Frac actinoporin-based biological nanopore for biopolymer sensing and sequencing

Abstract

To provide a system including novel nanopores having different structures and recognition sites that improve sequencing accuracy and / or provide different error profiles.SOLUTION: Provided are systems relating to the field of nanopores and their use in various applications, e.g., analysis of biopolymers and macromolecules, typically by making electrical measurements during translocation through the nanopore, comprising a funnel-shaped proteinaceous nanopore comprising alpha-helical pore-forming toxins that are members from the actinoporin family of proteins, more particularly Fraga Ceatoxin C (FraC), a variant FraC, a FraC paralog or a FraC homolog.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates generally to the field of nanopores and their use in various applications, such as the analysis of biopolymers and macromolecules, typically by making electrical measurements during translocation through the nanopore. [Background technology]

[0002] Nanopores represent an attractive method for analyzing biopolymers, for example, to determine the identity of polypeptides or polynucleotides or to infer the identity of individual building blocks in a polymer for sequencing, because the method is label-free, provides measurements that rely on few or even single molecules, and generates a highly scalable electrical signal.

[0003] In nanopore-based measurement systems, some property of the system depends on the nucleotides in the nanopore and is measured electrically. For example, a measurement system can be created by placing a nanopore in a membrane and measuring voltage-driven ionic flow through the nanopore in the presence of nucleotides of a polynucleotide.

[0004] Nanopores have emerged as a powerful approach for single-molecule monitoring of chemical and enzymatic reactions, protein detection, and nucleic acid sequencing [1], [2]. Phi29 [3] and ClyA [4] have been shown to enable the translocation of double-stranded DNA. Recently, aerolysin has been used to discriminate homopolymers of adenine but of different lengths [5]. To date, only αHL and MspA have been shown to discriminate nucleic acids. Both nanopores contain β-sheets in their transmembrane regions. When DNA passes through MspA, the level of blocked current is affected by four or more nucleotides [6], whereas αHL has three sensing zones in its barrel-shaped structure that accommodate approximately 20 nucleobases [7]. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides novel nanopores with different structures and recognition sites that improve sequencing accuracy and / or provide different error profiles. Herein, we describe the purification and pre-oligomerization of the α-helical pore-forming toxin Fragaceatoxin C (FraC), a member of the actinoporin protein family, complexed with sphingomyelin and its reconstitution into planar lipid bilayers composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). We further engineered a FraC mutant (e.g., ReFraC) that enables the capture and translocation of ssDNA and discriminates between homopolymers of adenine, thymine, and cytosine immobilized with neutravidin (NA). In particular, dsDNA can be translocated through FraC, most likely via the elastically deformed α-helical constriction of the nanopore.

[0006] Remarkably, the FraC nanopore was found to have an ideal geometry for protein sequencing and analysis of folded proteins. It is shown herein below that electroosmotic flow is the dominant force guiding the entry of proteins and polypeptides into the nanopore. By tailoring the nanopore's inner surface, either by precisely manipulating the nanopore constriction or by changing the solution pH, we were able to observe the translocation of both positively and negatively charged polypeptides. This is remarkable, since it has been shown that it is possible to induce electroosmotic flow strong enough to transport both positive and negative residues at a fixed applied potential. Remarkably, we found that a series of (unfolded) proteins of different sizes, e.g., ranging from 1.2 to 25 kDa, could be distinguished based on their individual blockages. Using a 20-amino acid model polypeptide, we demonstrated that even differences in a single amino acid residue could be observed by nanopore recording, indicating that the FraC nanopore enables the identification of specific sequence features in translocating polypeptides.

[0007] Furthermore, we devised a method to reconstitute a pre-oligomerized FraC nanopore in a sphingomyelin-free planar lipid bilayer. The ReFraC nanopore was engineered to enable electrophoretic DNA capture and demonstrated discrimination between ssDNA homopolymers. In contrast to other nanopores used for DNA sequencing (e.g., αHL and MspA), FraC possesses an α-helical V-shaped transmembrane region, which is advantageous for fine-tuning nucleobase discrimination. This is because amino acid substitutions at different positions within the transmembrane α-helix can modulate both the size and chemical composition of the constriction.

[0008] ReFraC is also shown herein to induce the unzipping of DNA duplexes at low applied potentials or to enable their translocation at high applied potentials. The unzipping of DNA hairpins or higher-order DNA structures has been investigated using the αHL nanopore

[20] ,

[21] . However, the cis vestibule of ReFraC (5.5 nm) is wider than that of αHL (2.6 nm)

[13] or MspA (4.8 nm)

[22] , indicating that ReFraC can be advantageously used to study larger, higher-order dsDNA structures, such as G-quadruplexes, or folded RNA structures, such as tRNA. [Means for solving the problem]

[0009] Thus, in a first aspect, the present invention provides a system comprising a funnel-shaped proteinaceous nanopore containing an α-helical pore-forming toxin that is a member of the actinoporin protein family. More specifically, the α-helical pore-forming toxin is Fragaceatoxin C (FraC), a mutant FraC, a FraC paralog, or a FraC homolog. FraC can be fused, preferably at its C-terminus, to a protein affinity tag such as a His tag or a Strep tag.

[0010] Very good results can be obtained using mutant FraC. For example, the mutant FraC contains at least one substitution of a negatively charged amino acid residue in the narrow part of the pore with a neutral or positively charged amino acid residue, and / or at least one substitution of a neutral amino acid residue in the narrow part of the pore with a positively charged amino acid residue. For example, the mutant contains at least one mutation in a transmembrane helix. Preferably, at least one negatively charged amino acid residue is changed to a positively charged amino acid residue. In a preferred embodiment, the mutant FraC contains a mutation at position 10, preferably the mutation AsplOArg or AsplOLys (residue numbering similar to that in the crystal structure PDB ID 4TSY). The mutant FraC may further comprise one or more compensatory mutations that restore the hemolytic activity of FraC, preferably at positions 2, 9, 34, 52, 112, 150, 153, and / or 159, and / or preferably at position 159. For example, the compensatory mutations are selected from the group consisting of A2S, I9T, A34V, F52Y, W112L, T150I, G153D, K159E, I171T, and any combination thereof. In a specific embodiment, the mutant FraC (further) comprises a mutation at position 159, preferably Lys159Glu. For example, the FraC double mutant D10R / K159E is used.

[0011] The nanopore can be located between a first liquid medium and a second liquid medium, at least one of the liquid media containing an analyte, and the system is operative to detect a property of the analyte.

[0012] In one embodiment, the system operates to migrate the analyte through the tunnel. In another embodiment, the system operates to detect a property of an analyte and includes subjecting the nanopore to an electric field such that the analyte interacts with the nanopore. An applied potential (creating an electric field) is necessary to have an electric current. The electric current is the output signal. For example, the system operates to detect a property of an analyte and includes subjecting the nanopore to an electric field such that the analyte translocates through the nanopore by electrophoresis and / or electroosmosis and / or is trapped in the nanopore. The property can be an electrical, chemical, or physical property of the analyte.

[0013] Preferably, the nanopore is comprised in a (planar) lipid bilayer. In a specific embodiment, the lipid bilayer comprises or consists of phosphatidylcholine (PC), preferably 1,2-diphytanoyl-sn-glycero-3-phosphocholine.

[0014] In a second aspect, the present invention provides a method for providing a system according to the present invention, comprising the steps of: providing a recombinant monomer of said α-helical pore-forming toxin from the actinoporin protein family; contacting the monomers with liposomes to assemble the monomers into oligomers; recovering the oligomers from the liposomes; and contacting the oligomer with a lipid bilayer, which may contain sphingomyelin, to allow formation of a nanopore; The present invention provides a method comprising:

[0015] In a third aspect, the present invention provides a method comprising applying an electric field to the system disclosed herein, wherein a funnel-shaped nanopore containing an α-helical pore-forming toxin is positioned between a first conductive liquid medium and a second conductive liquid medium. At least one of the conductive liquid media may contain an analyte. The method may further comprise detecting the analyte in a manner comprising measuring ionic current as the analyte interacts with the nanopore to obtain a current pattern, wherein the occurrence of a block in the current pattern indicates the presence of the analyte. The method may further comprise identifying the analyte, for example, by comparing the current pattern with known current patterns obtained under the same conditions using a known analyte. The analyte may be a nucleotide, nucleic acid, amino acid, peptide, protein, polymer, drug, ion, pollutant, nanoscopic object, or biological warfare agent.

[0016] In one embodiment, the analyte is a polymer, such as a protein, peptide, or nucleic acid. Preferably, the analyte is a nucleic acid, such as ssDNA, dsDNA, RNA, or a combination thereof. In another preferred embodiment, the analyte is a protein, polypeptide, or oligopeptide, for example, having a size ranging from about 1 to about 40 kDa, preferably from about 1 to about 30 kDa. In one embodiment, the analyte is an oligopeptide (about 10 or fewer amino acids), a polypeptide (>10 amino acids), or a folded protein (>50 amino acids).

[0017] In the methods of the present invention, the FraC nanopore is preferably a mutant FraC nanopore, the conductance through the tunnel of which is typically higher than the conductance through its corresponding wild-type FraC nanopore.

[0018] A still further aspect relates to a mutant Fragaceatoxin C (FraC) nanopore comprising at least a first mutant FraC monomer comprising at least one substitution of a negatively charged amino acid residue in the narrow part of the pore with a positively charged amino acid residue and / or at least one substitution of a neutral amino acid residue in the narrow part of the pore with a positively charged amino acid residue. For example, the mutant comprises at least one mutation in a transmembrane helix. Preferably, at least one negatively charged amino acid residue is changed to a positively charged amino acid residue. The mutant may comprise a substitution at position 3 or 10, or at both positions 3 and 10. In a preferred embodiment, the mutant FraC comprises a mutation at position 10, preferably an AsplOArg or AsplOLys mutation (residue numbering similar to that in the crystal structure PDB ID 4TSY). The mutant FraC may further comprise one or more compensatory mutations that restore the hemolytic activity of FraC; preferably, the compensatory mutations are present at positions 2, 9, 34, 52, 112, 150, 153, and / or 159, and / or preferably at position 159. For example, the compensatory mutations are selected from the group consisting of A2S, I9T, A34V, F52Y, W112L, T150I, G153D, K159E, I171T, and any combination thereof. In a specific embodiment, the mutant FraC (further) comprises a mutation at position 159, preferably Lys159Glu. Very good results can be obtained using the FraC double mutant D10R / K159E. Specific preferred mutants include those listed in Table 3 herein below.

