BIOLOGICAL NANOPORES HAVING TUNABLE PORE DIAMETERS AND USES THEREOF AS ANALYTICAL TOOLS

Abstract

The invention relates to the field of nanopores, in particular to engineered Fragaceatoxin C (FraC) nanopores and their application in analyzing biopolymers and other (biological) compounds, such as single-molecule (protein) sequencing. Provided is a system comprising oligomeric FraC nanopores comprised in a lipid bilayer, wherein the sum of the nanopore fraction in the heptameric (Type II) state and the nanopore fraction in the hexameric (Type III) state represents at least 60% of the total number of FraC nanopores.

Claims

1. A system comprising oligomeric Fragaceatoxin C (FraC) nanopores comprised in a lipid bilayer, wherein the sum of the nanopore fraction in the heptameric (Type II) state and the nanopore fraction in the hexameric (Type III) state represents at least 60% of the total number of FraC nanopores.

2. System according to claim 1, wherein the sum of the Type II and Type III state nanopores represents at least 65%, preferably at least 70%, of the total number of FraC nanopores.

3. System according to claim 1 or 2, wherein at least 60%, preferably at least 70%, of the FraC nanopores is in the Type II state.

4. System according to claim 1 or 2, wherein at least 60%, preferably at least 70%, of the FraC nanopores is in the Type III state.

5. System according to any one of the preceding claims, wherein the FraC nanopores comprise mutant FraC monomers comprising a mutation at position W112 and/or W116.

6. System according to claim 5, wherein said mutation(s) comprise substitution of W with S, T, A, N, Q or G, preferably with S or T.

7. System according to any one of the preceding claims, wherein the FraC nanopores comprise mutant FraC monomers comprising a mutation at position D109, preferably herein said mutation comprises the substitution of D with S or T, more preferably with S.

8. System according to any one of claims 5-7, wherein said mutant FraC monomer comprises mutation W112S, W116S and/or D109S, preferably wherein the FraC mutant is W112S/W116S or D109S/W116S.

9. System according to any one of the preceding claims, wherein the system is operative to detect a property of the analyte comprises subjecting the nanopore to an electric field such that the analyte electrophoretically and/or electroosmotically translocates through the nanopore.

10. System according to claim 9, wherein the analyte is a proteinaceous substance, preferably a peptide, more preferably a peptide up to about 30 amino acids in length.

11. A method for providing a system according to any one of claims 1 to 10, comprising the steps of: (i) providing recombinant FraC monomers; (ii) contacting said monomers with liposomes to assemble them into oligomers; (iii) recovering the oligomers from the liposomes; (iv) contacting the oligomers with a lipid bilayer to allow the formation of FraC nanopores; and (v) optionally isolating a fraction comprising FraC nanopores in the Type II state, and/or a fraction comprising FraC nanopores in the Type III state.

12. A mutant Fragaceatoxin C (FraC) polypeptide comprising a mutation at position W112 and/or W116 wherein the numbering corresponds to the FraC amino acid available under accession number B9W5G6 in UniProt, and wherein the W residue(s) is/are independently substituted with either S, T, A, N, Q or G.

13. A mutant Fragaceatoxin C (FraC) polypeptide comprising a mutation at position D109 wherein the numbering corresponds to the FraC amino acid available under accession number B9W5G6 in UniProt, and wherein the D residue is substituted with an uncharged residue, preferably with S or T, more preferably with S.

14. Mutant FraC polypeptide according to claim 12 or 13 comprising mutation W112S, W116S and/or D109S, preferably wherein the mutant FraC is W112S/W116S or D109S/W116S.

15. Mutant FraC polypeptide according to any one of claims 12 to 14, further comprising mutation D10C.

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

17. An expression vector comprising an isolated nucleic acid molecule according to claim 16.

18. A host cell comprising an expression vector according to claim 17.

19. The use of a system according to any one of claims 1 to 10, or a mutant FraC polypeptide according to any one of claims 12 to 15, in peptide analysis, preferably wherein peptide analysis comprises peptide mass detection and/or peptide sequencing.

20. The use of a system according to any one of claims 1 to 10, or a mutant FraC polypeptide according to any one of claims 12 to 15, in single molecule detection, preferably in combination with high throughput analysis.

21. The use according to claim 19 or 20, wherein the system is integrated in a portable device comprising a plurality of individual systems according to any one of claims 1-10.

Description

LEGEND TO THE FIGURES

[0059] FIG. 1. Preparation and characterization of type I, type II and type III FraC nanopores. a, Cut through of a surface representation of WT-FraC oligomer (PDB: 4TSY).sup.25 colored according to the vacuum electrostatic potential as calculated by Pymol. One protomer is shown as a carton presentation with tryptophans 112 and 116 displayed as spheres. b, Percentage of the distribution of type I, type II and type III for WT-FraC, W112S-FraC, W116S-FraC and W112S-W116S-FraC at pH 7.5 and 4.5. c, IV curves of type II nanopores formed by WT-FraC, W116S-FraC and W112S-W116S-FraC at pH 7.5 (15 mM Tris.HCl, 1 M KCl). d, Single nanopore conductance of W116S-FraC in 1 M KCl (0.1 M citric acid and 180 mM Tris base) at pH 4.5. e, Typical current traces for the three nanopore types of W116S-FraC in 1 M KCl at pH 4.5 under −50 mV applied potential. f, Reversal potentials measured under asymmetric condition of KCl (1960 mM cis, 467 mM trans) at pH 4.5 for the three W116S-FraC nanopore types. The ion selectivity was given by using the Goldman-Hodgkin-Katz equation (equation 1). g, Molecular models of the three type FraC nanopores constructed from the FraC crystals structure using the symmetrical docking function of Rosetta. The electrophysiology recordings were performed with 10 kHz sampling and 2 kHz filter. The error bars and color shadow in the I-V curves are standard deviations from three repeats at least.

