FraC actinoporin-based biological nanopores for biopolymer sensing and sequencing

Abstract

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 migration through nanopores. In one embodiment, a system is provided comprising a funnel-shaped protein nanopore, the nanopore comprising an alpha-helix pore-forming toxin, which is a member from the actinoporin protein family, more particularly, a Fragaceatoxin C (FraC), a mutant FraC, a FraC para-homolog, or a FraC homolog.

Description

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

[0002] Nanopores represent an attractive way to analyze biopolymers, for example to determine the identity of polypeptides or polynucleotides, or to estimate the identity of individual building blocks in a polymer for sequencing purposes. This is because the method is label-free, provides measurements that rely on small numbers or even single molecules, and produces highly scalable electrical signals.

[0003] In a measurement system utilizing a nanopore, some properties of the system depend on the nucleotides in the nanopore, and electrical measurements of the properties are employed. For example, a measurement system is created by placing a nanopore in an insulating membrane and measuring a voltage-driven ion flow through the nanopore in the presence of nucleotides of a polynucleotide.

[0004] Nanopores have emerged as an effective method 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 allow the migration of double-stranded DNA. Recently, aerolysin was used to distinguish between homopolymers composed of adenine but with different lengths [5]. To date, only αHL and MspA have been shown to distinguish nucleic acids. Both nanopores have β-folds in their transmembrane regions. When DNA passes through MspA, the current blockade level is affected by four or more nucleotides [6], while αHL has three sensing regions in its barrel structure and can accommodate about 20 nucleobases [7].

[0005] The present invention provides novel nanopores with different structures and recognition sites that provide improvements in sequencing accuracy and / or provide different error distributions. Here, we describe the purification and pre-oligomerization of Fragaceatoxin C (FraC), an α-helical pore-forming toxin from a member of the actinoporin protein family, complexed with sphingomyelin and reconstituted into a planar lipid bilayer composed of 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC). The inventors further designed a FraC mutant (e.g., ReFraC) that allows the capture and migration of ssDNA and the discrimination of homopolymers of adenine, thymine, and cytosine fixed with neutravidin (NA). Remarkably, dsDNA can migrate through FraC, most likely through the elastically deformed α-helical contraction of the nanopore.

[0006] Notably, the FraC nanopore was found to have an ideal shape for protein sequencing and analysis of folded proteins. It is shown below that electroosmotic flow is the dominant force that induces proteins and polypeptides to enter the nanopore. By precisely modifying the contraction of the nanopore or adjusting the inner surface of the nanopore by changing the solution pH, the migration of positively and negatively charged polypeptides can be observed. This is noteworthy because it shows that sufficiently strong electroosmotic flow can be induced to transport positive and negative residues under a fixed applied potential. A striking finding is that a range of (unfolded) proteins of different sizes, for example, proteins in the range of 1.2-25 kDa, can be distinguished based on individual blockages. Using a model polypeptide of 20 amino acids, it was demonstrated that even differences in single amino acid residues can be observed by nanopore recordings, indicating that the FraC nanopore allows the identification of specific sequence features in migrating polypeptides.

[0007] In addition, the inventors have designed a method for reconstructing pre-oligomerized FraC nanopores in a planar lipid bilayer without sphingomyelin. The ReFraC nanopore is modified to allow electrophoretic DNA capture and display the distinction between ssDNA homopolymers. In contrast to other nanopores used for sequencing DNA (such as αHL and MspA), FraC has a V-shaped transmembrane region of an α-helix, which is conducive to fine-tuning the discrimination of nucleobases. This is because amino acid substitutions at different positions within the transmembrane α-helix can adjust the size and chemical composition of the contraction.

[0008] ReFraC is also shown herein to induce the melting of DNA duplexes at low applied potentials or to allow their migration at high applied potentials. The melting of DNA hairpins or higher order DNA structures has been studied using αHL nanopores

[20] ,

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

[13] or MspA (4.8 nm)

[22] , suggesting that FraC could be advantageously used to study larger higher order dsDNA structures, such as G-quadruplexes, or folded RNA structures, such as tRNA.

[0009] Thus, in a first aspect, the present invention provides a system comprising a funnel-shaped protein nanopore, the nanopore comprising an α-helical pore-forming toxin, which 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 may preferably be fused 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, at least one of the negatively charged amino acid residues included in the mutant FraC in the narrow part of the hole is replaced by a neutral or positively charged amino acid residue, and / or at least one of the neutral amino acid residues in the narrow part of the hole is replaced by a positively charged amino acid residue. For example, the mutant comprises at least one sudden change in the transmembrane helix. Preferably, at least one negatively charged amino acid residue becomes a positively charged amino acid residue. In a preferred embodiment, the mutant FraC comprises a sudden change at position 10, preferably sudden changes Asp10Arg or Asp10Lys (residue numbering such as crystal structure PDB ID 4TSY). The mutant FraC can further comprise one or more compensatory sudden changes to restore the hemolytic activity of FraC, preferably, wherein the compensatory sudden changes are present at position 2, 9, 34, 52, 112, 150, 153 and / or 159, and / or preferably position 159. For example, the compensatory mutation is selected from A2S, I9T, A34V, F52Y, W112L, T150I, G153D, K159E, I171T and any combination thereof. In a specific aspect, 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 may be located between a first liquid medium and a second liquid medium, wherein at least one of the liquid media includes an analyte, and wherein the system is operable to detect a property of the analyte.

[0012] In one embodiment, the system is operable to allow an analyte to migrate through a channel. In another embodiment, the system is operable to detect a property of an analyte, comprising subjecting the nanopore to an electric field so that the analyte interacts with the nanopore. The applied potential (which produces an electric field) is necessary to generate an electric current. The electric current is an output signal. For example, the system is operable to detect a property of an analyte, comprising subjecting the nanopore to an electric field so that the analyte migrates through the nanopore and / or is captured in the nanopore by electrophoresis and / or electroosmosis. 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 aspect, the lipid bilayer comprises or consists of phosphatidylcholine (PC), preferably 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine.

[0014] In a second aspect, the present invention provides a method for providing the system of the present invention, comprising the following steps:

[0015] - providing a recombinant monomer of said α-helical pore-forming toxin from the actinoporin protein family;

[0016] - contacting the monomers with liposomes to assemble them into oligomers;

[0017] - recovering said oligomer from said liposome; and

[0018] - contacting the oligomer with a lipid bilayer, possibly containing sphingomyelin, to allow the formation of a nanopore.

[0019] In a third aspect, the present invention provides a method comprising applying an electric field to a system disclosed herein, wherein a funnel-shaped nanopore comprising an alpha-helical pore-forming toxin is located between a first conductive liquid medium and a second conductive liquid medium. At least one conductive liquid medium may include an analyte. The method may further include the step of detecting the analyte in a method comprising: measuring an ionic current when the analyte interacts with the nanopore to provide a current pattern, wherein the presence of a blockage in the current pattern indicates the presence of the analyte. The method may further include identifying the analyte, for example by comparing the current pattern with a known current pattern obtained using a known analyte under the same conditions. The analyte may be a nucleotide, a nucleic acid, an amino acid, a peptide, a protein, a polymer, a drug, an ion, a contaminant, a nano-object, or a biological warfare agent.

[0020] 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 aspect, the analyte is a protein, polypeptide or oligopeptide, such as a protein, polypeptide or oligopeptide having a size of about 1 to about 40 kDa, preferably about 1 to about 30 kDa. In one embodiment, the analyte is an oligopeptide (about 10 or less amino acids), a polypeptide (>10 amino acids) or a folded protein (>50 amino acids).

[0021] In the method of the present invention, the FraC nanopore is preferably a mutant FraC nanopore. The conductance of the passage through the mutant FraC nanopore is generally higher than the conductance through its corresponding wild-type FraC nanopore.

[0022] Yet another 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 portion of the pore to a positively charged amino acid residue and / or at least one substitution of a neutral amino acid residue in the narrow portion of the pore to 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 a substitution at position 3 and 10. In a preferred embodiment, the mutant FraC comprises a mutation at position 10, preferably a mutation Asp10Arg or Asp10Lys (residue numbering as in the crystal structure PDB ID 4TSY). Mutant FraC may further comprise one or more compensatory mutations to restore the hemolytic activity of FraC, preferably, wherein the compensatory mutation is present at position 2, 9, 34, 52, 112, 150, 153 and / or 159, and / or preferably position 159. For example, the compensatory mutation is selected from A2S, I9T, A34V, F52Y, W112L, T150I, G153D, K159E, I171T and any combination thereof. In a specific aspect, mutant FraC (further) comprises a mutation at position 159, preferably Lys159Glu. Very good results can be obtained using the FraC double mutant D10R / K159E. Particularly preferred mutants include those of Table 3 below.