[0019] The mutant FraC nanopore may further comprise at least a second monomer selected from the group consisting of a wild-type FraC monomer, a second mutant FraC monomer, a wild-type FraC paralog or homolog monomer, and a mutant FraC paralog or homolog monomer, where the second mutant FraC monomer may be the same as or different from the first mutant FraC monomer. For example, the second monomer is a wild-type FraC paralog or homolog monomer. In one embodiment, the first mutant FraC monomer of the mutant FraC nanopore comprises the mutation D10R, and preferably the mutation is selected from those shown in Table 3.

[0020] The mutant FraC nanopore preferably has a conductance through its tunnel that is higher than the conductance through the tunnel of its corresponding wild-type FraC nanopore.

[0021] The mutant FraC nanopore may further comprise a molecular motor capable of translocating the analyte into or through the nanopore at an average translocation rate that is lower than the average translocation rate at which the analyte translocates into or through the nanopore in the absence of the molecular motor.

[0022] For example, the molecular motor is an enzyme, such as a polymerase, an exonuclease, or the Klenow fragment.

[0023] For protein analysis, it is preferable to have electroosmotic flow (EOF) from the cis compartment to the trans compartment induced by the charged constriction. The migration of amino acids of the same charge as the constriction can be obtained by manipulating the pH of the analyte solution. It is advantageous to use one or more unnatural amino acids that maintain a negative charge at low pH values ​​(< about 4.5), e.g., sulfate (SO4). 2- ) or phosphate (PO4 2- The ) moiety is a suitable amino acid side chain group. Thus, nanopores engineered to have strong electroosmotic flow from the cis compartment to the trans compartment under a negative applied potential (pH 4.5 or less) are also provided.

[0024] The present invention also provides the use of a system disclosed herein or a mutant FraC nanopore according to the present invention for biopolymer sensing and / or biopolymer sequencing, wherein the biopolymer is, for example, a protein, peptide, or nucleic acid. Preferably, there is provided the use of the system disclosed herein or a mutant FraC nanopore according to the invention for sensing and / or sequencing nucleic acids, such as ssDNA, dsDNA, RNA, or proteins or polypeptides, or combinations thereof.

[0025] For example, the FraC nanopore can be advantageously used to recognize protein, polypeptide, and oligopeptide biomarkers. Once inside the nanopore, proteins and polypeptides of different sizes can be distinguished by ionic current. While individual amino acids could not be identified on the fly during translocation, we have shown that endothelin-1 and endothelin-2 (2.5 kDa), two small (unfolded) oligopeptides that differ by only a single tryptophan residue, can be distinguished by ionic current recordings. Thus, if polypeptide translocation can be controlled, for example, by the use of enzymes, the FraC nanopore allows for the identification of specific sequence features in translocating polypeptides.

[0026] In one embodiment, the (mutant) FraC nanopore is used as a sensor in single-molecule proteomics analysis. In the simplest implementation of nanopore proteomics, proteins are recognized amino acid by amino acid as they translocate linearly through the nanopore. Because the sequences of proteins and oligopeptides in organisms are known from genome analysis, proteins can be recognized by simply comparing specific protein blockades during unfolded translocation through the nanopore with a database of known protein blockades. Alternatively, folded proteins can be recognized as they reside inside the nanopore vestibule, with or without translocating through the nanopore. [Brief explanation of the drawings]

[0027] [Figure 1] Wild-type FraC (WtFraC) and D10R-K159E FraC (ReFraC) nanopores. (A) Cross-section of the octameric WtFraC showing Coulomb force surface coloring (red = negative charge, blue = positive charge). Aspartic acid residue 10, located in the constriction zone of WtFraC, is indicated. (B) Top views of WtFraC (top) and ReFraC (bottom). (C) Single-channel conductance histograms of WtFraC (blue) and ReFraC (red) at +50 mV in 1 M NaCl, 15 mM Tris.HCl pH 7.5 buffer. (D) Raw traces of WtFraC (top) and ReFraC (bottom) at +100 mV in 3 M NaCl, 15 mM Tris.HCl pH 7.5 buffer obtained using identical acquisition settings (2 kHz low-pass Bessel filter and 10 kHz sampling rate). [Figure 2] DNA discrimination using ReFraC. (A) Representative blockade of a homopolymeric DNA strand complexed with NA using ReFraC. A schematic diagram shows an illustration of the current blockade. (B) Representative distributions of residual currents obtained for A20, C20, and T20 homopolymeric strands using a ReFraC nanopore. (C) Current blockade of continuous traces induced by homopolymeric C20 and A20 nucleotides into the same ReFraC pore. The traces shown were digitally filtered using a 100 Hz cutoff. (D) Distribution of residual currents imposed by a mixture of C20 and A20 homopolymeric strands. (E) Continuous traces of an experiment to resolve a mixture of homopolymeric C20 and T20 nucleotides. (F) Distribution of residual currents imposed by a mixture of C20 and T20 homopolymeric strands. Traces were recorded in 3 M NaCl, 15 mM Tris.HCl, pH 7.5, using a 2 kHz low-pass Bessel filter and a 10 kHz sampling rate. Traces C and E were subjected to further 100 Hz Gaussian digital filtering. [Figure 3]Unzipping / Translocation of dsDNA by ReFraC. (A) Representative trace of ReFraC capturing the NA:A(dsDNA)C complex at +50 mV. The open pore current is shown as "1" and, for comparison, after complex capture. Two levels can be observed in this block: a lower level ("2") likely corresponding to homopolymeric cytosine, which transforms through an intermediate level (unzipping, brackets) to a higher level ("3") most likely corresponding to homopolymeric adenine. Upon potential reversal ("4"), the block is immediately released, indicating that the double-stranded region, the NA:A(dsDNA)C complex, has been stripped. (B) At +100 mV, in more than half of the cases (inset), a single block ("2") is observed after the dsDNA portion is pushed through (deformation, brackets); upon application of -30 mV, this block cannot be immediately released ("3"). At more negative potentials, this block can be released, indicating that a rotaxane has formed (further examples in Figure 8). Traces were recorded in 3 M NaCl, 15 mM Tris.HCl, pH 7.5, using a 2 kHz low-pass Bessel filter and a 10 kHz sampling rate. [Figure 4] Unitary channel conductance distributions and voltage-current dependences determined for WtFraC and ReFraC nanopores. A: Unitary channel conductance distributions measured for pre-oligomerized pores of WtFraC (top) and ReFraC (bottom) reconstituted in planar lipid bilayers. Conductance was measured at an applied potential of -50 mV. The orientation of each individual channel was verified according to the asymmetry in conductance. B: Voltage-current dependences measured for WtFraC and ReFraC nanopores. Experiments were repeated three times; error bars indicate the standard deviation between experimental values. Recordings were performed in 15 mM Tris.HCl pH 7.5 and 1 M NaCl. [Figure 5]Hemolytic activity of WtFraC, D10R FraC, and ReFraC. The hemolysis rate was calculated as the reciprocal of the time elapsed until a 50% decrease in turbidity (measured as optical density at 650 nm wavelength) was observed in a 1% suspension of horse red blood cells in 15 mM Tris.HCl pH 7.5, 150 mM NaCl. Protein was added at a concentration of 200 nM, and the hemolysis rate is expressed as a percentage of WtFraC. Experiments were repeated three times, and error bars indicate the standard deviation between experimental values. [Figure 6] Translocation and immobilization of the A(dsDNA)C DNA substrate recorded using a ReFraC nanopore. The A(dsDNA)C substrate (shown above the trace) was generated by annealing Oligo I (5'-biotinylated AAAAAAAAAAAAAAAAAAAAAGTGCTACGACTCTCTGTGTGCCCCCCCCCCCCCCCCCCCC) and Oligo II (CACACAGAGAGTCGTAGCAC). A: Blockade induced on the ReFraC nanopore by 1 μM A(dsDNA)C alone (left) and A(dsDNA)C complexed with 0.25 μM neutravidin (right). The substrate was added in cis under an applied potential of +50 mV. B: Blockade induced on the ReFraC nanopore by 1 μM A(dsDNA)C alone (left) and A(dsDNA)C complexed with 0.25 μM neutravidin (right). Substrate was added in cis at +70 mV. The two levels of residual current detected for free A(dsDNA)C blockade are shown as light purple dashed lines. Current levels corresponding to blocked and open pores are shown as light purple and gray dashed lines, respectively. The voltage stepping protocol is shown as a red line below the trace. Recordings were performed in 15 mM Tris.HCl pH 7.5 and 3 M NaCl, the sampling frequency was 10 kHz, and data were smoothed with a 2 kHz low-pass Bessel filter during acquisition. [Figure 7]Representative trace showing the stepwise increase in residual current within the A(dsDNA)C-neutravidin blockade induced on a ReFraC nanopore. 1 μM A(dsDNA)C and 0.25 μM neutravidin were present in cis at +50 mV. Within the blockade, the residual current switched from 8.8 ± 0.7% (initial level) to 12.5 ± 0.7% (final level). The voltage stepping protocol is shown below in red. Recordings were performed in 15 mM Tris.HCl pH 7.5 and 3 M NaCl, the sampling frequency was 10 kHz, and data were smoothed with a 2 kHz low-pass Bessel filter during acquisition. [Figure 8] Further example traces showing rotaxane formation by A(dsDNA)C-neutravidin driven into a ReFraC nanopore at an applied potential of +100 mV. 1 μM A(dsDNA)C and 0.25 μM neutravidin were added in cis. The voltage-stepping protocol is shown below in red. The rotaxane was disassembled by switching the applied potential to -40 mV. Recordings were performed in 15 mM Tris.HCl pH 7.5 and 3 M NaCl, the sampling frequency was 10 kHz, and the data were smoothed with a 2 kHz low-pass Bessel filter during acquisition. [Figure 9]Representative traces showing pseudorotaxane and rotaxane formation with oligonucleotide I-neutravidin immobilized within a ReFraC nanopore. A: Pseudorotaxane formation induced by 1 μM oligonucleotide I and 0.25 μM neutravidin in cis. B: Rotaxane formation with 1 μM oligonucleotide I and 0.25 μM neutravidin in cis, with 1 μM oligonucleotide II added in trans. The rotaxane was disassembled by switching the applied potential to -40 mV (red arrows above the trace indicate rotaxane disassembly). The transient states describing dsDNA unzipping are indicated in square brackets. The voltage-stepping protocol is shown below in red. Recordings were performed in 15 mM Tris.HCl pH 7.5 and 3 M NaCl. The sampling frequency was 10 kHz, and data were smoothed with a 2 kHz low-pass Bessel filter during acquisition. [Figure 10]Capture of an oligopeptide (endothelin-1) and a protein (chymotrypsin) using two FraC variants at two different pH conditions. a) Cross-sections of wild-type FraC (WtFraC, PDB:4TSY) and D10R-K159E-FraC (ReFraC). b-c) Representative traces induced by 1 μM endothelin-1 (b) and 200 nM chymotrypsin (c) on WtFraC (left) and ReFraC (right). Chymotrypsin (PDB:5CHA) and human endothelin-1 (PDB:1EDN) are shown as surface-displayed molecules. Endothelin-1 and chymotrypsin enter WtFraC under a negative applied potential but enter ReFraC under a positive applied potential. Chymotrypsin blockade of WtFraC was observed at -50 mV at pH 7.5 and 4.5, but the applied potential was increased to -100 mV to obtain sufficient blockade. At pH 7.5, chymotrypsin blockade of ReFraC under a positive applied bias required a higher potential than blockade of WtFraC under a negative applied bias. The pH 7.5 buffer contained 1 M KCl, 15 mM Tris, and the pH 4.5 buffer contained 1 M KCl, 0.1 M citric acid, and 180 mM Tris base. Endothelin-1 and chymotrypsin were added to the cis compartment. All traces were recorded using a 50 kHz sampling rate and a 10 kHz low-pass Bessel filter. The coloring indicates the electrostatic potential of the molecular surface calculated by APBS (13) (pH 7.5 in 1 M KCl), with red and blue corresponding to negative and positive potentials (range -4 to +4 kbT / ec), respectively. The structure was rendered using PyMOL. [Figure 11]Electrostatic distribution and ion selectivity of WtFraC and ReFraC. a) Monomer-averaged simulated electrostatic potentials reveal negatively and positively charged constrictions for WtFraC and ReFrac, respectively. For ReFrac, lowering the pH from 7.5 to 4.5 had only a small effect on the electrostatic potential, whereas for WtFraC, the peak value at the center of the constriction decreased by approximately 41%. All simulations were performed using APBS (13) in 1 M KCl, and the partial charges of each titratable residue were adjusted according to their average protonation state using a modified version of PDB2PQR software (14). Residue pKa values ​​were estimated using PROPKA (33, 34). b) Determination of reversal potentials indicates that WtFraC and ReFraC are cation- and anion-selective, respectively, as predicted by the electrostatic potentials at their constrictions. Decreasing the pH from 7.5 to 4.5 reduced the ion selectivity (P_(K^+) / P_([Cl]^-)) of WtFraC by approximately 43%, in accordance with the reduced magnitude of the simulated electrostatic potential. In contrast, the approximately 37% increase in ion selectivity of ReFraC at pH 4.5 was not predicted by the simulation. All reversal potentials were measured under asymmetric salt conditions (467 mM KCl in trans and 1960 mM KCl in cis), and ion selectivities were determined using the Goldman-Hodgkin-Katz equation. Detailed experimental procedures are given in the Supporting Information. The envelopes behind all current-voltage curves indicate their respective standard deviations. [Figure 12]Biomarker characterization using WtFraC at pH 4.5. a) From top to bottom: molecular surface and surface display with schematic representation (Pymol) of chymotrypsin (25 kD, PDB:5CHA), a representative trace obtained at an applied potential of -150 mV, heat plots showing the dwell time distribution versus Ires% at -150 mV, voltage dependence of Ires%, voltage dependence of dwell time, and capture frequency. b), c), d), and e) show the same information for β2-microglobulin (11.6 kD, PDB:1LDS), human EGF (6.2 kD, PDB:1JL9), endothelin-1 (2.5 kD, PDB:1EDN), and angiotensin I (1.3 kD), respectively. Angiotensin I is shown as a random structure drawn using Pymol. The concentrations of the biomarkers were as follows: 200 nM for chymotrypsin, 200 nM for β2-microglobulin, 2 μM for human EGF, 1 μM for endothelin-1, and 2 μM for angiotensin I, respectively. The isoelectric points of the biomarkers were obtained from the literature or using the online calculation tool Pepcalc. Error bars indicate the standard deviation obtained from at least three replicates and at least 300 events for each replicate. Data were fitted using a B-spline function (Origin 8.1). All recordings were collected using a 50 kHz sampling rate and a 10 kHz low-pass Bessel filter. [Figure 13]Discrimination of endothelin-1 and endothelin-2 using WtFraC at pH 4.5. a) Molecular surface display of endothelin-1 and endothelin-2 using electrostatic coloring (PyMOL). b) Top: Amino acid sequences of endothelin-1 and endothelin-2. Blue lines indicate disulfide bridges in each oligopeptide. Bottom: Ires% and residence time for endothelin-1 and endothelin-2 blockade at -50 mV in pH 4.5 buffer (1 M KCl, 0.1 M citric acid, 180 mM Tris. base). c) Representative endothelin-1 and endothelin-2 blockade into the same FraC nanopore at an applied potential of -50 mV. d) Histogram of residual current evoked by 2 μM endothelin-1 (left) and corresponding heat plot showing standard deviation of current amplitude versus Ires% (right). e) Same as (d), but after addition of 8 μM endothelin-2 to the same pore revealing a second population. Graphs were generated using custom R scripts. All recordings were performed using a 50 kHz sampling rate and a 10 kHz Bessel low-pass filter. DETAILED DESCRIPTION OF THE INVENTION