[0060] FIG. 2. Single channel conductance distributions of FraC nanopores at pH 7.5 and 4.5. a, The table reports the average conductance values which were obtained by fitting Gaussian functions to conductance histograms. S.D. represents the standard deviation of all single channels (number given as n). b-f, Each panel represents a different batch of FraC nanopores as indicated. Single channels were collected under −50 mV applied potential. pH 7.5 and 4.5 were obtained using 1 M NaCl, 15 mM Tris, or 1 M KCl, 0.1 M citric acid, 180 mM Tris base respectively.

[0061] FIG. 3. Discrimination of angiotensin peptides in mixture with type II W116S-FraC nanopores. a, (i) Sequences of angiotensin I, II, III and IV with corresponding Ires % measured at −30 mV. (ii) Typical blockades provoked by the four angiotensin peptides. (iii) Color density plot of the Ires % versus the standard deviation of the current amplitude for angiotensin I added to the cis compartment, and after the further addition of angiotensin II, angiotensin III, and angiotensin IV to the cis chamber (iv). b, Discrimination of angiotensin II and angiotensin A. (i) Table showing the sequences, the molecular weights and the Ires % of the peptides. The peptides differ by one amino acid as shown in red. (ii) Representative traces of the peptide blockades. Color density plot of the Ires % versus the standard deviation of the current amplitude for angiotensin II blockades prior (iii) and after (iv) the further addition of angiotensin A to the cis chamber. All measurements and recordings were performed in pH 4.5 buffer containing 1 M KCl, 0.1 M citric acid, 180 mM Tris base with a 50 kHz sampling and 10 kHz filter. Standard deviations were calculated from minimum three repeats. Color density plot was created with Origin.

[0062] FIG. 4. Evaluation of biological peptides having different chemical compositions. Relation between the molecular weight and Ires % of peptide using: (a) type I WT-FraC nanopores, (b) type II W116S-FraC nanopores and (c) type III W112S-W116S-FraC nanopores at pH 4.5. The solid line represents a second order polynomial fitting. Current blockades were measured at −30 mV for type I and II pore, and at −50 mV for type III pore. Error bars are standard deviations obtained from at least three measurements.

[0063] FIG. 5. A nanopore peptide mass spectrometer at pH 3.8. a, Amino acid sequences of four different peptides and their overall charge at different pH. The chargeable amino acids are underlined. b, pH dependence of the Ires % for the four peptides (cis) shown in a using type II W116S-FraC nanopores under −30 mV applied potential. c, Comparison of the Ires % versus the mass of peptides at pH 4.5 and 3.8. d, Voltage dependence of c-Myc dwell times at different pHs. All electrophysiology measurements were carried out in 1 M KCl, 0.1 M citric acid, and pH was adjusted with 1 M Tris base to desired values. 50 kHz sampling rate and 10 kHz filter was used for collecting the current events. Error bars are standard deviations obtained from at least three measurements. The charges of the peptides were calculated according to the pKa for individual amino acids.sup.36.

[0064] FIG. 6: Discrimination of short peptide mixture with type III FraC nanopores comprising mutant W112S-W116S-FraC. a, Sequence, Ires % (−50 mV) and MW of angiotensin IV, angiotensin 4-8, endomorphin I and leucine enkephalin. b, Typical blockades provoked by the different peptides. c, Color density plot showing the Ires % versus the standard deviation of the current blockade for the mixture of angiotensin IV, angiotensin 4-8, endomorphin I and leucine-enkephalin. All measurements and recordings were performed in pH 4.5 buffer containing 1 M KCl, 0.1 M citric acid, 180 mM Tris base with a 50 kHz sampling and 10 kHz filter. Standard deviations were calculated from three repeats at least.

[0065] FIG. 7. Characterization of type II FraC nanopores comprising an oxidized cysteine at position 10. Difference between the D10C/W116S type II pore (panel A) and the oxidized D10C/W116S type II pore (panel B). Recordings were performed in a buffer containing 1 M NaCl, pH 7.5, ±50 mV.