[0023] 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, wherein 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, preferably wherein the mutant is selected from the mutants shown in Table 3.

[0024] The mutant FraC nanopore preferably has a higher conductance through the channel than the conductance through the channel of its corresponding wild-type FraC nanopore.

[0025] The mutant FraC nanopore may further comprise a molecular motor, wherein the molecular motor is capable of moving an analyte into or through the nanopore at an average migration velocity that is less than an average migration velocity of the analyte into or through the nanopore in the absence of the molecular motor.

[0026] For example, the molecular motor is an enzyme, such as a polymerase, an exonuclease or a Klenow fragment.

[0027] For protein analysis, it is preferred to have an electroosmotic flow (EOF) from the cis to the trans compartment, which is induced by the charged contraction. The migration of amino acids of the same charge as the contraction can be obtained by manipulating the pH of the analyte solution. It is advantageous to use one or more unnatural amino acids that retain a negative charge at low pH values ​​(< about 4.5), such as sulfate (SO4 2- ) or phosphate (PO4 2- ) moiety is a suitable amino acid side chain group. Thus, also provided is a nanopore engineered to have a strong electroosmotic flow from the cis to the trans compartment under a negative applied potential (pH 4.5 or lower).

[0028] The present invention also provides the use of the system disclosed herein or the mutant FraC nanopore according to the present invention for biopolymer sensing and / or biopolymer sequencing. For example, the biopolymer is a protein, a peptide or a nucleic acid. Preferably, the use of the system disclosed herein or the mutant FraC nanopore according to the present invention for sensing and / or sequencing of nucleic acids, such as ssDNA, dsDNA, RNA or proteins or polypeptides, or a combination thereof is provided.

[0029] For example, FraC nanopores are advantageously used to identify protein, peptide and oligopeptide biomarkers. Once inside the nanopore, proteins and peptides of different sizes can be distinguished by ionic currents. Although it is not possible to identify individual amino acids in real time during migration, we show that two small (unfolded) oligopeptides, endothelin 1 and endothelin 2 (2.5 kDa), which differ by only one tryptophan residue, can be distinguished by ionic current recordings. Thus, if the migration of peptides can be controlled, for example, by using enzymes, FraC nanopores allow the identification of specific sequence features in migrating peptides.

[0030] In one embodiment, (mutant) FraC nanopores are used as sensors in single-molecule proteomic analysis. In the simplest implementation of nanopore proteomics, proteins are identified amino acid by amino acid as they migrate linearly through the nanopore. Since the sequences of proteins and oligopeptides in known organisms are from genomic analysis, proteins can be simply identified by comparing specific protein blockages during migration unfolded in the nanopore with a database of known protein blockages. Alternatively, folded proteins can be identified when they migrate in the nanopore or not in the nanopore within the nanopore cavity. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1. Wild-type FraC (WtFraC) and D10R-K159E FraC (ReFraC) nanopores, (A) Cross-section through the octameric WtFraC showing Coulomb surface coloring (red = negative charge, blue = positive charge). Aspartate residue 10 located in the constriction of WtFraC is indicated. (B) Top view 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. (D) Raw traces of WtFraC (top) and ReFraC (bottom) at +100 mV in 3 M NaCl, 15 mM Tris.HCl pH 7.5 buffer acquired under the same acquisition settings (2 kHz low-pass Bessel filter and 10 kHz sampling rate).

[0032] Figure 2 DNA discrimination using ReFraC. (A) Representative blockade of a homopolymeric DNA strand in complex with NA using ReFraC. This figure shows the interpretation of the current blockade. (B) A 20 , C 20 , T 20 Representative distribution of residual current obtained from homopolymer chains. (C) 20 and A 20 Nucleotide-induced current blockade of consecutive traces for the same ReFraC pore. The displayed traces were digitally filtered with a 100 Hz cutoff. (D) 20 and A 20 Distribution of residual current applied to a mixture of homopolymer chains. (E) For the analysis of homopolymer C 20 and T 20 Continuous traces of experiments with a mixture of nucleotides. (F) By C 20 and T 20 Distribution of residual current applied by a mixture of homopolymeric chains. Traces were recorded in 3M NaCl, 15mM Tris.HCl, pH 7.5 using a 2kHz low-pass Bessel filter and a 10kHz sampling rate. Traces C and E were subjected to an additional 100Hz Gaussian digital filtering.

[0033] Figure 3.Depolymerization / migration of dsDNA by ReFraC, (A) Representative trace of ReFraC capturing a NA:A(dsDNA)C complex at +50 mV. The open pore current is indicated as "1" and is shown after capturing the complex for comparison. Two levels can be observed in the blockade: first, a lower level ("2") likely corresponds to homopolymeric cytosine, which transforms through an intermediate level (melting, brackets) to a higher level ("3"), which most likely corresponds to homopolymeric adenine. Upon potential reversal ("4"), the blockade is immediately released, indicating that the double-stranded region NA:A(dsDNA)C complex is stripped. (B) At +100 mV, a single blockade ("2") is observed after the dsDNA portion is pushed through (deformation, brackets) for more than half of the case (inset), and when -30 mV is applied, the blockade cannot be immediately released ("3") . At higher negative potentials, the blockade can be released, indicating the formation of a rotaxane ( Figure 8 More examples in ). Traces were recorded in 3M NaCl, 15mM Tris.HCl, pH 7.5 using a 2kHz low-pass Bessel filter and 10kHz sampling rate.

[0034] Figure 4 .Determination of single channel conductance distribution and voltage-current dependence of WtFraC and ReFraC nanopores. A: Single channel conductance distribution measured for WtFraC (top) and ReFraC (bottom) pre-oligomerized pores reconstituted in a planar lipid bilayer. The conductance was measured at -50 mV applied potential. The orientation of each individual channel was verified based on the asymmetry of the conductance. B: Measurement of the voltage-current dependence of WtFraC and ReFraC nanopores. The experiments were repeated 3 times and the error bars represent the standard deviation between the experimental values. Recordings were performed in 15 mM Tris.HCl pH 7.5 and 1 M NaCl.

[0035] Figure 5 .Hemolytic activity of WtFraC, D10R FraC and ReFraC. The hemolytic rate was calculated as the reciprocal of the time elapsed for a 50% decrease in turbidity (measured as optical density at a wavelength of 650 nm) observed in a 1% suspension of horse erythrocytes in 15 mM Tris.HCl pH 7.5 150 mM NaCl. The proteins were added at a concentration of 200 nM and the hemolytic rate is expressed as a percentage of WtFraC. The experiments were repeated 3 times and the error bars represent the standard deviation between the experimental values.

[0036] Figure 6 . Migration and immobilization of A(dsDNA)C DNA substrate recorded using the ReFraC nanopore.

[0037] By oligonucleotide I (5' biotinylated AAAAAAAAAAAAAAAAAAAAA GTGCTACGACTCTCTGTGTGCCCCCCCCCCCCCCCCCCCCCC) and oligonucleotide II (CACACAGAGAGTCGTAGCAC) were annealed to prepare A(dsDNA)C substrate (depicted above the trace). A: Blockage was induced on the ReFraC nanopore by 1 μM A(dsDNA)C alone (left) and A(dsDNA)C complexed with 0.25 μM neutravidin (right), and the substrate was added in cis at an applied potential of +50 mV. B: Blockage was induced on the ReFraC nanopore by 1 μM A(dsDNA)C alone (left) and A(dsDNA)C complexed with 0.25 μM neutravidin (right), and the substrate was added in cis at +70 mV. The residual current at which two levels of blockage of free A(dsDNA)C were detected is represented by the lavender dashed line. The current levels corresponding to the blocked pore and the open pore are shown as lavender and gray dashed lines, respectively. The voltage step scheme is shown as the red line below the trace. Recordings were performed in 15 mM Tris.HCl pH 7.5 and 3 M NaCl with a sampling frequency of 10 kHz and data were smoothed by a 2 kHz low-pass Bessel filter during acquisition.