[0028] Experimental Section Section A material and method Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich. DNA was purchased from Integrated DNA Technologies (IDT). Enzymes were obtained from Fermentas and lipids from Avanti Polar Lipids. All errors in this study are given as standard deviations.

[0029] FraC cloning To enable cloning, an NcoI restriction site (CCATGG) was introduced at the beginning (5' end) of the DNA sequence corresponding to mature WtFraC from A. fragacea. To maintain the reading frame, two additional bases were inserted after the NcoI site, resulting in an additional alanine residue after the initiating methionine. For purification purposes, a His9 affinity tag was attached to the C-terminus of FraC via a flexible glycine-serine-alanine linker, and the open reading frame was terminated by two consecutive stop codons followed by a HindIII restriction site (3' end). 50 ng of the synthetic gene (IDT) with optimized codon composition served as template for the following PCR reaction: the gene was amplified with Phire Hot Start II DNA polymerase (Finnzymes) using 6 μM of primers Frf and Frr (Table 2) in a 300 μL volume. The PCR protocol was as follows: a preincubation step at 98°C for 30 seconds was followed by 30 cycles of denaturation at 98°C for 5 seconds and extension at 72°C for 1 minute. The resulting PCR product containing the Hi9-tagged WtFraC gene was purified using a QIAquick PCR Purification Kit (Qiagen) and digested with NcoI and HindIII (FastDigest, Fermentas). The gel-purified insert (QIAquick Gel Extraction Kit, Qiagen) was cloned under the control of the T7 promoter into the pT7-SC1 expression plasmid using sticky-end ligation (T4 ligase, Fermentas) via the NcoI (5') and HindIII (3') sites. 0.6 μL of the ligation mixture was transformed into 50 μL of E. cloni® 10G cells (Lucigen) by electroporation. Transformed bacteria were grown overnight on ampicillin (100 μg / ml) LB agar plates at 37° C. The identity of the clones was confirmed by sequencing.

[0030] Construction of 10R FraC One hundred nanograms of the pT7-SC1 plasmid containing the WtFraC gene served as a template for the PCR reaction: the gene was amplified with Phire Hot Start II DNA polymerase (Finnzymes) using 6 μM primer 10Rf (encoding D10R) and a T7 terminator (Table 2) in a volume of 300 μL. The PCR reaction cycling protocol was as follows: a pre-incubation step at 98°C for 30 seconds was followed by 30 cycles of denaturation at 98°C for 5 seconds and extension at 72°C for 1 minute. The PCR product was gel purified (QIAquick Gel Extraction Kit, Qiagen) and subjected to the MEGAWHOP procedure. [1] The PCR product was cloned into a pT7 expression plasmid (pT7-SC1) by the following procedure: approximately 500 ng of purified PCR product was mixed with approximately 300 ng of pT7-SC1 plasmid containing the WtFraC gene, and amplification was performed using Phire Hot Start II DNA polymerase (Finnzymes) in a final volume of 50 μL (preincubation at 98°C for 30 seconds, followed by 30 cycles of denaturation at 98°C for 5 seconds and extension at 72°C for 1.5 minutes). Circular templates were eliminated by incubation with Dpn I (1FDU) at 37°C for 2 hours. 0.6 μL of the resulting mixture was transformed into E. cloni® 10G cells (Lucigen) by electroporation. Transformed bacteria were grown overnight at 37°C on LB agar plates containing ampicillin (100 μg / ml). The identity of the clones was confirmed by sequencing.

[0031] Construction of a 10R FraC library by error-prone PCR The library was constructed by amplifying the D10R FraC gene from plasmid DNA using T7 promoter and T7 terminator primers (Table 2 ).

[0032] [Table 1] Table 2. Oligonucleotides used in this study. "5Biosg" indicates a biotin group conjugated to the 5' end of the DNA via a C6 linker (IDT).

[0033] In the first round of mutagenesis, we used a plasmid containing a synthetic gene encoding 10R FraC as a template. In the second round of mutagenesis, we used a pool of DNA plasmids derived from the clones with the highest activity identified in the first round of screening. DNA amplification was performed by error-prone PCR: 400 μL of PCR mix (200 μL of REDTaq ReadyMix, 6 μM T7 promoter and T7 terminator primers, approximately 400 ng of plasmid template) was divided into eight reaction volumes containing 0–0.2 mM MnCl2 and cycled 27 times (preincubation at 95°C for 3 min, followed by cycling: denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 3 min). These conditions typically generated 1–4 mutations per gene in the final library. The PCR products were pooled together, gel-purified (QIAquick Gel Extraction Kit, Qiagen), and cloned into the pT7 expression plasmid (pT7-SC1) by the MEGAWHOP procedure: approximately 500 ng of purified PCR product was mixed with approximately 300 ng of pT7-SC1 plasmid containing the 10R FraC gene, and amplification was performed with Phire Hot Start II DNA polymerase (Finnzymes) in a final volume of 50 μL (preincubation at 98°C for 30 seconds, followed by 30 cycles of denaturation at 98°C for 5 seconds and extension at 72°C for 1.5 minutes). Circular templates were eliminated by incubation with Dpn I (1FDU) at 37°C for 2 hours. 0.6 μL of the resulting mixture was transformed into E. cloni® 10G cells (Lucigen) by electroporation. Transformed bacteria are grown overnight at 37°C on ampicillin (100 μg / ml) LB agar plates, typically yielding >10 5Colonies were obtained and collected for plasmid DNA library preparation.

