Nanopore Proteomics

Abstract

The present invention relates to the field of engineered nanopores and their use in the analysis of biopolymers and other (biological) compounds.Provided is a proteinaceous nanopore comprising a mutant pore-forming toxin or a pore-forming fragment thereof, wherein the lumen-facing recognition region of the pore-forming protein or fragment thereof comprises one or more substitutions of lumen-facing amino acids in the recognition region corresponding to amino acids 10 to 20 of Fragaceatoxin C (FraC) with neutral or non-neutral aromatic amino acid residues.

Description

[Technical field]

[0001] The present invention relates generally to the field of nanopores and their use in the analysis of biopolymers and other (biological) compounds. In particular, the present invention relates to engineered nanopores and their improved performance in capturing and recognizing peptides. [Background technology]

[0002] Nanopores have become potential candidates for inexpensive, high-throughput, and / or portable protein detectors. Recently, it has been shown that nanopores function similarly to mass spectrometers for model analytes such as polyethylene glycol (PEG), as well as biological macromolecules such as peptides and proteins (Non-Patent Document 1). Biological nanopores have been shown to be particularly suitable for the detection and discrimination of small molecules based on the signal they generate when an analyte translocates through their recognition site. This method, known as nanopore spectrometry (Non-Patent Document 2), relies on the interaction of the analyte with the pore surface.

[0003] Pore ​​forming proteins (PFPs) can be broadly classified into two major groups, α-PFPs or β-PFPs, which form pores by a bundle of α-helices or a transmembrane β-barrel, respectively. Although members of both groups of PFPs share a common general mode of pore formation, several evolutionarily unrelated families can be distinguished depending on the structure of their soluble monomers.

[0004] The family of α-helical pore-forming toxins (actinoporins) produced by sea anemones are pore-forming proteins with a mass of approximately 20 kDa (Non-Patent Document 3). The sequence identity of the actinoporin family is high (60-80%) (Non-Patent Document 4), and the mechanism of pore formation is thought to be largely similar, with pore formation often depending on the presence of sphingomyelin in the lipid bilayer. The activity of actinoporins can be traced back to their α-helical transmembrane domain (Figure 1A), which is formed by the first 30-32 amino acids (Non-Patent Document 5). This domain also includes the constriction, which is the narrowest point of the pore (Non-Patent Document 6).

[0005] The physiological properties of the actinoporin Fragaceatoxin C (FraC), such as electro-osmotic flow (EOF) and recognition capacity (7, 6), can be manipulated, making FraC a prime target to be developed for single-molecule nanopore spectrometry. However, the interaction of biological analytes with the pore is poorly understood.

[0006] The three major families of β-PFPs are the α-hemolysin family found in Staphylococcus aureus, the MACPF / CDC protein superfamily, and PFPs showing similarity to aerolysin, a well-studied toxin from the pathogen Aeromonas hydrophila.

[0007] Analyte detection and DNA sequencing using biological nanopores has been a major advance in recent years. However, protein detection and sequencing by nanopores is complicated by the complex physiochemical structure of polypeptides and the lack of understanding of the mechanisms of polypeptide capture and recognition by nanopores.

[0008] The crystal structure of wild-type FraC (WtFraC) shows a multimeric (octameric) pore formed by eight identical subunits (Non-Patent Document 8). In previous studies, we have shown that FraC has the ability to form different multimeric forms, most notably octamers (T1) and heptamers (T2), with different pore volumes and different detectable peptide ranges (Patent Document 1, Non-Patent Document 8). In the same study, we also showed that the current observed from peptide translocation through WtFraC correlates with the mass of the peptide at 1 M KCl, pH 3.8. However, peptide blockade is rapid, i.e., on the order of a few microseconds (mean residence time for angiotensin 1 is 0.15±0.04 ms) (Non-Patent Document 6), which leads to a large proportion of translocation events remaining undetected and to inaccurate characterization of detected events. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2020 / 055246 [Non-patent literature]

[0010] [Non-Patent Document 1] Robertson et al. Proc Natl Acad Sci US A. 2007 [Non-Patent Document 2] Chavis et al., ACS Sens, 2017 [Non-Patent Document 3] Anderluh et al. Toxicon, 2002 [Non-Patent Document 4] Garcia-Ortega et al. Biochim Biophys Acta, 2011 [Non-Patent Document 5] Ros et al. Biochimie, 2015 [Non-Patent Document 6] Huang et al. Nat Commun, 2019 [Non-Patent Document 7] Huang et al. Nat Commun, 2017 [Non-Patent Document 8] Tanaka et al., Nat Commun, 2015 Summary of the Invention [Means for solving the problem]

[0011] In view of the above, the present invention aims to improve the accuracy of characterization of peptides captured individually by nanopore sensors. To this end, we engineered proteinaceous nanopores to improve capture of unlabeled peptides, increase the residence time of peptides in the nanopore sensor, and improve discrimination between peptide species. In the present invention, we engineered proteinaceous nanopores to improve peptide sensing under low pH conditions optimized for peptide detection.

[0012] Surprisingly, we found that the introduction of one or more "bulky" / aromatic amino acids at precise locations within the nanopore lumen can both increase the frequency of peptide capture and greatly improve discrimination between peptides. For example, we found that Fragaceatoxin C (FraC) nanopores containing subunits with tyrosine, phenylalanine, or tryptophan residues introduced into the lumen-facing region exhibit increased residence times of peptides within the pore. These "large aromatically modified" nanopores may also be able to detect and measure peptides in the tryptic digestion of lysozyme. Furthermore, we may be able to assign the individual unique spectra of individual proteins using advanced analytical methods such as spectral matching. Furthermore, we adapted our combination of mutational engineering discovery and detection conditions to other nanopore families with completely different structures but with similarly sized recognition regions within the nanopore lumen, e.g., beta-barrel pores, and found that the improvements in peptide characterization are universal.

[0013] These findings provide proof of concept that engineered nanopores can be used as single-molecule detectors capable of label-free protein detection and fingerprinting, providing the basis for enhancing peptide capture and improving recognition by nanopores, which is important for developing real-time, single-molecule analyzers for peptide recognition and identification.

[0014] The present invention therefore relates to a proteinaceous nanopore comprising a mutated (or "modified") transmembrane pore-forming toxin or pore-forming fragment thereof, for example of the actinoporin family, in which the lumen-facing recognition region of the pore-forming protein or fragment thereof comprises one or more mutations to neutral or non-neutral aromatic amino acid residues. The pore-forming toxin is typically multimeric. The pore is preferably composed of several repeating subunits, such as 6, 7 or 8 subunits. When inserted into a membrane, the pore comprises a central channel through which ions may flow, for example when a potential is applied across the membrane. The subunits of the pore typically contribute strands that surround the central axis and become a transmembrane α-helical bundle or channel. Thus, the modified proteinaceous nanopore of the present invention comprises a multimer (or "assembly") of a mutated pore-forming α-helical pore-forming subunit of the actinoporin family or a multimer of a pore-forming fragment thereof.

[0015] A "lumen-facing recognition region", also referred to herein as a "recognition area" or "water-facing region", is meant to refer to the portion of the nanopore involved in sensing the analyte across the pore. The recognition region is typically a portion (lumen) of the central water-filled channel that is formed through the nanopore from cis to trans when inserted into a membrane such as a lipid bilayer. The recognition region can typically be structurally identified from the dimensions of the central channel. Suitable structures or structural models can be obtained or constructed by means known in the art, including experimental X-ray diffraction structures, electron microscopy structures, and computer modeling. The recognition region should be a region of the channel through the nanopore where the electric field lines are concentrated and the presence of an analyte will disrupt most of the ionic current flowing through the nanopore under an applied potential. For small peptide detection, the recognition region will preferably include a section of the nanopore channel with an internal diameter of less than 2 nanometers, preferably less than 1 nanometer, to provide a significant deflection of the ionic current and sufficient residence / residence time during analyte interaction. Many nanopores may have one or more narrowed sections of small internal diameter (constrictions) within a longer recognition region. Maximum sensitivity / ionic current deflection to an analyte is typically achieved when the analyte interacts with the nanopore at or near the constriction. Thus, maximum control of analyte detection can often be achieved by protein mutagenesis / engineering at or near the residues that make up the constriction.

[0016] Alternatively, for proteins known to be nanopores (e.g., by homology searches or experimental determination) but without a suitable structural model, the recognition region can often be determined by homology mapping to the recognition regions of other known nanopores and / or computer modeling using means known in the art. For example, the recognition region often comprises or is entirely within the transmembrane section of a membrane protein nanopore (e.g., a transmembrane section composed of beta-barrel or alpha-helical multimers). Transmembrane beta-barrels and alpha-helices can be identified by means such as homology comparison with other known pores and by features such as amphipathic hydropathy maps.

[0017] Nanopore recognition regions can also be experimentally determined and / or confirmed by mutagenesis using means well known in the art. For example, the ionic current characteristics of different nanopores with different targeted mutations in candidate recognition regions can be compared in electrophysiological experiments with the nanopores inserted into a membrane. By varying the location of the mutations and, optionally, measuring the differences in response to an analyte of interest, the recognition regions can be mapped and characterized.

[0018] The pores of the invention are particularly characterized in that the recognition region facing the lumen of the pore is engineered (by one or more natural or unnatural amino acid substitutions) to manipulate the internal diameter / hydrophobicity / aromaticity of the pore and thereby increase the residence time and resolution of peptides traversing the nanopore.

[0019] Accordingly, the present invention provides a method for decreasing the translocation rate of a peptide analyte through a transmembrane (alpha-helical or beta-barrel) protein pore, comprising: (a) increasing the net aromaticity of the lumen of the pore by substituting one or more non-aromatic amino acids with one or more aromatic amino acids; and (b) passing a polypeptide through the pore, which increases the net aromaticity, thereby decreasing the translocation rate of the polypeptide through the pore; Including, Preferably, said substitution results in a proteinaceous nanopore as described herein. A method is also provided.

[0020] As will be appreciated by those skilled in the art, one or more mutations of the invention can be introduced into a nanopore or functional pore-forming fragment thereof in a number of configurations to produce a desired change in the recognition region of the assembled nanopore. For example, for a multimeric nanopore composed of multiple (e.g., 4, 5, 6, 7, 8, or more) monomeric units, one or more mutations are made in all monomers used to assemble the nanopore such that the assembled nanopore contains multiple rings of the same mutation in the recognition region that is flush with the membrane and perpendicular to the direction of analyte passage. Alternatively, mutant monomers may be mixed with monomers that do not contain mutations or contain different mutations during nanopore multimerization to create a "heteromultimeric" assembled nanopore with a controlled number of mutations. Thus, an assembled pore may contain one, two, three, four, five, six, seven, eight, nine, or more mutant monomers of the invention, depending on the number of multimeric units. Controlling the number of mutation units can help to reduce or otherwise modulate the degree / magnitude of changes to the recognition site. Means are known in the art for selectively purifying a population of heteromultimeric nanopores with a desired number of mutations from a mixture (e.g., Gouaux, et al Proc Natl Acad Sci USA 1994). For multimeric nanopores that contain more than one type of monomeric unit that both contribute to the recognition region in the assembled form (e.g., bicomponent leukocidins, Spaan et al Nat Rev Microbiol 2017), mutations may be made in one or both monomeric species. And, for pores in which the monomers are fused (e.g., genetically or by other chemical linkage means), e.g., in the case of dimers (Hammerstein et al, J Biol Chem 2011), one skilled in the art will understand that by selecting how many monomeric units to modify, a desired number of mutations can be introduced into the final assembly.For monomeric protein nanopores (e.g., outer membrane porins) in which a single protein strand constitutes the transmembrane channel, it is understood that mutations can be made along the sequence at a specific gap distance to include the required number of water-facing mutations, preferably on multiple beta strands of the transmembrane section of the pore, more preferably on all beta strand units, to achieve a ring-like mutation formation similar to that formed in homomultimeric nanopores. For beta-barrel nanopores, it is understood that mutations may be introduced on either or both the down or up strands. For example, aromatic or acidic substitutions of the invention may be added to both the up and down strands of a beta barrel such that they are roughly coplanar (at the same vertical position of the nanopore) and produce a stronger effect in the recognition region. In another embodiment, multiple mutations of the invention can be made along the alpha helices or beta strands of the monomers of the multimeric pore or fragments thereof (perpendicular to the membrane), for example, to produce a stronger change in the recognition region.

[0021] Natural and unnatural aromatic amino acid residues are known in the art. In one embodiment, the unnatural aromatic amino acid is selected from the group consisting of 3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine, L-thyroxine, phenylglycine (Phg), or nortyrosine (norTyr). Phg and norTyr. Suitable unnatural amino acids may include D-amino acids, homoamino acids (methylene), beta homoamino acids, N-methyl amino acids, alpha methyl amino acids. A wide variety of well-known unnatural amino acids are known in the art, preferably including derivatized Phe / Tyr / Trp amino acids, more preferably ring-substituted Phe / Tyr / Trp amino acids. Derivatives of Phe, Tyr, and Trp, such as halogens, -CH 3、Substitution with OH, -CH2NH3, -C(O)H, -CH2CH3, -CN, -CH2CH2CH3, -SH, or another group is also included. Exemplary unnatural aromatic amino acids include, but are not limited to, O-methyl-L-tyrosine, 3-methyl-phenylalanine, p-acetyl-L-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, amino-, isopropyl-, or O-allyl-containing phenylalanine analogs, p-(propargyloxy)phenylalanine, 3-nitro-tyrosine, 5-fluoro-tryptophan, 5-hydroxy-tryptophan, 5-methoxy-tryptophan, 5-methyl-tryptophan, trifluoromethyl-tryptamine ethyl ester.

[0022] Methods for introducing unnatural amino acids into proteins are also known in the art. For example, unnatural amino acids may be introduced by including synthetic aminoacyl-tRNA in the IVTT system used to express mutant monomers. Alternatively, unnatural amino acids may be introduced by expressing mutant monomers in E. coli auxotrophic for a particular amino acid in the presence of a synthetic (i.e., unnatural) analog of that particular amino acid. Unnatural amino acids may be introduced using synthetic peptide chemistry methods known in the art for peptide synthesis. The monomeric units of the nanopore may be formed entirely from synthetic peptides assembled using conjugation methods known in the art, such as native chemical ligation (Thapa et al., Molecules, 2014, 14461-83) or cysteine ​​coupling. Alternatively, the monomers of the nanopore may include partially synthetic units coupled to naturally expressed peptides using conjugation methods known in the art.

[0023] Mutant nanopores may be made by chemically linking a suitable aromatic molecule to either the precursor monomeric unit of the nanopore or to the assembled multimeric nanopore by means known in the art, for example by chemically linking a suitable molecule to one or more cysteines (cysteine ​​bond) or lysines already present in the wild-type protein or introduced by mutagenesis.

[0024] In alternative embodiments, the engineered nanopore comprises a mutation to a naturally occurring aromatic amino acid, preferably one or more mutations to Trp, Tyr, or Phe, more preferably one or more mutations to Phe.

[0025] In addition to aromatic amino acid mutations, one or more further mutations may be introduced into the lumen-facing recognition region that increase the net negative charge of the pore. For example, a mutant proteinaceous nanopore having mutations introduced into the lumen-facing recognition region to create a combination of aromatic residues that are "spatially close" to negative residues, the aromatic and negative residues being preferably within 4 nanometers, more preferably within 2 nanometers, of each other in the functional pore. The physical spacing between residue positions can be derived, for example, by measuring the C-alpha backbone distance between each mutant residue from a 3D model or crystal structure of the assembled multimeric pore protein using common molecular modeling software known in the art.

[0026] The transmembrane protein pore or fragment thereof for use according to the present invention may be derived from a beta barrel pore or an alpha helix bundle pore. A beta barrel pore comprises a barrel or channel formed from beta strands. A person skilled in the art will understand that a suitable nanopore of the present invention may be selected from transmembrane pores known in the art (Peraro et al. Nat Rev Microbiol 2016; Crnkovic´ et al. Life (Basel) 2021). A suitable nanopore will ideally have a recognition region dimension most suitable for measuring small analytes, including small peptides. A suitable nanopore will ideally have a transmembrane region, recognition region, or constriction with a diameter of less than 2 nanometers, most preferably less than 1.5 nanometers. Suitable beta barrel pores include, but are not limited to, beta toxins such as alpha hemolysin, erolysin, lysenin, cytolysin, cytolysin K, anthrax toxin and leukocidin, and bacterial outer membrane proteins / porins such as Mycobacterium smegmatis porin (Msp), e.g., MspA, MspB, MspC or MspD, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A (OMPLA), ferric hydroxamate uptake component A (FhuA), Curli production transport component CsgG, and Neisseria autotransporter lipoprotein (NalP). Alpha helix bundle pores comprise a barrel or channel formed from alpha helices. Suitable alpha-helical bundle pores include, but are not limited to, inner and outer membrane proteins such as actinoporins, the outer membrane core complex (OMCC) of the H. pylori Cag T4SS particle, and the transmembrane domain of the E. coli polysaccharide transporter Wza.

