60% of the total number of FraC nanopores.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates generally to the field of nanopores and the use of such pores in the analysis of biopolymers and other (biological) compounds. In particular, the present invention relates to a modified Fragaceatoxin C (FraC) nanopore and the use of such nanopores in single molecule analysis, such as single molecule peptide sequencing. [Background technology]

[0002] Biological nanopores are proteins that open nanoscale channels in biological or synthetic membranes. Under an external electric potential, an ionic current through a single nanopore is used to recognize an analyte that translocates through the nanopore. In particular, nanopores are currently being used to sequence nucleic acids at the single molecule level. In nanopore DNA sequencing, individual nucleic acid strings are passed through the nanopore base by base, and the ionic current is used to identify individual nucleic acid bases (Non-Patent Documents 1, 2).

[0003] Watanabe et al. (Analytical Chemistry 2017 89 (21), 11269-11277) describe the analysis of pore formation and detection of single protein molecules using large nanopores in five different pore-forming proteins, including FraC. It is shown that identification of pores suitable for nanopore sensing can be achieved by sorting the channel current signals and performing noise analysis.

[0004] However, protein sequencing with nanopores poses a new set of challenges: amino acids have greater chemical variability than nucleic acids, and they cannot be uniformly captured and pulled by the electrical potential inside the nanopore. Moreover, there are no known enzymes that process proteins or polypeptides amino acids one by one.

[0005] Another approach would be to first fragment the protein and identify the mass of individual peptides by nanopore current. This approach would be similar to conventional protein sequencing approaches using tandem mass spectrometry. However, nanopore peptide mass identifiers would have the advantages of being low-cost and portable, and of being single molecules. The latter advantage is important because it would allow the analysis of chemical heterogeneity in post-translational modifications and, especially when combined with high-throughput devices, would allow the detection of low-abundance proteins. Previous studies using PEG molecules (Non-Patent Documents 3, 4, 5, 6, 7, 8), neutral peptides (Non-Patent Document 9), or oligosaccharides (Non-Patent Document 10), uniformly charged peptides (Non-Patent Documents 11, 12, 13, 14, 15) and other peptides (Non-Patent Document 16) revealed that there may be a direct correlation between the depth of current blockade and the molecular weight of the polymer when the analyte composition is homogeneous. On the other hand, a wealth of other studies, including DNA (17, 18) and amino acid enantiomers (19), have revealed that the chemical identity of molecules, particularly the charge, inside the nanopore (20) has a strong and unpredictable effect on the ion current, suggesting that mass identification of complex biopolymers such as peptides may not be possible. A further concern is that peptides of opposite charge are not efficiently captured and analyzed at fixed potentials (16, 21, 22, 23, 24). Finally, the diameter and geometry of biological nanopores cannot be easily adapted to study a range of sizes, shapes, and chemical compositions of polypeptides in solution.

[0006] Recently, we have shown that the octameric Fragaceatoxin C (FraC) nanopore from the sea anemone Actinia fragacea (Non-Patent Document 25) can be used to study DNA (Non-Patent Document 26), proteins, and peptides (Non-Patent Document 27). See also US Pat. No. 5,999,333 in the name of the present applicant. The membrane-spanning region of FraC is unique in comparison to other nanopores used in biopolymer analysis in that it is formed by an α-helix with a sharp and narrow constriction at the trans-exit of the nanopore. Of particular importance, we have shown that the electroosmotic flow through the nanopore can be engineered to capture polypeptides at a fixed potential despite their charge composition (Non-Patent Document 27). However, peptides with sizes less than 1.6 kDa pass through the nanopore too quickly to be sampled. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2018 / 012963 [Non-patent literature]

[0008] [Non-Patent Document 1] Clarke, J. et al. Continuous base identification for single-molecule nanopore DNA sequencing. Nat. Nanotechnol. 4, 265-270 (2009). [Non-Patent Document 2] Derrington, IM et al. Nanopore DNA sequencing with MspA. Proc. Natl. Acad. Sci. 107, 16060-16065 (2010). [Non-Patent Document 3] Bezrukov, SM, Vodyanoy, I., Brutyan, RA & Kasianowicz, JJ Dynamics and free energy of polymers partitioning into a nanoscale pore. Macromolecules 29, 8517-8522 (1996). [Non-Patent Document 4] Robertson, JWF et al. Single-molecule mass spectrometry in solution using a solitary nanopore. Proc. Natl. Acad. Sci. 104, 8207-8211 (2007). [Non-Patent Document 5] Baaken, G. et al. High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore. ACS Nano 9, 6443-6449 (2015). [Non-Patent Document 6] Aksoyoglu, MA et al. Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin. Proc. Natl. Acad. Sci. 113, 9003-9008 (2016). [Non-Patent Document 7] Krasilnikov, OV, Rodrigues, CG & Bezrukov, SM Single polymer molecules in a protein nanopore in the limit of a strong polymer-pore attraction. Phys. Rev. Lett. 97, 1-4 (2006). [Non-Patent Document 8] Oukhaled, AG, Biance, AL, Pelta, J., Auvray, L. & Bacri, L. Transport of long neutral polymers in the semidilute regime through a protein nanopore. Phys. Rev. Lett. 108, 1-4 (2012).

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[0009] Based on the above studies, the inventors understood and appreciated that smaller diameter nanopores are needed to detect smaller molecular weight peptides, and therefore, the inventors aimed to provide a strategy that allows tuning the diameter of the FraC nanopore so that a wider range of peptide sizes can be identified.

[0010] Surprisingly, it was found that the FraC nanopore can be engineered to induce the formation of different nanopore types (herein referred to as type II and / or type III) when included in a lipid bilayer environment, thereby creating biological nanopores with sub-nanometer constrictions. Importantly, these novel narrow-type nanopores allow for the discrimination of (small) peptides differing by a single amino acid substitution with a resolution of about 40 Da, whereas previous nanopore studies reported differences of only about 200 Da. Furthermore, under selected pH conditions, the FraC nanopore signal was directly correlated with the mass of the peptide. The present invention provides a novel and unique approach for nanopore-based single molecule identification of proteins. [Means for solving the problem]

[0011] In one embodiment, the present invention relates to a system comprising multimeric Fragaceatoxin C (FraC) nanopores contained within a lipid bilayer, wherein the sum of the percentage of nanopores in the type II state and the percentage of nanopores in the type III state represents at least 60% of the total number of FraC nanopores.

[0012] For example, the sum of type II and type III state nanopores represents at least 65%, preferably at least 70%, of the total number of FraC nanopores.

[0013] As used herein, the term "Type II" state refers to a nanopore that has apparent heptameric properties and / or has a conductance of about 1.22-1.26 nS measured in 1 M NaCl solution at pH 7.5, or about 0.99-1.08 nS measured in 1 M KCl solution at pH 4.5. Conductance values ​​are suitably determined by collecting a single channel at an applied potential of -50 mV using 1 M NaCl, 15 mM Tris pH 7.5, or 1 M KCl, 0.1 M citrate, 180 mM Tris base pH 4.5.

[0014] The type II FraC nanopore is further characterized as having an apparent pore size of approximately 1.1 nm (at the narrowest constriction) as calculated by homology modeling.