[0034] Screening for hemolytic activity in crude lysates after FraC overexpression Overnight starter cultures from 600 clones (see above) were inoculated into 450 μL of fresh medium in new 96-deep-well plates, and the cultures were grown at 37°C to an OD of approximately 0.8. IPTG (0.5 mM) was then added to induce overexpression, and the temperature was reduced to 25°C for overnight incubation. The following day, bacteria were harvested by centrifugation at 3000 × g for 15 minutes at 4°C. The supernatant was discarded, and the pellet was frozen at -80°C for 2 hours to facilitate cell disruption. The cell pellet was then resuspended in 0.4 mL of lysis buffer (15 mM Tris.HCl pH 7.5, 1 mM MgCl, 10 μg / ml lysozyme, 0.2 units / mL DNAse I) and lysed by shaking at 1300 RPM for 30 minutes at 37°C. Next, 0.5–5 μL of the crude lysate was added to 100 μL of a ∼1% horse red blood cell suspension. The latter was prepared by centrifuging horse blood (bioMérieux Benelux) at 6,000 × g for 5 min at 4 °C and resuspending the pellet in 15 mM Tris.HCl pH 7.5, 150 mM NaCl. If the supernatant turned red, the solution was centrifuged again and the pellet resuspended in the same buffer. Hemolytic activity was monitored by the decrease in OD at 650 nm over time (Multiskan GO Microplate Spectrophotometer, Thermo Scientific).

[0035] Screening for mutations that compensate for the deleterious effects of the D10R amino acid substitution in FraC The D10R amino acid substitution resulted in an approximately five-fold decrease in the hemolytic activity of FraC (Figure 5). Although D10R FraC was still able to oligomerize and reconstitute in planar bilayers made with 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), we sought compensatory mutations that would restore the hemolytic activity of D10R FraC back to WtFraC levels. Maintaining the hemolytic activity of FraC is important for two reasons: first, it reflects its ability to assemble into oligomeric pores on targeted lipid bilayers and thus may lead to more efficient preparation of oligomeric nanopores. On the other hand, hemolytic activity provides a convenient method for screening the functionality of variants with any amino acid sequence changes, thus facilitating future engineering attempts on FraC nanopores. To identify compensatory mutations, we constructed a random mutagenesis library based on the D10R FraC gene, transformed it into Bl21 DE3 E. coli, and screened individual variants for hemolytic activity against horse erythrocytes in crude lysates after overexpression, using WtFraC as a reference. In the first round, we screened 600 variants and selected 12 clones as templates for the second round of random mutagenesis combined with hemolytic activity screening. Seven clones with the highest levels of hemolytic activity were then selected for further characterization. The sequence changes generated in the corresponding genes are summarized in Table 3.

[0036] [Table 2] Table 3. Sequence changes compensating for the deleterious effect of the D10R mutation in FraC

[0037] The purified variants were oligomerized in sphingomyelin:DPhPC (1:1) liposomes and solubilized in 0.6% LDAO. After detergent exchange with 0.02% DDM by Ni-NTA chromatography, the oligomeric proteins were tested for pore-forming activity in planar lipid bilayers composed of DPhPC. Initially, we identified variants 3, 4, and ReFraC (Table 3) as the most promising pore-forming entities. However, nanopores generated by variant 3 were heterogeneous (less than 50% produced octameric pores), and the pore-forming activity of variant 4 decayed within a few days when stored at 4°C. Oligomeric ReFraC maintained pore-forming activity for several months when stored at 4°C, remained capable of capturing ssDNA, and formed uniform nanopores nearly as uniform as WtFraC. Therefore, we chose ReFraC for further DNA analysis in this study. Furthermore, we replaced aspartate 10 in ReFraC with asparagine to obtain the D10N K159E variant, but no ssDNA entry was detectable.

[0038] WtFraC (protein sequence) MA SADVAGAVIDGAGLGFDVLKTVLEALGNVKRKIAVGIDNESGKTWTAMNTYFRSGTSDIVLPHKVAHGKALLYNGQKNRGPVATGVVGVIAYSMSDGNTLAVLFSVPYDYNWYSNWWNVRVYKGQKRADQRMYEELYYHRSPFRGDNGWHSRGLGYGLKSRGFMNSSGHAILEIHVTKA GSAHHHHHH ** WtFraC (DNA sequence) ATGGCGAGCGCCGATGTCGCGGGTGCGGTAATCGACGGTGCGGGTCTGGGCTTTGACGTACTGAAAACCGTGCTGGAGGCCCTGGGCAACGTTAAACGCAAAATTGCGGTAGGGATTGATAACGAATCGGGCAAGACCTGGACAG CGATGAATACCTATTTCCGTTCTGGTACGAGTGATATTGTGCTCCCACATAAGGTGGCGCATGGTAAGGCGCTGCTGTATAACGGTCAAAAAAATCGCGGTCCTGTCGCGACCGGCGTAGTGGGTGTGATTGCCTATAGTATGTC TGATGGGAACACACTGGCGGTACTGTTCTCCGTGCCGTACGATTATAATTGGTATAGCAATTGGTGGAACGTGCGTGTCTACAAAGGCCAGAAGCGTGCCGATCAGCGCATGTACGAGGAGCTGTACTATCATCGCTCGCCGTTT CGCGGCGACAACGGTTGGCATTCCCGGGGCTTAGGTTATGGACTCAAAGTCGCGGCTTTATGAATAGTTCGGGCCACGCAATCCTGGAGATTCACGTTACCAAAGCAGGCTCTGCGCATCATCACCACCATCACTGATAAGCTT

[0039] Overexpression and purification of FraC E. cloni® EXPRESS BL21(DE3) cells were transformed with the pT7-SC1 plasmid containing the FraC gene. Transformants were selected after overnight growth at 37°C on LB agar plates supplemented with 100 mg / L ampicillin. The resulting colonies were inoculated into 2xYT medium (Sigma) containing 100 mg / L ampicillin. The cultures were grown to an OD of approximately 0.8 with shaking at 200 rpm. 600FraC was grown at 37°C until reaching a concentration of 0.5 mM. Expression of FraC was then induced by the addition of 0.5 mM IPTG, and growth was continued at 25°C. The following day, bacteria were harvested by centrifugation at 6000 × g for 25 minutes, and the pellets were stored at -80°C. Pellets containing monomeric FraC (derived from 50–100 ml bacterial cultures) were thawed and resuspended in 40 ml of 15 mM Tris.HCl pH 7.5, 1 mM MgCl2, and 0.05 units / mL DNase I (Fermentas). To initiate cell disruption, the bacterial suspension was then supplemented with 0.2 mg / mL lysozyme and 2 M urea (to prevent debris formation) and subjected to vigorous shaking for 40 minutes at ambient temperature. Residual bacteria were disrupted by probe sonication. The crude lysate was clarified by centrifugation at 6000 × g for 20 minutes, and the supernatant was mixed with 200 μL (bead volume) of Ni-NTA resin (Qiagen) pre-equilibrated with wash buffer (10 mM imidazole, 150 mM NaCl, 15 mM Tris.HCl pH 7.5). After gentle mixing for 1 hour at ambient temperature, the resin was loaded onto a column (Micro Bio Spin, Bio-Rad) and washed with approximately 5 ml of wash buffer. FraC was eluted with approximately 0.5 mL of wash buffer containing 300 mM imidazole. Protein concentration was determined by Bradford assay. FraC monomer was stored at 4 °C until further use.

[0040] Hemolytic activity assay Defibriminated horse blood (bioMérieux Benelux) was washed with 150 mM NaCl, 15 mM Tris.HCl pH 7.5 until the supernatant was clear. The red blood cells were then resuspended in the same buffer to a concentration of approximately 1% (OD 650 nm of 0.6-0.8). The suspension was then mixed with 200 nM FraC. Hemolytic activity was measured by monitoring the decrease in OD 650 using a Multiskan™ GO Microplate spectrophotometer (Thermoscientific). The rate of hemolysis was determined integrally over the time period until a 50% decrease in turbidity was reached.

[0041] Preparation of sphingomyelin:DPhPC liposomes 20 mg of sphingomyelin (brain, porcine, Avanti Polar lipids) and DPhPC (1:1) mixture was dissolved in 4 ml of pentane supplemented with 0.5% ethanol (to aid in dissolving the sphingomyelin) and placed in a round-bottom flask. The solvent was evaporated by slowly rotating the flask to deposit a lipid film on the wall. After deposition of the lipid film, the flask was left open for 30 minutes to allow complete evaporation of the solvent. The lipid film was then resuspended in 150 mM NaCl, 15 mM Tris.HCl pH 7.5 (final concentration of total lipid: 10 mg / ml) using a sonication bath (5-10 minutes at ambient temperature). The resulting liposomes were stored at -20°C.

[0042] FraC oligomerization Monomeric FraC was mixed with liposomes (lipid / protein mass ratio 10:1) in 150 mM NaCl, 15 mM Tris.HCl pH 7.5 buffer. The mixture was briefly sonicated (bath sonicator) and incubated at 37°C for 30 minutes. The proteoliposomes were then solubilized with 0.6% LDAO and incubated for 5 minutes. The mixture was then diluted 20-fold with DDM-containing wash buffer (0.02% DDM, 150 mM NaCl, 15 mM Tris.HCl pH 7.5) and mixed with approximately 100 μl (bead volume) of Ni-NTA agarose resin (Qiagen) pre-equilibrated with DDM-containing wash buffer. After gentle mixing for 1 hour, the resin was loaded onto a column (Micro Bio Spin, Bio-Rad) and washed with approximately 2 ml of DDM wash buffer. FraC was eluted from the column with 50 μl of elution buffer (200 mM EDTA, 0.02% DDM, pH 8—alternatively, we could have used 1 M imidazole 0.02% DDM, but EDTA proved more efficient). Purified FraC oligomers were stored at 4°C.