EXPERIMENTAL SECTION

Materials and Methods

Chemicals

[0066] Endothelin 1 (≥97%, CAS#117399-94-7), endothelin 2 (≥97%, CAS#123562-20-9), dynorphin A porcine (≥95%, CAS#80448-90-4), angiotensin I (≥90%, CAS#70937-97-2), angiotensin II (≥93%, CAS#4474-91-3), c-Myc 410-419 (≥97%, # M2435), Asn1-Val5-Angiotensin II (≥97%, CAS#20071-00-5), Ile7 Angiotensin III (≥95%, #A0911), leucine enkephalin (≥95%, #L9133), 5-methionine enkephalin (≥95%, CAS#82362-17-2), endomorphin I (≥95%, CAS#189388-22-5), pentane (≥99%, CAS#109-66-0), hexadecane (99%, CAS#544-76-3), Trizma®hydrochloride (≥99%, CAS#1185-53-1), Trizma®base (≥99%, CAS#77-86-1), Potassium chloride (≥99%, CAS#7447-40-7), N,N-Dimethyldodecylamine N-oxide (LADO, ≥99%, CAS#1643-20-5) were obtained from Sigma-Aldrich. Pre angiotensin 1-14 (≥97%, #002-45), angiotensin 1-9 (≥95%, #002-02), angiotensin A (≥95%, #002-36), angiotensin III (≥95%, #002-31), angiotensin IV (≥95%, #002-28) were purchased from Pheonix Pharmaceuticals. Angiotensin 4-8 (≥95%) was synthesized by BIOMATIK. 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, #850356P) and sphingomyelin (Porcine brain, #860062) were purchased from Avanti Polar Lipids. Citric acid (99.6%, CAS#77-92-9) was obtained from ACROS. n-Dodecyl β-D-maltoside (DDM, ≥99.5%, CAS#69227-93-6) was bought from Glycon Biochemical EmbH. DNA primers were synthesized from Integrated DNA Technologies (IDT), enzymes from Thermo scientific. All peptides were dissolved with Milli-Q water without further purification and stored in −20° C. freezer. pH 7.5 buffer containing 15 mM Tris in this study was prepared by dissolving 1.902 g Trizma® HCl and 0.354 g Trizma® base in 1 litre Milli-Q water (Millipore, Inc).

FraC Monomer Expression and Purification

[0067] FraC gene containing NcoI and HindIII restriction site at the 5′ and 3′ ends, respectively, and a sequence encoding for a poly histidine tag at the 3′ terminus was cloned to a pT7-SC1 plasmid. Plasmids were transformed into BL21(DE3) E.cloni® competent cell by electroporation. Cells were grown on LB agar plate containing 100 μg/mL ampicillin overnight at 37° C. The entire plate was then harvested and inoculated into 200 mL fresh 2YT media and the culture was grown with 220 rpm shaking at 37° C. until the optical density at 600 nm of the cell culture reached 0.8. Then 0.5 mM IPTG was added to the media and the culture was transferred to 25° C. for overnight growth with 220 rpm shaking. The next day the cells were centrifuged (2000×g, 30 minutes) and the pellet stored at −80° C. Cell pellets harvested from 100 mL culture media were used to purify FraC monomer. 30 mL of cell lysis buffer (150 mM NaCl, 15 mM Tris, 1 mM MgCl.sub.2, 4 M urea, 0.2 mg/mL lysozyme and 0.05 unit/mL DNase) was added to resuspend the pellet and vigorously mixed for 1 hour. Cell lysate was then sonicated with Branson Sonifier 450 for 2 minutes (duty cycle 10%, output control 3). Then the crude lysate was centrifuged down at 4° C. for 30 minutes (5400×g), and the supernatant incubated with 100 μL Ni-NTA beads (Qiagen) for 1 hour with gentle shaking. Beads were spun down and loaded to a Micro Bio-spin column (Bio-rad). 10 mL of SDEX buffer (150 mM NaCl, 15 mM Tris, pH 7.5) containing 20 mM imidazole was used to wash the beads, and proteins were eluded with 150 μL elution buffer (SDEX buffer, 300 mM imidazole). The concentration of protein was measured by measuring the absorption at 280 nm with Nano-drop 2000 (Thermo scientific) using the elution buffer as blank. To further confirm the purity of monomer, monomeric FraC was diluted to 0.5 mg/mL using the elution buffer and 9 μL of the diluted sample was mixed with 3 μL of 4× loading buffer (250 mM Tris HCl, pH 6.8. 8% SDS, 0.01% bromophenol blue and 40% glycerol) and then loaded to 12% SDS-PAGE gel. Gels were run for 30 min with 35 mA constant applied current, and stained with coomassie dye (InstantBlue™, Expdedeon) for more than 1 hour before viewing using a gel imager (Gel Doc™, Bio-rad).

TABLE-US-00001 ≥FraC|B9W5G6 (amino acid sequence) SADVAGAVIDGAGLGFDVLKTVLEALGNVKRKIAVGIDNESGKTWTAMNTY FRSGTSDIVLPHKVAHGKALLYNGQKNRGPVATGVVGVIAYSMSDGNTLAV LFSVPYDYNWYSNWWNVRVYKGQKRADQRMYEELYYHRSPFRGDNGWHSRG LGYGLKSRGFMNSSGHAILEIHVTKA
>6×His-WtFraC (amino acid sequence) as used in the present invention. Bold residues indicate residues of the N- and C-terminal end that were added to the original sequence.