[0027] Those skilled in the art will appreciate that suitable nanopores of the present invention may be artificial nanopores, such as modified versions of known artificial nanopores, such as nanopores based on transmembrane beta barrels or alpha helices coupled to cyclic proteins (Zhang et al. BioRxiv 2020), proteins based on transmembrane peptides coupled to DNA origami (Spruijt et al. Nat Nanotechnol 2018), self-assembled nanopores based on engineered transmembrane peptides (Scott et al. Nat Chem 2021), and de novo designed ones (Vorobieva et al. Science 2021).

[0028] (Alpha helix pore) In one embodiment, the proteinaceous nanopore comprises a mutated actinoporin or its alpha-helical transmembrane region (aa 1-27). For example, the mutated actinoporin comprises a substitution of an aromatic amino acid residue in the recognition region corresponding to amino acids 10-20. For homologs with additional N-terminal sequence, the region corresponding to the transmembrane alpha-helix can be determined by homology mapping and other means known in the art. The actinoporin family of pore-forming toxins is well known in the art. See, e.g., Kristan et al. (Toxicon 2009).

[0029] Exemplary members of the actinoporin family for preparing mutants according to the invention include Fragaceatoxin A (FraA), Fragaceatoxin B (FraB), Fragaceatoxin C (FraC), Fragaceatoxin D (FraD), Fragaceatoxin E (FraE), Equinatoxin II (Eqt-II), Equinatoxin IV (Eqt-IV), Equinatoxin V (Eqt-V), Urticinatoxin (UcI), Actitoxin-Oor1b (Or-G), Actitoxin-Oor1a (Or-A), Gigantoxin-4 (Gigt4), Heteractis magnifica cytolysin III (HmgIII), Bandaporin (bp-1), Cribinopsis japonica toxin I (CJTOX I), Cribinopsis japonica Contains toxin II (CJTOX II), Sticholysin I (StI), Sticholysin II (StII), Stichotoxin Hcr4a (RTX-A), Stichotoxin Hcr4b (RTX-SII), and Stichotoxin I (Src I).

[0030] The wild-type sequences and SwissProt accession numbers of exemplary alpha-helical pore-forming proteins are as follows: >Fragaceatoxin C|B9W5G6 SADVAGAVIDGAGLGFDVLKTVLEALGNVKRKIAVGIDNESGKTWTAMNTYFRSGTSDIVLPHKVAHGKALLYNGQKNRGPVATGVVGVIAYSMSDGNTLAVLFSVPYDYNWYSNWWNVRVYKGQKRADQRMYEELYYHRSPFRGDNGWHSRGLGYGLKSRGFMNSSGHAILEIHVTKA >Fragaceatoxin A|P0DUW8| SAEVAGAVIEGAKLTFNVLQ >Fragaceatoxin B|A0A515MEN7 SLTFDVLQTVLKALGDVSRKIAVGIDNEPGMTWTAMNTYFRSGTSDVILPHTVPHSKALLYDGQKNRGPVTTGVVGVIAYAMSDGNTLAVLFSIPFDYNLYSNWWNVKVYKGHRRADQAMYEELYYDFSPFRGDNGWHTKSIGYGLKGRGFMNSSGKAILQIHVNKV >Fragaceatoxin D|P0DUW9 SVAVAGAVIKGAALTFNILQ >Fragaceatoxin E|A0A515MEM9 AGLGFDVLKTVLEALGNVKRKIAVGIDNESGRTWTAMNTYFRSGTSDIVLPHKVAHGKALLYNGQKNRGPVATGVVGVIAYSMSDGNTLAVLFSVPYDYNWYSNWWNVRVYKGQKRANQRMYEELYYHRSPFRGDNGWHSRSLGYGLKSRGFMNSSGHAILEIHVTKA >Equinatoxin II|P61914 SADVAGAVIDGASLSFDILKTVLEALGNVKRKIAVGVDNESGKTWTALNTYFRSGTSDIVLPHKVPHGKALLYNGQKDRGPVATGAVGVLAYLMSDGNTLAVLFSVPYDYNWYSNWWNVRIYKGKRRADQRMYEELYYNLSPFRGDNGWHTRNLGYGLKSRGFMNSSGHAILEIHVSKA >Equinatoxin IV|Q9Y1U9 SVAVAGAIIKGAALTFNVLQTVLKALGDISRKIAVGVDNESGKTWTALNTYFRSGTSDIVLPHKVPHGKALLYNGQKDRGPVATGAVGVLAYAMSDGNTLAVLFSVPYDYNWYSNWWNVRIFKGRRRADQRMYEQLYYYLSPFRGDNGWHERHLGYGLKSRGFMNSGGQAILEIHVTKA >Equinatoxin V|Q93109 SVAVAGAVIEGATLTFNVLQTVLKALGDISRKIAVGIDNESGMTWTAMNTYFRSGTSDVILPHTVPHGKALLYNGQKDRGPVATGVVGVLAYAMSDGNTLAVLFSIPFDYNLYSNWWNVKVYKGHRRADQRMYEELYYNLSPFRGDNGWHNRDLGYGLKGRGFMNSSGQSILEIHVTKA >Urticinatoxin|C9EIC7 SVAIAGAVIEGAKLTFGILEKILTVLGDINRKIAIGVDNESGREWTAQNAYFFSGTSDVVLPASVPNTKAFLYNAQKDRGPVATGVVGVLAYSLSNGNTLGILFSVPYDYNLYSNWWNIKLYKGIKRADRDMYNDLYYYAHPHKGDNGWHENSLGFGLKSKGFMTSSGQTILQIRVSRA >OrG|Q5I2B1 GAIIAGAALGFNVHQTVLKALGQVSRKIAIGVDNESGGTWTALNAYFRSGTTDVILPEFVPNQKALLYSGQKDTGPVATGAVGVLAYYMSDGNTLGVMFSVPFDYNLYSNWWDVKVYRGRRRADQAMYEGLLYGIPYGGDNGWHARKLGYGLKGRGFMKSSAQSILEIHVTKA >OrA|Q5I4B8 ATFRVLAKVLAELGKVSRKIAVGVDNESGGSWTALNAYFRSGTDTVILPDLVPNQKALLYRGGKDTGPVATGVVGVLAYAMSDGNTLAILFSVPYDYNLYSNWWNVKVYSGKRRADQGMSEDLSYGNPYGGDNGWHARKLAYGLKERGFMKSSAQSILEIHATKA >Gigantoxin4|H9CNF5 ASAVAGTIIEGASLTFQILDKVLTELGNVSRKIAIGIDNESGGSWTAMNAYFRSGTDTVILPEFVPNNKALLYSGRKDTGPVTTGAVGALAYYMSDGNTLAVMFSVPFDYNLYSNWWDVRVYSGKRRADQKMYEDLYNGSPFKGDNGWHQKNLGYGLRMKGIMTSAGEAKLQIKISR >HmgIII|Q9U6X1 SAALAGTIIEGASLGFQILDKVLGELGKVSRKIAVGVDNESGGSWTALNAIFRSGTDTVILPEFVPNQKALLYSGRKDTGPVATGAVAAFAYYMSNGHTLGVMFSVPFDYNFYSNWWDVKVYSGKRRADQGMYEDMYYGNPYRGDNGWHQKNLGYGLRMKGIMTSAGEAILQIRISR >Bandaporin|C5NSL2 SLAVAGAVIEGGNLVMSVLDRILEAIGDVNRKIAIGVENQSGKSWTAMNTYFRSGTSDVVLPHSVPSGKALLYDGQKTRGPVATGVVGVFAYAMSDGNTLAVMFSIPYDYNLYSNWWNVKTYSGMKRADQSMYEDLYYHASPFKGDNGWHSRNLGYGLKCRGFMNSSGAAKLEIHVSRA >CJTOX I|A0A2Z5Z9X0 LPMKEDISNEERPTSVNEKPVKKSVAVAGAVIQGAALAFQVLDKILTSLGGIGRKIAIGVDNESGMKWAARNVYFYSGTSDTVLPYSVPHSKAFLYGARKTRGSVRGAVGVLAYSMSDGNTLGILFSVPYDYNWYSNWWNIKVYRGYKRANKWMYHDLYYYARPHKGNNEWHEKSLGYGLKSKGFMTSSGQTKLEIRVSRA >CJTOX II|A0A2Z5Z9H5 LPMKEDISNDERPISVNEEPVKKNAAVAGAVIQGATLTFQVLDRILTVLGDISRKIAIGVDNESGRKWTAKNAYFFSGTSDVVLPYSVPNGKAFLYDGKKTRGPVATGAVGVLAYSMSDGNTLGILFSVPYDYNWYENWWNIKVYSGSKRANKWMYENLYYNASPHKGDNGWHEKSLGYGLKSRGYMASSGQTKLEIRVTRA >Sticholysin I|P81662 SELAGTIIDGASLTFEVLDKVLGELGKVSRKIAVGIDNESGGTWTALNAYFRSGTTDVILPEVVPNTKALLYSGRKSSGPVATGAVAAFAYYMSNGNTLGVMFSVPFDYNWYSNWWDVKIYPGKRRADQGMYEDMYYGNPYRGDNGWYQKNLGYGLRMKGIMTSAGEAKMQIKISR >Sticholysin II|P07845 ALAGTIIAGASLTFQVLDKVLEELGKVSRKIAVGIDNESGGTWTALNAYFRSGTTDVILPEFVPNTKALLYSGRKDTGPVATGAVAAFAYYMSSGNTLGVMFSVPFDYNWYSNWWDVKIYSGKRRADQGMYEDLYYGNPYRGDNGWHEKNLGYGLRMKGIMTSAGEAKMQIKISR >RTXA|P58691 ALAGAIIAGASLTFQILDKVLAELGQVSRKIAIGIDNESGGSWTAMNAYFRSGTTDVILPEFVPNQKALLYSGRKNRGPDTTGAVGALAYYMSNGNTLGVMFSVPFDYNLYSNWWDVKVYSGKRRADQAMYEDLYYSNPYRGDNGWHQKNLGYGLKMKGIMTSAGEAIMEIRISR >RTXSII|P0C1F8 SAALAGTITLGASLGFQILDKVLGELGKVSRKIAVGVDNESGGSWTALNAYFRSGTTDVILPEFVPNQKALLYSGRKDTGPVATGAVAAFAYYMSNGHTLGVMFSVPFDYNLYSNWWDVKIYSGKRRADQAMYEDMYYGNPYRGDNGWHQKNLGYGLKMKGIMTSAVEAILEIRISR >Src I|Q86FQ0 KISGGTVIAAGRLTLDLLKTLLGTLGSISRKIAIGVDNETGGLITGNNVYFRSGTSDDILPHRVETGEALLYTARKTKGPVATGAVGVFTYYLSDGNTLAVLFSVPFDYNFYSNWWNVKIYSGKRNADYDMYHELYYDANPFEGDDTWEYRYLGYGMRMEGYMNSPGEAILKITVMPD >Cytolysin AvtI|Q5R231 SAAVAGAVIAGGELALKILTKILDEIGKIDRKIAIGVDNESGLKWTALNTYYKSGASDVTLPYEVENSKALLYTARKSKGPVARGAVGVLAYKMSSGNTLAVMFSVPFDYNLYSNWWNVKIYDGEKKADEKMYNELYNNNNPIKPSTWEKRDLGKDGLKLRGFMTSNGDAKLVIHIEKS >Cytolysin PsTX20A|P0DL55 SAAVAGAVIAGGELALKILTKILDEIGKIDRKIAIGVDNESGLKWTALNTYYKSGASDVTLPYEVENSKALLY TARKSKGPVARGAVGVLAYKMSSGNTLAVMFSVPFDYNLYTNWWNVKIYDGEKKADEKMYNELYNNNNPIKPSIWEKRDLGQDGLKLRGFMTSNGDAKLVIHIEKS >Nigrelysin|A0A345GPN1 LPLEEKEDEKDEKRSLEVAGAVMEGANLGMSVLQTILQAIGDVSRKIAVGVDNESGRSWTAQNAYFRSGTSDVILPHTVPSGKALLYDGQKNRGPVATGVVGVITYTMGDGNTLAVMFSVPYDYNWYSNWWNVKIYHGKVRASQKMYEDLYYYRSPFKGDNGWHERNLGYGLKSKGFMNSSGAALLQIKVMKA

[0031] Pore-forming toxin homologs thereof having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any of these family members are also encompassed, provided that the pore-forming toxin retains the ability to form a multimeric nanopore in a membrane. This functionality can be readily tested in vitro using methods known in the art. For example, putative purified nanopores can be inserted into model membranes as described herein or by other means known in the art (e.g., vesicle insertion, detergent insertion, natural insertion, etc.) and characterized by electrophysiological means to determine their ability to pass ionic current and to detect the presence of model analytes added to the system.

[0032] As will be appreciated by those skilled in the art, the amino acid sequence of a family member will include one or more mutations within the recognition region of the pore, and thus reference to a pore comprising, for example, FraD, Eqt-IV or StII is meant to refer to FraD, Eqt-IV or StII mutants in which the degree of internal aromaticity has been engineered, optionally in conjunction with the introduction of negatively charged residues in accordance with the present invention.

[0033] For example, the proteinaceous nanopore according to the invention advantageously comprises: (i) FraC, FraE, or a functional homologue thereof that has at least 90% sequence identity thereto, which contains an aromatic residue at the position corresponding to Gly13 of FraC; (ii) FraB, Ten-C, Eqt-II, Gigt4, HmgIII, RTX-SII, Hmt, or a functional homologue thereof exhibiting at least 90% sequence identity, comprising an aromatic residue at the position corresponding to Ser13 and, optionally, an acidic residue at position 10; (iii) bp-1, or a functional homologue thereof that has at least 90% sequence identity, containing an aromatic residue at the position corresponding to Asn13; (iv) CJTOX I, or a functional homologue thereof exhibiting at least 90% sequence identity, containing an aromatic residue at the position corresponding to Thr36 and, optionally, an acidic residue at the position corresponding to Gln33; (v) CJTOX II, or a functional homologue thereof exhibiting at least 90% sequence identity, comprising an aromatic residue at the position corresponding to Ala36 and, optionally, an acidic residue at the position corresponding to Gln33; (vi) Cytolysin Avt-I, Cytolysin PsTX-20A, or a functional homologue thereof exhibiting at least 90% sequence identity, comprising an aromatic residue at the position corresponding to Glu13 and, optionally, an acidic residue at the position corresponding to Ala10; (vii) Eqt-IV, or a functional homologue thereof that has at least 90% sequence identity thereto, comprising an aromatic residue at the position corresponding to Ala13 and, optionally, an acidic residue at the position corresponding to Lys10; (viii) Eqt-V, or a functional homologue thereof that has at least 90% sequence identity, containing an aromatic residue at the position corresponding to Thr13; (ix) Nigrelysin or a functional homologue thereof that has at least 90% sequence identity, which contains an aromatic residue at the position corresponding to Asn27; (x) StII, RTX-A, or a functional homologue thereof exhibiting at least 90% sequence identity, comprising an aromatic residue at the position corresponding to Ser11 and, optionally, an acidic residue at the position corresponding to Ala8; (xi) Src I, or a functional homologue thereof that has at least 90% sequence identity, containing an aromatic residue at the position corresponding to Arg12 and, optionally, an acidic residue at the position corresponding to Ala9; (xii) StI, or a functional homologue thereof having at least 90% sequence identity, containing an aromatic residue at the position corresponding to Ser12; (xiii) UcI, or a functional homologue thereof that has at least 90% sequence identity, containing an aromatic residue at the position corresponding to Lys13; or (xiv) Or-G, or a functional homologue thereof having at least 90% sequence identity, comprising an aromatic residue at the position corresponding to Ala8 and, optionally, an acidic residue at the position corresponding to Ala5; or the alpha-helical transmembrane domain (aa1-27) of said polypeptide containing the aforementioned mutations; The mutant actinoporin includes a mutant actinoporin selected from the group consisting of:

[0034] In one embodiment, the mutant pore-forming toxin or pore-forming alpha-helical fragment is selected from Table 1, which exhibits one or more specific aromatic mutations in each of the actinoporins equivalent to mutations in the lumen-facing residues at positions 10, 13, 17, and 20 of FraC.

[0035] Also included are pore-forming toxin homologues that contain defined mutations and exhibit at least 85%, at least 87%, at least 90%, at least 93%, at least 95%, or at least 98% sequence identity.

[0036] Very good results may be obtained with proteinaceous nanopores containing mutated FraC or its pore-forming alpha-helical fragments containing mutations such as Gly13Tyr, Gly13Trp or Gly13Phe, particularly Gly13Phe.

[0037] [Table 1]

[0038] (Beta barrel pore) In another aspect, the invention relates to a proteinaceous nanopore comprising a mutated beta-barrel pore-forming protein or a pore-forming fragment thereof, wherein the lumen-facing recognition region of the pore-forming protein or fragment thereof comprises one or more mutations of lumen-facing non-aromatic residues to natural or non-natural aromatic amino acid residues, preferably to Trp, Tyr or Phe.