[0043] Alternatively, FraC oligomers can be formed by mixing FraC monomers with liposomes formed with sphingomyelin alone (1 hour at 37°C, then overnight at 4°C). The following day, 5 mM EDTA and 1% DDM (final) are added to the proteoliposomes and incubated at room temperature for 15 minutes. The solution is then diluted to a volume of 1 ml containing 5 mM EDTA, 0.05% DDM, 15 mM Tris HCl 7.5, and 150 mM NaCl. The solution is then concentrated to approximately 100 μl using a 100 kDa cutoff ultrafiltration device.

[0044] Electrical recording in planar lipid bilayers The applied potential refers to the potential of the trans electrode. The FraC nanopore was inserted into the lipid bilayer from the cis compartment, which was connected to the ground electrode. The two compartments were separated by a 25 μm-thick polytetrafluoroethylene film (Goodfellow Cambridge Limited) containing an orifice approximately 100 μm in diameter. The orifice was pretreated with approximately 5 μL of 10% hexadecane in pentane, and the bilayer was formed by adding approximately 10 μL of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) (10 mg / mL) in pentane to both electrophysiology chambers. Typically, the addition of 0.01–10 ng of oligomeric FraC to the cis compartment (0.5 mL) was sufficient to obtain a single channel. WtFraC nanopores exhibited higher open-pore currents at positive than negative applied potentials, providing a useful tool for determining pore orientation. Electrical recordings were performed in 1 M (initial characterization of the FraC nanopore) and 3 M NaCl (for polynucleotide analysis to amplify amplitude), 15 mM Tris.HCl pH 7.5.

[0045] Data recording and analysis Electrical signals from planar bilayer recordings were amplified using an Axopatch 200B patch clamp amplifier (Axon Instruments) and digitized using a Digidata 1440 A / D converter (Axon Instruments). Data were recorded using Clampex 10.4 software (Molecular Devices), and subsequent analysis was performed using Clampfit software (Molecular Devices). Electrical recordings were acquired by applying a 2 kHz low-pass Bessel filter and a 10 kHz sampling rate. Current transitions from level 0 to level 1 were analyzed using the "single channel search" function in Clampfit. Residual current values ​​(I res ) is the blocked pore current value (I B ) and open pore current (I O ) to I res =100 * I B / I O It was calculated as follows: I B and I O was determined from a Gaussian fit to the event amplitude histogram. For events showing a stepwise current buildup, the residual current level was calculated from a Gaussian fit to the Hall point current histogram. To determine event lifetime, the event dwell time (t off ) were binned together as a cumulative distribution and fitted to a single exponential function. The frequencies of events showing stepwise current enhancement (FIG. 3A) and rotaxane-forming blockade were calculated manually. Graphs were generated using Origin (OriginLab Corporation) or Clampfit software (Molecular Devices).

[0046] Graphical representation of the FraC nanopore Molecular graphics were performed using Chimera (http: / / www.cgl.ucsf.edu / chimera). [Example]

[0047] Reconstitution of wild-type FraC pore in planar lipid bilayers Recombinant wild-type FraC (WtFraC, Figure 1A; 1B, top) protein monomers genetically fused to a Hi9 tag at the C-terminus were expressed in the BL21(DE3) E. coli strain. Previous studies have established that actinoporin pore assembly is triggered by the presence of SM in lipid bilayers [9],

[10] ,

[11] , [8]. Consistently, water-soluble WtFraC monomers purified by Ni-NTA chromatography did not form pores in DPhPC planar lipid bilayers. Therefore, we pre-oligomerized the monomers with DPhPC:SM (1:1) liposomes. After solubilization of liposomes in 0.6% N,N-dimethyldodecylamine N-oxide (LDAO), to prevent oligomer dissociation

[12] , LDAO was exchanged for 0.02% β-dodecylmaltoside (DDM) by a second round of Ni-NTA chromatography (SI). Addition of submicrogram amounts of purified oligomeric WtFraC in 0.02% DDM to the cis side of DPhPC planar lipid bilayers readily generated pores. Distribution of unit channel conductances for WtFraC pores in 1 M NaCl, 15 mM Tris.HCl pH 7.5 buffer revealed predominantly a single conductance type (Figure 1C; 4A, top), likely corresponding to the octamer observed in the recently determined crystal structure [8]. Similar to other biological nanopores, the WtFraC channel exhibited an asymmetric current-voltage (IV) relationship (Fig. 4B), allowing for the determination of the pore orientation. An example trace obtained in 3 M NaCl, 15 mM Tris.HCl pH 7.5 buffer is shown in Fig. 1D, top. [Example]

[0048] Engineering WtFraC for nucleic acid analysis The crystal structure of octameric WtFraC suggests that the nanopore is large enough (1.2 nm constriction diameter) to allow ssDNA penetration [8]. However, in our initial experiments, we failed to observe ssDNA blockage, most likely due to the negatively charged constriction region of the WtFraC pore preventing DNA translocation

[13] ,

[14] . To induce ssDNA penetration through FraC, we substituted aspartic acid 10 with arginine to produce a nanopore with a positively charged constriction (Figure 1B, bottom). Because D10R FraC showed low pore-forming activity (Figure 5), we performed random mutagenesis against the D10R FraC genetic background and screened the resulting variants for hemolytic activity (SI). As a result, we identified a compensatory mutation of lysine 159 to glutamic acid (K159E), located at the outer edge of the wide vestibule (Figure 1A). The FraC double mutant D10R,K159E (ReFraC) exhibited near-wild-type hemolytic activity (Figure 5) and generated a uniform pore (Figure 1C), despite having an altered I-V relationship compared to wild-type FraC (Figure 4B and Figure 1D, bottom). The lower conductance of the ReFraC pore at ±50 mV in 1 M NaCl, 15 mM Tris.HCl pH 7.5 can be attributed to the narrower constriction, as arginine has a bulkier side chain than aspartic acid (Figure 1B). [Example]

[0049] Polynucleotide discrimination using ReFraC We followed established approaches using αHL [7],

[15] ,

[16] ,

[17] and MspA

[14] ,

[18] to immobilize DNA with neutravidin (NA). We evaluated the ability of ReFraC to translocate and discriminate DNA strands by complexing a 5'-end biotinylated A20 / C20 / T20 ssDNA homopolymer with tetrameric NA. We added premixed DNA (1 μM) and NA (0.25 μM) to the cis compartment of a planar lipid bilayer setup and performed DNA discrimination experiments in 3 M NaCl, 15 mM Tris.HCl, pH 7.5 buffer, and an applied potential of +70 mV (pointing toward the trans electrode). We observed a persistent current block induced by the pseudorotaxane, in which the ssDNA was stably threaded through the pore until the applied potential was reversed (Figure 2A, Figure 9A). The residual currents Ires, calculated by multiplying the percentage of the blocked and open pore current amplitudes by 100 ((IB / IO) × 100), were as follows: 13.1 ± 0.4% for NA:A20 (N = 5, n = 364, where N is the number of independent single-pore experiments and n is the analyzed blockage), 10.8 ± 0.3% for NA:C20 (N = 4, n = 920), and 14.0 ± 0.3% for NA:T20 (N = 5, n = 780) (Figure 2B). To eliminate the influence of pore-to-pore variability, we also resolved mixtures of homopolymers (Figures 2C–F). The relatively low residual currents suggest tight closure of the pore around the threaded ssDNA. [Example]

[0050] DNA unzipping and double-stranded DNA translocation by ReFraC nanopore The constriction of ReFraC (1.2 nm) is smaller than that of double-stranded DNA (dsDNA, approximately 2 nm) in the B-form. Therefore, to evaluate dsDNA as a stopper for DNA analysis, we designed two oligonucleotides: Oligo I, with a biotin group attached to the 5' end and the sequence biotin-5'-A20-GTGCTACGACTCTCTGTGTG-C20-3', and a shorter Oligo II, with a sequence reverse-complementary to the underlined portion of Oligo I. Annealing yielded the A(dsDNA)C substrate: a 20-base-pair-long central segment of dsDNA flanked by A20 and C20 ssDNA segments. Addition of 1 μM A(dsDNA)C to the cis compartment at +50 mV caused a transient blockade of the ReFraC pore (blockade lifetime 2 ± 5 s, Ires = 10.0 ± 0.2%, N = 3, n = 290, Figure 6A, left). Increasing the applied potential to +70 mV shortened the blockade lifetime to 2.9 ± 0.4 ms (note that the residual current showed two current levels: 12.8 ± 0.6% and 3.5 ± 0.5%, N = 3, n = 2700, Figure 6B, left). The decrease in blockade lifetime with potential suggests translocation of A(dsDNA)C through ReFraC. To demonstrate DNA translocation, we added NA to the cis chamber. The NA:A(dsDNA)C blockade became persistent at both +50 mV (Figure 6A, right) and +70 mV (Figure 6B, right), thus suggesting that the transient blockade in the absence of NA could not be induced by the retraction of A(dsDNA)C into the cis compartment. Curiously, at +50 mV, 31 ± 4% NA:A(dsDNA)C blockade (N = 3, n = 468) showed a gradual increase in the residual current, from a transient level (Fig. 3A, state "2", I = 8.8 ± 0.7%) to a stable level (Fig. 3A, state "3", I = 12.5 ± 0.7%, N = 4, n = 46; further examples in Fig. 7). The current level in state "2" was slightly lower than that of NA:C20 (I = 10.5 ± 0.7 at +50 mV; N = 3, n = 206). The current level in state "3" matched that of NA:A20.A plausible explanation for this current enhancement is that at +50 mV, the C20 segment of NA:A(dsDNA)C is lodged in the nanopore constriction (Figure 3A, state "2"), preventing further translocation of the duplex segment. However, after unzipping of the duplex, A20 occupies the ReFraC constriction, causing NA to stop translocation (Figure 3A, state "3"). Consistently, at +50 mV, the NA:A(dsDNA)C blockade is immediately released upon reversing the potential to -30 mV, indicating that at +50 mV, translocation of A(dsDNA)C is mediated by unzipping (Figure 3A, brackets).