[0038] Figure 7 .Representative traces show the step-wise enhancement of the residual current within the A(dsDNA)C-neutravidin blockade induced on the ReFraC nanopore. 1 μM A(dsDNA)C and 0.25 μM neutravidin are present in cis at +50 mV. Within the blockade, the residual current has shifted from 8.8±0.7% (initial level) to 12.5±0.7% (final level). The voltage step scheme is shown as the red line at the bottom. Recordings were performed in 15 mM Tris.HCl pH 7.5 and 3 M NaCl with a sampling frequency of 10 kHz and the data were smoothed by a 2 kHz low-pass Bessel filter during acquisition.

[0039] Figure 8 .Another example of a trace showing rotaxane formation from A(dsDNA)C-neutravidin driven into a ReFraC nanopore at +100 mV applied potential. 1 μM A(dsDNA)C and 0.25 μM neutravidin were added in cis. The voltage step scheme is shown as the red line at the bottom. The rotaxane was removed by switching the applied potential to -40 mV. Recordings were made in 15 mM Tris.HCl pH 7.5 and 3 M NaCl with a sampling frequency of 10 kHz and the data were smoothed by a 2 kHz low pass Bessel filter upon acquisition.

[0040] Fig. 9.Representative traces showing the formation of pseudorotaxanes and rotaxanes by oligonucleotide I-neutravidin immobilized within a ReFraC nanopore. A: Pseudorotaxane formation caused by 1 μM oligonucleotide I and 0.25 μM neutravidin present in cis. B: Rotaxane formation by 1 μM oligonucleotide I and 0.25 μM neutravidin present in cis, with simultaneous addition of 1 μM oligonucleotide II in trans. The rotaxane was removed by switching the applied potential to -40 mV (the red arrow above the trace indicates the removal of the rotaxane). The transients describing the unzipping of dsDNA are shown in brackets. The voltage step scheme is shown as the red line at the bottom. Recordings were performed in 15 mM Tris.HCl pH 7.5 and 3 M NaCl with a sampling frequency of 10 kHz and the data were smoothed by a 2 kHz low-pass Bessel filter during acquisition.

[0041] Fig.10 .Capture of oligopeptides (endothelin 1) and proteins (chymotrypsin) with two FraC variants under two different pH conditions. a) Cross-sections of wild-type FraC (WtFraC, PDB: 4TSY) and D10R-K159E-FraC (ReFraC). bc) Representative traces induced by 1 μM endothelin 1 (b) and 200 nM chymotrypsin (c) for WtFraC (left) and ReFraC (right). Chymotrypsin (PDB: 5CHA) and human endothelin 1 (PDB: 1EDN) are shown as surface representations. Endothelin 1 and chymotrypsin enter WtFraC at negative applied potentials, while they enter ReFraC at positive applied potentials. Chymotrypsin blockade was also observed for WtFraC at pH 7.5 and 4.5 at -50 mV, however, the applied potential was increased to -100 mV to obtain a sufficient number of blockades. At pH 7.5, chymotrypsin requires a higher potential for blocking ReFraC under a positive bias than for blocking WtFraC under a negative bias. The buffer at pH 7.5 contains 1M KCl, 15mM Tris, and the buffer at pH 4.5 contains 1M KCl, 0.1M citric acid, 180mM Tris.Base. Endothelin 1 and chymotrypsin are added to the cis compartment. All traces are recorded using a 50kHz sampling rate and a 10kHz low-pass Bessel filter. Coloring represents the electrostatic potential of the molecular surface calculated by APBS (13) (pH 7.5, 1M KCl), where red and blue correspond to negative and positive potentials, respectively (range -4 to +4kbT / ec). The structure is colored using PyMOL.

[0042] Fig.11Electrostatic profiles and ion selectivity of WtFraC and ReFraC. a) Average simulated electrostatic potentials of monomers reveal negative and positive charge constrictions of WtFraC and ReFrac, respectively. Lowering the pH from 7.5 to 4.5 has only a small effect on the electrostatic potential for ReFrac, while for WtFraC the peak at the center of the constriction decreases by about 41%. All simulations were performed in 1 M KCl using APBS (13), and the partial charge of each titratable residue was adjusted according to their average protonation state using a modified version of PDB2PQR software (14). The pKa values ​​of the residues were estimated using PROPKA (33, 34). b) Determination of reversal potentials shows that, as expected from their electrostatic potentials during constriction, WtFraC and ReFrac are cation and anion selective, respectively. Based on the amplitude of the decrease in the simulated electrostatic potential, lowering the pH from 7.5 to 4.5 decreases the ion selectivity of WtFraC (P_(K^+) / P_(〖Cl〗^-)) by about 43%. 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 for trans and 1960 mM KCl for cis), and the ion selectivity was determined using the Goldman-Hodgkin-Katz equation. The detailed experimental procedures are given in the Supporting Information. The envelopes following each current-voltage curve represent their respective standard deviations.

[0043] Fig.12. Biomarker characterization at pH 4.5 using WtFraC. a) From top to bottom: surface representation and pictorial representation (Pymol) of the molecular surface of chymotrypsin (25 kD, PDB: 5CHA), representative traces obtained at -150 mV applied potential, heatmap depicting the distribution of dwell time versus Ires% at -150 mV, voltage dependence of Ires%, voltage dependence of dwell time and capture frequency. b), c), d), 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 depicted as a random structure drawn with Pymol. The concentrations of biomarkers were 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. The isoelectric points of biomarkers can be obtained from the literature or the online calculation tool Pepcalc. Error bars represent the standard deviation obtained from at least 3 replicates and at least 300 events per replicate. The data were fitted using a B-spline function (Origin 8.1). All records were collected using a 50 kHz sampling rate and a 10 kHz low-pass Bessel filter.

[0044] Fig.13 .Discrimination of endothelin 1 and 2 at pH 4.5 using WtFraC. a) Molecular surface representation of endothelin 1 and endothelin 2 using electrostatic coloring (PyMOL). b) Top: Amino acid sequences of endothelin 1 and 2. Blue lines indicate disulfide bonds in each oligopeptide. Bottom: Ires% and residence time of endothelin 1 and endothelin 2 blockage at -50 mV in pH 4.5 buffer (1M KCl, 0.1M citric acid, 180mM Tris.Base). c) Representative endothelin 1 and endothelin 2 blockage of the same FraC nanopore at an applied potential of -50 mV. d) Histogram of residual current induced by 2 μM endothelin 1 (left) and corresponding heat map, which depicts the standard deviation of current amplitude relative to Ires% (right). e) Same as in (d), but showing a second population after adding 8 μM endothelin 2 to the same well. Graphs were created using custom R scripts. All recordings were made with a 50 kHz sampling rate and a 10 kHz Bessel low-pass filter.

[0045] Experimental Section

[0046] Section A

[0047] Materials and methods

[0048] 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 were obtained from Avanti Polar Lipids. All errors in this work are given as standard deviations.

[0049] FraC cloning

[0050] In order to clone, Nco I restriction site (CCATGG) was introduced at the starting point (5' end) of the DNA sequence corresponding to the ripe WtFraC from A. fragacea. In order to maintain the reading frame, two other bases were inserted after the Nco I site, thereby producing other alanine residues after the start methionine. For purification purposes, at the C-terminus of FraC, His9 affinity tag was connected by a flexible glycine-serine-alanine joint, and the open reading frame was terminated by two continuous stop codons, followed by Hind III restriction site (3' end). 50ng of synthetic gene with optimized codon composition (IDT) was used as template for the following PCR reaction: 6 μM primers Frf and Frr (Table 2) were used in 300 μL volumes, and the gene was amplified by Phire Hot Start II DNA polymerase (Finnzymes). PCR protocol was as follows: pre-incubated at 98°C for 30 seconds, then denatured at 98°C for 5 seconds, extended at 72°C for 1 minute, 30 cycles. The resulting PCR product containing the WtFraC gene with a Hi9 tag was purified using the QIAquick PCR purification kit (Qiagen) and digested with Nco I and Hind III (FastDigest, Fermentas). The gel-purified insert (QIAquick Gel Extraction Kit, Qiagen) was cloned into the pT7-SC1 expression plasmid under the control of the T7 promoter via the Nco I (5') and Hind III (3') sites using sticky end ligation (T4 ligase, Fermentas). 0.6 μL of the ligation mixture was transformed into 50 μL of E. 10G cells (Lucigen). The transformed bacteria were grown on ampicillin (100 μg / ml) LB agar plates at 37° C. overnight. The identity of the clones was confirmed by sequencing.