[0039] Preferably, the beta barrel pore-forming toxin has an internal diameter (pore diameter, constriction) in the recognition region ranging from 0.2 to 2.0 nanometers, and most preferably has a minimum internal diameter of 0.5 to 1.5 nanometers. For example, the beta barrel pore-forming toxin is selected from the group consisting of alpha hemolysin, erolysin, lysenin, epsilon toxin (ETX), hemolytic lectin (LSL), cytolysin K (cytK), and functional homologs thereof that show at least 80%, preferably at least 85%, more preferably at least 90% sequence identity. Preferably, the beta barrel pore-forming toxin is selected from the group consisting of erolysin, lysenin, and cytolysin K (cytK), and functional homologs thereof that show at least 90%, preferably at least 95%, more preferably at least 98% sequence identity.

[0040] As described herein above for the alpha-helical pore-forming proteins, the pores contain one or more mutations that increase the net negative charge or decrease the net positive charge of the pore barrel or channel, with the aim of increasing the flux of cations through the nanopore, particularly under acidic pH conditions (pH<4.5) (Table 4).

[0041] The wild-type sequences and SwissProt accession numbers of exemplary beta-barrel pore-forming proteins are as follows: >Aerolysin|P09167 MQKIKLTGLSLIISGLLMAQAQAAEPVYPDQLRLFSLGQGVCGDKYRPVNREEAQSVKSNIVGMMGQWQISGLANGWVIMGPGYNGEIKPGTASNTWCYPTNPVTGEIPTLSALDIPGDEVD VQWRLVHDSANFIKPTSYLAHYLGYAWVGGNHSQYVGEDMDVTRDGGDGWVIRGNNDGGCDGYRCGDKTAIKVSNFAYNLDPDSFKHGDVTQSDRQLVKTVVGWAVNDSDTPQSGYDVTLRYDT ATNWSKTNTYGLSEKVTTKNKFKWPLVGETELSIIEIAANQSWASQNGGSTTTSLSQSVRPTVPARSKIPVKIELYKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDNRPNWNHTFVIG PYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLSTMQNNLARVLRPVRAGITGDFSAESQFAGNIEIGAPVPLAADSKVRRARSVDGAGQGLRLEIPLDAQELSGLGFNNVSLSVTPAANQ >CytK|Q937V2 MKRSKTYLKCLALSAVFASSALALSTPAAYAQTTSQVVTDIGQNAKTHTSYNTFNNDQTDNMTMSLKVTFIDDPSADKQIAVINTTGSFLKANPTISSAPIDNYPIPGASATLRYPSQYDIAFNLQDNSARFFNVAPTNAVEETTVTSSVSYQLGGSVKASATPNGLS AEAGATGQVTWSDSVSYKQTSYKTNLIDQTNKNVKWNVFFNGYNNQNWGIYTRDSYHSLYGNQLFMYSRTYLYESDAKGNLIPMDQLPALTNSGFSPGMIAVVISEKNTDQSNLQVAYTKHADDYQLRPGFTFGTANWVGNNVKDVDQKTFNKSFTLDWKNKKLVEKNR >Alpha-hemolysin|P09616.2 MKTRIVSSVTTTLLLGSILMNPVAGAADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEM >Gamma-hemolysin component B|P0A075.1 MNKLVKSSVATSMALLLLSGTANAEGKITPVSVKKVDDKVTLYKTTATADSDKFKISQILTFNFIKDKSYDKDTLVLKATGNINSGFVKPNPNDYDFSKLYWGAKYNVSISSQSNDSVNVVDYAPKNQNEEFQVQNTLGYTFGGDISISNGLSGGLNGNTAFSETINYKQESYRTTLSRNTNYKNVGWGVEAHKIMNNGWGPYGRDSFHPTYGNELFLAGRQSSAYAGQNFIAQHQMPLLSRSNFNPEFLSVLSHRQDGAKKSKITVTYQREMDLYQIRWNGFYWAGANYKNFKTRTFKSTYEIDWENHKV >Leukocidin-F|P31715.2 MNKLVKSSVATSMALLLLSGTANAEGKITPVSVKKVDDKVTLYKTTATADSKFKISQILTFNIKSYDKDTLVLKATGNINSGFVKPNPNDYDFSKLYWGAKYNVSISSQSNDSVNAVDYAPKNQNEEFQVQNTLGYTFGGDISISNGLSG GLNGNTAFSETINYKQESYRTLSRNTNYKNVGWGVEAHKIMNGWGPYGRDSFHPTYGNELFLAGRQSSYAGQNFIAQHQMPLLSRSNFNPEFLSVLSHRQDRAKKSKITVTYQREMDLYQIRWNGFYWAGANYKNFKTRTFKSTYEIDWENHKV >Leucotoxin LukD|O54082.1 IEKLGKSSVASSIALLLLSNTVDAAQNITPKREKKVDDKITLYKTTATSDNDKLNIFQILTFNFIKDKSYDKDTLVLKAAGNINSGYKNSNPKDYNYSQFYWGGKYNVSVSSESNDAVNVVDYAPKNQNEEFQVQQTLGYSYGGDINISNGLSGGLNGSKSFSETINYKQESYRTTIDRKTNHKSIGWGVEAHKIMNNGWGPYGRDSYDPTYGNELFLGGDKSSSNAGQNFLPTHQIPLLARGNFNPEFISVLSHKLFDTKKSKIKVTYQREMDRYTNQWNRSHWVGNNYKNQNTVTFTSTYEVDWQN >Gamma-hemolysin component A|P0A071.1 IKNKILTATLAVGLIAPLANPFIEISKAENKIEDIGQGAEIIKRTQDITSKRLAITQNIQFDFVKDKKYNKDALVVKMQGFISSRTTYSDLKKYPYIKRMIWPFQYNISLKTKDSNVDLINYLPKNKIDSADVSQKLGYNIGGNFQSAPSIGGSGSFNYSKTISYNQKNYVTEVESQNSKGVKWGVKANSFVTPNGQVSAYDQYLFAQDPTGPAARDYFVPDNQLPPLIQSGFNPSFITTLSHERGKGDKSEFEITYGRNMD >Leukocidin-S subunit|P31716.1 DIGKGSDIEIIKRTEDKTSNKWGVTQNIQFDFVKDTKYNKDALILKMQGFISSRTTYYNYKKTNHVKAMRWPFQYNIGLKTNDKYVSLINYLPKNKIESTNVSQTLGYNIGGNFQSAPSLGGNGSFNYSKSISYTQQNYVSEVEQQNSKSVLWGVKANSFATESGQKSAFDSDLFVGYKPHSKDPRDYFVPDSELPPLVQSGFNPSFIATVSHEKGSSDTSEFEITYGRNMDVTHAIKRSTHYGNSYLDGHRVHNAFVNRNYTVKYEVNWKTHEI >Gamma-hemolysin component C|Q5HDD4.1 DIGKGSDIEIIKRTEDKTSNKWGVTQNIQFDFVKDKKYNKDALILKMQGFISSRTTYYNYKKTNHVKAMRWPFQYNIGLKTNDKYVSLINYLPKNKIESTNVSQILGYNIGGNFQSAPSLGGNGSFNYSKSISYTQQNYVSEVEQQNSKSVLWGVKANSFATESGQKSAFDSDLFVGYKPHSKDPRDYFVPDSELPPLVQSGFNPSFIATVSHEKGSSDTSEFEITYGRNMDVTHAIKRSTHYGNSYLDGHRVHNAFVNRNYTVKYEVNWKTHEI >Leucotoxin LukEv|Q2FXB0.2 VTQNVQFDFVKDKKYNKDALIVKMQGFINSRTSFSDVKGSGYELTKRMIWPFQYNIGLTTKDPNVSLINYLPKNKIETTDVGQTLGYNIGGNFQSAPSIGGNGSFN YSKTISYTQKSYVSEVDKQNSKSVKWGVKANEFVTPDGKKSAHDRYLFVQSPNGPTGSAREYFAPDNQLPPLVQSGFNPSFITTLSHEKGSSDTSEFEISYGRNLD >Lysenin Lys|O18423 MSAKAAEGYEQIEVDVVAVWKEGYVYENRGSTSVDQKITITKGMKNVNSETRTVTATHSIGSTISTGDAFEIGSVEVSYSHSHEESQVSMTETEVYESKVIEHTITIPPTSKFTRWQLNADVGGADIEYMYLIDEVTPIGGTQSIPQV ITSRAKIIVGRQIILGKTEIRIKHAERKEYMTVVSRKSWPAATLGHSKLFKFVLYEDWGGFRIKTLNTMYSGYEYAYSSDQGGIYFDQGTDNPKQRWAINKSLPLRHGDVVTFMNKYFTRSGLCYDDGPATNVYCLDKREDKWILEVV

[0042] The following list shows the lumen-facing amino acid positions in an exemplary beta-barrel pore that will be mutated to aromatic residues: >Aerolysin(pdb:5jzt, uniprot:UniProtKB-P09167):222, 224, 226, 228, 230, 232, 236, 234, 238, 240, 242, 244, 246, 252, 253, 254, 256, 258, 260, 262, 264, 266, 268, 272, 270, 274 >Alpha-hemolysin(pdb:7ahl、uniprot:UniProtKB-P09616):109、111、113、115、117、121、119、123、125、127、129、131、135、133、139、137、141、145、143、147 >NetB(pdb:4h56、uniprot:UniProtKB-A8ULG6):114、116、118、120、124、122、128、126、130、132、135、141、143、145、147、149、151 >HlyA(pdb:3o44、uniprot:UniProtKB-P09545):279、281、283、285、289、287、293、291、297、295、299、301、304、306、308、310、312、314、316、318、320 >Hemolytic lectin(pdb:3w9t、uniprot:UniProtKB-Q868M7):305、307、309、311、313、315、317、319、323、321、325、327、329、331、338、340、342、344、346、348、350、352、354、356、358、360、362 >Bacillus protective antigen(pdb:3j9c);278、280、282、284、286、288、290、292、294、296、298、300、302、304、306、308、310、312、313、314、315、319、317、321、325、323、327、329、331、333、335、337、339、341、343、345、347 >Lysenin(pdb:5gaq、uniprot:UniProtKB-P13423):35、37、39、41、45、43、47、49、51、53、55、57、59、63、61、65、68、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104 >Epsilon-toxin B(pdb:6rb9, uniprot:UniProtKB-Q02307):99, 101, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 122 , 126, 128, 130, 132, 136, 137, 141, 139, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 166, 168

[0043] In one aspect, the present invention provides a mutant proteinaceous nanopore comprising a mutant of the aerolysin-like β-PFP (aβ-PFP) subfamily.

[0044] For example, the invention provides an erolysin (Aer) comprising aromatic amino acid substitutions in the water-facing regions of the pore, the regions spanning residues 212-242 and 256-284. In one aspect, the aromatic mutations are in at least region 212-242. For example, one or more basic residues are substituted with aromatic residues to reduce the net positive charge. In certain embodiments, the variant is either W, Y, or F substituted at one or more positions including Q212, G214, D216, T218, R220, D222, A224, N226, S228, T230, T232, G234, S236, K238, T240, or K242. This may include substituting K238 with W, Y, or F. In certain embodiments, the variant is Aer-K238F or Aer-K238W.

[0045] In another embodiment, the aromatic mutations are at least in the region 256-284. For example, the positions corresponding to S256, E258, A260, N262, S264, A266, Q268, G270, S272, T274, S276, S278, S280, R282, and / or T284 are mutated to Trp, Tyr, or Phe. The aromatic substitutions are preferably combined with acidic substitutions, for example, an acidic substitution at position K238. In one embodiment, the mutant erolysin comprises the mutation K238D. In a particular embodiment, the nanopore comprises the erolysin mutants A260F, S264F, Q268F, S272F, and / or the mutation K238D. Preferred mutants include Aer-K238D, Aer-K238D-A260F, Aer-K238D-S264F, Aer-K238D-Q268F and Aer-K238D-S272F. These mutant pores are suitably used for analyte detection, preferably for (unlabeled) peptide detection, at pH≦4.5, e.g. pH 3.8 or pH 3.0. See Example 7 herein below.

[0046] In another embodiment, the present invention provides a mutant proteinaceous nanopore comprising a mutant lysenin or a pore-forming fragment thereof. Although their water-soluble monomeric structures are fundamentally different, the lysenin pore resembles the mushroom-shaped pore complex of the α-hemolysin family of small β-PFTs. In one particular embodiment, the Lys mutant comprises an aromatic substitution at Glu76, e.g., Lys-E76F.

[0047] In another aspect, the present invention provides a mutant proteinaceous nanopore comprising a mutant cytolysin K (CytK) or a pore-forming fragment thereof. CytK is a pore-forming toxin of Bacillus cereus (Hardy et al. FEMS Microbiol Lett. 2001). Although it has been identified as a nanopore with suitable properties for sensing, currently no structure exists to aid in mutagenesis of the CytK nanopore. Those skilled in the art will appreciate that the beta-barrel region of the protein, and thus the putative recognition region for sensing an analyte, can be determined by homology modeling to a suitable similar structure (e.g., alpha-hemolysin from Staphylococcus aureus), and then experimentally confirmed by mutagenesis at the candidate region. An exemplary CytK mutant of the present invention contains an aromatic amino acid substitution in the lumen-facing region of the pore, the region spanning from residue 112 to residue 155. Residues identified as most suitable for aromatic substitution, optionally in combination with one or more acidic substitutions, include one or more (e.g., up to five, preferably up to four, more preferably up to three or two) of the following lumen-facing non-aromatic transmembrane residues: E112 / T114 / T116 / S118 / S120 / Q122 / G124 / S126 / K128 / S130 / T132 / S134 / S137 / E139 / G141 / T143 / Q145 / T147 / S149 / S151 / S153 / K155, and preferably include one or more of E112 / T114 / T116 / S118 / S120 / Q122 / G124 / S126.

[0048] These aromatic mutations are advantageously combined with one or more acidic substitutions of neutral and / or positively charged residues such as K128E or K128D, or any of the lumen-facing residues that can be substituted with an aromatic residue except those that are already negative. Such mutations ideally render the barrel of the pore net negative (if they are not already), thereby altering the electroosmotic flow through the pore.

[0049] In a particular embodiment, the mutant pore or fragment thereof comprises CytK having an aromatic amino acid at position S126 or K128, e.g. comprising a Ser126Tyr, Ser126Trp, Ser126Phe, Lys128Tyr, Lys128Trp, Lys128Phe mutation, preferably a Ser126Phe or Lys128Phe mutation.

[0050] In another particular embodiment, the mutant pore or fragment thereof comprises a CytK mutant having an aromatic amino acid substitution "up" in the barrel, for example at position Ser120, Gln122, or Gly124. See Figure 20. Exemplary CytK mutant nanopores according to the invention include the following mutations: S120W / F / Y+K128D, Q122W / F / Y+K128D, G124W / F / Y+K128D, or S126W / F / Y+K128D.

[0051] As described herein above for alpha-helical and beta-barrel pore-forming proteins, the pores contain one or more mutations that increase the net negative charge or decrease the net positive charge of the pore barrel or channel, with the goal of increasing the flux of cations through the nanopore, particularly under acidic pH conditions (pH<4.5) (Table 3). The mutations K128F or K128D are illustrated (Figures 19 and 20).

[0052] In particular embodiments, as known for the pore-forming proteins described above, the mutant pore comprises a CytK mutant having an aromatic amino acid in the water-facing region of the nanopore, e.g., S126F, and further comprising a mutation that increases the negative charge of the water-facing region, e.g., K128D, which improves the analysis of peptides.

[0053] (Analysis System) A further aspect of the invention relates to an analytical system comprising a mutant proteinaceous nanopore according to the invention. For example, the analytical system comprises a hydrophobic membrane separating a fluid chamber into a cis side and a trans side, the mutant proteinaceous nanopore being inserted into the membrane. A nanopore sensor system may comprise i) a fluid-filled compartment separated by a membrane into a first cis chamber and a second trans chamber, the fluid being an ionic solution, ii) an engineered mutant pore of the invention inserted into the membrane, and iii) electrodes configured to measure an ionic current through the nanopore and, optionally, to create a potential difference across the membrane to facilitate ionic flow through the pore from the first chamber to the second chamber and vice versa.

[0054] The system thereby provides a pore-based sensor. In one particular embodiment, the assay system comprises a mutant alpha-helical pore-forming toxin of the actinoporin family, preferably FraC. In another particular embodiment, the assay system comprises a mutant beta-barrel pore-forming toxin, preferably erolysin-like βPFP or cytolysin K.

[0055] When a system according to the invention is in use, the nanopore is typically located between a first liquid medium and a second liquid medium, at least one of which contains an analyte of interest, and the system is operable to detect a property of the analyte, hi one embodiment, the system is operable to detect a property of the analyte by applying an electric field to the nanopore such that the analyte translocates electrophoretically and / or electroosmotically through the nanopore.

[0056] As exemplified herein below, the system provided herein is particularly suitable for the analysis of proteinaceous materials, preferably peptides, more preferably peptides of about 30 amino acids or less in length. More particularly, the system of the present invention is defined for the capture of peptides of 20, 15, 10, 5, 3, or 2 amino acids or less in length. As exemplified herein below, the mutant nanopore can detect peptides with a wide variation in amino acid composition. Thus, the system is broadly applicable without being limited to a particular structure and / or nature. However, in one embodiment, the peptide comprises at least 50%, preferably at least 60%, more preferably at least 70% hydrophobic charged amino acids. For example, the peptide contains 40% or less, preferably 30% or less, more preferably 25% or less aromatic amino acids (Tyr, Trp, and Phe).