[0015] As used herein, the term "type III" state refers to a nanopore that has apparent hexameric nature and / or has a conductance of about 0.37-0.43 nS measured in 1 M KCl solution at pH 4.5.

[0016] The type III FraC nanopore is further characterized as having a pore size of approximately 0.8 nm (at the narrowest constriction) as shown by homology modeling.

[0017] Thus, in one embodiment, the invention provides a system comprising multimeric FraC nanopores contained within a lipid bilayer, wherein the sum of the percentage of nanopores in the heptameric (type II) state and the percentage of nanopores in the hexameric (type III) state represents at least 60% of the total number of FraC nanopores.

[0018] In another embodiment, the invention provides a system comprising multimeric FraC nanopores contained within a lipid bilayer, wherein the sum of (i) a proportion of nanopores exhibiting a conductance of about 0.99-1.08 nS measured in 1 M KCl solution at pH 4.5 (Type II), and (ii) a proportion of nanopores exhibiting a conductance of about 0.37-0.43 nS measured in 1 M KCl solution at pH 4.5 (Type III) represents at least 60% of the total number of FraC nanopores.

[0019] Furthermore, the present invention provides a system comprising multimeric FraC nanopores contained within a lipid bilayer, wherein the sum of the proportion of nanopores having an apparent pore size of about 1.1 nm (Type II) and the proportion of nanopores having an apparent pore size of about 0.8 nm (Type III) represents at least 60% of the total number of FraC nanopores.

[0020] The relative amounts of type II and type III nanopores can vary as desired. In one embodiment, at least 40%, preferably at least 50%, of the FraC nanopores are in the type II state. Alternatively or additionally, at least 15%, preferably at least 20%, of the FraC nanopores are in the type III state.

[0021] In one embodiment, at least 60%, preferably at least 70%, of the FraC nanopores are in the type II state. In another embodiment, at least 60%, preferably at least 70%, of the FraC nanopores are in the type III state.

[0022] Systems containing substantially one multimeric form / type of FraC are also encompassed. For example, in one embodiment, at least 90%, preferably at least 95%, of the FraC nanopores are in the type II state. In certain aspects, all of the FraC nanopores are in the type II state. In another embodiment, at least 90%, preferably at least 95%, of the FraC nanopores are in the type III state. In certain aspects, all of the FraC nanopores are in the type III state. The different multimeric forms of FraC can be readily isolated using liquid chromatography methods, including size exclusion, affinity, reversed phase, or ion exchange chromatography.

[0023] In certain embodiments, the FraC nanopore comprises or consists of a mutant FraC monomer that contains one or more mutations that weaken the interaction between the nanopore and the lipid bilayer (i.e., the lipid interface).

[0024] Very good results are obtained when FraC is mutated at W112 and / or W116. For example, in one embodiment, FraC is mutated at W112, preferably W116 is not mutated, or FraC is mutated at W116, preferably W112 is not mutated. In a further embodiment, the FraC mutant comprises mutations at both W112 and W116. According to the invention, the W residue is substituted with either S, T, A, N, Q, or G, preferably S or T. FraC comprises 179 amino acids with a relative molecular weight of 20 kDa. The cDNA of FraC is available in GenBank under the accession number FM958450. The polypeptide sequence of FraC is available in UniProt under the accession number B9W5G6. Furthermore, the crystal structure of FraC was solved in 2010 and deposited in the RCSB PDB under the accession number 3LIM. In the variants according to the invention, the numbering of the residues corresponds to the numbering of the residues in the FraC sequence of UniProtKB Accession No. B9W5G6.

[0025] The importance of (conserved) tryptophan residues for the function of pore-forming toxins has been previously studied. Tanaka et al. (25) revealed the structures of FraC corresponding to four different stages of the FraC activation pathway: the aqueous form, the lipid-bound form, the assembly intermediate form, and the transmembrane pore form. Mutational analysis revealed that the W112R / W116F mutant lost the ability to bind lipid membranes and was completely inactive. Garci´a-Linares et al. (2016) studied the role of tryptophan residues in the specific interaction of the actinoporin sticholysin II (StnI) of the sea anemone Stichodactyla helianthus with biomembranes. It was elucidated that residues W110 and W114 (corresponding to W112 and W116 of FraC) support the hydrophobic effect, which is one of the main driving forces for membrane binding in the presence of cholesterol. In particular, the authors state that "the results support a major role for the Trp residues in actinoporin in membrane recognition and binding," but conclude that "these residues only have a minor effect on the diffusion and multimerization steps required to assemble a functional pore." Thus, our discovery that W112 and / or W116 are appropriately modified to modulate the multimeric state of FraC was not reasonably predicted in the prior art.

[0026] Thus, in one aspect, the invention provides a system comprising a multimeric FraC nanopore contained within a lipid bilayer, the FraC nanopore comprising mutant FraC monomers comprising mutations at positions W112 and / or W116, preferably wherein one or more of said W residues are independently substituted with S, T, A, N, Q, or G, preferably with S or T. In one embodiment, the system comprises or consists of a W112S or W112T FraC mutant. In another embodiment, the system comprises or consists of a W116S or W116T mutant. In yet another embodiment, the system comprises or consists of a W112S / W116S, W112T / W116S, or W112S / W116T FraC mutant.

[0027] We noted that type II FraC nanopores inserted into lipid bilayers are more efficient at low pH. Therefore, to increase the production of type II nanopores at physiological pH, we replaced the aspartic acid at position 109, located at the lipid interface, with an uncharged residue. As expected, the proportion of type II nanopores at pH 7.5 increased from 23.0±4.9% to 48±3.6%, while only a small proportion of type III nanopores was observed.

[0028] The invention also provides a system comprising a multimeric FraC nanopore contained within a lipid bilayer, the FraC nanopore comprising a mutant FraC monomer comprising a mutation at position D109, preferably wherein the mutation comprises a substitution of D with an uncharged residue such as S or T, more preferably with S. Concomitant substitution of tryptophan at position 116 with serine showed a small further increase in the proportion of type I and type II nanopores at pH 7.5.

[0029] Thus, the present invention further relates to a system comprising a multimeric FraC nanopore contained within a lipid bilayer, the FraC nanopore comprising a mutant FraC monomer comprising the D109S and one or both of the W112S and W116S mutations.

[0030] The nanopore-containing system of the present invention can accommodate peptides ranging from 22 to 4 amino acids in length. Smaller peptides can be detected using finer tuning of the membrane-spanning region of the nanopore, for example by the introduction of amino acids with bulky side chains. The inventors have also shown that the nanopore can distinguish the difference between alanine and glutamic acid (about 40 Da) in a mixture of peptides. Furthermore, the inventors have found that just at pH 3.8, the ion signal of a peptide depends on the mass of the analyte, while at higher pH values ​​the current signal of a negatively charged peptide is higher than the current signal predicted from its mass alone.

[0031] Without wishing to be bound by any theory, the inventors' explanation is that while the peptide to be analyzed loses its charge, the constriction of the nanopore still maintains a sufficient negative charge to recognize the charge of the peptide. The most likely explanation is that the negatively charged constriction is important for creating an electrophoretic environment for peptide mass recognition. At the same time, electrostatic interactions between the constriction and the negatively charged analyte may interfere with the correct positioning of the analyte within the reading frame of the nanopore.