TABLE-US-00002 MASADVAGAVIDGAGLGFDVLKTVLEALGNVKRKIAVGIDNESGKTWTAMN TYFRSGTSDIVLPHKVAHGKALLYNGQKNRGPVATGVVGVIAYSMSDGNTL AVLFSVPYDYNWYSNWWNVRVYKGQKRADQRMYEELYYHRSPFRGDNGWHS RGLGYGLKSRGFMNSSGHAILEIHVTKAGSAHHHHHH >6xHis-WtFraC (DNA sequence) ATGGCGAGCGCCGATGTCGCGGGTGCGGTAATCGACGGTGCGGGTCTGGGC TTTGACGTACTGAAAACCGTGCTGGAGGCCCTGGGCAACGTTAAACGCAAA ATTGCGGTAGGGATTGATAACGAATCGGGCAAGACCTGGACAGCGATGAAT ACCTATTTCCGTTCTGGTACGAGTGATATTGTGCTCCCACATAAGGTGGCG CATGGTAAGGCGCTGCTGTATAACGGTCAAAAAAATCGCGGTCCTGTCGCG ACCGGCGTAGTGGGTGTGATTGCCTATAGTATGTCTGATGGGAACACACTG GCGGTACTGTTCTCCGTGCCGTACGATTATAATTGGTATAGCAATTGGTGG AACGTGCGTGTCTACAAAGGCCAGAAGCGTGCCGATCAGCGCATGTACGAG GAGCTGTACTATCATCGCTCGCCGTTTCGCGGCGACAACGGTTGGCATTCC CGGGGCTTAGGTTATGGACTCAAAAGTCGCGGCTTTATGAATAGTTCGGGC CACGCAATCCTGGAGATTCACGTTACCAAAGCAGGCTCTGCGCATCATCAC CACCATCACTGATAAGCTT

FraC Mutation Preparation

[0068] FraC mutants were prepared according to MEGAWHOP method. 25 μL REDTaq® ReadyMix™ was mixed with 4 μM primer (see Table 1) containing the desired mutation with 50 ng plasmid (pT7-SC1 with wild type FraC gene) as template and the final volume was brought to 50 μL with MilliQ water.

TABLE-US-00003 TABLE 1 Primer sequences used in this study for preparing FraC mutants. Primer name DNA sequences T7 promoter 5′ TAATACGACTCACTATAGGG 3′ T7 terminator 5′ GCTAGTTATTGCTCAGCGG 3′ W112S Fw 5′ ACGATTATAATAGCTATAGCAATTGGTGG 3′ W116S Fw 5′ ATTGGTATAGCAATAGCTGGAACGTG 3′ W112116S Fw 5′ GTACGATTATAATAGCTATAGCAATAGCTGGA ACGTGC 3′ D109S ReV 5′ TGCTATACCAATTATAGCTGTACGGCA 3′

[0069] The PCR protocol was initiated by 150 seconds denature step at 95° C., followed by 30 cycles of denaturing (95° C., 15 s), annealing (55° C., 15 s), and extension (72° C., 60 s). The PCR products (MEGA primer) were combined and purified using a QIAquick PCR purification kit with final DNA concentration around 200 ng/μL. The second PCR was performed for whole plasmid amplification. 2 μL of MEGA primer, 1 μL Phire II enzyme, 10 μL 5× Phire buffer, 1 μL 10 mM dNTPs, were mixed with PCR water to 50 μL final volume. PCR started with pre-incubated at 98° C. (30 s) and then 25 cycles of denaturing (98° C., 5 s), annealing (72° C., 90 s), extension (72° C., 150 s). When the PCR was completed, 1 μL Dpn I enzyme was added and the mixture kept at 37° C. for 1 hour. Then the temperature was raised to 65° C. for 1 minute to inactivate the enzyme. Products were then transformed into E. cloni® 10G cells (Lucigen) competent cell by electroporation. Cells were plated on LB agar plates containing 100 μg/mL ampicillin and grew at 37° C. overnight. Single clones were enriched and sent for sequencing.

Sphingomyelin-DPhPC Liposome Preparation

[0070] 20 mg sphingomyelin and 20 mg DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) were dissolved in 4 mL pentane with 0.5% v/v ethanol and brought to a 50 mL round flask. The solvent was then evaporated by rotation and using a hair-dryer to warm-up the flask. After evaporation, the flask was kept at ambient temperature for an additional 30 minutes. The lipid film was resuspended with 4 mL SDEX buffer (150 mM NaCl, 15 mM Tris, pH 7.5) and the solution immersed in a sonication bath for 5 minutes. Liposome suspensions were stored at −20° C.

FraC Oligomerization

[0071] FraC oligomerization was triggered by incubation of FraC monomers with sphingomelyin-DPhPC liposomes. Frozen liposome were thawed and sonicated in a water bath for one minute. FraC monomers were diluted to one mg/mL using SDEX buffer, and then 50 μL of FraC monomers were added to 50 μl of a 10 mg/mL liposome solution to obtain a mass ratio of 10:1 (liposome:protein). The lipoprotein solution was incubated at 37° C. for 30 min to allow oligomerization. Then 10 μl of 5% (w/v, 0.5% final) LADO was added to the lipoprotein solution to solubilize the liposomes. After clarification (typically 1 minute) the solution was transferred to a 50 mL Falcon tube. Then 10 mL of SDEX buffer containing 0.02% DDM and 100 μL of pre-washed Ni-NTA beads were added to the Falcon tube and mixed gently in shaker for 1 hour at room temperature. The beads were then spun down and loaded to a Micro Bio-spin column. 10 mL wash buffer (150 mM NaCl, 15 mM Tris, 20 mM imidazole, 0.02% DDM, pH 7.5) was used to wash the beads and oligomers eluded with 100 μL elution buffer (typically 200 mM EDTA, 75 mM NaCl, 7.5 mM Tris pH 7.5, 0.02% DDM). The FraC oligomers were stored at 4° C. Under these conditions the nanopores are stable for several months.