[0051] 10R FraC Construction

[0052] 100 ng of the pT7-SC1 plasmid containing the WtFraC gene was used as a template for the PCR reaction: the gene was amplified by Phire Hot Start II DNA polymerase (Finnzymes) using 6 μM primer 10Rf (encoding D10R) and T7 terminator (Table 2) in a volume of 300 μL. The PCR reaction cycle protocol was as follows: pre-incubation at 98°C for 30 seconds, followed by denaturation at 98°C for 5 seconds and extension at 72°C for 1 minute, for 30 cycles. The PCR product was gel purified (QIAquick Gel Extraction Kit, Qiagen) and purified by the MEGAWHOP program. [1] It was cloned into the pT7 expression plasmid (pT7-SC1): about 500 ng of the purified PCR product was mixed with about 300 ng of the pT7-SC1 plasmid containing the WtFraC gene and amplified with Phire Hot Start II DNA polymerase (Finnzymes) in a final volume of 50 μL (pre-incubation at 98°C for 30 seconds, followed by denaturation at 98°C for 5 seconds and extension at 72°C for 1.5 minutes, 30 cycles). The circular template was eliminated by incubation with Dpn I (1FDU) at 37°C for 2 hours. 0.6 μL of the resulting mixture was transformed into E. 10G cells (Lucigen). The transformed bacteria were grown overnight on LB agar plates containing ampicillin (100 μg / ml) at 37°C. The identity of the clones was confirmed by sequencing.

[0053] Construction of 10R FraC library by error-prone PCR

[0054] The library was constructed by amplifying the D10R FraC gene from plasmid DNA using T7 promoter and T7 terminator primers (Table 2).

[0055] Table 2. Oligonucleotides used in this study

[0056]

[0057] "5Biosg" represents a biotin group conjugated to the 5' end of DNA via a C6 linker (IDT)

[0058] 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 DNA plasmid pool from the clones with the highest activity identified in the first round of screening. DNA amplification was performed by error-prone PCR: 400 μL PCR mixture (200 μl REDTaq ReadyMix, 6 μM T7 promoter and T7 terminator primers, about 400 ng plasmid template) was divided into 8 reaction volumes containing 0 to 0.2 mM MnCl2 and cycled 27 times (pre-incubation at 95°C for 3 minutes, then cycle: denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 3 minutes). These conditions usually produce 1-4 mutations per gene in the final library. The PCR products were pooled together, gel purified (QIAquick GelExtraction Kit, Qiagen) and cloned into the pT7 expression plasmid (pT7-SC1) by the MEGAWHOP procedure: About 500 ng of the purified PCR product was mixed with about 300 ng of the pT7-SC1 plasmid containing the 10R FraC gene and amplified with Phire HotStart II DNA polymerase (Finnzymes) in a final volume of 50 μL (pre-incubation 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). The circular template was eliminated by incubation with Dpn I (1FDU) at 37°C for 2 hours. 0.6 μL of the resulting mixture was transformed into E. 10G cells (Lucigen). Transformed bacteria were grown overnight at 37°C on ampicillin (100 μg / ml) LB agar plates and typically produced >10 5 Colonies were harvested and used for plasmid DNA library preparation.

[0059] Screening of crude lysates for hemolytic activity after FraC overexpression

[0060] Overnight seed cultures from 600 clones (see above) were inoculated into 450 μL of fresh medium in a new 96-deep-well plate, and the cultures were grown at 37°C to OD 600 ~ 0.8. Then, IPTG (0.5 mM) was added to induce overexpression, and the temperature was lowered to 25°C for overnight incubation. The bacteria were harvested the next day 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 MgCl2, 10 μg / ml lysozyme, 0.2 units / mL DNAse I) and lysed by shaking at 1300 RPM for 30 minutes at 37°C. 0.5-5 μL of the crude lysate was then added to 100 μL of approximately 1% horse erythrocyte suspension. The latter was prepared by centrifuging horse blood (bioMérieux Benelux) at 6000×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 was red, the solution was centrifuged again and the pellet was resuspended in the same buffer. The hemolytic activity was monitored by the decrease in OD at 650 nm over time (Multiskan GO Microplate Spectrophotometer, Thermo Scientific).

[0061] Screening for mutations that compensate for the deleterious effects of the D10R amino acid substitution in FraC

[0062] The D10R amino acid substitution resulted in a 5-fold decrease in the hemolytic activity of FraC ( Figure 5). Although D10R FraC can still oligomerize and reconstitute in planar bilayers prepared from 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC), we searched for compensatory mutations that restored the hemolytic activity of D10R FraC to the level of WtFraC. Maintaining the hemolytic activity of FraC is important for two reasons: first, it reflects the ability to assemble into oligomeric pores on targeted lipid bilayers and can therefore translate into the preparation of more efficient oligomeric nanopores. On the other hand, the hemolytic activity provides a convenient way to screen the functionality of variants with arbitrary amino acid sequence changes and will therefore aid future engineering efforts on FraC nanopores. To identify compensatory mutations, we constructed a random mutagenesis library based on the D10R FraC gene, transformed it into Bl21 DE3 Escherichia coli, and screened the hemolytic activity of individual variants 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. Then, 7 clones with the highest hemolytic activity levels were selected for further characterization. Table 3 summarizes the sequence changes that occurred in the corresponding genes.

[0063] Table 3. Sequence changes that compensate for the deleterious effects of the D10R mutation in FraC

[0064]

[0065] The purified variants were oligomerized in sphingomyelin:DPhPC (1:1) liposomes and solubilized in 0.6% LDAO. After exchanging the detergent to 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 the variants designated 3, 4 and ReFraC (Table 3) as the most promising pore formers. However, the nanopores prepared by variant 3 were heterogeneous (less than 50% produced octameric pores), while the pore-forming activity of variant 4 decayed after being stored at 4°C for several days. When stored at 4°C, oligomeric ReFraC maintained pore-forming activity for several months and formed nanopores that were almost as homogeneous as WtFraC while being able to capture ssDNA. Therefore, we chose ReFraC for further DNA analysis in this study. In addition, we replaced aspartic acid 10 with asparagine in ReFraC, generating the D10N K159E variant, but no ssDNA entry was detected.

[0066] WtFraC (protein sequence)

[0067] MASADVAGAVIDGAGLGFDVLKTVLEALGNVKRKIAVGIDNESGKTWTAMNTYFRSGTSDIVLPHKVAHGKALLYNGQKNRGPVATGVVGVIAYSMSDGNTLAVLFSVPYDYNWYSNWWNVRVYKGQKRADQRMYEELYYHRSPFRGDNGWHSRGLGYGLKSRGFMNSSGHAILEIHVTKA GSAHHHHHH **

[0068] WtFraC (DNA sequence)

[0069] ATGGCGAGCGCCGATGTCGCGGGTGCGGTAATCGACGGTGCGGGTCTGGGCTTTGACGTACTGAAAACCGTGCTGGAGGCCCTGGGCAACGTTAAACGCAAAATTGCGGTAGGGATTGATAACGAATCGGGCAAGACCTGGACAGCGATGAATACCTATTTCCGTTCTGGTACGAGTGATATTGTGCTCCCACATAAGGTGGCGCATGGTAAGGCGCTGCTGTATAACGGTCAAAAAAATCGCGGTCCTGTCGCGACCGGCGTAGTGGGTGTGATTGCCTATAGTATGTCTGATGGGAACACACTGGCGGTACTGTTCTCCGTGCCGTACGATTATAATTGGTATAGCAATTGGTGGAACGTGCGTGTCTACAAAGGCCAGAAGCGTGCCGATCAGCGCATGTACGAGGAGCTGTACTATCATCGCTCGCCGTTTCGCGGCGACAACGGTTGGCATTCCCGGGGCTTAGGTTATGGACTCAAAAGTCGCGGCTTTATGAATAGTTCGGGCCACGCAATCCTGGAGATTCACGTTACCAAAGCAGGCTCTGCGCATCATCACCACCATCACTGATAAGCTT

[0070] Overexpression and purification of FraC

[0071] Transform E. with the pT7-SC1 plasmid containing the FraC gene. EXPRESSBL21 (DE3) cells. 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 culture was grown at 37°C with shaking at 200 rpm until OD600 reached about 0.8. The expression of FraC was then induced by adding 0.5 mM IPTG and allowed to continue growing at 25°C. The next day, the bacteria were harvested by centrifugation at 6000×g for 25 minutes and the pellet was stored at -80°C. The pellet containing monomeric FraC (from 50-100 ml bacterial culture) was 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). Then, in order to cause cell disruption, 0.2mg / ml lysozyme and 2M urea (to prevent debris formation) were supplemented in the bacterial suspension and vigorously shaken for 40 minutes at ambient temperature. The remaining bacteria were destroyed by probe ultrasound. The crude lysate was clarified by centrifugation at 6000xg for 20 minutes, and the supernatant was mixed with 200 μL (bead volume) Ni-NTA resin (Qiagen) pre-equilibrated with washing buffer (10mM imidazole 150mM NaCl, 15mM 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 about 5ml washing buffer. FraC was eluted with about 0.5mL of washing buffer containing 300mM imidazole. Protein concentration was determined by the Bradford method. FraC monomers were stored at 4°C before further use.