[0057] However, this should in no way be construed as limiting the invention to applications related to peptide analysis. For example, other analytes that can be detected using the system of the invention include (non-proteinaceous) biomarkers, antibiotics or other drugs, DNA, metabolites, and small biological and non-biological molecules. Exemplary analytes include various subclasses of small molecule biomarkers, such as steroids, carbohydrates, amino acids, nucleotides, hormones, fatty acids, vitamins, flavins, protein cofactors, lipids, phenolic compounds, etc. In one embodiment, the analyte of interest is a biopolymer, preferably a biopolymer selected from the group consisting of proteins, polypeptides, and oligopeptides. In one aspect, the analyte is a substance having a mass in the range of 200 to 5000 Da, e.g., in the range of 200-500 Da or in the range of 500 to 1700 Da. See in particular FIG. 22, which illustrates the capture and detection of non-proteinaceous discrete small molecules (flavins, vitamins) by the aromatic engineered pore-forming toxins of the invention. Vitamin B12, also known as cyanocobalamin, is a non-proteinaceous molecule with a molecular weight of 1355 Da.

[0058] The present invention also provides a method for providing a system according to the present invention. Typically, the method comprises the steps of: providing a recombinant monomer of said mutant pore-forming toxin or pore-forming fragment thereof; contacting the monomers with liposomes and / or surfactants to assemble them into oligomers; recovering the oligomer from the liposomes and / or surfactant; and contacting the oligomer with a membrane that may contain sphingomyelin to allow formation of a nanopore; Includes.

[0059] Further embodiments of the present invention relate to means and methods for recombinantly preparing mutant protein pores, including nucleic acid molecules encoding the nanopores of the present invention and expression vectors comprising said nucleic acid molecules, as well as host cells, preferably bacterial host cells, comprising the nucleic acid-containing expression vectors.

[0060] The analytical system of the invention may be integrated into a device, for example in the form of an array of multiple systems. The device may be any conventional device for analyte analysis, such as an array or a chip. A device is provided that comprises multiple analytical systems (sensors) according to the invention. The multiple sensors may be based on the same proteinaceous nanopore (i.e. the same variant) or on separate nanopores inserted into multiple membranes, which may be connected, for example, to multiple electrical circuits to individually address and measure each nanopore sensor. Preferably, a single pore is present in each membrane. The pores may differ in terms of their type, family, mutations, etc. In one embodiment, the device comprises multiple pores that generate different characteristic signals from peptides, which can be compared or combined to improve their identification and characterization.

[0061] The protein nanopore of the present invention may be present within a membrane or, if desired, inserted into a membrane. The protein nanopore is typically asymmetric and may be directionally inserted into the cis and trans compartments of the analytical system by various means known in the art. Typically, the nanopore is inserted from the cis compartment unless otherwise noted in the examples herein.

[0062] Any membrane may be used according to the invention. Suitable membranes are well known in the art. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed from amphiphilic molecules such as phospholipids that have both hydrophilic and hydrophobic / lipophilic properties. The amphiphilic layer may be a monolayer or a bilayer. The membrane is preferably formed from a bilayer of phospholipids. The membrane is preferably formed from lipids or amphiphilic molecules that are chemically stable under low pH conditions, for example, from ether-linked phospholipids. The amphiphilic molecules may be synthetic or of natural origin. Non-natural amphiphiles that form monolayers are known in the art and include, for example, block copolymers (diblock, triblock, tetrablock, etc.) of various polymeric components.

[0063] (Analysis method) The analytical system or device finds its application in various analytical methods. For example, it is advantageously used in single molecule analysis. Thus, a further embodiment of the present invention relates to a method for single molecule analysis, comprising adding a substance or mixture of substances to be analyzed to a chamber of the analytical system(s) provided herein, allowing the substance to contact the (lumen-facing region of) the nanopore, and detecting / characterizing at least one property of the substance (also referred to herein as "analyte" or "analyte of interest"). The substance / analyte can be detected by a change in the current through the nanopore. For example, various properties of the substance can be detected, including but not limited to volume, shape, charge, structure, cross-linking, post-translational modifications (phosphorylation, glycosylation, rhamnosylation, etc.), damage, (D / L) chirality, and sequence.

[0064] Preferably, the method comprises identification and / or sequencing of the substance.The analytical system or method is surprisingly suitable for the analysis of analytes having a mass in the range of 200 to 5000 Da, for example 500-1700 Da.

[0065] The substance, e.g., a peptide analyte, is typically present in any suitable sample. The invention is typically performed on a sample known or suspected to contain the analyte. Alternatively, the invention may be performed on a sample to confirm the identity of an analyte known or expected to be present in the sample. The sample may be a biological sample. The invention may be performed in vitro with a sample obtained or extracted from any organism or microorganism (e.g., an archaeal, prokaryotic or eukaryotic sample). The sample is preferably a fluid sample. The sample may include a patient's body fluid (e.g., urine, lymph, mucus or amniotic fluid, or preferably sweat, saliva, blood, plasma or serum). The sample may be of human origin, or alternatively from another animal, such as a commercially farmed animal, or from a plant origin. The sample may be a non-biological sample. Examples of non-biological samples include surgical fluids, water, such as drinking water, sea water or river water, and clinical laboratory test reagents.

[0066] Samples may be treated (pretreated) before being used in the present invention, for example by various purification means known in the art to isolate a mixture of proteins / peptides / molecules or a target protein / peptide / molecule. These means may include, for example, affinity binding methods such as antibodies or chromatographic methods to isolate and purify specific components of the sample or to remove unwanted background impurities. For protein samples, the proteins contained therein are preferably fragmented into peptides (preferably into defined populations), for example by enzymatic means (e.g. proteases) or other degradative means known in the art. The analytical methods of the present invention may include one or more sample preparation steps, for example a pre-filtration step and / or other modifications as are made for other methods (e.g. mass spectrometry).

[0067] Those skilled in the art will appreciate that many of the sample preparation methods employed in classical mass spectrometry can be used here as well. For example, proteins in the sample may be denatured by physical (e.g., temperature) or chemical (e.g., chaotropic agents, detergents) means prior to processing and nanopore sensing. Alternatively, cross-links such as disulfide bonds can be disrupted to disrupt certain secondary structures. Alternatively, modifications, such as large glycans, may be altered, truncated, or removed prior to nanopore sensing. Alternatively, some of the amino acids in the peptide sample may be modified to change the signal, e.g., cysteines or lysines may be chemically labeled with additional tags to modulate the signal in nanopore sensing to provide further insight into the analyte. In some embodiments of the invention, peptide analytes may be subjected to reactions that alter the N- or C-terminus of the molecule using methods known in the art, for example, to add molecular labels or tags (e.g., to add barcodes to register precursor samples or to facilitate capture and detection in nanopore systems). Such molecular labels / tags may be peptide-based, polynucleotide-based, or composed of other chemical entities.

[0068] In one aspect, the present invention provides a method for single molecule analysis comprising adding a substance or mixture of substances to be analyzed to a chamber of an analytical system as provided herein, allowing the substance to contact the nanopore (a region facing the lumen), and detecting / characterizing at least one property of the substance, wherein the substance is a proteinaceous substance, preferably a peptide, more preferably a peptide of about 30, 20, 15, 10, 5, 3, or 2 amino acids in length or less.

[0069] The method may involve detecting mutations and / or post-translational modifications of the substance, e.g., detecting peptide fragments that differ in terms of a single amino acid residue, degree of phosphorylation, and / or degree of glycosylation. In certain embodiments, the nanopore detects peptides resulting from a protein mixture subjected to denaturing conditions or fragmentation conditions including, for example, protease digestion as typically used in MS analysis. In one aspect, the fragmentation conditions result in positively charged peptide fragments. The method of the invention has the ability to quantify absolute or relative amounts of proteins in an original mixture from peptide spectra.

[0070] In some embodiments of the present invention, the optimal conditions for peptide detection are performed under low pH conditions, preferably below pH 4.5, preferably below pH 4.0. Under physiological pH conditions, naturally occurring peptides have a wide range of charge distributions and net charges (e.g., both net positive and net negative charges) as a result of the highly variable composition of acidic, basic, and natural amino acids. This charge diversity significantly complicates the ability to capture and detect all peptides in a diverse mixture of different peptides in a nanopore sensing system when a fixed applied potential is applied, since not all peptides will experience the same net electrophoretic force. Depending on the polarity of the applied potential and the specific charge composition of each peptide, some peptides will experience a net electrophoretic force entering the nanopore, while other peptides of opposite charge will experience a net electrophoretic force exiting the nanopore. By implementing low pH conditions on the side of the nanopore sensing system containing the complex peptide analyte mixture, preferably on both the cis and trans sides of the membrane, the amino acids of the peptide analytes are rendered protonated. Increased protonation serves both to 1) increase the net positive charge of all peptides within a diversity mixture and 2) produce a more uniform distribution of charge in the peptide mixture. The increased net positive charge allows for improved electrophoretic capture of peptides in nanopore systems that are held under an appropriately polarity applied potential (e.g., when a negative potential is applied to the electrode on the opposite side of the membrane to the peptide analyte).

[0071] Improved uniformity of charge in a peptide mixture is also highly advantageous, since all peptide molecules should experience more similar electrophoretic forces acting on them under an applied potential. This reduces capture efficiency bias between different peptide compositions within a mixture, since electrophoresis is a key component determining the efficiency of capture of analytes into the nanopore. This highly advantageous feature reduces the likelihood that a peptide population with inefficient capture will be overlooked or lost in the background of a peptide population with higher capture efficiency.

[0072] Implementing low pH conditions also alters the charge characteristics of the nanopore in the sensing system by partially protonating some of the water-facing amino acids. Notably, the increased positive charge inside the nanopore channel and inside the lumen recognition region alters the capture and subsequent detection of peptide analytes. The increased positive charge inside the nanopore under low pH conditions can electrostatically repel mostly positively charged peptide analytes, which in turn can decrease the residence time of the peptide inside the nanopore and / or decrease the capture efficiency. This can decrease the ability to detect and characterize certain peptide analytes.

[0073] A variety of different types of measurements can be made on nanopore systems, including but not limited to electrical and optical measurements. Possible electrical measurements include field effect transistor (FET) measurements of local voltage changes, as well as tunneling, impedance, and current measurements. Optical and electrical measurements can be combined to provide additional information (Heron et al. J Am Chem Soc 2009). Optical measurements can employ dye systems that are reporters of ion currents (Heron et al. J Am Chem Soc 2009).

[0074] The method is preferably carried out with a potential applied to the membrane. The applied potential may be a voltage potential. The applied voltage allows electrophoretic and / or electroosmotic flow through the nanopore to facilitate capture and detection of the analyte. Following convention, unless otherwise defined herein for data in the present invention, the active electrode is defined as being in the trans compartment and having the aforementioned polarity of potential applied (e.g., relative to the ground electrode in the cis compartment). Those skilled in the art will appreciate that alternative electrode configurations are known in the art and can be employed, for example, to control electrophoretic and / or electroosmotic analyte capture in nanopore systems via application of an applied potential and / or to measure changes in ionic current or local voltage. The applied potential may be kept constant for a period of time (milliseconds, seconds, minutes, hours). Alternatively, the voltage may be changed in unobtrusive steps to change the sensing conditions and / or obtain different information from the analyte. The voltage may be constantly varying, for example, one skilled in the art will appreciate that waveforms of various patterns (e.g., square wave, triangle wave, sine, etc.) may be employed to control analyte capture and obtain different properties from the analyte. Alternatively, the applied potential may be a chemical potential (e.g., a salt gradient across the membrane). The voltage used is typically +50V to -50V or +100V to -100V. The voltage used is preferably within a range having a lower limit selected from -300mV, -300mV, -150mV, -100mV, -50mV, -20mV, and 0mV, and an upper limit independently selected from +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300mV. More preferably, the voltage used is within the range of +-50mV to +-150mV, more preferably within the range of +-50mV to +-100mV.

[0075] The method is typically carried out with well-known charge carriers present in the aqueous solution in the chamber, such as metal salts or ionic liquids. For example, alkali metal salts, halide salts, such as chloride salts, alkali metal chloride salts, or ionic fluids, or organic salts, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3-methylimidazolium chloride. The salt is preferably potassium chloride (KCl), sodium chloride (NaCl), or lithium chloride (LiCl). The solution may contain a redox salt that mediates electron transfer at a suitable electrode, such as potassium ferrocyanide and potassium ferricyanide or other well-known redox couples. The salt concentration may range from 0.1 to 3M, or up to the saturation point of a given salt type. The salt concentration is preferably 0.1 to 1.5M, most preferably 0.15 to 1.0M. The method is typically carried out in the presence of a buffer. In the exemplary device described above, a buffer is present in the aqueous solution in the chamber. Any buffer may be used in the method of the invention. Typically, the buffer is a Bis-Tris buffer, a citrate buffer, a phosphate buffer, a HEPES buffer, or a Tris-HCl buffer. The method is typically carried out at a pH below 8.0, preferably below pH 4.5, and most preferably below pH 4.0, using a buffer suitable for this range (e.g., a citrate buffer). The method may typically be carried out at a temperature between 0°C and 100°C, preferably between about 20°C and about 40°C.

[0076] Electrical measurements may be made using standard single-channel recording equipment such as those described herein, or alternatively, electrical measurements may be made using multi-channel systems known in the art that are capable of acquiring signals simultaneously from multiple independent nanopore systems (e.g., multiple membranes containing inserted nanopores).

[0077] The method of the present invention may involve measuring multiple characteristics of the electrical signal, most preferably the event blockade resulting from the capture and detection of the analyte. The one or more characteristics are preferably selected from open pore current, average or median current of the event blockade, duration (dwell) time of the event blockade, frequency of the event blockade, number of event blockades, noise in the event blockade, and shape of the event blockade (including step changes). Those skilled in the art will appreciate that various analytical tools can be used to extract advanced information from the event blockade and from other parts of the current signal. For example, edge detection algorithms can be used to segment the event blockade to simplify the data. Alternatively, the raw data may be directly analyzed, with or without the application of filters, for example, using algorithms with long-range memory and sliding window features to extract characteristic metrics.

[0078] The method of the invention may involve determining one, two, three, four, five, or more properties of the analyte from the property metrics of the signal. The one or more properties are preferably selected from the length of the analyte, the volume of the analyte, the mass of the analyte, the shape of the analyte, the charge distribution of the analyte, the identity of the analyte, the sequence of the analyte, any chemical modifications of the analyte. The properties of the analyte may be determined by any number of a wide range of analytical methods known in the art, including, for example, statistical or machine learning methods. These methods may be trained or optimized, for example, by training the system with model analytes, or may be constructed from first principles. For example, the identity of a peptide may be determined by comparison with previously obtained data using training data. Also provided herein is an analytical method for determining the identity of a raw protein from a peptide fingerprint by comparing the spectrum to theoretical or pre-trained data. Those skilled in the art will appreciate that the multi-metric data obtained from each nanopore event can be exploited in high dimensional analysis (e.g., by combinatorial comparison of two, three, four, five, six, or more separate event metrics) to identify different analytes that may not be separable by any single metric. Those skilled in the art will also appreciate that a collection (spectrum) of multiple analyte events can be analyzed as a population collection for discreet populations of analytes in a sample, and that discreet populations may be decomposed (e.g., in multiple dimensions using multiple metrics as axes) and identified using any number of advanced fitting and classification tools. Furthermore, unique data from the populations, e.g., fingerprints, may be used in analytical methods to identify analyte compositions and thus to identify and / or quantify precursor protein(s), for example, for a digested peptide mixture.

[0079] As exemplified later herein, the inventors have found that for certain protein nanopores, such as erolysin and CytK, to enable efficient capture and recognition of peptide analytes under low pH conditions, it is essential or highly advantageous to reduce the net positive charge in the nanopore channel, preferably in the recognition region, most preferably at or near the constriction, preferably in combination with aromatic mutations, for example by introducing acidic residue(s) (Asp / Glu) by substitution close to the aromatic mutation(s) (Phe / Tyr / Trp). Thus, in one embodiment, the pore comprises one or more mutations to Glu and / or Asp residues in the water-facing region. Alternatively, it is understood that the net positive charge can be reduced by substituting basic residue(s) (Arg / Lys / His) with neutral or acidic residue(s) and, optionally, with aromatic residues (e.g., examples included herein such as CytK-K128F, Aer-K238F), which also individually additionally improves peptide capture and discrimination.

[0080] Increased positive charge within the nanopore channel under low pH conditions also alters the ion selectivity of the nanopore. Increased positive charge within the nanopore channel favors increased transport of anionic species and decreased transport of cationic species, which in turn alters the net electroosmotic flow of hydrated ions through the nanopore under an applied potential. Increased anionic electroosmotic flow through the nanopore will act against the electrophoretic forces acting on mostly positively charged peptide analytes under low pH conditions.