W112S-W116S-FraC Oligomer Separation with His-Trap Chromatography

[0072] 200 μL of W112S-W116S-FraC monomers (3 mg/mL) were incubated with 300 μL of Sphingomyelin-DPhPC liposome (10 mg/mL) and kept at 4° C. for 48 hours after which 0.5% LADO (final concentration) was added to solubilize the lipoprotein. Then the buffer was exchanged to the 500 mM NaCl, 15 mM Tris, 0.01% DDM, 30 mM imidazole, pH 7.5 (binding buffer) using a PD SpinTrap G-25 column. W112S-W116S-FraC oligomers were then loaded to Histrap HP 1 mL column (General Electric) using an ÄKTA pure FPLC system (General Electric). The loaded oligomers were washed with 10 column volumes of 500 mM NaCl, 15 mM Tris, 0.01% DDM, 30 mM imidazole, pH 7.5, prior applying an imidazole gradient (from 30 mM to 1 M imidazole, 500 mM NaCl, 15 mM Tris, 0.01% DDM, pH 7.5) over 30 column volumes. The signal was monitored with the absorbance at 280 nm and fractions were collected when the absorbance was higher than 5 mAu.

Electrophysiology Measurement and Data Analysis

[0073] Electrical recordings were performed as explained in details previously.sup.27,37. I.sub.O, referring to open pore current, were measured by fitting Gaussian functions to event amplitude histograms. Residual current values (Ires %) were calculated by dividing the blockade current (I.sub.B) by open pore current (I.sub.B/I.sub.O×100%). Dwell times and inter-event times were measured by fitting single exponentials to histograms of cumulative distribution.

Ion Permeability Measurement

[0074] In order to measure reversal potentials, a single channel was obtained under symmetric conditions (840 mM KCl, 500 μL in each electrophysiology chamber) and the electrodes were balanced. The 400 μL of a buffered stock solution of 3.36 M KCl was slowly added to cis chamber, while 400 μL of salt free buffered solution was added to the trans chamber to obtain a total volume of 900 μL (trans:cis, 467 mM KCl:1960 mM KCl). After the equilibrium was reached, IV curves were collected from −30 to +30 mV. The resulting voltage at zero current is the reversal potential (Vr). The ion selectivity (P.sub.K.sup.+/P.sub.Cl.sup.−) was then calculated using the Goldman-Hodgkin-Katz equation (equation 1) where [a.sub.K.sub.+.sub./Cl.sub.−].sub.cis/trans is the activity of the K.sup.+ or Cl.sup.− in the cis or trans compartment, R the gas constant, T the temperature and F the Faraday's constant.

[00001]$\begin{array}{cc}\frac{P_{K^{+}}}{P_{C{l}^{-}}}=\frac{{\left[a_{{\mathrm{Cl}}^{-}}\right]}_{\mathrm{trans}}-{\left[a_{{\mathrm{Cl}}^{-}}\right]}_{cis}{e}^{V_{r}F/RT}}{{\left[a_{K^{+}}\right]}_{\mathrm{trans}}{e}^{V_{r}F/RT}-{\left[a_{K^{+}}\right]}_{\mathrm{cis}}}& \left(1\right)\end{array}$

[0075] The activity of ions was calculated by multiplying the molar concentration of the ion for the mean ion mobility (0.649 for 500 mM KCl, and 0.573 for 2000 mM). Ag/AgCl electrodes were surrounded by 2.5% agarose bridge in 2.5 M NaCl.

Molecular Models of Type I, II and III FraC Nanopores

[0076] The 3D models with different multimeric order, ranging from five to nine monomers, were constructed with the symmetrical docking function of Rosetta.sup.38. A monomer without lipids was extracted from the crystal structure of FraC with lipids (PDB_ID 4tsy). Symmetrical docking arranged this monomer around a central rotational axis ranging in order form 5 to 9. In total Rosetta generated and scored 10 000 copies for each symmetry. In all cases, a multimeric organization with a symmetry similar to the crystal structure could be identified as a top scoring solution. However, in the pentameric assembly the multimer interface was not fully satisfied as compared to the crystal structure, with large portions left exposed. The 9-fold symmetric model however exhibited a significant drop in Rosetta score compared to the 6-7- and 8-fold symmetric models indicating an unfavored assembly of the nonameric assembly with the 6-7- and 8-fold assemblies as the most plausible. To create lipid bound models, the crystal structure with lipids was superimposed on each monomer of the generated models, allowing the lipid coordinates to be transferred. The residues within 4.5 angstrom of the lipids were minimized with the Amber10 forcefield.