[0072] Hemolytic activity assay

[0073] Defibrinated horse blood (bioMérieux Benelux) was washed with 150 mM NaCl, 15 mM Tris.HCl pH 7.5 until the supernatant was clear. The erythrocytes were then resuspended to about 1% concentration (OD 650nm 0.6-0.8) with the same buffer. The suspension was then mixed with 200 nM FraC. Hemolytic activity was measured by monitoring the reduction of OD650 using a MultiskanTM GO microplate spectrophotometer (Thermoscientific). The hemolysis rate was determined as 1 divided by the time it took for the turbidity to decrease by 50%.

[0074] Preparation of sphingomyelin:DPhPC liposomes

[0075] 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 help dissolve the sphingomyelin) and placed in a round-bottom flask. The solvent was evaporated while slowly rotating the flask to deposit a lipid film on the wall. After the lipid film was deposited, the flask was kept 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 lipids 10 mg / ml) using an ultrasonic bath (5-10 minutes at ambient temperature). The obtained liposomes were stored at -20°C.

[0076] Oligomerization of FraC

[0077] 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 (ultrasonic bath) 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 diluted 20-fold with DDM-containing wash buffer (0.02% DDM 150 mM NaCl, 15 mM Tris.HCl pH 7.5) and mixed with about 100 μl (bead volume) 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 (MicroBio Spin, Bio-Rad) and washed with about 2 ml 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, however, EDTA proved to be more effective). The purified FraC oligonucleotide was stored at 4°C.

[0078] Alternatively, FraC oligonucleotides can be formed by mixing FraC monomers with liposomes formed only from sphingomyelin (1 hour at 37°C, then overnight at 4°C). The next day, 5mM EDTA and 1% DDM (final) were added to the proteoliposomes and incubated at room temperature for 15 minutes. The solution was then diluted to a volume of 1 ml (containing 5mM EDTA, 0.05% DDM 15mM Tris HCl 7.5 150mM NaCl). The solution was then concentrated to approximately 100 μl using a 100 kDa cut-off ultrafiltration device.

[0079] Electrical recordings in planar lipid bilayers

[0080] The applied potential refers to the potential of the trans electrode. The FraC nanopore was inserted into the lipid bilayer from the cis compartment connected to the ground electrode. The two compartments were separated by a 25 μm thick polytetrafluoroethylene membrane (Goodfellow Cambridge Limited) containing a pore with a diameter of approximately 100 μm. The pores were pretreated with approximately 5 μl of pentane containing 10% hexadecane, and the bilayer was formed by adding approximately 10 μL of pentane containing 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC) (10 mg / mL) to both electrophysiological 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. The WtFraC nanopore showed a higher open pore current at a positive applied potential than at a negative applied potential, which provides a useful tool for determining the orientation of the pore. Electrical recordings were performed in 1 M (initial characterization of FraC nanopores) and 3 M NaCl (for increased amplitude of polynucleotide analysis), 15 mM Tris.HCl pH 7.5.

[0081] Data recording and analysis

[0082] Electrical signals from planar bilayer recordings were amplified using an Axopatch 200B patch clamp amplifier (Axon Instruments) and digitized with a Digidata 1440 A / D converter (Axon Instruments). Data were recorded using Clampex 10.4 software (Molecular Devices) and subsequently analyzed using Clampfit software (Molecular Devices). Electrical recordings were obtained 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. The blocked pore current values ​​(I B ) and the open pore current value I O )Calculate the residual current value (I res ) is I res =100*I B / I O I was determined from a Gaussian fit to the event amplitude histogram B and I O In the case of events showing a stepwise current increase, the residual current level was calculated from the Gaussian fit to the integral current histogram. To determine the event lifetime, the event dwell time (t off ) were combined into cumulative distributions and fitted to a single exponential. The stepwise current enhancement ( Figure 3A) The frequency of rotaxane blockade events was calculated manually and plotted using Origin (OriginLab Corporation) or Clampfit software (Molecular Devices).

[0083] Schematic representation of the FraC nanopore

[0084] Molecular mapping was performed using Chimera (http: / / www.cgl.ucsf.edu / chimera).

[0085] Example 1: Reconstitution of wild-type FraC pores in planar lipid bilayers.

[0086] The recombinant wild-type FraC (WtFraC, Figure 1 A; 1B, top) Protein monomers. Previous work established that pore assembly of actinoporin is initiated by the presence of SM in the lipid bilayer [9],

[10] ,

[11] , [8]. Consistently, water-soluble monomers of WtFraC 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 solubilizing the liposomes in 0.6% N,N-dimethyldodecylamine N-oxide (LDAO) to prevent oligonucleotide dissociation

[12] , LDAO was exchanged for 0.02% dodecyl-β-maltoside (DDM) by a second round of Ni-NTA chromatography (SI). Submicrogram amounts of oligomeric WtFraC purified in 0.02% DDM were readily added to the cis-side of the DPhPC planar lipid bilayer to generate pores. The distribution of single channel conductance of WtFraC pores in 1 M NaCl, 15 mM Tris.HCl pH 7.5 buffer showed a predominantly single conductance type ( Figure 1 C; 4A, top), which may correspond to the octamer observed in a recently determined crystal structure [8]. Similar to other biological nanopores, the WtFraC channel exhibits an asymmetric current-voltage (IV) relationship ( Figure 4 B), allowing the orientation of the pores to be determined. Figure 1 D top shows an exemplary trace obtained in 3M NaCl, 15mM Tris.HCl pH 7.5 buffer.

[0087] Example 2: Modification of WtFraC for nucleic acid analysis

[0088] The crystal structure of the octameric WtFraC showed that this nanopore is large enough to pass ssDNA (1.2 nm constriction diameter) [8]. However, in our initial experiments, we were unable to observe ssDNA blockage, most likely because the negatively charged constriction of the WtFraC pore prevented DNA migration

[13] ,

[14] . To induce ssDNA to pass through FraC, we replaced aspartate 10 with arginine, thereby preparing a nanopore with a positively charged constriction ( Figure 1 B, bottom). Because D10R FraC showed low pore-forming activity ( Figure 5 ), we performed random mutagenesis on the D10R FraC gene background and screened the obtained variants (SI) for hemolytic activity. As a result, we identified a compensatory mutation of lysine 159 to glutamic acid (K159E), which is located at the outer edge of the wide pore cavity ( Figure 1 A). The double mutant of FraC, D10R, K159E (ReFraC), showed hemolytic activity close to the wild-type level ( Figure 5 ) and produces uniform pores ( Figure 1 C), despite having an altered IV relationship compared to WtFraC ( Figure 4 B and Figure 1 D, 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 a narrower constriction, as arginine has a more bulky side chain than aspartate ( Figure 1 B).

[0089] Example 3: Polynucleotide Identification Using ReFraC

[0090] We followed established methods using αHL[7],

[15] ,

[16] ,

[17] and MspA

[14] ,

[18] to immobilize DNA using neutravidin (NA). We complexed 5'-end biotinylated A20 / C20 / T20 ssDNA homopolymers with tetrameric NA to evaluate the ability of ReFraC to migrate and discriminate DNA strands. We added premixed DNA (1 μM) and NA (0.25 μM) to the cis compartment of a planar lipid bilayer device and performed DNA discrimination experiments in 3 M NaCl, 15 mM Tris.HCl, pH 7.5 buffer and +70 mV applied potential (see trans electrode). We observed a permanent current blockade, which was caused by the pseudorotaxane, where the ssDNA stably passed through the pore until the applied potential was reversed ( Figure 2 The residual current Ires, which is the percentage of the blocked and open pore current amplitudes multiplied by 100 (I B / I O)×100) were as follows: NA:A20 was 13.1±0.4% (N=5, n=364, where N is the number of multiple independent single-well experiments and n is the blockage analyzed), NA:C20 was 10.8±0.3% (N=4, n=920), and NA:T20 was 14:0±0.3% (N=5, n=780) ( Figure 2 B). To exclude the effect of pore-to-pore variations, we also analyzed a mixture of homopolymers ( Figure 2 CF). The relatively low residual current indicates that the pore around the traversed ssDNA is tightly closed.