[0081] The direction and magnitude of the electroosmotic component for a nanopore system can be determined by ion selectivity measurements known in the art. For example, the ion selectivity of a nanopore can be measured in an in vitro electrophysiological system by measuring the reversal potential under asymmetric salt conditions (e.g., 2M KCl in the trans compartment, 0.5M KCl in the cis compartment). Table 3 below contains the measured reversal potentials and ion selectivities for selected aerolysin and CytK nanopores. FraC ion selectivity at low pH has been previously determined (Huang et al. Nat. Commun. 2019).

[0082] For nanopore sensing systems that are designed to allow analyte capture by electrophoresis (e.g., in systems where a negative potential is applied to an electrode in a compartment opposite a compartment containing a peptide analyte), such as the FraC and CytK nanopore examples contained herein, it is advantageous to ensure that excess electroosmotic forces do not act against the electrophoretic capture of the analyte under the selected sensing conditions. Thus, in some embodiments of the invention (e.g., for the CytK nanopore examples contained herein) where ion selectivity and electroosmotic flow are increased by implementing low pH conditions, it may be advantageous to reduce the net ion selectivity and electroosmotic flow, preferably to a level where electrophoretic forces dominate analyte capture, preferably approaching zero net ion selectivity. Electroosmosis can be reduced by reducing the net charge inside the nanopore channel (e.g., by mutagenesis) (see Table 4). For example, anion ion selectivity bias and the resulting net anionic electroosmotic flow can be reduced by introducing acidic residues by substitution near aromatic mutations. Thus, in one embodiment, the pore comprises one or more mutations to Glu and / or Asp residues in the water-facing region. Alternatively, it will be appreciated that the net positive charge can also be reduced by substituting basic residues with neutral or acidic residue(s) and, optionally, with aromatic residues (e.g., examples included herein such as CytK-K128F, Aer-K238F), which also individually additionally improve peptide capture and discrimination.

[0083] In some proteins, such as FraC, FraE, or FraB, the wild-type pore already contains sufficient negative charges within the nanopore channel / lumen or recognition region facing the water under low pH conditions for optimal ion selectivity and electroosmosis and optimal interaction with mostly positively charged analytes, and does not require mutations to add additional negative charges in spatial combination with the introduced aromatic residue(s). In contrast, removing the acidic residues in the FraC example herein increased the net positivity of the nanopore but dramatically reduced peptide capture and discrimination under electrophoretic conditions.

[0084] In certain embodiments of the invention, efficient peptide analyte capture and detection can be achieved under conditions designed to result in predominantly electroosmotic capture. For example, for the example of the erolysin nanopore system herein, the implementation of low pH conditions increases the net positive charge inside the nanopore channel, which results in increased anion selectivity and a stronger net anion selective nanopore (see Table 3) as well as increased electrostatic repulsion of mostly positively charged analytes. The resulting strong electroosmotic flow through the nanopore (e.g., by a positive applied potential at the trans electrode for a system with mostly positively charged peptides in the cis solution) can be exploited to capture the analyte against the direction of the electrophoretic force acting on the analyte (see the erolysin example herein). For some embodiments of the invention, exploiting the electroosmotic force to capture the analyte can be highly advantageous due to its lower sensitivity to charge composition. This can therefore be highly advantageous for capturing and detecting a diverse composition of unlabeled peptides (e.g., neutral, net positive, net negative). In some embodiments of the invention, the strength of the electroosmotic force acting on the analyte can be further tuned (e.g., by mutagenesis). For example, in some embodiments, it may be useful to reduce the electroosmotic force to increase the duration that the analyte is retained in the nanopore. For example, anion ion selectivity bias resulting from low pH conditions and the resulting anionic electroosmotic flow can be reduced by introducing acidic residues, preferably by substitution near aromatic mutations. Acidic mutation substitutions that reduce the net positive charge will also reduce the electrostatic repulsion of mostly positively charged analytes. Thus, in one embodiment, the pore comprises one or more mutations to Glu and / or Asp residues in the water-facing region. Alternatively, it is understood that the net positive charge can also be reduced by substitution of basic residues with neutral or acidic residues, and optionally by substitution with aromatic residues (e.g., examples included herein such as CytK-K128F, Aer-K238F), which also individually additionally improves peptide capture and discrimination.It will also be appreciated that mutagenesis can be combined with changes to system conditions (e.g., pH, salt type, salt asymmetry) to control the direction and magnitude of the electroosmotic effect, which can be determined experimentally as described above, for example by measuring reverse voltage.

[0085] Thus, described herein are methods that minimize the electrophoretic and electroosmotic components of a nanopore sensing system for the capture and characterization of unlabeled peptides with the aim of discriminating between different peptides. Optimal "characterization parameters" for effective peptide sensing, or sensing of other molecules, can be determined experimentally in a nanopore system by measurements with model or native peptides.

[0086] (Kit of parts) The invention also provides a kit of parts, comprising (i) a mutant proteinaceous nanopore, analytical system and / or device according to the invention, and (ii) an analyte-handling enzyme, for use, e.g., in characterizing an analyte of interest. Preferably, the analyte-handling enzyme is a protein-handling enzyme, such as a protease. Of particular interest is trypsin or other proteases, such as chymotrypsin or Lys-C protease. The use of trypsin may be advantageous since it preferentially cleaves after K / R amino acids, and since most peptides should have a positive charge next to the zwitterionic charge on the peptide, this results in an additional net charge of +1 under the acidic to neutral pH conditions employed in the analysis. Lys-C protease has high activity and specificity for lysine residues, which results in larger peptides and lower sample complexity (i.e. fewer peptides) compared to trypsin. Unlike trypsin, Lys-C protease can cleave lysine before proline, making it ideal for sequential protein digestion followed by trypsin to reduce leakage. When used alone or prior to trypsin digestion, these unique properties of Lys-C protease ensure high digestion efficiency.

[0087] As the skilled person will appreciate, devices or kits comprising the mutant proteinaceous pores described herein find many uses and applications, for example in the field of molecular analysis and identification, including single molecule analysis, preferably identification and / or sequencing of biomolecules or biopolymers, more preferably label-free proteins or protein fingerprints.

[0088] Further embodiments of the present invention are as follows. <1> A proteinaceous nanopore comprising a mutant beta-barrel pore-forming toxin or a pore-forming fragment thereof, the lumen-facing recognition region of said pore-forming protein or fragment thereof comprises one or more substitutions of lumen-facing non-aromatic amino acids with neutral or non-neutral aromatic amino acid residues; Proteinaceous nanopores. <2> a mutant pore-forming toxin comprising one or more substitutions of lumen-facing amino acids to Trp, Tyr, or Phe; The proteinaceous nanopore described in <1>. <3> The beta-barrel pore-forming toxins have an internal diameter (pore system, constriction) in the recognition region in the range of 0.2 to 2.0 nanometers, preferably a minimum internal diameter of 0.5 to 1.5 nanometers. A proteinaceous nanopore according to <1> or <2>. <4> The mutant pores are selected from the group consisting of alpha-hemolysin (SwissProt P09616.2), erolysin (SwissProt P09167), gamma-hemolysin component B (SwissProt P0A075.1), lysenin (SwissProt O18423), epsilon toxin (ETX), hemolytic lectin (LSL, SwissProt Q868M7), cytolysin K (cytK, SwissProt Q937V2), and functional homologs thereof exhibiting at least 80%, preferably at least 85%, more preferably at least 90% sequence identity thereto. A proteinaceous nanopore according to any one of <1> to <3>. <5> Selected from the group consisting of erolysin, lysenin, and cytolysin K (cytK), and functional homologs thereof that show at least 90%, preferably at least 95%, more preferably at least 98% sequence identity thereto. The proteinaceous nanopore described in <4>. <6> erolysin (Aer) containing aromatic amino acid substitutions in the water-facing regions of the pore, the regions spanning residues 212 to 242 and 256 to 284 of erolysin; Preferably, the aromatic mutation is at least within region 212-242; More preferably, the variant is either W, Y, or F substituted at one or more positions including Q212, G214, D216, T218, R220, D222, A224, N226, S228, T230, T232, G234, S236, K238, T240, or K242. A proteinaceous nanopore according to any one of <1> to <5>. <7> mutated CytK, preferably comprising an aromatic amino acid substitution in the lumen-facing region of the pore, the region spanning residues 112 to 155, more preferably one or more (e.g. up to 5, preferably up to 4, more preferably up to 3 or 2) of the lumen-facing non-aromatic transmembrane residues E112 / T114 / T116 / S118 / S120 / Q122 / G124 / S126 / K128 / S130 / T132 / S134 / S137 / E139 / G141 / T143 / Q145 / T147 / S149 / S151 / S153 / K155, such as one or more of E112 / T114 / T116 / S118 / S120 / Q122 / G124 / S126; A proteinaceous nanopore according to any one of <1> to <5>. <8> Preferably, the mutated lysenin (UniProtKB-P13423) or a pore-forming fragment thereof comprises an aromatic substitution at positions 35, 37, 39, 41, 45, 43, 47, 49, 51, 53, 55, 57, 59, 63, 61, 65, 68, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, and / or 104, More preferably, the Lys variant comprises an aromatic substitution at position Glu76, e.g. the mutation Lys-E76F. A proteinaceous nanopore according to any one of <1> to <5>. <9> one or more further mutations that increase the net negative charge of the pore were introduced into amino acids of the recognition region facing the lumen; A proteinaceous nanopore according to any one of <1> to <8>. <10> comprising one or more mutations to Glu and / or Asp residues, The proteinaceous nanopore described in <9>. <11> (i) Aer-K238D, Aer-K238D-A260F, Aer-K238D-S264F, Aer-K238D-Q268F, and Aer-K238D-S272F, (ii) CytK-Ser126Tyr, CytK-Ser126Trp, CytK-Ser126Phe, (ii) CytK-Lys128Tyr, CytK-Lys128Trp, CytK-Lys128Phe, S120W / F / Y+K128D, Q122W / F / Y+K128D, G124W / F / Y+K128D, S126W / F / Y+K128D, and (iii) Lys-E76F, A mutant beta barrel pore or a fragment thereof selected from the group consisting of: A proteinaceous nanopore according to any one of <1> to <10>. <12> An analytical system comprising a hydrophobic membrane that separates a fluid chamber into a cis side and a trans side, the hydrophobic membrane comprising a mutant proteinaceous nanopore described in any one of <1> to <11>. <13> providing a recombinant monomer of said mutant pore-forming toxin or pore-forming fragment thereof; contacting the monomers with liposomes and / or surfactants to assemble them into oligomers; recovering the oligomer from the liposomes and / or surfactant; and contacting the oligomer with a membrane that may contain sphingomyelin to allow formation of a nanopore; Including, A method for providing the system described in <12>. <14> Adding a target analyte to a chamber of the analytical system according to item 12; allowing the analyte to contact the nanopore; and Detecting / characterizing at least one property of said analyte; Including, A method for single molecule analysis, preferably for identification and / or sequencing of an analyte of interest. <15> applying an electric field to the nanopore such that the analyte is electrophoretically and / or electroosmotically trapped in the nanopore. The method described in <14>. <16> The analyte of interest has a mass in the range of 200 to 5000 Da, preferably in the range of 200 to 500 Da or 500 to 1700 Da; The method according to <14> or <15>. <17> The analyte of interest is a biopolymer, preferably a biopolymer selected from the group consisting of proteins, polypeptides, and oligopeptides; A method according to any one of <14> to <16>. <18> The analyte of interest is a proteinaceous material, preferably a peptide, more preferably a peptide of about 30, 20, 15, 10, 5, 3, or 2 amino acids or less in length. The method described in <17>. <19> Detecting mutations and / or post-translational modifications of the analyte, including, for example, detecting peptide fragments that differ in single amino acid residues, amino acid chirality, degree of phosphorylation, and / or degree of glycosylation; The method according to <17> or <18>. <20> The detection is carried out at a pH ≦ 4.5, preferably below pH 4.0; A method according to any one of <14> to <19>. <twenty one> 1. A method for decreasing the translocation rate of a peptide analyte through a beta-barrel transmembrane pore, comprising: (a) increasing the net aromaticity of the lumen of the pore by substituting one or more lumen-facing non-aromatic amino acids with one or more aromatic amino acids; and (b) passing the polypeptide through the pore, increasing the net aromaticity thereby decreasing the translocation rate of the polypeptide through the pore; A method comprising: <twenty two> Step (a) comprises providing a proteinaceous nanopore according to any one of <1> to <11>; The method described in <21>. <twenty three> A device comprising a plurality of the analytical systems according to <12>, preferably each of the analytical systems comprising a distinct pore type. <twenty four> (i) a mutant proteinaceous nanopore according to any one of <1> to <11>, an analysis system according to <12>, or an apparatus according to <23>; and (ii) an analyte-manipulating enzyme, preferably a protease; Including, A kit of parts for characterizing an analyte of interest. <twenty five> Use of the analytical system described in <12>, the device described in <23>, or the kit described in <24> for single molecule analysis, preferably for identification and / or sequencing of biomolecules or biopolymers, more preferably for label-free protein fingerprinting. [Brief description of the drawings]