Example 1: Engineering the Size of FraC Nanopores

[0077] One of the main challenges in biological nanopores analysis is to obtain nanopores with different size and shape. Most of biological nanopores are formed by multiple repeats of individual monomers. Hence, different nanopore sizes might be obtained by engineering the protein oligomeric composition.sup.28. We noticed that at pH 7.5 a small fraction of Wild Type FraC (WT-FraC) nanopores showed a lower conductance (1.26±0.08 nS, −50 mV, type II WT-FraC, FIG. 1b) compared to the dominant fraction (2.26±0.08 nS, −50 mV, type I WT-FraC), suggesting that FraC might be able spontaneously to assemble into nanopores with smaller size. At pH 4.5 yet a smaller nanopore conductance was observed (0.42±0.03 nS, type III WT-FraC, −50 mV, FIG. 1b). We noticed that the reconstitution of lower conductance nanopores depended to several purification conditions. In particular, we observed that the occurrence of type II and type III nanopores increased when the oligomers were stored in solution for several weeks or when the concentration of monomeric WT-FraC was reduced during oligomerisation.

[0078] In an attempt to enrich for type II and type III FraC nanopores, the interaction between the nanopore and the lipid interface was weakened by substituting W112 and W116 at the lipid interface of FraC (FIG. 1a) with serine. The inventors reasoned that a lower concentration of monomers during liposome-triggered oligomerisation would increase the population of lower molecular mass oligomers. Surprisingly, it was found that at pH 4.5 using W116S-FraC and W112S-W116S-FraC oligomers, type II and type III FraC nanopores were the dominant species, respectively (FIG. 1b, FIG. 2). Conveniently, the different nanopore types could be separated by Ni-NTA affinity chromatography using an imidazole gradient. Furthermore, it was found that enrichment of type II and type III FraC nanopores could also be obtained at pH 7.5 by replacing aspartate 109 at the lipid interface with serine (see FIG. 2e, Table 2).

TABLE-US-00004 TABLE 2 relative amounts of Type I, Type II and Type III for each of the FraC nanopores investigated at neutral and acidic pH. Type I(%) SD Type II(%) SD Type III(%) SD pH 7.5 Wild type 85.7 3.8 14.3 3.8 0.0 0.0 W112S 61.9 4.3 38.1 4.3 0.0 0.0 W116S 61.1 5.7 38.9 5.7 0.0 0.0 W112116S 27.1 3.9 72.9 3.9 0.0 0.0 D109S 50.3 3.8 48.0 3.6 1.7 1.5 D109SW116S 29.3 9.5 66.7 8.4 4.0 4.0 pH 4.5 Wild type 42.5 10.6 51.9 7.7 5.6 7.9 W116S 29.0 4.0 47.0 4.6 24.0 2.3 W112SW116S 21.7 4.7 38.0 8.5 40.3 9.3 D109S 35.7 2.1 56.3 9.1 8.0 7.0 D109SW116S 19.3 8.4 64.3 6.7 16.3 3.2

[0079] Among FraC nanopores of the same type, the lipid interface modifications caused by W112S and W116S substitutions did not alter the conductance and ion selectivity as compared to that of wild type (FIG. 1c, FIG. 2, Table 3) suggesting that the modifications did not altered the overall fold of the nanopores. When characterised in lipid bilayers, type I, type II and type III nanopores showed a well-defined single conductance distribution and a steady open pore current (FIG. 1d-e). Interestingly, Type I, Type II and Type III nanopores showed increasing cation selectivity (from 2.0 for type I to 4.2 for type III W116S-FraC nanopores at pH 4.5 (FIG. 1f, Table 3), most likely reflecting a larger overlap of the electrical double layer in the nanopores with a narrower constriction.

TABLE-US-00005 TABLE 3 Ion selectivity of different FraC pores at pH 7.5 and 4.5. pH 7.5 pH 4.5 Reversal Reversal potential (mV) P.sub.K.sup.+/P.sub.Cl.sup.− potential (mV) P.sub.K.sup.+/P.sub.Cl.sup.− WT-FraC Type I 17.2 ± 1.2 3.6 ± 0.4 10.5 ± 1.4 2.1 ± 0.2 Type II 20.8 ± 1.6 5.2 ± 0.9 12.3 ± 1.2 2.4 ± 0.2 Type III / / 20.6 ± 1.1 5.0 ± 0.6 W116S-FraC Type I / / 10.1 ± 0.9 2.0 ± 0.1 Type II / / 12.8 ± 0.7 2.5 ± 0.2 Type III / / 18.8 ± 0.5 4.2 ± 0.2 W112S-W116S-FraC Type I / /  8.8 ± 1.2 1.9 ± 0.2 Type II / / 14.0 ± 0.1 2.8 ± 0.1 Type III / / 20.1 ± 0.6 4.8 ± 0.3 [0080] The ion selectivity (P.sub.K.sup.+/P.sub.Cl.sup.−) was calculated from the reversal potential according to the Goldman-Hodgkin-Katz equation:

[00002]$\frac{P_{K^{+}}}{P_{{\mathrm{Cl}}^{-}}}=\frac{{\left[a_{{\mathrm{Cl}}^{-}}\right]}_{\mathrm{trans}}-{\left[a_{{\mathrm{Cl}}^{-}}\right]}_{\mathrm{cis}}{e}^{V_{r}F/\mathrm{RT}}}{{\left[a_{K^{+}}\right]}_{\mathrm{trans}}{e^{V_{r}F/\mathrm{RT\_}}\left[a_{K^{+}}\right]}_{\mathrm{cis}}},$  where Vr is the reversal potential, P.sub.K.sup.+/P.sub.Cl.sup.− the ion selectivity, a the activity of ions and F the Farady constant. Electrophysiology recordings were carried out with 1960 mM KCl in the cis solution and 467 mM KCl in the trans solution. The activity of ions was calculated by multiplying the molar concentration of the ion for the mean ion mobility (0.649 for 500 mM KCl, and 0.573 for 2000 mM).sup.3. Errors are given as standard deviations calculated from 3 experiments at least.