[0032] Presumably, the peptides need to be uniformly charged, which can be achieved by lowering the pH of the solution, but at the same time, the constriction of the nanopore should be negatively charged for optimal mass recognition, and it may be difficult to achieve both effects, since lowering the pH also makes the constriction less charged.

[0033] Thus, in addition to one or more amino acid substitutions described herein above, the mutant FraC monomer may contain one or more unnatural amino acids that contain a moiety that retains a negative charge at low pH, preferably a sulfate or phosphate group. In one embodiment, a residue (e.g., the residue at position 10) is mutated to cysteine ​​and then oxidized, thus introducing a sulfonic acid (or sulfinic acid) group at that position. The charge of such a group remains negative over the entire pH range. This approach may improve recognition of the peptide. Alternatively, the peptide may be chemically modified (e.g., by esterification) to neutralize the negative charge.

[0034] In the system according to the invention, the FraC nanopore is contained in a lipid bilayer. The reconstitution of the FraC nanopore in a lipid bilayer is described in the art. Typically, the lipid bilayer contains phosphatidylcholine (PC), preferably 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), optionally in combination with sphingomyelin (SM). Very good results are obtained when DPhPC and SM are present in approximately equal masses.

[0035] When the system according to the invention is in use, the nanopore is typically located between a first liquid medium and a second liquid medium, at least one of which contains an analyte, and the system is operable to detect a property of the analyte. In one embodiment, the system is operable to detect a property of the analyte by applying an electric field to the nanopore such that the analyte moves electrophoretically and / or electroosmotically through the nanopore. As exemplified herein below, the system provided herein is particularly suitable for the analysis of proteinaceous materials, preferably peptides, more preferably peptides up to about 30 amino acids in length. In particular, the system of the invention also provides the capture of peptides with different charges, recognition of the mass of the peptides, and the differentiation capability of only up to 40 Da.

[0036] However, the present invention should not be understood to be limited in its application to peptide analysis, for example, other analytes that can be detected using the system of the present invention include (non-proteinaceous) biomarkers, antibiotics or other drugs, DNA, metabolites, and small biological molecules.

[0037] The present invention further relates to a mutant Fragaceatoxin C (FraC) polypeptide comprising one or more of the above mentioned mutations. Such a polypeptide is advantageously used in the (analysis) system described herein. Also provided is an isolated nucleic acid molecule encoding a mutant FraC polypeptide according to the invention and an expression vector comprising said isolated nucleic acid molecule. The present invention also further provides a host cell comprising the expression vector as described above.

[0038] In one embodiment, the mutant FraC polypeptide comprises a mutation at D109, W112, and / or W116. For example, the mutant FraC polypeptide comprises a mutation at W112 (optionally, W116 is not mutated) or a mutation at W116 (optionally, W112 is not mutated). In one embodiment, the mutant FraC polypeptide comprises a mutation at both W112 and W116. As described herein above, the one or more mutations may comprise a substitution of D or W with S, T, A, N, Q, or G, preferably with S or T. In a particular aspect, the invention provides a FraC W112S, FraC W116S, or FraC W112S / W116S mutant, or a nucleic acid molecule encoding same, or a vector comprising said nucleic acid molecule. Furthermore, the present invention provides a polypeptide comprising a D109S mutation, preferably wherein the mutation is D109S / W116S, or a nucleic acid molecule encoding same, or a vector comprising said nucleic acid molecule.

[0039] Any of the foregoing mutations may be supplemented with one or more unnatural amino acids that contain a moiety that retains a negative charge at low pH, where, for example, the moiety is a sulfate, sulfonate, or phosphate group. Preferred positions for the introduction of such one or more negatively charged residues include one or more of positions 10, 17, and 24.

[0040] In a particular embodiment, the W116 mutation is complemented by a D10C mutation. The thiol group of the cysteine ​​is then oxidized to sulfonic acid, for example by incubation of the FraC double mutant monomer with 10% (v / v) hydrogen peroxide. It has been found that the introduction of a sulfonic acid moiety at position 10 of FraC results in multimerized pores that exhibit less noisy signals in electrophysiological recordings compared to the noisier signals observed in nanopores that have not been subjected to the oxidative treatment. Thus, in one embodiment, the invention provides mutants that include a D10C substitution, preferably in combination with one or more of W112S, W116S, and D109S, more preferably in combination with at least W116S.

[0041] A further embodiment is a method for providing a system according to the present invention, comprising the steps of: providing a recombinant FraC monomer; contacting the monomers with the liposomes to assemble the monomers and the liposomes into multimers; recovering the multimers from the liposomes; contacting the multimer with a lipid bilayer (which may contain sphingomyelin) to allow formation of a FraC nanopore; Including, It concerns the method. In one embodiment, contacting with the lipid bilayer is performed at a pH of less than 4.5, preferably less than 4.0.

[0042] The method of the invention may further comprise isolating a fraction comprising a FraC nanopore in the type II state and / or a fraction comprising a FraC nanopore in the type III state. In one embodiment, the method of the invention comprises isolating the different multimeric forms of FraC using liquid chromatography methods, including size exclusion, affinity, reverse phase, or ion exchange chromatography.

[0043] The peptide mass detection FraC nanopore system of the present invention is advantageously incorporated into a real-time protein sequencing system, for which reason the system preferably includes one or more further modifications.

[0044] In one embodiment, a protease-unfoldase pair is directly bound above (i.e., on its cis side) the FraC nanopore. Then, the cleaved peptides are continuously recognized and translocated through the nanopore. For example, the barrel-shaped ATP-dependent ClpXP protease is an ideal candidate, as it encases the digested peptides and prevents their release into the solution. Another approach is based on the protein complex that constitutes the proteasome, or any other protease. For example, the complex includes the 20S α / β subunits of the proteasome and the 19S regulatory particle, of which the ATPase is the minimally required unit. However, other proteases could be used. The protease cleaves the polypeptide specifically (e.g., after positively or negatively charged residues or aromatic residues, etc.) or is engineered to cleave specifically or at a non-specific position within the polypeptide chain. The protease ideally encases the substrate and also allows for the docking of other components (eg, an unfoldase) to deliver the polypeptide to the unfoldase active site.

[0045] The attachment can be covalent or non-covalent, for example, attachment can be achieved by chemical bonding, by genetic fusion, or by the introduction of a binding loop into the FraC nanopore that can non-covalently interact with the peptide.

[0046] The inventors showed that peptides that enter the cis side of the nanopore are more likely to exit the nanopore into the trans chamber, thus preventing the double detection phenomenon. Furthermore, the inventors showed that at low pH, peptides can be captured by the nanopore at a fixed applied potential and their mass recognized, regardless of the chemical composition of the peptide. If such low pH values ​​are not compatible with enzyme activity, asymmetric solutions can be used on both sides of the nanopore (34, 35). In such a system, the conditions on the cis side can be adjusted to optimize the ATPase activity of the unfoldase-peptidase, while the pH and ionic strength on the trans side can be optimized to capture and recognize individual peptides.