[0089] [Figure 1A] Actinoporin consensus sequence alignment and wild-type Fragaceatoxin C. (A) Consensus sequence alignment of several known actinoporins. Dots represent amino acids that are the same as the consensus sequence, and other amino acids that differ between pores are represented by their one-letter code. (B) Artistic model of Fragaceatoxin C (PDB: 4TSY) inserted into a lipid bilayer to which a voltage is applied. Some non-conserved positions are shown enlarged. (C) Representative traces of the octameric (T1) and heptameric (T2) forms of wild-type Fragaceatoxin C under an applied potential of -50 mV in 1 M KCl, 50 mM citrate (titrated with bis-trispropane to pH 3.8). Traces were collected at a sampling frequency of 50 kHz with a 10 kHz Bessel filter and a 5 kHz Gaussian filter. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Diagram 2] Alignment between homologues of Fragaceatoxin C. The positions in the homologues that correspond to D10 and G13 in Fragaceatoxin C are highlighted. [Figure 3A]Electrophysiological recordings of (mutated) Fragaceatoxin C with trypsin-digested lysozyme. (A) Representative electrochemical ionic current traces of (mutated) Fragaceatoxin C under an applied potential of -50 mV combined with equal units of trypsin-digested lysozyme added to the cis side. The current traces show representative sections of ionic current data for different pores. The lowest current level is the open pore current (IO) of the pore, and the step-up events are the result of trapped analyte preventing a portion of the ionic current flowing through the nanopore (blockade events, IB). (B)-(D) Representative traces of octameric Fragaceatoxin C (T1, B), heptameric Fragaceatoxin C (T2, C), and the Fragaceatoxin C mutant G13F (D). The raw current data of the traces are overlaid with fit lines derived from application of an edge-detection event detection algorithm. The blocks above the traces are aligned with the event lengths to indicate the duration of the pulse. Traces were collected in 1 M KCl, 50 mM citric acid (titrated with bis-tris propane to pH 3.8) with a 10 kHz Bessel filter and a 5 kHz Gaussian filter at a sampling frequency of 50 kHz. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 4A]Event counting and signal correlation of (mutated) Fragaceatoxin C with trypsin-digested lysozyme. (A)-(D) Observed post-exclusion current (Iex%) spectra from trypsin digestion of lysozyme. (A) Octameric wild-type Fragaceatoxin C (T1), (B) Heptameric wild-type Fragaceatoxin C (T2), (C) Fragaceatoxin C mutant G13F, and (D) Fragaceatoxin C mutant G13N. Traces were collected at -50 mV in 1 M KCl, 50 mM citric acid (titrated with Bis-Trispropane to pH 3.8) at a sampling frequency of 50 kHz with a Bessel filter of 10 kHz and a Gaussian filter of 5 kHz. (E) Squared first derivative Euclidean cosine correlation of residual current spectra of (mutated) Fragaceatoxin C combined with equal units of trypsin-digested lysozyme. Black boxes around multiple mutants represent similar signals. Traces were collected in 1M KCl, 50 mM citric acid (titrated with bis-tris propane to pH 3.8) at a sampling frequency of 50 kHz with a 10 kHz Bessel filter and a 5 kHz Gaussian filter. The external bias was -50 mV except for D10R# and G13H#, which were tested at +50 mV. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Diagram 5]Peptide recognition of (mutated) Fragaceatoxin C. (A) The location of the mutation in the lumen of Fragaceatoxin C (modeled in PDB: 4TSY) is indicated by an arrow. (B) Gaussian fitting to histograms of post-exclusion currents derived from clustered event blockade for capture and detection of angiotensin IV [1], angiotensin III [2], angiotensin I [3], and angiotensinogen [4] recorded under an applied potential of -50 mV. (C) Scatter plot of dwell time versus post-exclusion current % (IEX%) of single molecule peptide event blockade detected by different pore types. Traces were collected in 1 M KCl, 50 mM citrate (titrated with bis-tris propane to pH 3.8) at a sampling frequency of 50 kHz with a Bessel filter of 10 kHz and a Gaussian filter of 5 kHz. [Figure 6] Peptide recognition by (mutated) Fragaceatoxin C. Peptide recognition by additional pore types including heptameric and hexameric Fragaceatoxin C. (Top panel) Residual current fits are shown for leucine-enkephalin (YGGFL) [Leu-enk], angiotensin II(4-8) (YIHPF) [AngII], and kemptide (LRRASLG) [kemptide] at a concentration of 10 μM each, recorded at an applied potential of -70 mV. (Bottom panel) Scatter plot of dwell time versus current after exclusion of single molecule peptide event blockade (IEX%) for different pore types. Traces were collected in 1 M KCl, 50 mM citrate (titrated with bis-tris propane to pH 3.8) at a sampling frequency of 50 kHz with a Bessel filter at 10 kHz and a Gaussian filter at 5 kHz. This figure shows that the aromatic nanopore can identify and discriminate different peptides better than wild-type Fragaceatoxin C. [Figure 7]Electrophysiological setup of an analytical system containing a nanopore. This schematic shows an example of one type of system that can be used with a nanopore sensor for electrical detection of an analyte. Other types of systems, such as an array of nanopore sensors on a microchip, are also suitable. This schematic shows a chamber consisting of two sections made of Delrin separated by a Teflon film containing one 100 μm hole. Both compartments are filled with buffer, and electrodes (e.g., Ag / AgCl electrodes) are connected to each chamber to facilitate electrical detection. A lipid membrane is formed across the hole inside the Teflon film using the Langmuir-Blodgett technique to separate the two compartments. The nanopore is typically added from the cis chamber and allowed to insert into the membrane. The analyte is typically added to the cis chamber for detection. [Figure 8A] Conceptual diagram of bottom-up nanopore-based proteomics. (A) Artistic representation of a protease protein digestion that digests proteins into a mixture of peptide fragments. (B) Artistic representation of the experimental setup where peptides from the resulting peptide fragment mixture are captured and translocated through the FraC nanopore by applying an electric field to the membrane. (C) Artistic representation of the resulting ionic currents for peptides detected from a nanopore-based electrophysiological experiment. (D) Artistic representation of the standard deviation spectra for the resulting residual currents obtained from the analysis of individual single molecule blockades showing distinct clusters of different peptide populations. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 9A]Calibration of post-exclusion current-mass with peptides and spectra obtained from tryptic lysozyme peptides. Characterization of the G13F-FraC-T1 nanopore using synthetic model peptides predicted to arise from intact trypsin-digested chicken lysozyme. (A) Mass of synthetic model peptides (circles) plotted against the average measured post-exclusion current (%) for each peptide when added to the G13F-FraC-T1 nanopore system (obtained from multiple separate experiments with n>3 for each model peptide in separate pores). The dashed line represents a logistic function fit through the data, showing a clear correlation between post-exclusion current and molecular weight, which can be used for characterization of trapped peptides and for predictive purposes when testing unknown peptides. (B) Post-exclusion current spectra recorded from addition of a mixture of all model peptides to the G13F-FraC-T1 pore. Peaks are labeled based on the experimentally determined predictions in A and correspond to the same positions observed in the separate experiments. [Figure 9B] See legend to Figure 9A. [Figure 10A] Nanopore experiments compared to electrospray ionization mass spectrometry. (A) Residual current spectra obtained by nanopore electrophysiology using tryptic digests of G13F-FraC-T1 and chicken lysozyme. (B) Mass spectrometry results from the same tryptic digests as in A but measured by mass spectrometry (ESI-MS). The resulting peptide masses are mapped to the residual currents using the logistic function predictions shown in Figure 9A with a standard deviation of 0.5Iex%. [Figure 10B] See legend to Figure 10A. [Figure 11A]Reproducibility of nanopore protein spectra. Each column shows three independent replicates of sensing proteolytic digests of BSA (A), DHFR (B), and EFP (C). Each replicate was obtained from a separate nanopore experiment with a fresh nanopore, using the same digested sample in each replicate. The left panel shows post-exclusion current histograms at 100% normalized area, obtained from a scatter plot of post-exclusion current vs. dwell time for all event blockades shown in the respective right panel. All measurements were made with G13F-FraC-T1 in 1 M KCl buffered to pH 3.8 with 50 mM citric acid spiked with bis-tris propane, under an applied potential of -70 mV. Recordings were made at 50 kHz with a 10 kHz analog Bessel filter and a 5 kHz digital Gaussian filter. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11C. [Figure 12A] Spectral matching using squared first difference correlation coefficients. (A) Exemplary representative baseline-corrected residual currents from measurements of a peptide fragment mixture from nine trypsin-digested proteins show that unique spectra are observed for each protein type. The right panel shows post-exclusion current histograms at 100% normalized area, derived from scatter plots of post-exclusion currents versus dwell time for all event blockades shown in the respective left panel. (B) Leave-one-out spectral matching of baseline-corrected residual current spectra using Euclidean cosine cross-correlation. [Figure 12B] See legend to Figure 12A. [Figure 13]Detection of phosphorylated proteins. 2.5 μM kemptide (LRRASLG) and 2.5 μM phosphorylated kemptide (LRRA{pS}LG) were added to the cis chamber of a system containing a FraC_G13F nanopore. Measurements were performed in 1 M KCl, 50 mM citrate (buffered with bis-tris propane to pH 3.8). Recordings were made at a frequency of 50 kHz with a 10 kHz low-pass filter and an applied potential of -70 mV. The graph shows that the peptides can be detected as two separate clusters when the residence time is plotted against the residual current (Ires = blocking current / open pore current). [Figure 14] Detection of glycopeptides. 2.5 μM of the unmodified peptide (ANVTLNTAG), 2.5 μM of the peptide with one glycan (ANVT(Glc)LNTAG), and 2.5 μM of the peptide with two glycans (ANVT(Glc)LNTT(Glc)G) were added to the cis chamber of a system containing a FraC_G13F-T1 nanopore (3 M LiCl, 50 mM citrate (buffered with bis-tris propane to pH 3.8), -50 mV, 50 kHz frequency with a 10 kHz low-pass filter). The figure shows the residual current blockade histograms derived from all detected capture events when a mixture containing all three glycosylated peptides was measured. [Figure 15A] Detection of rhamnosylated proteins. 25 μg of unmodified elongation factor P (EF-P) (A) and 75 μg of rhamnosylated EF-P (B) were digested with Lys-C to peptide fragments. After digestion, in a separate experiment, 8 μg of digested protein was added to the cis chamber of a nanopore sensing system containing a FraC_G13F-T1 nanopore for peptide analysis (3 M LiCl, 50 mM citric acid (buffered with bis-trispropane to pH 3.8), -50 mV, 50 kHz frequency with 10 kHz low-pass filter). The rhamnosylated modification is on the SGRNAAVVK peptide fragment. The rhamnosylated modification is clearly identified by a large shift in the residual current (Ires) between the modified peptide [SGR{rham}NAAVVK] and the unmodified peptide [SGRNAAVVK]. [Figure 15B] See legend to Figure 15A. [Figure 16A] Discrimination between single amino acid changes. (Panel A) Detection of two forms of enkephalin with sequences YGGFL and YdAGFdL (where d represents a D-amino acid and all other amino acids are L-amino acids) added to the cis chamber of a G13F-FraC-T1 pore. Measurements were performed in 1 M KCl, 50 mM citrate (with dropwise addition of bis-trispropane (pH 3.8)) solution with a G13F-FraC-T1 pore, sampled at 50 kHz and filtered to 10 kHz, at an applied potential of -100 mV. The figure plots the standard deviation of the noise in the blockage against the blockage magnitude for the recorded event blockages, illustrating that a difference of at least 4 Da can be distinguished as two clear clusters. (Panels B and C) Difference in nanopore signal due to the presence of D-amino acids. A mixture of 10 μM [Ala2]-Leu enkephalin and 10 μM DADLE ([D-Ala2,D-Leu5]-enkephalin) was added to the cis compartment (FraC-G13F, panel B) or to the trans compartment (CytK-K128F, panel C). Measurements were performed in 3 M LiCl, 50 mM citric acid (buffered with bis-tris propane to pH 3.8). Data were recorded at a sampling frequency of 50 kHz and a filter of 10 kHz. [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 17A]Detection of trypsinized lysozyme in erolysin nanopores. Representative electrical ionic current traces from (mutated) erolysin nanopores with 4 μg trypsinized lysozyme added to the cis chamber (+150 mV) of a nanopore sensing system. Current traces show representative sections of ionic current data for select pores including Wt-Aer at pH 7.5 (A), WT-Aer at pH 3.8 (B), Aer-K238F at pH 3.8 (C), and Aer-K238D-S264F at pH 3.0 (D). Open pore current (IO) and exemplary step-wise current block (IB) resulting from peptide capture are marked. Traces were taken in 1 M KCl in cis and trans with 50 mM citrate buffered to approximately pH 3.8 or pH 3.0 with bis-tris propane or 50 mM Tris buffered to pH 7.5 as indicated. [Figure 17B] See legend to Figure 17A. [Figure 17C] See legend to Figure 17A. [Figure 17D] See legend to Figure 17A. [Figure 18A]Detection of trypsinized lysozyme in erolysin nanopores. Structure or schematic of erolysin nanopore with indicated position of modifications and spacing between modifications and scatter plot of residence time versus residence time of individual peptide blockades induced by 4 μg trypsinized lysozyme added to the cis chamber of nanopore sensing systems containing either WT-erolysin at pH 7.5 (A), WT-erolysin at pH 3.8 (B), K238F erolysin at pH 3.8 (C), K238D erolysin at pH 3.0 (D), K238D-A260F erolysin at pH 3.0 (E), K238D-S264F erolysin at pH 3.0 (F), K238D-Q268F erolysin at pH 3.0 (G), K238D-S272F erolysin at pH 3.0 (H). Measurements were performed in 1 M KCl in cis and trans with 50 mM citrate buffered to about pH 3.8 or pH 3.0 with bis-tris propane for low pH experiments or 50 mM Tris buffered to pH 7.5 as indicated. Recordings were made at a frequency of 50 kHz with a low-pass filter of 10 kHz and an applied potential of +150 mV. The figure shows that aromatic mutations, especially in combination with modifications that increase the negative charge of the pore, improve peptide recognition, especially at pH values ​​below 4. (I) Measurements of 4 μg trypsinized lysozyme (final concentration 10 ng / μl) added to the cis compartment of a nanopore system containing Aer-K238W. Measurements were performed in 1 M KCl, 50 mM citrate (buffered with bis-tris propane to pH 3.8) solution under an applied potential of +150 mV. Data were recorded with a sampling frequency of 50 kHz and a filter of 10 kHz. [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 18D] See legend to Figure 18A. [Figure 18E] See legend to Figure 18A. [Figure 18F] See legend to Figure 18A. [Figure 18G] See legend to Figure 18A. [Figure 18H] See legend to Figure 18A. [Figure 18I] See legend to Figure 18A. [Figure 19A] Detection of trypsinized lysozyme in a cytolysin K (CytK) nanopore. Representative electrical ionic current traces from a (mutated) cytolysin K nanopore with 4 μg of trypsinized lysozyme added to the trans chamber (+100 mV) of a nanopore sensing system. The current traces show representative sections of ionic current data for selected pores containing either WT-CytK at pH 3.8 (A), CytK-K128F at pH 3.8 (B), or CytK-S126F-K128D at pH 3.8 (C). The open pore current (IO) and an exemplary step-like current block (IB) resulting from peptide capture are marked. Traces were acquired with 1 M KCL in the chamber and 50 mM citrate buffered to pH 3.8. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 20A]Detection of trypsinized lysozyme in a cytolysin K (CytK) nanopore. (A) Homology model of CytK mapped onto the structure of the alpha-hemolysin nanopore from Staphylococcus aureus (left) and predicted beta strands showing the inward water-facing amino acids for the beta-barrel lumen of the nanopore (right). (B)-(G) Scatter plots of residence time versus residence time of individual peptide blockade induced by 4 μg trypsinized lysozyme added to the trans chamber of systems containing either (B) wild type (WT-CytK) at pH 3.8, (C) K128F CytK nanopore at pH 3.8, (D) S126F-K128D CytK nanopore at pH 3.8, (E) S120F-K128D CytK nanopore at pH 3.0, (F) Q122F-K128D CytK nanopore at pH 3.0, (G) G124F-K128D CytK nanopore at pH 3.0, or (H) two peptides (10 μM Lys4 and 10 μM Lys7) added to the trans compartment of a system containing a K128W CytK nanopore. Measurements were performed in 1 M KCl, 50 mM citric acid (buffered with bis-tris propane to pH 7.5) solution at an applied potential of +100 mV. Data were recorded with a sampling frequency of 50 kHz and a filter of 10 kHz. Schematic positions of the substituted amino acids are shown to the left of each panel. Recordings were made at an applied potential of +100 mV at a frequency of 50 kHz with a low-pass filter of 10 kHz. This figure shows that aromatic mutations, especially in combination with modifications that increase the negative charge of the pore, allow recognition of peptides, especially at pH values ​​below 4. [Figure 20B] See legend to Figure 20A. [Figure 20C] See legend to Figure 20A. [Figure 20D] See legend to Figure 20A. [Figure 20E] See legend to Figure 20A. [Figure 20F] See legend to Figure 20A. [Figure 20G] See legend to Figure 20A. [Figure 20H] See legend to Figure 20A. [Figure 21A] Detection of Lys-C digested lysozyme in lysenin nanopores. Measurements of 0.5 μg of Lys-C digested lysozyme (final concentration 1.25 ng / μl) added to the trans compartment of systems containing either (A) wild-type (WT-Lys) or (B) mutant Lys-E76F nanopores. Measurements were performed in 1 M KCl, 50 mM citric acid (buffered with bis-tris propane to pH 3.8) solution under an applied potential of -70 mV. Data were recorded at a sampling frequency of 50 kHz and a filter of 10 kHz. [Figure 21B] See legend to Figure 21A. [Figure 22A] Detection of non-proteinaceous small molecules. Analytes were added to the cis chamber (thioflavin, 2.0 μM) or trans chamber (vitamin B12, 10.0 μM) of systems containing heptameric (A) wild-type FraC or (B, C) mutant FraC_G13F nanopores (thioflavin) or octameric (D) wild-type FraC or (E, F) mutant FraC_G13F nanopores (vitamin B12). Measurements in 1 M KCl, 50 mM Tris-HCl pH 7.5. Recordings were made at an applied potential of -70 mV (vitamin B12) or -50 mV (thioflavin) at a frequency of 50 kHz with a 10 kHz low-pass filter. The graph shows that the molecules can be detected as separate clusters when the residence time is plotted against the residual current (Ires = blocking current / open pore current). [Figure 22B] See legend to Figure 22A. [Figure 22C] See legend to Figure 22A. [Figure 22DE] See legend to Figure 22A. [Figure 22F] See legend to Figure 22A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] (Experimental Section) Materials and Methods (chemical substance) Sphingomyelin (porcine brain, ≥99%, CAS number 383907-91-3) and diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, ≥99%, CAS number 207131-40-6) were obtained from Avanti Polar Lipids. Ni-NTA resin was obtained from Qiagen. Lysozyme (albumin-free for trypsin digestion, CAS number 12650-88-3), glucose (≧99%, CAS number 50-99-7), sodium chloride (≧99.5%, CAS number 7647-14-5), potassium chloride (≧99%, CAS number 7447-40-7), dithiothreitol (DTT, ≧99.0%, 3483-12-3), Trizma(R) hydrochloride (≧99%, CAS number 1185-53-1), Trizma(R) base (≧99.9%, CAS number 77-86-1), imidazole (≧99%, CAS number 288-32-4), n-dodecyl β-D-maltoside (DDM, ≥99%, CAS no. 69227-93-6), hydrochloric acid (1M, CAS no. 7647-01-0), urea (≥99.5%, CAS no. 57-13-6), magnesium chloride (≥98.5%, CAS no. 7786-30-3), LB medium (Luria / Miller), agar, and 2xYT medium were obtained from Carl Roth. Ampicillin sodium salt (CAS no. 69-52-3), isopropyl β-D-1-thiogalactopyranoside (IPTG, ≥99%, CAS no. 367-93-1), ethanol (≥99.8%, CAS no. 64-17-5), and all enzymes were received from Fisher Scientific. Hen egg white lysozyme (for lysis, CAS no. 12650-88-3), N,N-dimethyldodecylamine N-oxide (LDAO, ≥99.0%, CAS no. 1643-20-5), pentane (≥99%, CAS no. 109-66-0), iodoacetamide (IAA, ≥99%, CAS no. 144-48-9), bis-trispropane (≥99.0%, CAS no. 64431-96-5) were purchased from Sigma-Aldrich. n-Hexadecane (99%, CAS no. 544-76-3), and citric acid (99.6%, CAS no. 77-92-9) were purchased from Acros.Trypsin (bovine pancreas, CAS number 9002-07-7) was obtained from Alfa Aesar.