[0081] These findings strongly suggest that the three types of FraC nanopores represent nanopores with different protomeric compositions. Molecular modelling allowed predicting the diameter of type II (1.1 nm) and type III (0.8 nm) nanopores (FIG. 1g); and revealed that type III FraC is most likely the biological nanopore with the smallest constriction known to date.

Example 2: Identification of Peptides Containing Single Amino Acid Substitutions Using Type II or Type III FraC Nanopores as Sensor

[0082] Type II FraC nanopores were used to sample a series of angiotensin peptides (which in blood regulate blood pressure and fluid balance. The peptides were added to the cis side of type II W116S-FraC nanopores and the induced ionic current blockades (I.sub.B) was measured. Residual currents percent (Ires %, defined as I.sub.B/I.sub.O×100) were used instead of current blockades because they provided more reliable values when comparing different nanopores. Results are shown in FIG. 3a and Table 4.

[0083] Angiotensin I (DRVYIHPFHL, 1296.5 Da), showed the deepest blockade (Ires %=8.8±0.2) and angiotensin IV (VYIHPF, 774.9 Da) the shallowest blockade (Ires %=38.9±4.0). The residual current of angiotensin II (DRVYIHPF, 1046.2 Da, Ires %=17.9±1.3) and angiotensin III (RVYIHPF, 931.1 Da, Ires %=22.1±0.5) fell at intermediate values. When the four peptides were tested simultaneously, individual peptides could be readily discriminated (FIG. 3a).

TABLE-US-00006 TABLE 4 Peptide analysis using different types of FraC nanopores at pH 4.5 Molecular Ires % weight pH (I.sub.B/I.sub.O) % Dwell time Peptide Sequence (g/mol) 7.5 pH 4.5 pH4.5 (ms) WT-FraC type I pore, −30 mV Endothelin 2 CSCSSWLDKECVYFCHLDIIW 2546.9 −2.15 0.36  6.1 ± 1.8 104.0 ± 29.9 Endothelin 1 CSCSSLMDKECVYFCHLDIIW 2491.9 −2.15 0.36  7.5 ± 0.5 19.73 ± 1.95 Dynorphin A YGGFLRRIRPKLKWDNQ 2147.5 3.76 4.48 15.1 ± 2.6  3.68 ± 0.76 Pre angiotensinogen DRVYIHPFHLVIHN 1758.9 0.03 3.45 24.6 ± 2.3  0.29 ± 0.04 Angiotensin I DRVYIHPFHL 1296.5 −0.06 2.46 43.4 ± 0.9  0.15 ± 0.04 W116S-FraC type II pore, -30 mV Angiotensin I DRVYIHPFHL 1296.5 −0.06 2.46  8.8 ± 0.2  0.54 ± 0.01 c-Myc 410-419 EQKLISEEDL 1203.3 −3.24 −1.19 30.0 ± 3.4  0.12 ± 0.01 Angiotensin 1-9 DRVYIHPFH 1183.3 −0.06 2.46 14.0 ± 0.2  0.37 ± 0.04 Angiotensin II DRVYIHPF 1046.2 −0.15 1.47 17.9 ± 1.3  0.37 ± 0.04 AsnlVal5 Angiotensin II NRVYVHPF 1031.2 0.85 2.03 19.6 ± 0.2  0.34 ± 0.06 Angiotensin A ARVYIHPF 1002.2 0.85 2.03 21.0 ± 0.6  0.34 ± 0.02 Angiotensin III RVYIHPF 931.1 0.85 2.03 22.1 ± 0.5  0.35 ± 0.04 Ile7 Angiotensin III RVYIHPI 897.1 0.85 2.03 24.3 ± 0.4  0.19 ± 0.05 Angiotensin IV VYIHPF 774.9 −0.15 1.02 38.9 ± 4.0  0.15 ± 0.06 W112S-W116S-FraC type III pore, −50 mV Angiotensin IV VYIHPF 774.9 −0.15 1.02  1.1 ± 0.8  0.61 ± 0.07 Angiotensin 4-8 YIHPF 675.8 −0.15 1.02  8.2 ± 0.4  0.40 ± 0.04 Endomorphin I YPWF 610.7 −0.24 0.04 19.2 ± 0.5  0.32 ± 0.04 Met5 Enkephalin YGGFM 573.7 −0.24 0.04 33.5 ± 0.7  0.16 ± 0.02 Leucine Enkephalin YGGFL 555.6 −0.24 0.04 34.5 ± 2.4  0.20 ± 0.05 The electrophysiology solution contained 1M KCl, 0.1M citric acid, 180 mM Tris base at pH 4.5. Recordings were performed using a 50 kHz sampling and applying 10 kHz Bassel filter. Standard deviations were obtained for at least three measurements. The charges of the peptides were calculated according to the pK.sub.a for individual amino acid.sup.36.