[0091] Example 4: DNA melting and double-stranded DNA migration through ReFraC nanopores

[0092] The contraction of ReFraC (1.2 nm) is smaller than that of B-form double-stranded DNA (dsDNA, about 2 nm). Therefore, to evaluate dsDNA as a terminator for DNA analysis, we designed two oligonucleotides: oligonucleotide I with the sequence bio-5'-A20-GTGCTACGACTCTCTGTGTG-C20-3' with a biotin group attached to the 5'-end and a short oligonucleotide II with the reverse complementary sequence of the underlined portion of oligonucleotide I. Annealing produced 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 resulted in transient blockage of the ReFraC pore (blockage lifetime 2 ± 5 sec, Ires = 10.0 ± 0.2%, N = 3, n = 290, Figure 6 A, left). Increasing the applied potential to +70 mV shortens the blockade lifetime to 2.9 ± 0.4 ms (note that the residual current shows two current levels: 12.8 ± 0.6% and 3.5 ± 0.5% N = 3n = 2700 Figure 6 B, left). The decrease in blockade lifetime with potential indicates that A(dsDNA)C migrates through ReFraC. To demonstrate DNA migration, we added NA to the cis chamber. The NA:A(dsDNA)C blockade at |+50 mV ( Figure 6 A, right side) and +70mV( Figure 6 B, right) are permanent, thus indicating that in the absence of NA, the transient blockade cannot be triggered by the contraction of A(dsDNA)C to the cis compartment. Curiously, at +50 mV, 31 ± 4% of the NA:A(dsDNA)C blockade (N = 3, n = 468) showed that the residual current was reduced from the transient level ( Figure 3 A, state "2", Ires = 8.8 ± 0.7%) gradually increased to a stable level ( Figure 3A, state “3”, Ires = 12.5 ± 0.7%, N = 4, n = 46; Figure 7 More examples in ). The current level of state “2” is slightly lower than that of NA:C20 (Ires=10.5±0.7; N=3, n=206, +50 mV). The current level of state “3” is similar to that of NA:A20. A possible explanation for the above current enhancement is that at +50 mV, the C20 segment of NA:A(dsDNA)C stays in the constriction of the nanopore ( Figure 3 A, state "2"), where the duplex segment blocks further migration. However, after the duplex melts, A20 occupies the constriction region of ReFraC, where NA blocks migration ( Figure 3 A, state “3”). Consistently, at +50 mV, the NA:A(dsDNA)C blockade was immediately relieved when the potential was reversed to −30 mV, indicating that at +50 mV, the migration of A(dsDNA)C was mediated by unzipping ( Figure 3 A, brackets).

[0093] In contrast, at +70mV, a large portion of the blockage is not immediately released at -30mV ( Figure 3 B, inset), indicating the formation of an interlocked state ( Figure 3 B, states “2” and “3”). Moreover, 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 3 B, inset). Considering that the blockade of oligonucleotide I alone in complex with NA is immediately released at -30 mV ( Fig. 9 A), we attribute this interlocked state to the rotaxane, in which the duplex DNA fragments of NA and A(dsDNA)C serve as cis and trans terminators, respectively ( Figure 3 B, right). It is expected that this rotaxane can also be formed from NA:oligonucleotide I cis blocking by adding oligonucleotide II in trans ( Fig. 9 B) Switching the potential to -40 mV rapidly disassembles the rotaxane, probably through the unzipping of the trans-dsDNA terminator ( Figure 8 and 9 B). Rotaxane formation from cis-present NA:A(dsDNA)C requires deformation of the ReFraC pore to allow migration of the duplex segment of the A(dsDNA)C substrate ( Figure 3 B, brackets).

[0094] This structural flexibility may be a general feature of α-helical pores. Previously, we observed blockage of type I ClyA-CS nanopores (~3.3 nm constricted diameter) by human thrombin (~4.2 nm in diameter), followed by a transient increase in the open pore current

[19] . This phenomenon was interpreted as allowing protein migration through deformation and constriction of ClyA.

[0095] References for Part A

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[11] P.Schon,AJGarcia-Saez,P.Malovrh,K.Bacia,G.Anderluh,P.Schwille,Biophys J2008,95,691-698.

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[12] K.Tanaka,JMCaaveiro,K.Tsumoto,Biochemistry 2015,54,6863-6866.

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[14] T.Z. Butler, M. Pavlenok, I.M. Derrington, M. Niederweis, J.H. Gundlach, Proc Natl Acad Sci U S A 2008, 105, 20647 - 20652.

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[15] R.F. Purnell, J.J. Schmidt, ACS Nano 2009, 3, 2533 - 2538.

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[16] D. Stoddart, A.J. Heron, J. Klingelhoefer, E. Mikhailova, G. Maglia, H. Bayley, Nano Lett 2010, 10, 3633 - 3637.

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

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

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[19] M. Soskine, A. Biesemans, M. De Maeyer, G. Maglia, J Am Chem Soc 2013, 135, 13456 - 13463.

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[20] J. Mathe, H. Visram, V. Viasnoff, Y. Rabin, A. Meller, Biophys J 2004, 87, 3205 - 3212.

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

[0117]

[22] M. Faller, M. Niederweis, G.E. Schulz, Science 2004, 303, 1189 - 1192.

[0118] Part B

[0119] Part A above shows that an alpha-helical pore-forming toxin from the actinoporin protein family, Fragaceatoxin C (FraC), is advantageously used for polynucleotide analysis.

[0120] Part B demonstrates that the FraC nanopore is also suitable for identifying biomarkers in the form of proteins, such as oligopeptides (about 10 or fewer amino acids), polypeptides (>10 amino acids), and folded proteins (>50 amino acids).

[0121] Material

[0122] 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), Hydrochloride (Lot#SLBG8541V) and Base (lot #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-phosphatidylcholine (DPhPC, 850356P) and sphingomyelin (Brain, Porcine, 860062) were purchased from Avanti Polar Lipids. Potassium chloride (≥99%, lot #BCBL9989V) was purchased from Fluka. Citric acid (≥99%, lot #A0365028) was obtained from ACROS. All peptide biomarkers and chemicals were used directly without further purification.

[0123] Note: Use the Recipe for the protocol Prepare the 15 mM Tris, pH 7.5 buffer used below: 1.902 g HCl and 0.354 g The base was dissolved in 1 liter H2O to give 15 mM Tris, pH 7.5.

[0124] method

[0125] FraC monomer expression and purification

[0126] 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. 1 For expression, the plasmid was transformed into E. EXPRESS BL21 (DE3) competent cells. After overnight incubation at 37°C, transformants were harvested from LB agar plates containing 100 mg / l ampicillin 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 220 rpm until the optical density at 600 nm was 0.8, and then 0.5 mM IPTG was added to the cell culture. The temperature was lowered to 25°C to induce FraC protein expression for 12 hours. Cells were recovered by centrifugation at 4,000 RP 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 with 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 for 1 hour using a vertex shaker. To completely disrupt the cells, the suspension was sonicated for 2 minutes (duty cycle 10%, output control 3 of Branson Sonifier 450). The crude lysate was then centrifuged at 6,500 RPM for 20 minutes at 4°C. The supernatant (containing FraC monomers) was transferred to a 50 ml falcon tube containing 100 μl Ni-NTA resin (Qiagen, stored at 4°C and suspended before removing 100 μl) and incubated for 1 hour at room temperature with gentle mixing, wherein the falcon tube was pre-washed with 3 ml wash buffer (10 mM imidazole, 150 mM NaCl, 15 mM Tris, pH 7.5). The resin was rotated at 4,000 RPM for 5 minutes at 4°C. Most of the supernatant was discarded and the particles containing Ni-NTA resin in about 5 ml buffer were transferred to a Micro Bio Spin column (Bio-Rad) at room temperature. The Ni-NTA beads were washed with 10 ml of washing buffer and the proteins were eluted with 500 μl of 300 mM imidazole. The protein concentration was determined using NanoDrop 2000 (Thermo Scientific). The monomers were stored at 4°C.