[0047] The FraC nanopore or mutant FraC polypeptides provided herein are advantageously used for any type of analyte analysis, including peptide or DNA analysis, preferably peptide analysis including peptide mass detection and / or peptide sequencing. However, although the advantageous properties of the systems provided herein are illustrated in the context of peptide analysis, one skilled in the art will appreciate that the systems can be used in a variety of applications. Other applications of the invention include: Post-peptide modification detection (e.g. glycosylation), proteomics, High-precision DNA sequencing or post-DNA modification detection (e.g. methylation), Other small analyte detection or polymer analysis at higher resolution, Single molecule protein and DNA sequencing, Detection of peptides, biomarkers, antibiotics, and small molecules directly in human samples; Trapping proteins of different sizes for binding ligand analysis, such as glucose.

[0048] Mass spectrometry is a major contribution to the field of proteomics. Currently, nanopore systems do not reach the resolution of commercially available mass spectrometers. A peptide mass analyzer based on the FraC nanopore as described herein has unique advantages over conventional mass spectrometers, which are expensive, highly complex, bulky, and not single molecule. In contrast, nanopores can be integrated into portable and low-cost devices that contain hundreds of thousands of individual sensors. Thus, in one embodiment, the FraC nanopore system is integrated into a portable device that includes a plurality of individual FraC nanopore systems as described herein.

[0049] The electrical nature of the signal also allows for real-time sampling of biological samples. Moreover, because the nanopore reads individual molecules, the signal contains additional information that is not available in ensemble techniques. In particular, single molecule detection, especially when combined with high-throughput analysis, allows for the detection of low abundance peptides to unravel chemical heterogeneity in post-translational modifications, a problem that is difficult to solve with conventional mass spectrometry. For example, the present invention also provides the use of the FraC nanopore system or mutant FraC polypeptides in single molecule detection, preferably combined with high-throughput analysis. [Brief description of the drawings]

[0050] [Figure 1]Preparation and analysis of type I, type II, and type III FraC nanopores. a. Cutaway of the surface representation of WT-FraC multimers (PDB: 4TSY) (Non-Patent Document 25) color-coded based on the vacuum electrostatic potential calculated by Pymol. One protomer is shown in carton notation with tryptophans 112 and 116 depicted as spheres. b. Percentage distribution of types I, II, and III for WT-FraC, W112S-FraC, W116S-FraC, and W112S-W116S-FraC at pH 7.5 and 4.5. c. IV curves of type II nanopores formed by WT-FraC, W116S-FraC, and W112S-W116S-FraC at pH 7.5 (15 mM Tris-HCl, 1 M KCl). d. Single nanopore conductance of W116S-FraC in 1 M KCl (0.1 M citrate and 180 mM Tris base) at pH 4.5. e. Typical current traces for three nanopore types of W116S-FraC in 1 M KCl at pH 4.5 under an applied potential of -50 mV. f. Reversal potentials measured for three nanopore types of W116S-FraC under asymmetric conditions of KCl (1960 mM cis, 467 mM trans) at pH 4.5. Ion selectivities were determined by using the Goldman-Hodgkin-Katz equation (Eq. 1). g. Molecular models of the three FraC nanopores constructed from the FraC crystal structure using the symmetric docking function of Rosetta. Electrophysiological recordings were performed at a sampling frequency of 10 kHz and a filter frequency of 2 kHz. Error bars and color shading on IV curves are standard deviations determined from at least three replicates. [Diagram 2] Distribution of single-channel conductance of FraC nanopores at pH 7.5 and 4.5. a. The table reports the average conductance values ​​obtained by fitting a Gaussian function to the conductance histograms. SD represents the standard deviation over all single channels (number indicated by n). bf. Each panel represents a different batch of FraC nanopores as indicated. Single channels were collected under an applied potential of -50 mV. pH 7.5 and 4.5 were obtained using 1 M NaCl, 15 mM Tris, or 0.1 M citrate, 180 mM Tris base, respectively. [Diagram 3] Discrimination of angiotensin peptides in a mixture by type II W116S-Frac nanopore. a. (i) Sequences of angiotensin I, II, III, and IV shown with the corresponding Ires% measured at -30 mV. (ii) Typical block caused by four angiotensin peptides. (iii) Color density plot of Ires% versus standard deviation of current amplitude for angiotensin I added to the cis compartment, and color density plots after further addition of angiotensin II, angiotensin III, and angiotensin IV to the cis chamber are shown in (iv). b. Discrimination of angiotensin II from angiotensin A. (i) Table showing the sequence, molecular weight, and Ires% of the peptides. The peptides differ by one amino acid as indicated in red. (ii) Representative traces of peptide block. Color density plots of Ires% versus standard deviation of current amplitude before (iii) and after (iv) further addition of angiotensin A to the cis chamber for blockade of angiotensin II are shown. All measurements and recordings were performed in a pH 4.5 buffer containing 1 M KCl, 0.1 M citric acid, 180 mM Tris base at a sampling frequency of 50 kHz and a filter frequency of 10 kHz. Standard deviations were calculated from a minimum of three replicates. Color density plots were generated using Origin. [Figure 4] Evaluation of biological peptides with different chemical compositions. Relationship between peptide molecular weight and Ires% at pH 4.5 using (a) type I WT-FraC nanopore, (b) type II W116S-FraC nanopore, and (c) type III W112S-W116S-FraC nanopore. Solid lines represent second order polynomial fits. Current blockade was measured at -30 mV for type I and type II pores and at -50 mV for type III pores. Error bars and standard deviations are obtained from at least three measurements. [Diagram 5]Nanopore peptide mass spectrometry. a. Amino acid sequences of four different peptides and their overall changes at different pH. Charged amino acids are underlined. b. pH dependence of Ires% for the four peptides (cis) shown in a using type II W116S-FraC nanopore under an applied potential of -30 mV. c. Comparison of Ires% to peptide mass at pH 4.5 and 3.8. d. Voltage dependence of c-Myc residence time at different pH. All electrophysiological measurements were performed in 1 M KCl, 0.1 M citric acid, and the pH was adjusted to the desired value with 1 M Tris base. A sampling frequency of 50 kHz and a filter frequency of 10 kHz were used to collect the current events. Error bars and standard deviations were obtained from at least three measurements. Peptide changes were calculated based on the pKa for the individual amino acids (36). [Figure 6] Discrimination of a mixture of short peptides by a type III Frac nanopore containing mutant W112S-W116S-FraC. a. Sequence, Ires% (-50 mV), and MW for angiotensin IV, angiotensin 4-8, endomorphin I, and leucine-enkephalin. b. Typical blocks caused by different peptides. c. Color density plot of Ires% versus standard deviation of current block for a mixture of angiotensin IV, angiotensin 4-8, endomorphin I, and leucine-enkephalin. All measurements and recordings were performed in a pH 4.5 buffer containing 1 M KCl, 0.1 M citric acid, 180 mM Tris base at a sampling frequency of 50 kHz and a filter frequency of 10 kHz. Standard deviations were calculated from a minimum of three replicates. [Figure 7] Analysis of a type II FraC nanopore containing an oxidized cysteine ​​at position 10. Differences between the D10C / W116S type II pore (panel A) and the oxidized D10C / W116S type II pore (panel B) are shown. Recordings were made in a buffer containing 1 M NaCl, pH 7.5, ±50 mV. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] <Experimental Section> Materials and Methods (Chemicals) Endothelin 1 (≧97%, CAS#117399-94-7), endothelin 2 (≧97%, CAS#123562-20-9), dynorphin A (porcine) (≧95%, CAS#80448-90-4), angiotensin I (≧90%, CAS#70937-97-2), angiotensin II (≧93%, CAS#4474-91-3), c-Myc 410-419 (≧97%, #M2435), Asn1-Val5-angiotensin II (≧97%, CAS#20071-00-5), Ile7-angiotensin III (≧95%, #A0911), leucine enkephalin (≧95%, #L9133), 5-methionine enkephalin (≧95%, CAS#82362-17-2), endomorphin I (≧95%, CAS#189388-22-5), pentane (≧99%, CA S#109-66-0), hexadecane (99%, CAS#544-76-3), Trizma® hydrochloride (≧99%, CAS#1185-53-1), Trizma® base (≧99%, CAS#77-86-1), potassium chloride (≧99%, CAS#7447-40-7), and N,N-dimethyldodecylamine N-oxide (LADO, ≧99%, CAS#1643-20-5) were obtained from Sigma-Aldrich. The precursors angiotensin 1-14 (≧97%, #002-45), angiotensin 1-9 (≧95%, #002-02), angiotensin A (≧95%, #002-36), angiotensin III (≧95%, #002-31), and angiotensin IV (≧95%, #002-28) were obtained from Pheonix Pharmaceuticals. Angiotensin 4-8 (≧95%) was synthesized by BIOMATIK. 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, #850356P) and sphingomyelin (Porcine brain, #860062) were purchased from Avanti Polar Lipids. Citric acid (99.6%, CAS#77-92-9) was obtained from ACROS. n-Dodecyl-β-D-maltoside (DDM, ≥99.5%, CAS#69227-93-6) was purchased from Glycon Biochemical EmbH.DNA primers were synthesized by Integrated DNA Technologies (IDT) and enzymes were from Thermo scientific. All peptides were dissolved in Milli-Q water without further purification and stored in a freezer at -20°C. In this study, a pH 7.5 buffer solution containing 15 mM Tris was prepared by dissolving 1.902 g Trizma® HCl and 0.354 g Trizma® Base in 1 liter Milli-Q water (Millipore).