[0084] The resolution limit of the nanopore sensor was challenged by sampling a mixture of peptides. Remarkably, angiotensin II and angiotensin A, having an identical composition except for the initial amino acid (aspartate in angiotensin II vs. alanine in angiotensin A), appeared as distinctive peaks in Ires % plots (FIG. 3b). Smaller differences in peptide mass, e.g. the 34 Da difference between phenylalanine and leucine in angiotensin III and Ile7 angiotensin III, were observed but not easily detected (data not shown), indicating the resolution of our system at ˜40 Da. Smaller peptides such as angiotensin II 4-8 (YIHPF, 675.8 Da), endomorphin I (YPWF, 610.7 Da) or leucine enkephalin (YGGFL, 555.6 Da) translocated too quickly across type II W116S-FraC nanopores to be sampled. However, they could be readily measured using type III W112S-W116S-FraC nanopores (Table 4; FIG. 6).

Example 3: A Nanopore Mass Spectrometer for Peptides

[0085] Although the ability of biological nanopores to distinguish between known analytes is useful, a more powerful application would be the identification of peptide masses directly from ionic current blockades without holding prior knowledge of the analyte identity. In an effort to assess FraC nanopores as peptide mass analyzer, additional peptides were tested at pH 4.5 and 1 M KCl using type I, type II and type III FraC nanopores (FIG. 4a-c, Table 4). Crucially, analytes with largely different charge compositions were included.

[0086] It was found that for most of peptides there was a direct correlation between the size and the residual current (FIG. 4a-c). A notable exception was c-Myc 410-419 (1203.3 Da), an intentionally selected peptide because it includes a long stretch of negatively charged residues (FIG. 5a). The overall negative charge of the peptide at pH 4.5 (see Table 4) was expected to have an effect on both peptide capture and recognition. c-Myc 410-419 could be readily captured at negative applied potentials (trans), indicating that the cis to trans electroosmotic flow across the nanopore can overcome the electrostatic energy barrier opposing peptide capture. However, the Ires % of c-Myc 410-419 (30.0±3.4) was significantly higher than the expected value (FIG. 4b).

[0087] We reasoned that such anomaly might be due to the interaction between the acidic amino acids of the peptide and the negatively charged constriction of FraC nanopores. Thus, we lowered the pH solution to values where the aspartate and glutamate side chains in the peptides are expected to be protonated, hence become neutral (FIG. 5a). Rewardingly, at pH 3.8, the signal corresponding to c-Myc 410-419 (1203.3 Da) fell between the signal of angiotensin I (1296.5 Da), and angiotensin 11 (1046.2 Da, FIG. 5b, c). This indicates that, after losing its negative charges, the peptide blockades scaled with the expected mass of the peptides.

[0088] It has been assumed.sup.1,30,31 and experimentally proven.sup.32 that the voltage dependence of the average dwell time (τ.sub.off) can report on the translocation of a molecule across a nanopore. Under a negative bias (trans) for positively charged peptides (added in cis) both electrophoretic and electroosmotic forces (from cis to trans) promote the entry and translocation.sup.27 across the nanopore. For negatively charged peptides, such as c-Myc 410-419 at pH 4.5 (FIG. 5a), the electroosmotic driving force must be stronger than the opposing electrophoretic force. The voltage dependence of τ.sub.off was examined for c-Myc 410-419 at different pH values (FIG. 5d). At pH 4.5 the peptide exhibited a maximum in τ.sub.off at −50 mV, suggesting that at low potentials c-Myc 410-419 returns to the cis chamber (<50 mV), and at higher potentials (>50 mV) c-Myc 410-419 exits to the trans chamber. At pH 3.8 and lower, we observed a decrease in τ.sub.off at higher potentials, indicating that c-Myc 410-419 crosses the membrane to the trans chamber.

[0089] As shown in FIG. 6, type III FraC nanopore can detect differences in peptide length down to 4 amino acids (mass around 500 Dalton) in a peptide mixture. It was also found that the residual current signal correlated well with the mass of peptides, suggesting that Type III can be used as a detector for a peptide having a mass down to ˜500 Da.

Example 4: Peptide Mass Identifier at pH 3

[0090] This example shows that mutation D10C can be used as additional mutation to obtain a FraC pore showing a quiet signal in electrophysiology recordings.

[0091] Using mutant W116S as exemplary mutant, the aspartic acid at position 10 of FraC was converted to cysteine by site-directed mutagenesis. The thiol group of cysteine was then oxidized to sulfonic acid by incubation of FraC monomers with 10% hydrogen peroxide (v/v), which was dissolved in regular buffer (e.g. 10 mM Tris buffer pH 7.5, 150 mM NaCl). As a control, the double mutant was left without oxidation.

[0092] D10C/W116S FraC was oligomerized, and the oligomers tested in electrical recordings. FIG. 7 shows the trace comparison between the D10C/W116S pore and oxidized D10C/W116S pore to demonstrate the difference after oxidization. Oligomerised pores from oxidized D10C/W116S FraC monomers showed a quiet signal in electrophysiology recordings, as compared to a more noisy signal observed for nanopores that had not been subjected to oxidation.

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