[0091] (Expression and purification of Fragaceatoxin C (FraC) monomer) The His6-tagged FraC plasmid-containing pT7-SC1 vector was electrochemically inserted into E. coli BL21(DE3) cells and grown overnight at 37°C on LB agar plates (supplemented with 100 mg / l ampicillin and 1% glucose). Colonies were used to inoculate 200 ml of 2xYT medium (supplemented with 100 mg / l ampicillin) and the optical density at 600 nm (OD 600The cells were grown at 37°C until the chromatin ratio (K) reached 0.6, after which expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and allowed to continue growing overnight at 21°C. Cell pellets were collected by centrifugation (6,000g, 20 min, 4°C) and stored at -80°C for at least 1 h. The pellets were resuspended in 10 ml of lysis buffer per 50 ml of culture, which consisted of 150 mM NaCl, 15 mM Tris base solution (pH 7.5) supplemented with 1 mM MgCl2, 2 M urea, 20 mM imidazole, 0.2 mg / ml lysozyme, and 0.2 units / ml DNase. The solution was mixed for 1 h at room temperature (21°C) using a rotary mixer at 15 RPM. Cells were disrupted by sonication using a Branson Sonifier 450 applying 30 sweeps (duty cycle 30%, output control 3) three times. The lysate was centrifuged at 6000 g for 20 minutes at 4°C. The supernatant was incubated with 100 μL of resuspended Ni-NTA resin (resuspended in 150 mM NaCl, 15 mM Tris base (pH 7.5) with 20 mM imidazole) for 1 hour under constant rotation speed (15 RPM). The solution was loaded onto a pre-washed Micro Bio-Spin column (Bio-Rad). The Ni-NTA beads were extensively washed with 20 ml of WB (containing 150 mM NaCl, 15 mM Tris base (pH 7.5) with 20 mM imidazole). The column was inserted into a microfuge tube and spun down using a centrifuge (13,300 g, 1 min) to remove residual wash buffer. 150 μl of 150 mM NaCl, 15 mM Tris base solution (pH 7.5, with 300 mM imidazole (EB)) was added and left to incubate for 5 min before elution. This step was repeated four times to obtain four fractions containing FraC monomer. The presence and purity of FraC monomer was estimated using SDS-PAGE. Pure fractions were pooled and stored at 4°C.The concentration of FraC monomer was estimated using the elution buffer Nano Drop 2000 UV-Vis Spectrophotometer (Thermo Scientific) as a blank.

[0092] (Sphingomyelin-DPhPC liposome preparation) 25 mg of sphingomyelin (brain, porcine) was mixed with 25 mg of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and dissolved in 4 ml of pentane (containing 0.5% v / v ethanol). The lipid mixture was evaporated by swirling inside a round-bottom flask with the application of a hot air stream to produce a thin lipid film on the surface of the flask. The film was reconstituted in 10 ml of Sdex buffer (150 mM NaCl, 15 mM Tris, pH 7.5) using a bath sonicator. The liposome solution (5 mg / ml) was frozen and stored at -20°C.

[0093] (Fragaceatoxin C oligomerization) Liposomes were thawed and added to FraC monomer at a lipid:protein mass ratio of 10:1. The mixture was incubated at 37°C for 30 min, after which N,N-dimethyldodecylamine N-oxide (LDAO) was added to a final concentration of 0.6 v / v% to dissolve the liposomes. The solution was diluted 10-fold with 150 mM NaCl (containing 15 mM Tris, pH 7.5, and 0.02 v / v% n-dodecyl β-D-maltoside (DDM)). The diluted solution was combined with 100 μl of Ni-NTA prewashed with WB2 (containing 150 mM NaCl, 15 mM Tris base, pH 7.5, 20 mM imidazole, and 0.02 v / v% DDM). The mixture was left to incubate for 30 min with constant mixing at a constant rotation speed (15 RPM). The solution was loaded onto a Micro Bio-Spin column (Bio-Rad) that had been prewashed with 500 μl of WB2. The Ni-NTA beads were washed extensively with 10 ml of WB2. The column was spun in a microfuge using a centrifuge (13,300 g, 1 min) to remove residual wash buffer. 150 μl of elution buffer (containing 150 mM NaCl, 15 mM Tris base, 1 M imidazole, and 0.02% v / v DDM) was added to the column and left to stand for 10 min before eluting into a clean microfuge tube by centrifugation (13,300 g, 2 min). Multimers are stable at 4°C for several months and can be frozen at -80°C for long-term storage.

[0094] (Construction of Fragaceatoxin C mutants) Fragaceatoxin C mutant DNA was analyzed by MEGAWHOP method. 6A megaprimer was constructed using a forward primer synthesized by Integrated DNA Technologies and a T7 reverse primer (5'-GCTAGTTATTGCTCAGCGG-3'). Six reactions were performed per mutation (to receive enough DNA for the secondary PCR) using 25 μl of REDTag® ReadyMix™ PCR Reaction Mix (Sigma-Aldrich) combined with 22 μl of PCR grade water (Sigma-Aldrich), 1 μl of each forward and reverse primer, and 1 μl of His6-tagged Fragaceatoxin C template DNA. The PCR procedure consisted of a 90 s denaturation step at 95°C followed by 30 cycles of denaturation at 95°C (15 s), annealing at 55°C (15 s), and extension at 72°C (120 s). The six PCR reactions were pooled and purified using a GeneJET PCR Purification Kit (Thermo Scientific). For the secondary PCR, 10 μl of 5× Phire Buffer (Thermo Scientific) was combined with 1 μl of template DNA, 1 μl dNTPs (10 mM), 2 μl of Mega Primer (primary PCR), 35 μl of PCR grade water (Sigma-Aldrich), and 1 μl of Phire II Hot Start DNA Polymerase (Thermo Scientific). The PCR procedure consisted of an initial pre-denaturation step at 98°C (30 s) followed by 25 cycles of denaturation at 98°C (5 s) and extension at 72°C (90 s). 5.7 μl of 5× FD green buffer (Thermo Scientific) and 1 μl of Dpn1 enzyme (Thermo Scientific) were added to the PCR mixture and left to digest at 37°C for 1–3 h. 0.5 μl of the digestion product was electrochemically introduced into 50 μl of E. cloni 10G® (Lucigen) competent cells and grown on LB agar plates (containing 100 mg / l ampicillin and 1% glucose).Single colonies were enriched using the GeneJET Plasmid Miniprep Kit (Thermo Scientific) and sequences were confirmed using Macrogen Europe's sequencing services.

[0095] (Amino acid sequence of His6-tagged wild-type Fragaceatoxin C) MASADVAGAVIDGAGLGFDVLKTVLEALGNVKRKIAVGIDNESGKTWTAMNTYFRSGTSDIVLPHKVAHGKALLYNGQKNRGPVATGVVGVIAYSMSDGNTLAVLFSVPYDYNWYSNWWNVRVYKGQKRADQRMYEELYYHRSPFRGDNGWHSRGLGYGLKSRGFMNSSGHAILEIHVTKAGSAHHHHHH

[0096] (Non-specific lysozyme digestion) Lysozyme (Carl Roth, from chicken egg white, albumin-free) was dissolved in 8 M urea (with 15 mM Tris, pH 9.5) at a final concentration of 20 mg / ml and left to denature at 95°C for 5 min. 200 μl of the denatured lysozyme solution was incubated with 20 mM dithiothreitol (DTT) at 37°C for 30 min to reduce cysteine ​​residues. Iodoacetamide (IAA) was added to the mixture at a final concentration of 45 mM to react with the reduced cysteines and incubated at room temperature in the dark for 30 min. The mixture was diluted 5-fold with 100 mM Tris, pH 8.5, and trypsin (Alfa Aesar™ Trypsin, bovine pancreas) was added at a ratio of 1:50 (trypsin:protein). The mixture was left to digest overnight (approximately 18 hours) at 37°C. The next day, the final mixture was denatured at 95° C. for 10 min to denature and inactivate any remaining trypsin, and the pH was lowered (to approximately pH 4) by adding HCl. The mixture was then frozen at −20° C. until use.

[0097] (Planar lipid bilayer electrophysiological recordings) The electrophysiology chamber consisted of two compartments separated by a 25 μm thick Teflon (Goodfellow Cambridge Ltd) membrane. The Teflon membrane contained one aperture of approximately 100-200 μm diameter. A lipid membrane was formed by first applying 5 μl of a 5% solution of hexadecane (Sigma Aldrich) in pentane (Sigma Aldrich) to the Teflon membrane near the aperture. The pentane was left to dry and 400 μl of buffer (1 M KCl, 50 mM citric acid (dropped with bis-tris propane to pH 3.8)) was added to both sides. 20 μl of a 6.25 mg / ml solution of DPhPC dissolved in pentane was added on top of the buffer on each side of the chamber. The chamber was left to dry for approximately 2 min to allow evaporation of the pentane. A silver / silver chloride electrode was attached to each compartment. The cis compartment was connected to the ground electrode and the trans compartment was connected to the working electrode. Planar lipid bilayers were developed by Maglia et al. 7 The nanopores were fabricated using the Langmuir-Blodgett technique as described. The integrity of the FraC nanopores was determined by the asymmetry of the current-voltage relationship. A 2-min baseline was recorded for each of the recording pores. Analytes were added to the cis compartment of the chamber.

[0098] (Data Recording) The ionic current recording was performed according to the previous study. 1、2 Similar to the above, the data were acquired using an Axopatch 200B (Axon Instruments) coupled to a Digidata 1550B A / D converter (Axon instruments). The sampling frequency was set to 50 kHz for analyte recording, and the analog Bessel filter was set to 10 kHz. Data were recorded using a Clampex 10 (Molecular Devices).

[0099] (Standard Data Analysis and Event Detection) Many well-known means of analyzing stepwise current blockade measured from nanopore electrophysiology are known in the art, and a variety of methods can be employed for the types of events we observed to extract useful data, including but not limited to, block magnitude, block duration, block shape, block noise, and other sub-characteristics of the block (such as mini-steps).

[0100] For basic data analysis, a custom Python script was used to analyze the raw electrical data. The open pore current (I O ) and standard deviation were determined by calculating the average current of three independent measurements, bootstrapped with 100 replicates of a 10 second snippet for each measurement. For event detection, the baseline current and standard error of the recorded trace were determined from the total current histogram of a blank nanopore measurement containing no analyte. The value for the baseline is then used to determine events when analyte is added. All data points above the baseline current and standard error, separated by at least two sampling periods, are detected as events. The current (I ex %) was calculated as the difference between the open pore current Io and the blocking current Ib divided by the open pore current Io (Iex%=[Io-Ib] / Io).

[0101] (Unbiased Event Detection) To improve the analysis, an unbiased event detector method was employed. We found that short-lived events with dwell times close to the sampling frequency tend to form spikes or Gaussian profiles due to undersampling and filtering effects, while longer-lived events follow a flat-top shape. Therefore, we introduced a parameter describing the shape of the current blockade to unbiasedly compare the performance of mutant pores. We assumed that the profile of the ionic current blockade can be described by a generalized flat-top normal distribution function (gNDF, Eq. 3). Due to the non-polynomial nature of the function, each observation block was fitted to Eq. 1 using the least squares method.

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[0102] (Spectral Matching) Because some of the residual current spectra we acquired are expected to contain random events induced by factors other than the analyte (gating), to reduce the baseline slope and maintain high sensitivity, we used the squared first derivative Euclidean cosine correlation (Equation 5): 9 This comparison is sensitive to the positions of the peaks observed in the spectra, but is not sensitive to shifting baselines.

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[0103] We performed hierarchical clustering on the resulting correlation coefficients to determine which spectra were most similar, using the Ward distance as implemented in SciPy version 1.4.1. 10 Essentially, this metric orders the data so that the variance between neighbors is minimal, thus constructing a map of similar spectra.

[0104] (Example 1: Fragaceatoxin C Mutant Screening) (FraC mutation) The sequence of WtFraC from the sea anemone Actinia fragacea was aligned with other actinoporins to identify sequence homologies (Fig. 1A). A number of generally non-conserved positions that were likely to be amenable to mutations were identified (Fig. 1B), including D10, G13, G15, D17, and K20. These positions were engineered to introduce different mutations to improve the pore's ability to detect and discriminate between different peptides.

[0105] Position D10 was mutated to arginine (R) and glycine (G) to test for changes in electroosmotic capture of analytes. Each of the positions near the recognition site (G13) was modified to basic (K, R, or H) or acidic (D or E) residues as well as amino acids with neutral (G or Q) or aromatic (W, Y, and F) groups. In FraC, a glycine residue is located at residue 15, whereas the most common amino acid in other actinoporins is threonine. The mutation G15T was introduced to test whether increased hydrophobicity facing outward into the membrane would stabilize and improve the behavior of the FraC nanopore.

[0106] Sequence alignment (Figure 1A) showed a common pair of opposite charges at positions 20 / 21, therefore two mutations with similar properties were constructed: T21D and the double mutation K20D / T21K. Altering the charge at position 20 by introducing a glutamic acid (K20D) was also tested.

[0107] (multimer formation) WtFraC can exist in three multimeric forms, predicted to correspond to octamers, heptamers, and hexamers. We tested the octameric pore (or type I pore, T1), the heptameric pore (or type II pore, T2), and the hexameric pore (or type III pore, T3).

[0108] The octamer was identified as the nanopore with the highest conductance. Some mutations significantly reduced the open pore current (I0) relative to WtFraC-T1 (95 ± 1 pA), and some even reduced I0 to a similar extent to WtFraC-T2 (47 ± 3 pA). Notably, reduced I0 was observed when residues with larger volume were introduced, especially when aromatic residues (W / F / Y) were introduced at position 13 (I0 = 64 ± 8 pA, 77 ± 4 pA, and 82 ± 3 pA, respectively), suggesting that a smaller recognition region can be achieved that may be advantageous for detecting smaller analytes such as small peptides. The introduction of a threonine residue at position 15 increased the open pore current I0 flowing through the pore (100 ± 3 pA), a property that is useful for nanopore analysis, since the increased current is generally more sensitive to changes due to analyte binding.

[0109] (Peptide mixture) To ensure a fair comparison between the pores, a mixture of peptides was generated from a non-specific tryptic digest of lysozyme (chicken). We used trypsin or other proteases such as chymotrypsin or Lys-C protease. The use of trypsin may be advantageous since it preferentially cleaves after K / R amino acids and since the majority of peptides should have a positive charge next to the zwitterionic charge on the peptide, this results in a net charge of +1 under the low pH conditions employed. All pores were tested with the same proteolytic mixture.

[0110] (Blocking Event Analysis) Events due to nanopore current blockade were analyzed by fitting to a flat-top shape using the least-squares Levenberg-Marquardt method and a generalized flat-top normal distribution function. The fit yields a β value that allows events to be classified as either spikes with β<1, normal distribution with β=1, or flat-top distribution with β>1. All events with β>1 were used for further analysis. For each blockade, a number of characteristic metrics are extracted. These are the post-exclusion current (I ex%) (percentage of current blocked during a translocation event relative to the open pore current (I ex %=[Io-Ib] / Io), the duration of the interruption (called the dwell time), the shape of the interruption, noise in the interruption current, etc.

[0111] (Experimental conditions) Peptide capture and discrimination in the FraC nanopore was studied under a wide range of conditions. Peptide capture was observed over a wide range of voltages, e.g., from as low as +-10mV to +-200mV. The majority of sensing was performed at +-50mV to +-100mV, as this voltage was generally found to be optimal for peptide capture and characterization. Peptide detection was observed over a wide range of salt types, concentrations, and asymmetries across the membrane, all of which may combine with the pore type to alter the capture and detection properties of the system. The preferred salt conditions are about 1M KCl (or NaCl or LiCl) at pH<4.5 (e.g., pH 3.8).

[0112] (result) Wild-type FraC-T1 and wild-type FraC-T2 reacted at pH 3.8 for approximately 10–13 events·s -1The peptide was captured at a frequency of 0.01 and 0.02. When the charge at position 10 or 17 was removed (D10G-FraC-T1 or D17Q-FraC-T1 mutations), the capture frequency decreased by 3.4 and 7.2 times relative to WtFraC-T. It has been shown that electroosmotic flow (EOF) is a critical component for efficient capture of peptides in nanopores and can act together or against the electrophoretic force acting on the analyte. It has also been shown that the strength and manner of EOF depends on the charge at the constriction (Table 4). At low pH, which protonates the water-facing residues and generally increases the net positive charge inside the pore (increasing anion selectivity), we found that the intrinsically negative residues in wild-type FraC result in near-zero net ion selectivity (Table 4) and thus near-zero net electroosmotic flow across the nanopore (versus the extremely high cation selectivity at pH 7.5). Removing the negative charge at position 10 further increases anion selectivity at low pH, resulting in a stronger EOF component acting against the capture of mostly positively charged peptides, thus resulting in lower capture efficiency. Furthermore, we showed that pores with positively charged constrictions, such as D10R-FraC-T1, exhibit a destabilized baseline current under an applied bias of -50 mV, but a stable baseline current under an applied bias of +50 mV, thereby behaving oppositely to WtFraC. However, the D10R mutation showed good capture of peptide analytes in the cis chamber at positive applied voltages (similar capture to that of native D10 in WT at negative voltages). The increased capture under this polarity is the result of a stronger net anion-selective electroosmotic bias (flowing from cis to trans) caused by the positive mutation, which dominates over the weaker electrophoretic forces acting against peptide capture at this polarity.