[0127] Preparation of sphingomyelin-DPhPC liposomes

[0128] 20 mg of sphingomyelin (brain, porcine, Avanti Polar lipids) was mixed with 20 mg of 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC, Avanti Polar lipids) and dissolved in 4 ml of pentane (Sigma) containing 0.5% v / v ethanol. The lipid mixture was placed in a round flask and slowly rotated near a hair dryer to allow the lipids to be fully dispersed around the wall to evaporate the solvent. The flask was opened for another 30 minutes at room temperature to allow the solvent to evaporate completely. Then 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 an ultrasonic bath for 5 minutes. The liposome solution was stored at -20 ° C.

[0129] Oligomerization of FraC

[0130] Frozen liposomes were sonicated after thawing and mixed with monomeric FraC at a mass ratio of 1:1. The FraC and liposome mixture was sonicated in a water bath for about 30 seconds and then kept at 37°C for 30 minutes. The protein-liposomes were dissolved with 0.6% LADO (N,N-dimethyldodecylamine N-oxide, 5% w / v stock in water) and then transferred to a 50ml falcon tube and diluted 20 times with buffer (150mM NaCl, 15mM Tris, pH 7.5, 0.02% DDM). 100μl 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 10ml buffer (150mM NaCl, 15mM Tris, pH7.5). FraC oligonucleotides were eluted with 300 μl elution buffer (200 mM EDTA, 75 mM NaCl, 7.5 mM Tris, pH 8, 0.02% DDM). The oligonucleotides were stable at 4°C for several weeks.

[0131] Electrical recordings in planar lipid bilayers

[0132] Electrical recordings were performed as previously described 2. Briefly, the two chambers were separated by a 25 μm polytetrafluoroethylene membrane (GoodfellowCambridge Limited) containing pores of approximately 100 μm in diameter. Two silver / silver chloride electrodes were immersed in each compartment of the electrophysiological chamber, which was filled with 0.5 ml of buffer. The ground electrode was connected to the cis compartment and the working electrode was connected to the trans side. To form the lipid bilayer, approximately 5 μl of a hexadecane solution (10% v / v hexadecane in pentane) was added to the polytetrafluoroethylene membrane. After approximately 2 minutes, 10 μl of a pentane solution containing 10 mg / ml 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC) was added directly to the buffer in both compartments. The lipid bilayer was then spontaneously formed by reducing the buffer above and below the pores in the Teflon membrane. The FraC oligonucleotide was added to the cis side. Under applied potential, the ionic current of FraC was asymmetric, which helped to assess the orientation of the FraC nanopores in the lipid bilayer. When higher conductance was measured at negative applied potentials, the FraC nanopore showed Fig.10 The orientation shown in . The analyte was then added to the cis chamber. Depending on the pH, two buffer solutions were used for electrophysiological recordings in this study. At pH 7.5, 1 M KCl and 15 mM Tris were used for recordings. When the pH was changed from 7.5 to 4.5, the buffer used contained 1 M KCl, 0.1 M citric acid, and 180 mM Tris.Base. From pH 4.5 to 7.5, FraC and ReFraC oligonucleotides can insert into the lipid bilayer.

[0133] Data recording and analysis

[0134] Planar bilayer recordings were collected using a patch clamp amplifier (Axopatch 200B, Axon Instruments) and the data were digitized with a Digidata 1440 A / D converter (Axon Instruments). Data were acquired using Clampex 10.4 software (Molecular Devices) and subsequently analyzed using Clampfit software (Molecular Devices). Event duration (dwell time), time between two events (inter-event time), blocked current level, and open pore level were detected by the “single channel search” function. The blocked current level is referred to as I B , and the open pore current is called I O Ires%, defined as (I B / I O )*100, which is used to describe the degree of blockage caused by different biomarkers. The mean inter-event time was calculated by combining the inter-event times and applying a single exponential fit to the cumulative distribution.

[0135] Ion selectivity measurements

[0136] The ion permeability (K) was calculated using the Goldman-Hodgkin-Katz equation (Equation (1) below). + / Cl - ), which uses a reverse potential (V r ) was input as a variable. V was measured by extrapolation from the IV curve using asymmetric salt concentration conditions as follows r : A single FraC nanopore was reconstructed using the same buffer in both chambers (symmetric conditions: 840mM KCl, 15mM Tris, pH7.5, 500μl) to evaluate the orientation of the nanopore. 400μl of a solution containing 3.36M KCl, 15mM Tris, pH7.5 was slowly added to the cis chamber, and 400μl of a KCl-free buffer solution (15mM Tris, pH7.5) was added to the trans solution (trans: cis, 467mM KCl: 1960mM KCl). The solutions were mixed and IV curves were collected from -30mV to 30mV in 1mV steps. The same method was used, but the experiment was performed at pH4.5 using 0.1M citric acid buffer solution. Initially, 500μl of 840mM KCl, 0.1M citric acid, 180mM Tris.Base buffer was added to both chambers, and a single FraC channel was obtained. Then, 400 μl of pH 4.5 solution containing 3.36M KCl, 0.1M citric acid, 180mM Tris.Base was slowly added to the cis chamber, and 400 μl of a buffer solution (0.1M citric acid, 180mM Tris.Base, pH 4.5) without KCl was added to the trans solution (thus producing a trans: cis ratio of 467mM KCl: 1960mM KCl). The solution was mixed and IV curves were collected from -30mV to 30mV with 1mV steps. The directionality of ion selectivity was also tested by using high KCl concentrations in the trans chamber and low KCl concentrations in the cis chamber. The Ag / AgCl electrode was surrounded by a 2.5% agarose bridge containing 2.5M NaCl.

[0137] Example 5: Capture of peptides and proteins using FraC nanopores

[0138] To evaluate FraC nanopores as sensors for oligopeptide biomarkers, we first selected endothelin 1 (21 amino acid oligopeptide, 2.5 kD) and α-II-chymotrypsin (hereafter referred to as chymotrypsin, a 245 amino acid globular protein, 25 kD) ( Fig.10Analytes were added to the wild-type FraC (WtFraC) nanopore using 1 M KCl, 15 mM Tris, pH 7.5 solution ( Fig.10 A) on the cis side and applied an external potential to the "working" electrode located in the trans compartment. Because WtFraC exhibits gating above approximately +50 mV but is stable at potentials up to -300 mV, we applied potentials between these limits. Addition of 1 μM endothelin 1 to the cis compartment does not cause gating at ±50 mV ( Fig.10 B) and up to -300 mV. Since the constriction of ClyA is lined with aspartate residues ( Fig.10 A), we reasoned that protonation of these residues under more acidic conditions should reduce the energy barrier for endothelin 1 (carrying a net charge of -2) to migrate through the WtFraC constriction. At the same time, under negative applied potentials, the less negatively charged endothelin 1 would also migrate more easily toward the trans electrode. Blockage of endothelin 1 began to occur at pH 6.4, and their capture frequency increased linearly with decreasing pH (from 0.6 ± 0.2 events s at pH 6.4 to 1.5 ± 0.1 events s at pH 6.4). -1 μM -1 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), blockade of WtFraC by endothelin-1 was observed at -50 mV (Ires%: 9.1 ± 0.1%, residence time: 5.6 ± 2.0 ms, inter-event time: 5.8 ± 0.7 ms), but not at +50 mV ( Fig.10 B).

[0139] Inspired by the more active contraction effect under acidic conditions, we next investigated the effects of D10 R , K159 E FraC( Re Endothelin-1 was captured by a WtFraC nanopore that has an arginine residue in the constriction region engineered for DNA analysis purposes in Part A above. In contrast to WtFraC, ReFraC is stable under positive applied potentials but shows gating at potentials around -50 mV. Therefore, we only applied voltages from -50 mV to +200 mV to ReFraC. At pH 7.5, addition of 1 μM endothelin-1 to the cis compartment caused blockage at +50 mV (residence time: 3.3 ± 2.2 ms, inter-event time: 1413 ± 223 ms) but no blockage was observed at -50 mV ( Fig.10 B). Lowering to pH 4.5 (1M KCl, 0.1M citric acid, 180mM Tris.Base) resulted in an increase in capture frequency at +50mV ( Fig.10 B, residence time: 8.5 ± 1.8 ms, inter-event time: 402 ± 79 ms), despite a reduced electrophoretic mobility toward the counter electrode.