[0052] (Expression and purification of FraC monomer) The FraC gene, containing NcoI and HindIII restriction enzyme sites at the 5' and 3' ends, respectively, and a sequence encoding a polyhistidine tag at the 3' end, was cloned into the pT7-SC1 plasmid. The plasmid was transformed into BL21(DE3) E. cloni® competent cells by electroporation. The cells were grown overnight at 37°C on LB agar plates containing 100 μg / mL ampicillin. The entire plate was then harvested and plated on 200 mL of fresh 2YT medium, and the culture was grown at 37°C with shaking at 220 rpm until the optical density at 600 nm of the cell culture was 0.8. Then, 0.5 mM IPTG was added to the medium, and the culture was transferred to 25°C and grown overnight with shaking at 220 rpm. The next day, the cells were centrifuged (2000×g, 30 min) and the pellet was stored at −80°C. FraC monomer was purified using cell pellets harvested from 100 mL of culture medium. 30 mL of cell lysis buffer (150 mM NaCl, 15 mM Tris, 1 mM MgCl2, 4 M urea, 0.2 mg / mL lysozyme, and 0.05 units / mL DNase) was added, the pellet was resuspended, and vigorously mixed for 1 h. The cell lysate was then sonicated for 2 min in a Branson Sonifier 450 (duty cycle 10%, output control 3). The crude lysate was then centrifuged (5400 × g) for 30 min at 4 °C, and the supernatant was incubated with 100 μL of Ni-NTA beads (Qiagen) for 1 h with gentle shaking. The beads were spun down and loaded onto a Micro Bio-spin column (Bio-rad). The beads were washed with 10 mL of SDEX buffer (150 mM NaCl, 15 mM Tris, pH 7.5) containing 20 mM imidazole, and the protein was eluted with 150 μL of elution buffer (SDEX buffer, 300 mM imidazole). The protein concentration was measured by measuring the absorbance at 280 nm using Nano-drop 2000 (Thermo Scientific) with the elution buffer as a blank.To further confirm the purity of the monomer, monomeric FraC was diluted to 0.5 mg / mL in elution buffer and 9 μL of the diluted sample was mixed with 3 μL of 4× loading buffer (250 mM Tris HCl, pH 6.8, 8% SDS, 0.01% bromophenol blue, and 40% glycerol) and then loaded onto a 12% SDS-PAGE gel. The gel was subjected to a constant applied current of 35 mA for 30 min and stained with Coomassie dye (InstantBlue™, Expdedeon) for >1 h before being viewed using a gel imager (Gel Doc™, Bio-rad).

[0053] >FraC|B9W5G6(amino acid sequence) SADVAGAVIDGAGLGFDVLKTVLEALGNVKRKIAVGIDNESGKTWTAMNTYFRSGTSDIVLPHKVAHGKALLYNGQKNRGPVATGVVGVIAYSMSDGNTLAVLFSVPYDYNWYSNWWNVRVYKGQKRADQRMYEELYYHRSPFRGDNGWHSRGLGYGLKSRGFMNSSGHAILEIHVTKA

[0054] >6xHis-WtFraC (amino acid sequence), as used in the present invention The bolded residues indicate the N- and C-terminal residues that were added to the original sequence. MA SADVAGAVIDGAGLGFDVLKTVLEALGNVKRKIAVGIDNESGKTWTAMNTYFRSGTSDIVLPHKVAHGKALLYNGQKNRGPVATGVVGVIAYSMSDGNTLAVLFSVPYDYNWYSNWWNVRVYKGQKRADQRMYEELYYHRSPFRGDNGWHSRGLGYGLKSRGFMNSSGHAILEIHVTKAGSAHHHHHH

[0055] >6xHis-WtFraC (DNA sequence) ATGGCGAGCGCCGATGTCGCGGGTGCGGTAATCGACGGTGCGGGTCTGGGCTTTGACGTACTGAAAACCGTGCTGGAGGCCCTGGGCAACGTTAAACGCAAAATTGCGGTAGGGATTGATAACGAATCGGGCAAGACCTGGACAG CGATGAATACCTATTTCCGTTCTGGTACGAGTGATATTGTGCTCCCACATAAGGTGGCGCATGGTAAGGCGCTGCTGTATAACGGTCAAAAAAATCGCGGTCCTGTCGCGACCGGCGTAGTGGGTGTGATTGCCTATAGTATGTC TGATGGGAACACACTGGCGGTACTGTTCTCCGTGCCGTACGATTATAATTGGTATAGCAATTGGTGGAACGTGCGTGTCTACAAAGGCCAGAAGCGTGCCGATCAGCGCATGTACGAGGAGCTGTACTATCATCGCTCGCCGTTT CGCGGCGACAACGGTTGGCATTCCCGGGGCTTAGGTTATGGACTCAAAGTCGCGGCTTTATGAATAGTTCGGGCCACGCAATCCTGGAGATTCACGTTACCAAAGCAGGCTCTGCGCATCATCACCACCATCACTGATAAGCTT

[0056] (FraC mutant preparation) FraC mutants were prepared according to the MEGAWHOP method: 25 μL of REDTaq® ReadyMix™ was mixed with 4 μM of the primers containing the desired mutations (Table 1) and 50 ng of plasmid (pT7-SC1 carrying the wild-type FraC gene) as template, and the final volume was brought to 50 μL with Milli-Q water.