[0051] In contrast, at +70 mV, a significant proportion of the blockade was not immediately released at -30 mV (Figure 3B, inset), indicating the formation of interlocked states (Figure 3B, states "2" and "3"). Furthermore, these interlocked states were generated more frequently with increasing potential (e.g., from 7 ± 4% of all blockades at +70 mV to 54 ± 14% at +100 mV, N = 3, n = 739; Figure 3B, inset). Considering that the blockade of Oligo I alone complexed with NA was immediately released at -30 mV (Figure 9A), we attributed such interlocked states to the rotaxane, in which the NA and double-stranded DNA segments A(dsDNA)C function as cis and trans stoppers, respectively (Figure 3B, right). As expected, such a rotaxane could also be formed from the NA:Oligo I cis blockade by adding Oligo II in trans (Figure 9B). Switching the potential to -40 mV rapidly disassembled the rotaxane, likely via unzipping the dsDNA stopper in trans (Figures 8 and 9B). Formation of a rotaxane from NA:A(dsDNA)C present in cis requires deformation of the ReFraC pore to allow translocation of the duplex segment of the A(dsDNA)C substrate (Figure 3B, brackets).

[0052] This structural flexibility may be a general feature of α-helical pores. Previously, we observed that blocking of human thrombin (diameter ∼4.2 nm) into a type I ClyA-CS nanopore (constriction diameter ∼3.3 nm) resulted in a transient increase in the open pore current

[19] . This phenomenon was interpreted as protein translocation through the deformed constriction of ClyA.

[0053] Section A References [1] JJ Kasianowicz, E. Blandin, D. Blanton, D.W. Deamer, Proc Natl Acad Sci USA 1996, 93, 13770-13773. [2] M. Acheson, D. Blanton, J.J. Kasianowicz, E. Blandin, D.W. Deamer, Biophys J 1999, 77, 3227-3233. [3] D. Wendel, P. Jin, J. Geng, V. Subramaniam, T.J. Li, C. Montemagno, P. Guo, Nature nanotechnology 2009, 4, 765-772. [4] L. Franceschini, M. Soskin, A. Beesemans, G. Maria, Nature communications 2013, 4, 2415. [5] C. Zhao, Y. L. Yin, Z. L. Fu, D. F. Liao, H. Tian, ​​Y. T. Long, Nature nanotechnology 2016. [6] A.H. Laszlo, I.M. Derrington, B.C. Ross, H. Brinkerhoff, A. Adie, I.C. Nova, J.M. Craig, K.W. Langford, J.M. Samson, R. Dasa, K. Doering, J. Shendur, J.H. Gundlach, Nat Biotechnol 2014, 32, 829-833. [7] D. Stoddart, A. J. Heron, E. Mikhailova, G. Maria, H. Bailey, Proc Natl Acad Sci USA 2009, 106, 7702-7707. [8] K. Tanaka, J.M. Caaveiro, K. Morante, J.M. González-Manas, K. Tsumoto, Nature communications 2015, 6, 6337. [9] A. Barlick, I. Gutierrez-Aguirre, J.M. Caaveiro, A. Cruz, M.B. Ruiz-Arguello, J. Pérez-Hill, J.M. González-Manas, J. Biol Chem 2004, 279, 34209-34216.

[10] B. Bacharach, I. Gutierrez-Aguirre, Z. Podlesek, A.F. Sonnen, R.J. Gilbert, P. Matzek, J.H. Lakey, G. Anderloo, J Biol Chem 2008, 283, 18665-18677.

[11] P. Schon, A.J. Garcia-Saez, P. Marabou, K. Basia, G. Anderloo, and P. Schwille, Biophys J 2008, 95, 691-698.

[12] K. Tanaka, J.M. Caaveiro, and K. Tsumoto, Biochemistry 2015, 54, 6863-6866.

[13] G. Maria, M.R. Restrepo, E. Mikhailova, H. Bailey, Proc Natl Acad Sci USA 2008, 105, 19720-19725.

[14] TZ Butler, M. Pavlenok, IM Derrington, M. Niederweiss, JH Gundlach, Proc Natl Acad Sci USA 2008, 105, 20647-20652.

[15] RF Parnell, JJ Schmidt, ACS Nano 2009, 3, 2533-2538.

[16] D. Stoddart, A. J. Heron, J. Klingelhöfer, E. Mikhailova, G. Maria, and H. Bailey, Nano Lett 2010, 10, 3633-3637.

[17] D. Stoddart, G. Maria, E. Mikhailova, A. J. Heron, H. Bailey, Angew Chem Int Ed Engl 2010, 49, 556-559.

[18] E.A. Manrao, I.M. Derrington, M. Pavlenok, M. Niederweiss, and J.H. Gundlach, PLoS One 2011, 6, e25723.

[19] M. Soskin, A. Biesemans, M. de Meyer, G. Maria, J Am Chem Soc 2013, 135, 13456-13463.

[20] J. Maté, H. Vislam, V. Viasnov, Y. Rabin, and A. Möller, Biophys J 2004, 87, 3205-3212.

[21] N. Ahn, A. M. Fleming, E. G. Middleton, and C. J. Burrows, Proc Natl Acad Sci USA 2014, 111, 14325-14331.

[22] M. Farrar, M. Niederweiss, and G. E. Schultz, Science 2004, 303, 1189-1192.

[0054] Section B Section A herein above demonstrates that Fragaceatoxin C (FraC), an alpha-helical pore-forming toxin from the actinoporin protein family, is advantageously used for polynucleotide analysis.

[0055] Section B demonstrates that the FraC nanopore is also suitable for recognizing proteins, e.g., biomarkers, in the form of oligopeptides (approximately 10 amino acids or less), polypeptides (>10 amino acids), and folded proteins (>50 amino acids).

[0056] material Chymotrypsin (from bovine pancreas, ≥85%, C4129), β2-microglobulin (from human urine, ≥98%, M4890), endothelin-1 (≥97%, E7764), endothelin-2 (≥97%, E9012), angiotensin I (≥90%, A9650), pentane (≥99%, 236705), and hexadecane (99%, H6703), Trizma® hydrochloride (lot no. SLBG8541V) and Trizma® base (lot no. SLBK4455V), N,N-dimethyldodecylamine N-oxide (LADO, ≥99%, 40234), and n-dodecyl β-D-maltoside (DDM, ≥98%, D4641) were obtained from Sigma-Aldrich. Human EGF (≥98%, CYT-217) was obtained from PROSPEC. 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, 850356P) and sphingomyelin (brain, porcine, 860062) were purchased from Avanti Polar lipids. Potassium chloride (≥99%, lot number BCBL9989V) was purchased from Fluka. Citric acid (≥99%, lot number A0365028) was obtained from ACROS. All polypeptide biomarkers and chemicals were used directly without further purification.

[0057] Note: The 15 mM Tris, pH 7.5 buffer used below was prepared using the recipe from the Trizma® protocol: 1.902 g Trizma® HCl and 0.354 g Trizma® base were dissolved in 1 liter of HO to make 15 mM Tris, pH 7.5.

[0058] method Expression and purification of FraC monomers The gene encoding FraC with a C-terminal His6 tag was cloned into the pT7-SC1 expression plasmid using NcoI and HindIII restriction digestion sites. 1The plasmid was cloned into E. cloni® EXPRESS BL21(DE3) competent cells for expression by electroporation. Transformants were harvested from LB agar plates containing 100 mg / L ampicillin after overnight incubation at 37°C and inoculated into 200 ml of fresh liquid 2-YT medium containing 100 mg / L ampicillin. The cell culture was grown at 37°C with shaking at 200 rpm to an optical density at 600 nm of 0.8, and then 0.5 mM IPTG was added to the cell culture. The temperature was reduced to 25°C to induce expression of the FraC protein for 12 hours. The cells were harvested by centrifugation at 4,000 RPM for 30 minutes at 4°C, and the cell pellet was kept at -80°C. 50-100 ml of cell culture pellet was thawed at room temperature, resuspended in 30 ml of lysis buffer (15 mM Tris pH 7.5, 1 mM MgCl2, 4 M urea, 0.2 mg / ml lysozyme, and 0.05 units / ml DNase), and vigorously mixed on a vortex shaker for 1 hour. To completely disrupt the cells, the suspension was sonicated for 2 minutes (duty cycle 10%, output control 3 on a Branson Sonifier 450). The crude lysate was then centrifuged at 6,500 RPM for 20 minutes at 4°C. The supernatant (containing FraC monomer) was transferred to a 50 ml Falcon tube containing 100 μl of Ni-NTA resin (Qiagen, stored at 4°C, suspended, and then pipetted off 100 μl) pre-washed with 3 ml of wash buffer (10 mM imidazole, 150 mM NaCl, 15 mM Tris, pH 7.5) and incubated at room temperature for 1 hour with gentle mixing. The resin was spun down at 4,000 RPM for 5 minutes at 4°C. Most of the supernatant was discarded, and the pellet containing Ni-NTA resin in approximately 5 ml of buffer was transferred to a Micro Bio Spin column (Bio-Rad) at room temperature. The Ni-NTA beads were washed with 10 ml of wash buffer, and the protein was eluted with 500 μl of 300 mM imidazole. Protein concentration was determined using a NanoDrop 2000 (Thermo Scientific). The monomer was stored at 4°C.

[0059] Preparation of sphingomyelin-DPhPC liposomes 20 mg of sphingomyelin (brain, porcine, Avanti Polar lipids) was mixed with 20 mg of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, Avanti Polar lipids) and dissolved in 4 ml of pentane (Sigma) containing 0.5% v / v ethanol. The lipid mixture was transferred to a round-bottom flask and slowly rotated near a hair dryer to thoroughly distribute the lipids throughout the walls and evaporate the solvent. The flask was left open at room temperature for another 30 minutes to allow complete evaporation of the solvent. Next, 4 ml of buffer (150 mM NaCl, 15 mM Tris, pH 7.5) was added to the dried lipids, and the flask was placed in a sonication bath for 5 minutes. The liposome solution was kept at -20°C.

[0060] FraC oligomerization Frozen liposomes were sonicated after thawing and mixed with monomeric FraC at a 1:1 mass ratio. The FraC and liposome mixture was bath sonicated for approximately 30 seconds and then maintained at 37°C for 30 minutes. Proteoliposomes were solubilized with 0.6% LADO (N,N-dimethyldodecylamine N-oxide, 5% w / v stock solution in water) and then transferred to a 50 ml Falcon tube and diluted 20-fold with buffer (150 mM NaCl, 15 mM Tris, pH 7.5, 0.02% DDM). 100 μl of pre-washed Ni-NTA resin (Qiagen) was added to the diluted protein / liposome mixture. After incubation with gentle shaking for 1 hour, the beads were loaded onto a column (Micro Bio Spin, Bio-Rad) and washed with 10 ml of buffer (150 mM NaCl, 15 mM Tris, pH 7.5). FraC oligomers were eluted with 300 μl of elution buffer (200 mM EDTA, 75 mM NaCl, 7.5 mM Tris, pH 8, 0.02% DDM). Oligomers can be stable for several weeks at 4° C.