[0140] Next, the protein chymotrypsin (pI 8.75, Sigma) was tested as an example of a relatively large protein analyte. In pH 7.5 buffer (1 M KCl, 15 mM Tris), protein blockages were observed at -50 mV, but they became uniform when we increased the potential to -100 mV (45.2 ± 19.1 events s -1 μM -1 , residence time: 12.0 ± 5.7 ms), while no capture was observed at positive applied potential ( Fig.10 C). In contrast to that 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 ; pH 6.4, 50 ± 22.6 events s -1 μM -1 ; At pH 5.5, 45.2 ± 20.6 events s -1 μM- 1 ), and when the pH dropped to 4.4, the capture frequency decreased (at pH 4.4, 20.8 ± 5.3 events s -1 μM -1 Using ReFraC at pH 7.5, we observed little blockade at high positive applied potentials (at +200 mV, dwell time: 0.2 ± 0.1 ms, inter-event time: 174.3 ± 22.9 ms), but none at -50 mV ( Fig.10 Lowering the pH to 4.5 resulted in an increase in capture frequency (residence time: 1.3 ± 0.7 ms, 112.5 ± 9.5 events s -1 μM -1 , Fig. 9 B). It is noteworthy that ReFraC often shows weak gating events when negatively applied potentials are applied under acidic conditions, e.g. Fig.10 C, bottom right. In summary, both nanopores can capture analytes with a 10-fold difference in molecular weight (2.5 kDa vs. 25 kDa).

[0141] Example 6: Ion selectivity and electrostatic potential of FraC nanopores

[0142] To better understand the effect of pH on the electrostatic environment and the effect of electroosmotic flow on the entry of peptides into the FraC nanopore, we used the Adaptive Poission-Boltzmann Solver (APBS) (13) and a modified version of the PDB2PQR software (14) to estimate the electrostatic potentials in the homology models of WtFraC and ReFraC at pH 7.5 and 4.5 in 1 M KCl. The simulations showed that the constriction region at the center of the nanopore of WtFraC and ReFraC exhibited extremely high negative and positive potentials, respectively ( Fig.11 A). Interestingly, although for WtFraC, a decrease in pH from 7.5 to 4.5 resulted in a decrease in the reduction potential of the contractile center from -1.2 to -0.7 k B T / e c (298K, 1k B T / e c =25.6 mV), but no such effect was observed for ReFraC.

[0143] The contribution of electroosmotic flow to analyte capture with WtFraC and ReFraC pores was estimated by measuring the ion selectivity of the two pores using asymmetric KCl concentrations (1960 mM and 467 mM) on either side of the nanopore. r ), that is, the potential at which the current is zero ( Fig.11 B), Calculation of the ion selectivity of the two nanopores together with the Goldman-Hodgkin-Katz equation

[0144]

[0145] where [ax] comp is the activity of ion X in the cis / trans compartment, R is the gas constant, T is the temperature, and F is the Faraday constant. We found that the ion selectivity of FraC nanopores is controlled by the charge of the constriction, with WtFrac having strong cation selectivity ReFraC is anion selective Lowering the pH to 4.5 reduced the cation selectivity of WtFraC ( However, this improves the anion selectivity of ReFraC

[0146] Example 7: Detection of biomarkers using WtFraC nanopores

[0147] Following evaluation of the capture of chymotrypsin (25 kD, 245 amino acids) and endothelin 1 (12.5 kD, 21 amino acids), protein biomarkers for pancreatic cysts (15) and bronchiolitis obliterans (16), respectively, the WtFraC nanopore was used to detect a wider range of protein biomarkers, including the peripheral arterial disease biomarker β2-microglobulin (11.6 kDa, 99 amino acids (17)), the chronic kidney disease biomarker human EGF (6.2 kDa, 53 amino acids (18)), and the hypertensive crisis biomarker angiotensin I (1.3 kD, 10 amino acids (19)).

[0148] All biomarkers were evaluated at negative applied potentials and pH 4.5 (except chymotrypsin). The capture frequency of all biomarkers increased with the applied potential. All other parameters tested showed non-uniform voltage dependence. The residence time of the biomarkers within WtFraC increased (chymotrypsin), decreased (β2-microglobulin and angiotensin 1) or showed a biphasic behavior with the applied potential (EGF and endothelin 1) See Fig.12 The voltage dependence of the residual current percentage (Ires%) of chymotrypsin decreased with potential, the Ires% of endothelin-1 increased with potential, and the Ires% of β2-microglobulin, EGF, and angiotensin-1 remained unchanged. Despite the complex voltage dependence of the current blockade, our results show that the WtFraC nanopore is able to discriminate oligopeptide and protein biomarkers of different sizes solely by their current blockade Ires% ( Fig.12 ).

[0149] Example 8: Discrimination of similar isoform oligopeptides

[0150] To challenge our experimental system, we sought to identify highly similar analytes. We chose endothelin-1 (ET-1) and endothelin-2 (ET-2), near-isomeric oligopeptides that differ in only one of their 21 amino acids ( Fig.13 At -50 mV, we observed discernible blockades with 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) having distinct Ires% and dwell time ( Fig.12 B). This enables them to discriminate already at a single blocking level ( Fig.13 C).

[0151] Surprisingly, when we sequentially added first 2 μM ET-1 ( Fig.13 D), and then 8 μM ET-2 was added to the same well ( Fig.13 E) We can also separate the mixture of two different populations by plotting the standard deviation of the amplitude of the events relative to their corresponding Ires%. This observation suggests that highly similar (oligo)peptides or other analytes can be discriminated using FraC nanopores.

Claims

1. A system comprising a funnel-shaped protein nanopore, wherein the nanopore comprises an α-helical pore-forming toxin, wherein the α-helical pore-forming toxin is a member from 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. The system of claim 1, wherein the FraC is a mutant FraC, preferably, wherein the mutant FraC comprises at least one of the negatively charged amino acid residues in the narrow portion of the pore being substituted with a neutral or positively charged amino acid residue, and / or at least one of the neutral amino acid residues in the narrow portion of the pore being substituted with a positively charged amino acid residue.

3. The system according to claim 2, wherein the mutant FraC comprises a mutation at position 10, preferably a mutation to Asp10Arg or Asp10Lys.

4. The system of claim 2 or 3, wherein the mutant FraC further comprises one or more compensatory mutations to restore the hemolytic activity of FraC, preferably, wherein the compensatory mutations are present at position 2, 9, 34, 52, 112, 150, 153 and / or 159, preferably position 159.

5. A method for providing a system as claimed in any one of claims 1 to 4, 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 them into oligomers; - recovering said oligomer from said liposome; as well as - contacting the oligomer with a lipid bilayer, possibly containing sphingomyelin, to allow the formation of a nanopore.

6. A method comprising applying an electric field to a system as described in any one of claims 1 to 4, wherein a funnel-shaped nanopore containing an α-helical pore-forming toxin is located between a first conductive liquid medium and a second conductive liquid medium, preferably, wherein at least one of the conductive liquid media contains an analyte, more preferably, the method further comprises detecting the analyte in a method comprising: measuring an ionic current to provide a current pattern when the analyte interacts with the nanopore, wherein the presence of a blockage in the current pattern indicates the presence of the analyte, preferably, 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 nano-object or a biological warfare agent; the polymer is preferably selected from proteins, polypeptides, oligopeptides, unfolded peptides, unfolded oligopeptides and unfolded proteins; or preferably is a nucleic acid, more preferably, wherein the nucleic acid is ssDNA, dsDNA, RNA or a combination thereof.

7. The method of claim 6, wherein the FraC nanopore is a mutant FraC nanopore.

8. A mutant Fragaceatoxin C (FraC) nanopore comprising at least a first mutant FraC monomer, wherein the first mutant FraC monomer comprises at least one substitution of the negatively charged amino acid residues in the narrow part of the pore by a positively charged amino acid residue, and / or at least one substitution of the neutral amino acid residues in the narrow part of the pore by a positively charged amino acid residue, preferably comprising a substitution at position 3 or 10, or a substitution at position 3 and 10, preferably D10R.

9. Use of the system of any one of claims 1 to 4 or the mutant FraC nanopore of claim 8 for biopolymer sensing and / or biopolymer sequencing, preferably, wherein the biopolymer is a protein, a peptide or a nucleic acid.

10. The use according to claim 9, wherein the biopolymer is a nucleic acid, preferably, wherein the nucleic acid is ssDNA, dsDNA, RNA or a combination thereof; or, wherein the biopolymer is a protein, a polypeptide, an oligopeptide, an unfolded peptide, an unfolded oligopeptide or an unfolded protein.

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