[0057] Table 1: Primer sequences used in this study to prepare FraC mutants. [Table 1]

[0058] The PCR protocol consisted of an initial denaturation step at 95°C for 150 seconds, followed by 30 cycles of denaturation (95°C, 15 seconds), annealing (55°C, 15 seconds), and extension (72°C, 60 seconds). The PCR products (MEGA primers) were pooled and purified with a QIAquick PCR purification kit to a final DNA concentration of approximately 200 ng / μL. A second PCR was performed for total plasmid amplification. 2 μL MEGA primer, 1 μL Phire II enzyme, 10 μL 5× Phire buffer, and 1 μL 10 mM dNTPs were mixed with PCR water to a final volume of 50 μL. PCR began with a preincubation at 98°C (30 seconds), followed by 25 cycles of denaturation (98°C, 5 seconds), annealing (72°C, 90 seconds), and extension (72°C, 150 seconds). After PCR was completed, 1 μL of DpnI enzyme was added and the mixture was kept at 37° C. for 1 hour. The temperature was then raised to 65° C. for 1 minute to inactivate the enzyme. The products were then transformed into competent E. cloni® 10G cells (Lucigen) by electroporation. The cells were plated on LB agar plates containing 100 μg / mL ampicillin and grown overnight at 37° C. Single clones were expanded and sent for sequencing.

[0059] (Sphingomyelin-DPhPC liposome preparation) 20 mg of sphingomyelin and 20 mg of DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) were dissolved in 4 mL of pentane containing 0.5% v / v ethanol and placed in a 50 mL round-bottom flask. The solvent was then evaporated by rotation and using a hair dryer to warm the flask. After evaporation, the flask was kept at ambient temperature for an additional 30 min. The lipid film was resuspended in 4 mL of SDEX buffer (150 mM NaCl, 15 mM Tris, pH 7.5) and the solution was placed in a sonication bath for 5 min. The liposome suspension was stored at -20 °C.

[0060] (FraC oligomerization) FraC monomer was incubated with sphingomyelin-DPhPC liposomes to induce FraC multimerization. Frozen liposomes were thawed and sonicated in a water bath for 1 min. FraC monomer was diluted to 1 mg / mL using SDEX buffer, and then 50 μL of FraC monomer was added to 50 μL of 10 mg / mL liposome solution, resulting in a mass ratio of 10:1 (liposome:protein). The lipoprotein solution was incubated at 37 °C for 30 min to allow multimerization. Then, 10 μL of 5% (w / v, final concentration 0.5%) LADO was added to the lipoprotein solution to solubilize the liposomes. After clarification (usually 1 min), the solution was transferred to a 50 mL Falcon tube. Then, 10 mL of SDEX buffer containing 0.02% DDM and 100 μL of pre-washed Ni-NTA beads were added to the Falcon tube and gently mixed on a shaker for 1 h at room temperature. The beads were then centrifuged and loaded onto a Micro Bio-spin column. The beads were washed with 10 mL of washing buffer (150 mM NaCl, 15 mM Tris, 20 mM imidazole, 0.02% DDM, pH 7.5), and the multimer was eluted with 100 μL of elution buffer (usually 200 mM EDTA, 75 mM NaCl, 7.5 mM Tris, pH 7.5, 0.02% DDM). The FraC multimer was stored at 4°C. Under these conditions, the nanopore is stable for several months.

[0061] (W112S-W116S-FraC multimer separation by His-Trap chromatography) 200 μL of W112S-W116S-FraC monomer (3 mg / mL) was incubated with 300 μL of sphingomyelin-DPhPC liposomes (10 mg / mL) and kept at 4 °C for 48 h, after which 0.5% LADO (final concentration) was added to solubilize the lipoproteins. The buffer was then exchanged into 500 mM NaCl, 15 mM Tris, 0.01% DDM, 30 mM imidazole, pH 7.5 (binding buffer) using a PD SpinTrap G-25 column. The W112S-W116S-FraC multimer was then loaded onto a Histrap HP 1 mL column (General Electric) using an A¨KTA pure FPLC system (General Electric). The loaded multimer was washed with 10 column volumes of 500 mM NaCl, 15 mM Tris, 0.01% DDM, 30 mM imidazole, pH 7.5 before being subjected to an imidazole gradient (30 mM to 1 M imidazole, 500 mM NaCl, 15 mM Tris, 0.01% DDM, pH 7.5) over 30 column volumes. The signal was monitored by absorbance at 280 nm and fractions were collected when the absorbance exceeded 5 mAu.

[0062] (Electrophysiological measurements and data analysis) Electrical recordings were performed as previously described (Non-Patent Documents 27, 37). O (refers to the open pore current) was measured by fitting a Gaussian function to the event amplitude histogram. The residual current value (Ires%) was calculated as the blocking current (I B ) divided by the open pore current (I B / I O × 100%). Residence times and inter-event times were determined by fitting single exponential functions to the cumulative distribution histograms.

[0063] (Ion permeability measurement) To measure the reversal potential, a single channel was acquired under symmetrical conditions (840 mM KCl, 500 μL in each electrophysiological chamber) and the electrodes were balanced. 400 μL of 3.36 M KCl buffer stock was slowly added to the cis chamber, while 400 μL of salt-free buffer was added to the trans chamber to a total volume of 900 μL (trans:cis, 467 mM KCl:1960 mM KCl). After equilibration, IV curves were collected from -30 to +30 mV. The resulting voltage at zero current was defined as the reversal potential (V r ) and then ion selectivity (P K+ / P Cl- ) was calculated using the Goldman-Hodgkin-Katz equation (Equation 1). In the equation, [a k+ / cl- ] cis / trans is the K in the cis or trans compartment + Or Cl - where R is the gas constant, T is the temperature, and F is the Faraday constant.

number

[0064] (Molecular models of types I, II, and III FraC nanopores) 3D models with different numbers of multimerization ranging from 5 to 9 monomers were constructed by the symmetrical docking function of Rosetta (38). Lipid-free monomers were extracted from the crystal structure of FraC with lipids (PDB_ID 4tsy). The monomers were arranged around a central rotation axis by symmetrical docking, with numbers from 5 to 9. In total, Rosetta generated and stored 10,000 copies of each symmetry. In all cases, multimer configurations with symmetries similar to the aforementioned crystal structures could be identified as the best-scoring solutions. However, the pentameric assemblies remained largely exposed and the multimer interfaces were not completely satisfactory compared to the aforementioned crystal structures. On the other hand, the 9-fold symmetric model showed a significant decrease in Rosetta scores compared to the 6-, 7-, and 8-fold symmetric models, which showed unfavorable assemblies of the nonameric assemblies with the 6-, 7-, and 8-fold assemblies as the most likely. To generate lipid-bound models, the aforementioned crystal structures with lipids were superimposed onto each monomer of the generated model, and lipid coordinates were transferred. Residues within 4.5 Å of the lipid were minimized with the Amber10 forcefield.