[0061] Electrical recording in planar lipid bilayers Electrical recordings were performed as previously described. 2 Briefly, the two chambers were separated by a 25 μm polytetrafluoroethylene film (Goodfellow Cambridge Limited) containing an opening with a diameter of approximately 100 μm. Two silver / silver chloride electrodes were immersed in each compartment of the electrophysiology chamber, which was filled with 0.5 ml of buffer solution. The ground electrode was connected to the cis compartment, and the working electrode was connected to the trans compartment. To form a lipid bilayer, approximately 5 μl of a hexadecane solution (10% v / v hexadecane in pentane) was added to the polytetrafluoroethylene film. After approximately 2 min, 10 μl of a 10 mg / ml solution of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) in pentane was added directly to the buffer in both compartments. Lipid bilayers were then spontaneously formed by lowering the buffer above and below the opening in the Teflon film. FraC oligomers were added to the cis side. Under an applied potential, the ionic current of FraC is asymmetric, which helps assess the orientation of the FraC nanopore in the lipid bilayer. The FraC nanopore exhibited an orientation as shown in Figure 10 when a higher conductance was measured at a negative applied potential. The analyte was then added to the cis chamber. Two buffer solutions, depending on the pH, were used for electrophysiological recordings in this study. At pH 7.5, recordings were performed using 1 M KCl and 15 mM Tris. When the pH was varied from 7.5 to 4.5, the buffer used contained 1 M KCl, 0.1 M citric acid, and 180 mM Tris base. FraC and ReFraC oligomers could insert into lipid bilayers at pH 4.5–7.5.

[0062] Data recording and analysis Planar bilayer recordings were collected using a patch clamp amplifier (Axopatch 200B, Axon Instruments), and data were digitized using a Digidata 1440 A / D converter (Axon Instruments). Data were acquired using Clampex 10.4 software (Molecular Devices), and subsequent analysis was performed using Clampfit software (Molecular Devices). Event duration (dwell time), time between two events (interevent time), blocked current level, and open pore level were detected by the "single channel search" function. The current level of block was calculated as I B and the open pore current is called I o (I B / I O ) * Ires%, defined as 100, was used to describe the degree of blockage caused by different biomarkers. The mean inter-event time was calculated by binning the inter-event times and applying a single exponential fit to the cumulative distribution.

[0063] Ion selectivity measurements Ion permeability ratio (K + / Cl - ) as a variable input and the reversal potential (V r ) was calculated using the Goldman-Hodgkin-Katz equation (hereinafter, equation (1)). rwas measured by extrapolation from IV curves using the following asymmetric salt concentration conditions: Individual FraC nanopores were reconstituted in both chambers using the same buffer (symmetric condition, 840 mM KCl, 15 mM Tris, pH 7.5, 500 μl) to assess nanopore orientation. 400 μl of a solution containing 3.36 M KCl, 15 mM Tris, pH 7.5 was slowly added to the cis chamber, and 400 μl of a KCl-free buffer solution (15 mM Tris, pH 7.5) was added to the trans chamber (trans:cis, 467 mM KCl:1960 mM KCl). The solutions were mixed, and IV curves were collected from -30 mV to 30 mV in 1 mV increments. Experiments at pH 4.5 were performed using the same method but with 0.1 M citrate buffer. First, 500 μl of a buffer solution containing 840 mM KCl, 0.1 M citric acid, and 180 mM Tris. base was added to both chambers to create a single FraC channel. Then, 400 μl of a solution containing 3.36 M KCl, 0.1 M citric acid, and 180 mM Tris. base, pH 4.5, was slowly added to the cis chamber, and 400 μl of a KCl-free buffer solution (0.1 M citric acid, 180 mM Tris. base, pH 4.5) was added to the trans solution (thus resulting in a trans:cis ratio of 467 mM KCl:1960 mM KCl). The solutions were mixed, and IV curves were collected from -30 mV to 30 mV in 1 mV increments. The directionality of ion selectivity was also examined by using a high KCl concentration in the trans chamber and a low KCl concentration in the cis chamber. The Ag / AgCl electrode was surrounded by a 2.5% agarose bridge containing 2.5 M NaCl. [Example]

[0064] Trapping polypeptides and proteins using FraC nanopores To evaluate the FraC nanopore as a sensor for oligopeptide biomarkers, we first selected endothelin-1, a 21-amino acid, 2.5 kD oligopeptide, and α-II-chymotrypsin (hereafter referred to as chymotrypsin), a 245-amino acid, 25 kD globular protein (Figure 10). Analytes were added to the cis side of the wild-type FraC (WtFraC) nanopore (Figure 10A) using a 1 M KCl, 15 mM Tris, pH 7.5 solution, and an external potential was applied to the "working" electrode located in the trans compartment. Because WtFraC exhibits gating above approximately +50 mV but is stable at potentials as high as -300 mV, we applied potentials between these limits. Addition of 1 μM endothelin-1 to the cis compartment did not induce blockade at ±50 mV (Figure 10B) or down to -300 mV. Because the ClyA constriction is covered with aspartic acid residues (Figure 10A), we reasoned that protonation of these residues in more acidic conditions should weaken the energy barrier for translocation of endothelin-1 (which carries a net charge of -2) through the WtFraC constriction. At the same time, less negative endothelin-1 would also migrate more easily toward the trans-electrode under negative applied potentials. Endothelin-1 blockade began to occur at pH 6.4, and their capture frequency increased linearly with decreasing pH (0.6 ± 0.2 events / s at pH 6.4). -1 / μM -1 to 10.8±2.3 events / s at pH 4.4 -1 / μM -1 At pH 4.5 (1 M KCl, 0.1 M citric acid, 180 mM Tris. base), endothelin-1 blockade of WtFraC was observed at −50 mV (Ires%: 9.1 ± 0.1%, dwell time: 5.6 ± 2.0 ms, inter-event time: 5.8 ± 0.7 ms) but not at +50 mV (Figure 10B).

[0065] Prompted by the effect of more positive constriction under acidic conditions, we next investigated the D10 R ,K159 E FraC( ReWe investigated the capture of endothelin-1 using a ReFraC nanopore, a pore with an arginine residue at the constriction, engineered in Section A above for DNA analysis purposes. Contrary to wtFraC, ReFraC is stable under positive applied potentials but exhibits gating at potentials of approximately -50 mV. Therefore, we applied only voltages between -50 mV and +200 mV to ReFraC. Addition of 1 μM endothelin-1 to the cis compartment elicited a block at pH 7.5 at +50 mV (dwell time: 3.3 ± 2.2 ms, inter-event time: 1413 ± 223 ms) but not at -50 mV (Figure 10B). A decrease to pH 4.5 (1 M KCl, 0.1 M citric acid, 180 mM Tris. base) resulted in an increase in capture frequency at +50 mV despite reduced electrophoretic mobility toward the trans electrode (Figure 10B, dwell time: 8.5 ± 1.8 ms, inter-event time: 402 ± 79 ms).

[0066] Next, the protein chymotrypsin (pI 8.75, Sigma) was tested as an example of a relatively large protein analyte. Protein blockades were observed at -50 mV in pH 7.5 buffer (1 M KCl, 15 mM Tris), but these leveled off (45.2 ± 19.1 events / s) when we increased the potential to -100 mV. -1 / μM -1 , dwell time: 12.0 ± 5.7 ms), and no capture was observed at positive applied potentials (Figure 10C). In contrast to what was observed with endothelin-1, the capture frequency of chymotrypsin remained constant between pH 7.5 and 5.5 (45.2 ± 19.1 events / s at pH 7.5). -1 / μM -1 , 50.5±22.6 events / s at pH 6.4 -1 / μM -1 , 45.2±20.6 events / s at pH 5.5 -1 / μM -1 ), which decreased when the pH was lowered to 4.4 (20.8 ± 5.3 events / s at pH 4.4). -1 / μM -1). Using ReFraC at pH 7.5, we observed only a slight blockade at highly positive applied potentials (dwell time at +200 mV: 0.2 ± 0.1 ms, inter-event time: 174.3 ± 22.9 ms), but none at -50 mV (Fig. 10C). Decreasing the pH to 4.5 resulted in an increase in capture frequency (dwell time: 1.3 ± 0.7 ms, 112.5 ± 9.5 events / s). -1 / μM -1 , Figure 9B). Notably, ReFraC frequently exhibited shallow gating events at negative applied potentials under acidic conditions, as shown in Figure 10C, bottom right. Combined, both nanopores can capture analytes with a 10-fold difference in molecular weight (2.5 kD vs. 25 kDa). [Example]

[0067] Ion selectivity and electrostatic potential of FraC nanopores To gain better insight into the effect of pH on the electrostatic environment and the effect of electroosmotic flow on polypeptide entry inside the FraC nanopore, we estimated the electrostatic potentials inside homologous models of WtFraC and ReFraC at pH 7.5 and 4.5 in 1 M KCl using the Adaptive Poission-Boltzmann Solver (APBS) (13) and a modified version of PDB2PQR software (14). Simulations showed that the constriction regions of WtFraC and ReFraC in the center of the nanopore exhibited highly negative and positive potentials, respectively (Figure 11A). Interestingly, for WtFraC, decreasing the pH from 7.5 to 4.5 reduced the electrostatic potentials at the center of the constriction by −1.2 to −0.7 kJ, respectively. B T / e c (298K for 1k B T / e c = 25.6 mV), whereas no such effect was observed for ReFraC.