[0065] Example 1: Altering the size of the FraC nanopore One of the main challenges in biological nanopore analysis is obtaining nanopores of different sizes and shapes. The majority of biological nanopores are formed by multiple repeats of individual monomers. Thus, different nanopore sizes may be obtained by modifying the protein multimer composition (28). We noticed that at pH 7.5, a minor fraction of wild-type FraC (WT-FraC) nanopores (1.26 ± 0.08 nS, -50 mV, WT-FraC type II, Fig. 1b) showed a lower conductance than the major fraction (2.26 ± 0.08 nS, -50 mV, WT-FraC type I), suggesting that FraC may spontaneously assemble into nanopores with smaller sizes. At pH 4.5, an even lower nanopore conductance was observed (0.42 ± 0.03 nS, WT-FraC type III, -50 mV, Fig. 1b). The inventors noticed that the reconstitution of lower conductance nanopores depended on several purification conditions: in particular, they observed that the incidence of type II and type III nanopores increased when the multimers were stored in solution for several weeks or when the concentration of monomeric WT-FraC was reduced during multimerization.

[0066] In an attempt to increase the number of types II and III of FraC nanopores, we weakened the interaction between the nanopore and the lipid interface by substituting W112 and W116 (Fig. 1a) at the lipid interface of FraC with serine. We assumed that lowering the concentration of monomers during liposome-induced multimerization would increase the population of multimers with lower molecular mass. Surprisingly, we found that types II and III of FraC nanopores were the predominant species at pH 4.5 with multimers of W116S-FraC and W112S-W116S-FraC, respectively (Fig. 1b, Fig. 2). Conveniently, the different nanopore types could be separated by Ni-NTA affinity chromatography with an imidazole gradient. Furthermore, we found that an increase in types II and III of FraC nanopores could also be obtained at pH 7.5 by substituting aspartic acid 109 at the lipid interface with serine (Fig. 2e, see Table 2).

[0067] Table 2: Relative amounts of types I, II, and III for each of the FraC nanopores examined at neutral and acidic pH. [Table 2]

[0068] Within the same type of FraC nanopore, lipid interface modifications resulting from W112S and W116S substitutions did not alter conductance and ion selectivity compared to the wild type (Fig. 1c, Fig. 2, Table 3), suggesting that the modifications did not change the overall folding of the nanopore. When analyzed within lipid bilayers, type I, type II, and type III nanopores showed a clear single conductance distribution and steady-state open pore current (Fig. 1d-e). Interestingly, type I, type II, and type III nanopores showed increasing cation selectivity (from 2.0 for type I to 4.2 for type III at pH 4.5, W116S-FraC) (Fig. 1f, Table 3), likely reflecting a greater overlap of the electrical double layer in nanopores with narrower constrictions.

[0069] Table 3: Ion selectivity of different FraC pores at pH 7.5 and 4.5 [Table 3] Ion selectivity (P K+ / P Cl- ) was calculated from the reversal potential according to the Goldman-Hodgkin-Katz equation:

number

[0070] These findings strongly suggest that the three types of FraC nanopores represent nanopores with distinct protomer compositions. Molecular models allow us to predict the diameters of the types II (1.1 nm) and III (0.8 nm) nanopores (Fig. 1g), revealing that type III FraC is likely to be the biological nanopore with the smallest constriction known to date.

[0071] Example 2: Identification of peptides containing single amino acid substitutions using type II or type III FraC nanopores as sensors A series of angiotensin peptides, which regulate blood pressure and fluid balance in the blood, were sampled using the type II FraC nanopore. The peptides were added to the cis side of the type II W116S-FraC nanopore and induced ionic current blockade (I B ) was measured. The residual current percentage (Ires%, I B / I O × 100) was used instead of current block. The results are shown in Figure 3a and Table 4.

[0072] Angiotensin I (DRVYIHPFHL, 1296.5 Da) showed the deepest block (Ires%=8.8±0.2), whereas angiotensin IV (VYIHPF, 774.9 Da) showed the shallowest block (Ires%=38.9±4.0). Residual currents of angiotensin II (DRVYIHPF, 1046.2 Da, Ires%=17.9±1.3) and angiotensin III (RVYIHPF, 931.1 Da, Ires%=22.1±0.5) were intermediate. When the four peptides were tested simultaneously, the individual peptides were easily distinguishable (Fig. 3a).

[0073] Table 4: Peptide analysis using different types of FraC nanopores at pH 4.5. The electrophysiological solution contained 1 M KCl, 0.1 M citric acid, 180 mM Tris base at pH 4.5. Recordings were performed with a sampling frequency of 50 kHz and a Bessel filter of 10 kHz. Standard deviations were obtained from at least three measurements. Charges of peptides were calculated based on the pKa for individual amino acids (36). [Table 4]

[0074] The resolution limit of the nanopore sensor was tested by sampling a mixture of peptides. Notably, angiotensin II and angiotensin A, which are identical in composition except for the first amino acid (aspartic acid in angiotensin II and alanine in angiotensin A), showed distinctive peaks in the Ires% plot (Figure 3b). Smaller differences in peptide mass, for example the 34 Da difference between phenylalanine in angiotensin III and leucine in Ile7 angiotensin III, were observed but not easily detectable (not shown), indicating that the resolution of our system is about 40 Da. Smaller peptides, such as angiotensin II4-8 (YIHPF, 675.8 Da), endomorphin I (YPWF, 610.7 Da), or leucine enkephalin (YGGFL, 555.6 Da), pass through the type II W116S-FraC nanopore too quickly to be sampled. On the other hand, these could be easily measured using the type III W112S-W116S-FraC nanopore (Table 4, Figure 6).

[0075] Example 3: Nanopore mass spectrometer for peptides Although the ability of biological nanopores to distinguish between known analytes is useful, a more compelling application would be the identification of peptide masses directly from ion current intercepts without prior knowledge of the analyte's identity. To evaluate the FraC nanopore as a peptide mass analyzer, additional peptides were tested at pH 4.5, 1 M KCl using FraC nanopores type I, type II, and type III (Figure 4a-c, Table 4). Importantly, analytes with widely differing charge compositions were included.

[0076] We found a direct correlation between size and residual current for most of the peptides (Fig. 4a-c). A notable exception was c-Myc 410-419 (1203.3 Da), a peptide that was purposely selected because it contains a long stretch of negatively charged residues (Fig. 5a). The overall negative charge of this peptide at pH 4.5 (see Table 4) was likely to affect both peptide capture and recognition. c-Myc 410-419 was readily captured at negative applied potentials (trans), indicating that cis-to-trans electroosmotic flow through the nanopore can overcome the electrostatic energy barrier that opposes peptide capture. On the other hand, the Ires% of c-Myc 410-419 (30.0 ± 3.4) was significantly higher than expected (Fig. 4b).

[0077] We speculated that these anomalies were due to interactions between the acidic amino acids of the peptide and the negatively charged constriction of the FraC nanopore. We then lowered the pH of the solution to a value where the aspartic and glutamic acid side chains of the peptide were protonated and neutral (Fig. 5a). As expected, at pH 3.8, the signal corresponding to c-Myc 410-419 (1203.3 Da) fell between the signals of angiotensin I (1296.5 Da) and angiotensin II (1046.2 Da) (Fig. 5b, c). This indicated that after losing the negative charge, the blockade of the peptide increased or decreased depending on its expected mass.

[0078] average dwell time(τ off It has been speculated (1, 30, 31) and demonstrated (32) that the voltage dependence of τ can inform the translocation of molecules through a nanopore. Under a negative bias (trans) for a positively charged peptide (applied in cis), both the electrophoretic and electroosmotic forces (from cis to trans) drive the entry and translocation through the nanopore (27). For negatively charged peptides (such as c-Myc 410-419 at pH 4.5 (Figure 5a)), the electroosmotic driving force must be stronger than the electrophoretic force that opposes it. τ offThe voltage dependence of τ was examined for c-Myc 410-419 at different pH values ​​(Figure 5d). At pH 4.5, the peptide exhibited a maximum τ at -50 mV. off This suggests that at low potentials (<50 mV), c-Myc 410-419 returns to the cis chamber, whereas at high potentials (>50 mV), c-Myc 410-419 escapes to the trans chamber. At pH 3.8 or lower, the higher the potential, the greater the τ off There was a decrease in the expression of c-Myc 410-419 across the membrane and into the trans chamber.

[0079] As shown in Figure 6, the type III FraC nanopore can discriminate peptide lengths of up to four amino acids (masses of approximately 500 Da) in a peptide mixture. We also found that the residual current signal correlates well with the peptide mass, suggesting that type III can be used as a detector for peptides with masses up to approximately 500 Da.

[0080] Example 4: Peptide mass identifier at pH 3 This example shows that the D10C mutation can be used as an additional mutation to obtain a FraC pore that exhibits a less noisy signal in electrophysiological recordings.

[0081] Using W116S as an exemplary mutant, the aspartic acid at position 10 of FraC was converted to a cysteine ​​by site-directed mutagenesis. The thiol group of the cysteine ​​was then oxidized to a sulfonic acid by incubating the FraC monomer with 10% hydrogen peroxide (v / v) in a standard buffer (e.g., 10 mM Tris buffer, pH 7.5, 150 mM NaCl, etc.). As a control, a double mutant that was not oxidized was used.

[0082] D10C / W116SFraC was multimerized and the multimers were tested in electrical recordings. Figure 7 shows a comparison of traces between D10C / W116S and oxidized D10C / W116S pores to show the differences after oxidation. Multimerized pores consisting of oxidized D10C / W116SFraC monomers showed a less noisy signal in electrophysiological recordings compared to the noisier signal observed in nanopores that were not subjected to oxidation.

[0083] [Note] [Appendix 1] A system comprising multimeric Fragaceatoxin C (FraC) nanopores contained within a lipid bilayer, wherein the sum of the percentage of nanopores in the heptameric (type II) state and the percentage of nanopores in the hexameric (type III) state represents at least 60% of the total number of said FraC nanopores.

[0084] [Appendix 2] The system of claim 1, wherein the sum of type II and type III state nanopores represents at least 65%, preferably at least 70%, of the total number of FraC nanopores.

[0085] [Appendix 3] The system of claim 1 or 2, wherein at least 60%, preferably at least 70%, of the FraC nanopores are in type II state.

[0086] [Appendix 4] The system of claim 1 or 2, wherein at least 60%, preferably at least 70%, of the FraC nanopores are in type III state.

[0087] [Appendix 5] 5. The system of any one of claims 1 to 4, wherein the FraC nanopore comprises a mutant FraC monomer comprising a mutation at positions W112 and / or W116.

[0088] [Appendix 6] The system of claim 5, wherein the one or more mutations include replacement of W with S, T, A, N, Q, or G, preferably with S or T.

[0089] [Appendix 7] The system of any one of claims 1 to 6, wherein the FraC nanopore comprises a mutant FraC monomer comprising a mutation at position D109, preferably wherein the mutation comprises a substitution of D with S or T, preferably with S.

[0090] [Appendix 8] 8. The system of any one of claims 5 to 7, wherein the mutant FraC monomer comprises a W112S, W116S, and / or D109S mutation, preferably the FraC mutant is W112S / W116S / W116S or D109S / W116S.

[0091] [Appendix 9] A system described in any one of appendix 1 to 8, wherein the system has the function of detecting properties of the analyte by applying an electric field to the nanopore so that the analyte moves electrophoretically and / or electroosmotically through the nanopore.

[0092] [Appendix 10] 10. The system of claim 9, wherein the analyte is a proteinaceous substance, preferably a peptide, more preferably a peptide up to about 30 amino acids in length.

[0093] [Appendix 11] A method for providing a system according to any one of claims 1 to 10, comprising the steps of: (i) providing a plurality of recombinant FraC monomers; (ii) contacting the monomer with the liposome to assemble the monomer and the liposome into multimers; (iii) recovering the multimers from the liposomes; (iv) contacting the multimer with a lipid bilayer to allow formation of a FraC nanopore; Including, Optionally, (v) further comprising isolating a fraction comprising a FraC nanopore in the type II state and / or a fraction comprising a FraC nanopore in the type III state; method.

[0094] [Appendix 12] A mutant Fragaceatoxin C (FraC) polypeptide comprising a mutation at position W112 and / or W116, where the numbering corresponds to the FraC amino acid sequence available in UniProt under accession number B9W5G6, and one or more of the W residues are independently substituted with S, T, A, N, Q, or G.

[0095] [Appendix 13] 1. A mutant Fragaceatoxin C (FraC) polypeptide comprising a mutation at position D109, where the numbering corresponds to the FraC amino acid sequence available in UniProt under accession number B9W5G6, and where the D residue has been replaced with an uncharged residue, preferably with S or T, more preferably with S.

[0096] [Appendix 14] 14. A mutant FraC polypeptide according to claim 12 or 13, comprising a W112S, W116S, and / or D109S mutation, preferably the mutant FraC is W112S / W116S or D109S / W116S.

[0097] [Appendix 15] 15. The mutant FraC polypeptide of any one of appendices 12 to 14, further comprising a D10C mutation.

[0098] [Appendix 16] 16. An isolated nucleic acid molecule encoding a mutant FraC polypeptide according to any one of claims 12 to 15.

[0099] [Appendix 17] 17. An expression vector comprising the isolated nucleic acid molecule of claim 16.

[0100] [Appendix 18] A host cell comprising the expression vector of appendix 17.

[0101] [Appendix 19] Use of a system according to any one of appendices 1 to 10 or a mutant FraC polypeptide according to any one of appendices 12 to 15 in peptide analysis, preferably including peptide mass detection and / or peptide sequencing.

[0102] [Appendix 20] Use of a system according to any one of appendices 1 to 10 or a mutant FraC polypeptide according to any one of appendices 12 to 15 in single molecule detection, preferably combined with high throughput analysis.

[0103] [Appendix 21] 21. The use of claim 19 or 20, wherein the system is integrated into a portable device comprising a plurality of the individual systems of any one of claims 1 to 10.

Claims

[Claim 1] A system comprising multimeric Fragaceatoxin C (FraC) nanopores contained within a lipid bilayer, wherein the sum of the percentage of nanopores in a heptameric (type II) state and the percentage of nanopores in a hexameric (type III) state represents at least 60% of the total number of said FraC nanopores.

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