[0068] The contribution of electroosmotic flow to analyte capture by the WtFraC and ReFraC pores was estimated by measuring the ion selectivity of both pores using asymmetric KCl concentrations (1960 mM and 467 mM) on either side of the nanopore. The reversal potential (V r ), i.e., the potential at which the current is zero (FIG. 11B), was used together with the Goldman-Hodgkin-Katz equation to calculate the ion selectivity (P K+ / P Cl- ) was calculated:

[0069]

number

[0070] In the formula, [a x ] compartment is the activity of ion X in the cis / trans compartment, R is the gas constant, T is temperature, and F is the Faraday constant. We show that the ion selectivity of the FraC nanopore is dominated by the charge at the constriction, with WtFrac being strongly cation selective (P K+ / P Cl- =3.55±0.30, pH 7.5), and ReFraC is anion selective (P K+ / P Cl- = 0.57 ± 0.04, pH 7.5). Lowering the pH to 4.5 decreased the cation selectivity of WtFraC (P K+ / P Cl- = 2.02 ± 0.15, pH 7.5), which increased the anion selectivity of ReFraC (P K+ / P Cl- = 0.36 ± 0.08, pH 4.5, Figure 11B). [Example]

[0071] Biomarker detection using WtFraC nanopore After assessing the capture of chymotrypsin (25 kD, 245 amino acids) and endothelin-1 (12.5 kD, 21 amino acids), protein biomarkers for pancreatic cysticercosis (15) and bronchiolitis obliterans (16), respectively, the WtFraC nanopore was used to detect a broader range of protein biomarkers, including β2-microglobulin, an 11.6 kDa (99 amino acids) biomarker for peripheral arterial disease (17), human epidermal growth factor (EGF), a 6.2 kDa (53 amino acids) biomarker for chronic kidney disease (18), and angiotensin I, a 1.3 kD (10 amino acids) biomarker for hypertensive crisis (19).

[0072] All biomarkers were evaluated under negative applied potentials and at pH 4.5, except for chymotrypsin. The capture frequency of all biomarkers increased with applied potential. All other parameters tested showed nonuniform voltage dependence. The residence time of the biomarkers inside WtFraC increased (chymotrypsin), decreased (β2-microglobulin and angiotensin 1), or showed biphasic behavior with applied potential (EGF and endothelin 1). See Figure 12. The voltage dependence of the residual current percentage (Ires%) for chymotrypsin decreased with potential, and the Ires% for endothelin 1 increased with potential, whereas the Ires% for β2-microglobulin, EGF, and angiotensin 1 remained constant. Despite the complex voltage dependence of current blockade, our results demonstrated that the WtFraC nanopore is capable of discriminating between oligopeptide and protein biomarkers of different sizes by virtue of the Ires% of their current blockade alone (Figure 12). [Example]

[0073] Identification of near-isoform oligopeptides To challenge our experimental system, we sought to identify highly similar analytes. We selected endothelin-1 (ET-1) and endothelin-2 (ET-2), nearly isomeric oligopeptides differing by only one of 21 amino acids (Figures 13A and 13B). At -50 mV, we observed distinct blockade with unique Ires% and dwell times for ET-1 (Ires% 8.9 ± 0.1%, dwell time 5.6 ± 2.0 ms, N = 3, n = 600) and ET-2 (6.1 ± 1.4%, dwell time 19.0 ± 5.3 ms, N = 3, n = 384) (Figure 12B). This already enabled their identification by individual blockade levels (Figure 13C).

[0074] Surprisingly, when we first added 2 μM ET-1 (FIG. 13D) and then 8 μM ET-2 (FIG. 13E) to the same pore, we were also able to separate both mixtures of two distinct populations by plotting the standard deviation of event amplitudes across their corresponding Ires%. This observation indicates that highly similar (oligo)peptides or other analytes can be distinguished using the FraC nanopore.

Claims

1. A system comprising a funnel-shaped proteinaceous nanopore containing an α-helical pore-forming toxin that is a member of the actinoporin protein family, wherein the α-helical pore-forming toxin is Fragaceatoxin C (FraC), a mutant FraC, a FraC paralog, or a FraC homolog.

2. 10. The system of claim 1, wherein the nanopore is located between a first liquid medium and a second liquid medium, at least one of the liquid media containing an analyte, and the system is operative to detect a property of the analyte.

3. 3. The system of claim 1, wherein the system is operable to translocate and / or trap an analyte in the pore.

4. 4. The system of claim 1, operative to detect a property of an analyte, comprising subjecting the nanopore to an electric field such that the analyte interacts with the nanopore.

5. 5. The system of claim 1 , operative to detect a property of an analyte, comprising subjecting the nanopore to an electric field such that the analyte translocates through the nanopore by electrophoresis and / or electroosmosis.

6. The system of claim 4 or 5, wherein the property is an electrical, chemical or physical property of the analyte.

7. The system of claim 1 , wherein the nanopore is comprised in a (planar) lipid bilayer.

8. The system of claim 7, wherein the lipid bilayer comprises or consists of phosphatidylcholine (PC), preferably 1,2-diphytanoyl-sn-glycero-3-phosphocholine.

9. The system of claim 1 , wherein the FraC is a mutant FraC.

10. 10. The system of claim 9, wherein the mutant FraC comprises at least one substitution of a negatively charged amino acid residue in the narrow part of the pore with a neutral or positively charged amino acid residue and / or at least one substitution of a neutral amino acid residue in the narrow part of the pore with a positively charged amino acid residue.

11. The system according to claim 10, wherein the mutant FraC comprises a mutation at position 10, preferably the mutation AsplOArg or AsplOLys.

12. 12. The system of claim 10, wherein the mutant FraC further comprises one or more compensatory mutations that restore the hemolytic activity of the FraC, preferably wherein the compensatory mutations are present at positions 2, 9, 34, 52, 112, 150, 153 and / or 159, preferably at position 159.

13. 13. The system of any one of claims 1 to 12, comprising FraC fused, preferably at its C-terminus, to a protein affinity tag, such as a His tag or a Strep tag.

14. A method for providing a system according to any one of claims 1 to 13, comprising the steps of: providing a recombinant monomer of said α-helical pore-forming toxin from the actinoporin protein family; contacting the monomers with liposomes to assemble the monomers into oligomers; recovering the oligomer from the liposome; contacting the oligomer with a lipid bilayer, which may contain sphingomyelin, to allow formation of a nanopore; A method comprising:

15. 14. A method comprising the step of applying an electric field to the system described in any one of claims 1 to 13, wherein a funnel-shaped nanopore containing the α-helical pore-forming toxin is positioned between a first conductive liquid medium and a second conductive liquid medium.

16. The method of claim 15 , wherein at least one of the conducting liquid media comprises an analyte.

17. 17. The method of claim 15 or 16, further comprising detecting the analyte in a method comprising measuring ionic current as the analyte interacts with the nanopore to obtain a current pattern, wherein the occurrence of a block in the current pattern indicates the presence of the analyte.

18. 18. The method of any one of claims 15 to 17, further comprising identifying the analyte.

19. 20. The method of claim 18, wherein identifying the analyte comprises comparing the current pattern to known current patterns obtained using known analytes under the same conditions.

20. 20. The method of any one of claims 16 to 19, wherein the analyte is a nucleotide, a nucleic acid, an amino acid, a peptide, an oligopeptide, a protein, a polymer, a drug, an ion, a pollutant, a nanoscopic entity, or a biological warfare agent.

21. 20. The method of any one of claims 16 to 19, wherein the analyte is a polymer.

22. 22. The method of claim 21, wherein the polymer is selected from the group consisting of a protein, a polypeptide, an oligopeptide, an unfolded peptide, an unfolded oligopeptide, and an unfolded protein.

23. 23. The method of claim 22, wherein the polymer is a nucleic acid.

24. 24. The method of claim 23, wherein the nucleic acid is ssDNA, dsDNA, RNA, or a combination thereof.

25. 25. The method of any one of claims 15 to 24, wherein the FraC nanopore is a mutant FraC nanopore.

26. 26. The method of claim 25, wherein the conductance through the tunnel of the mutant FraC nanopore is higher than the conductance through its corresponding wild-type FraC nanopore.

27. A mutant Fragaceatoxin C (FraC) nanopore comprising at least a first mutant FraC monomer comprising at least one substitution of a negatively charged amino acid residue in the narrow part of the pore with a positively charged amino acid residue and / or at least one substitution of a neutral amino acid residue in the narrow part of the pore with a positively charged amino acid residue.

28. 28. The variant of claim 27, comprising a substitution at position 3 or 10, or at both positions 3 and 10.

29. 29. The mutant FraC nanopore of claim 28, comprising a mutation at position 10, preferably D10R.

30. 30. A mutant FraC according to any one of claims 27 to 29, further comprising one or more compensatory mutations that restore the hemolytic activity of FraC, preferably wherein said compensatory mutations are present at positions 2, 9, 34, 52, 112, 150, 153, 159, and / or preferably at position 159, more preferably wherein said mutant FraC comprises the mutations D10R and K159E.

31. 31. The mutant FraC nanopore of any one of claims 27 to 30, further comprising at least a second monomer selected from the group consisting of a wild-type FraC monomer, a second mutant FraC monomer, a wild-type FraC paralog or homolog monomer, and a mutant FraC paralog or homolog monomer, wherein the second mutant FraC monomer can be the same as or different from the first mutant FraC monomer.

32. 32. The mutant FraC nanopore of claim 31 , wherein the second monomer is a wild-type FraC paralog or homolog monomer.

33. 33. A mutant FraC nanopore according to any one of claims 27 to 32, wherein the first mutant FraC monomer comprises mutation D10, preferably wherein the mutant is selected from those shown in Table 3.

34. 34. The mutant FraC nanopore of any one of claims 27 to 33, having a conductance through the tunnel that is higher than the conductance through the tunnel of the corresponding wild-type FraC nanopore.

35. 35. The mutant FraC nanopore of any one of claims 27 to 34, further comprising a molecular motor capable of translocating an analyte into or through the nanopore at an average translocation rate that is lower than the average translocation rate at which the analyte translocates into or through the nanopore in the absence of the molecular motor.

36. 36. The mutant FraC nanopore of claim 35, wherein the molecular motor is an enzyme.

37. 37. The mutant FraC nanopore of claim 36, wherein the enzyme is a polymerase, an exonuclease, or a Klenow fragment.

38. 38. Use of a system according to any one of claims 1 to 13 or a mutant FraC nanopore according to any one of claims 27 to 37 for biopolymer sensing and / or biopolymer sequencing.

39. 39. The use according to claim 38, wherein the biopolymer is a protein, peptide or nucleic acid.

40. 40. The use according to claim 39, wherein the biopolymer is a nucleic acid, preferably the nucleic acid is ssDNA, dsDNA, RNA or a combination thereof.

41. 40. The use according to claim 39, wherein the biopolymer is a protein, polypeptide, oligopeptide, unfolded peptide, unfolded oligopeptide or unfolded protein.

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