[0113] Removing the charge on residue K20 by substituting it with glutamine increased the capture frequency by 1.4 times relative to wild-type FraC. Substituting the charge on K20 by introducing an aspartic acid decreased the capture frequency by 1.5 times relative to wild-type FraC. These relatively small changes illustrate how the EOF can be finely tuned to control the capture frequency and / or event residence time.

[0114] Interestingly, we found that the introduction of an aromatic residue (Y, F, or W) increased the capture frequency by approximately four times relative to wild-type FraC-T1 and FraC-T2 pores for all three mutations.

[0115] Furthermore, we found that aromatic mutations also increased the duration of peptide event blockade in nanopores. Most of the blockades in pores with an aromatic residue on G13 were flat-topped with relatively long residence times (e.g., Fig. 3D). Indeed, the median residence times of events in these aromatic pores increased to 0.32 ± 0.06 ms, 0.18 ± 0.03 ms, and 0.22 ± 0.06 ms for G13Y-FraC-T1, G13F-FraC-T1, and G13W-FraC-T1, respectively, compared to 0.09 ± 0.06 ms for WtFraC-T1 and 0.10 ± 0.01 ms for WtFraC-T2.

[0116] To compare the different mutants, we filtered out all events with β>1 (Gaussian filter at 5 kHz, see Methods) and then calculated the post-exclusion current (I ex We constructed post-exclusion current spectra (four pores are shown in Figure 4A-D) by creating histograms of Iex%. We normalized the spectra and observed distinct patterns for WtFraC-T1 and T2 (Figure 4A / B) and a clear Gaussian-shaped peak for G13F-FraC-T1 (Figure 4C). Most of the peaks for G13N-FraC-T1 were at low Iex% (Figure 4D), reflecting faster translocation of the peptide across the nanopore. We found that the peaks were mostly at low Iex% (Figure 4E), reflecting faster translocation of the peptide across the nanopore. ex ​With post-exclusion current spectra that were %<95%, the post-exclusion current spectra were compared using a point-to-point spectral matching algorithm.

[0117] Example 2: Characterization of Fragaceatoxin C Mutants We selected five mutants for further characterization. Specifically, they had a slightly increased I O We chose G15T-FraC-T1 because it was comparable to WtFraC-T1 with a β-amino acid sequence, K20D-FraC-T1 because it had a higher SNR and better capture frequency, and aromatic mutations at G13 (G13Y / F / W-FraC-T1) because of their increased residence time and capture frequency compared to WtFraC-T2. For the characterization of these pores, we used mixtures of well-defined peptides (i.e., mixtures were prepared by adding the individual peptides in equimolar concentrations). The mixture consisted of four peptides, angiotensinogen (DRVYIHPFHLVIHN, 1758.9 Da, charge = +3.96), angiotensin 1 (DRVYIHPFHL, 1296.5 Da, charge = +2.96), angiotensin 3 (RVYIHPF, 931.1 Da, charge = +2.16), and angiotensin 4 (VYIHPF, 774.9 Da, charge = +1.16), abbreviated as angiotensinogen, Ang-I, Ang-III, and Ang-IV, respectively. The resolution of the nanopore was quantified by measuring the separation between the peptides using the difference between the peak centers and their average standard deviations as shown in Equations 1 and 2.

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[0118] Figure 5 shows a comparison between WtFraC-T2 and selected engineered FraC pores. The aromatic pore G13F / Y / W showed a significant improvement in the ability to discriminate between peptides. The aromatic pore shows significantly longer blockage event durations relative to WtFraC-T2. Longer duration events (with more raw data points at a given acquisition frequency) allow the magnitude of post-exclusion currents for individual event blockages to be determined with greater precision. This could at least partially explain the reduced spread in post-exclusion currents observed for each peptide cluster for the aromatic pore.

[0119] Example 3: Peptide analysis with T2 nanopore We tested the resolution of the aromatic heptameric (T2) nanopore and compared it with the hexameric (T3) WtFraC-T3 and WtFraC-T2 nanopores using leucine-enkephalin (Leu-enk, YGGFL, 555.6 Da), angiotensin II (4-8) {Ang-II (4-8), YIHPF, 675.8 Da}, and Kemptide (LRRASLG, 771.9 Da). For WtFraC-T3, we used a version of FraC with two altered membrane-contacting modifications, W112S-W116S, that allowed the formation of a hexameric nanopore. WtFraC-T2 showed no blockage (Figure 5), suggesting that the majority of peptides translocated through the pore were not detected. FraC-T3 and G13W-FraC-T2 showed leucine-enkephalin blockade and angiotensin II(4-8) blockade, whereas Kemptide blockade was not observed. This is surprising considering that Kemptide has a larger molecular weight than leucine-enkephalin and angiotensin II(4-8). Perhaps the two arginine residues in Kemptide induce a faster electrophoretic migration across these nanopores. Interestingly, we found that Kemptide induces a blockade to G13F-FraC-T2, indicating that this aromatic modification is paramount for detecting this class of peptides. A possible explanation is that the cation-π interaction between the ring of the phenylalanine residue and the two arginine residues is crucial to reduce the residence time of the peptide inside the nanopore. Table 3: Differences between peptide peak centers (ΔI ex %) and observed baseline separation (R s ) [Table 3]

[0120] Example 4: Analytical system including nanopores A nanopore is a nanometer-sized opening in a thin membrane through which analytes that migrate are detected. An exemplary analytical system of the present invention is depicted diagrammatically in FIG. 7. It consists of two chambers filled with an electrolyte solution and separated by a membrane. The chambers are connected through a nanopore formed in the membrane. When a potential is applied to the membrane through electrodes in either chamber, ions will migrate through the pore, generating a small ionic current that is amplified and measured. When an analyte enters the nanopore, the ionic current flowing through the open pore is altered due to the movement of ions by the analyte, typically resulting in a decrease in the ionic current (blocking event). The characteristics of the current block (e.g., magnitude, duration, shape, noise, etc.) depend on the nature of the captured analyte and the conditions (e.g., applied potential, buffer conditions, temperature, etc.) and can be used to provide information about the nature of the captured analyte.

[0121] (Example 5: FraC nanopore as a next-generation single-molecule protein analyzer) This example shows that an engineered sub-nanometer biological nanopore of mutant Fragaceatoxin C (FraC) has the ability to identify multiple trypsin-digested proteins. Calibration via several synthetic peptides allowed us to find the relationship between the residual current spectrum and the mass spectrum, which allowed the identification of the proteins. Figure 8 illustrates the conceptual diagram of such a "bottom-up" nanopore-based proteomics.

[0122] (Protein Digestion) 100 μg of protein stock was taken and the volume was adjusted to 50 μl with 20 mM Tris buffer (pH 7.5). Dithiothreitol (DTT) was added to a final concentration of 20 mM to reduce any disulfide bonds. The sample was incubated at 37°C for 15 min, followed by a denaturation step at 95°C for 15 min. Then, 20 mM iodoacetamide (IAA) was added and the sample was left to incubate at room temperature in the dark for 15 min to alkylate the reduced cysteine ​​residues. Finally, the total volume was adjusted to 100 μl with 100 mM Tris buffer (pH 8.5).

[0123] For trypsin digestion, we used a kit containing proteomics-grade trypsin purchased from Sigma-Aldrich. 50 μl of sample (containing 50 μg protein) was added to 1 μg mass spectrometry-grade trypsin (enzyme:protein 1:50), and the sample was then incubated overnight at 37°C. Finally, large (>2000 Da) peptides were removed using a centrifugal filter (Amicon) with a molecular weight cutoff of 3000 Da. Filtered samples were stored at -20°C before use.

[0124] Trypsin is a sequence-dependent protease that cleaves primarily at the carboxyl side chains of arginine (R) and lysine (K) residues unless these residues are followed by proline (P). Thus, trypsinization of a given protein results in a peptide mixture that contains a specific population of peptide fragments resulting from specific cleavages, which also contain some levels of other peptide fragments resulting from incomplete digestion or off-target cleavage.

[0125] (Expression of proteins for trypsin digestion) For nanopore sensor testing, five model proteins were expressed and / or purified: DHFR (dihydrofolate reductase), BSA (bovine serum albumin, Sigma-Aldrich), PAN (PAN unfoldase), ThpA (thiamine binding protein), and HMWI_Act (C-terminal fragment of Haemophilus influenzae high-molecular weight adhesin protein, residues 1205-1536).

[0126] (DHFR / PAN / ThpA / HMWI_Act protein expression) All proteins were expressed using a similar procedure. Briefly, a plasmid containing the gene of interest was electrochemically introduced into BL21(DE3) competent E. coli cells. The cells were grown on LB agar plates (supplemented with 100 mg / L ampicillin and 1% glucose) at 37°C overnight. The next day, the grown LB plates were solubilized in 200 mL of 2xYT medium (supplemented with 100 mg / L ampicillin). The cultures were incubated at 37°C for 1 h at an optical density (OD) of 0.6. 600 The cells were grown at 37°C with constant shaking until they reached a final yield of 0.5 mM. Then, 0.5 mM isopropyl β-D-1-thiogalactopyranoside was added for incubation and growth was continued overnight at 21°C. The bacterial cells were pelleted using centrifugation and stored at -80°C for at least 1 h.

[0127] (DHFR / PAN / ThpA / HMWI_Act protein purification) Cell pellets were first processed by resuspending in lysis buffer and lysing by sonication (Branson Sonifier 450) in the presence of a protease inhibitor cocktail (Roche). Cell debris was removed by centrifugation, and the supernatant was processed through a Ni affinity chromatography column to collect purified protein fractions. For PAN, additional purification was performed by anion exchange-mediated protein purification using a HiTrap Q HP anion exchange column (GE Healthcare Life Sciences). Purity was confirmed by SDS-PAGE, and the fractions with the highest protein concentration were pooled and concentrated using a 10 kDa molecular weight cutoff spin filter (Amicon). For HMWI_Act, fractions containing the protein of interest were collected and dialyzed against storage buffer (50 mM HEPES, 100 mM NaCl, 10% glycerol, pH 7.5) using a SnakeSkin dialysis system (10 kDa molecular weight cutoff, Thermo Fischer Scientific). The dialyzed protein was aliquoted and stored at −80°C until further use.

[0128] (Protein purification of BSA) BSA was purchased from Acros Organics. The purity of BSA was increased by anion exchange chromatography (Akta Pure) by running 10 mg of BSA (1 ml of 50 mM Tris, pH 7.5) on a HiTrap Q HP anion exchange column (GE Healthcare Life Sciences). Eluted protein fractions were assessed by SDS-PAGE, and the fractions with the highest protein concentrations were pooled and concentrated using a 10 kDa molecular weight cutoff spin filter (Amicon).

[0129] (result) (Detection of model protein digestion) Protein detection and identification using (standard) mass spectrometry-based techniques relies heavily on (tryptic digest) peptide fingerprints. To mimic a properly digested protein, we employed a model peptide system (Sigma Aldrich and Genscript) containing seven synthetic peptides with masses between 700 and 1700 Da that would be predicted to result from complete trypsinization of lysozyme, i.e., the protein was cleaved in silico at all arginine (R) and lysine (K) residues except those followed by proline (P).

[0130] Seven model peptides were added individually to separate nanopore experiments (G13F-FraC-T1 pore, 1 M KCl, pH 3.8, -50 mV) giving rise to unique clusters of events when plotted against post-exclusion current and residence time. For each single experiment, the mean post-exclusion current for the event blockade was calculated by fitting a Gaussian curve to the histogram of clustered events. The mean post-exclusion current for each peptide species was calculated by averaging over n>3 experiments performed for each peptide. A strong correlation between the molecular weight of the peptides and their respective mean post-exclusion current blockade was observed (Figure 9A). The data were fitted with a logistic function (Equation 1, Figure 9A), which allows prediction of peptide mass from post-exclusion current measurements.

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[0131] Figure 9B shows a histogram of post-exclusion current blockade events measured from a mixture of all seven model peptides in a nanopore system (G13F-FraC-T1 pore, 1 M KCl, pH 3.8, -50 mV). Peaks are labeled based on predictions from the logistic function and correspond to the same post-exclusion current positions observed in the individual experiments.

[0132] (Detection of digested lysozyme proteins and comparison with mass spectrometry) Lysozyme protein was digested by trypsinization as described above. The resulting peptide fragment mixture was then analyzed using both nanopore sensing (G13F-FraC-T1 pore, 1 M KCl, pH 3.8, -50 mV) and mass spectrometry (LC ESI-MS). A histogram of post-exclusion current blockade measured from the mixture using nanopore is plotted in Figure 10A. For comparison, mass data obtained from mass spectrometry spectra were transformed onto the pseudo post-exclusion current axis using predictions from the parameters when fitting to the logistic equation determined in Eq. 1 (Figure 10B). Of note, although the methods cannot be directly compared due to differences in detection efficiency, for example, we observed a remarkable correlation between the observed electrospray ionization (ESI) mass spectra and the nanopore mass spectra.

[0133] (Detection of trypsin-digested proteins) Nine additional proteins with large and diverse compositions were examined by nanopore spectrometry. The nine proteins were bovine serum albumin (BSA), dihydrofolate reductase (DHFR), high molecular weight adhesin 1 (HMW1Act), PAN unfoldase, thiamine binding protein (TbpA), β-casein, cytochrome C, lysozyme, and trypsin. The proteins were digested by trypsinization as described. The resulting peptide fragment mixtures were examined separately in multiple separate nanopore experiments (G13F-FraC-T1 pore, 1 M KCl, pH 3.8, -50 mV). Similar to what was observed for the digested lysozyme peptide mixture, separate clusters of blocking events were observed from the peptide mixture for each of the digested proteins (Figures 11 and 12), where the clusters of blocking events were reduced by the current I after their exclusion. ex Figure 11 shows that for three representative protein samples, a high degree of consistency for each unique spectrum is observed between separate nanopore experiments.

[0134] To account for pore-to-pore variability in baseline current, we use I ex Residual current spectra were aligned to the reference spectrum using a sliding window on the percent. Figure 12 plots the "post-exclusion current spectra", which are clumped histograms derived from fitting to a scatter plot of post-exclusion current vs. dwell time of individual peptide blockade events for each protein sample. As expected, the post-exclusion current spectra for each protein show a unique pattern of peaks that depend on the unique composition of digested peptides in each system (with fragments that differ in terms of mass, length, and amino acid composition). Interestingly, despite the large amount of fragments predicted from the in silico digestion, the spectra of PAN and BSA show distinct peptide clusters. This indicates that even large (50 kDa) proteins yield distinct spectra that may enable fingerprinting of precursor proteins.

[0135] (Protein fingerprinting and spectral matching) The characteristic post-exclusion current spectra of tryptic digests (Figure 12A) can be used to fingerprint proteins. The most straightforward method of fingerprinting is spectral matching, where the measured spectrum is compared to a database of known previously measured spectra. After taking into account baseline shifts resulting from pore-to-pore variability in separate replicates, the different data sets showed a high degree of reproducibility (see, for example, Figure 11).

[0136] Spectral uniqueness and reproducibility were determined using spectral correlation using the squared first derivative Euclidean cosine correlation (DEuc) (Equation 2).

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[0137] To ensure representativeness for the spectral matching, we performed a leave-one-out comparison, where a comparative database was constructed from all spectra except one that were matched. The probability P(X)% was calculated from the DEuc score relative to the sum of all DEuc scores (Fig. 12B). We found that eight of the nine tryptic digests were correctly assigned to known proteins (diagonal line), with the exception of DHFR, which was incorrectly assigned to lysozyme. From visual inspection of the DHFR and lysozyme spectra (Fig. 12A), the incorrect assignment was easily explained as both digests share some peak similarity for the post-exclusion currents. This analysis uses only one 'metric' of the events, their post-exclusion currents. We found that further spectral analysis using other metrics, e.g., the standard deviation of the noise in each event, showed that clusters / peaks that are not easily separable in one metric dimension are often separable by another metric dimension.

[0138] (Detection of Amino Acid Changes) To evaluate the resolution of the analytical detection system for discriminating between peptides that differ by only 4 daltons, two different forms of enkephalin peptides, YGGFL and Y d AGF d L (where d represents a D-amino acid and all other amino acids are in the L-configuration) was tested. Figure 16A shows that two distinct clusters are observed for the different peptides, illustrating that mass differences of at least 4 Da can be distinguished with chirality differences using the exemplary FraC G13F nanopore. Detection of peptide chirality for peptides of the same mass was confirmed in Figures 16B and 16C, which showed differences in the nanopore signal due to the presence of D-amino acids. A mixture of 10 μM [Ala2]-Leu enkephalin and 10 μM DADLE ([D-Ala2,D-Leu5]-enkephalin) was added to either the cis compartment (FraC-G13F, Figure 16B) or the trans compartment (CytK-K128F, Figure 16C).

[0139] Example 6: Detection of post-translationally modified peptides This example shows that mutant proteinaceous nanopores can detect post-translationally modified peptides. An analytical system comprising the FraC-G13F nanopore described herein above was used to distinguish between phosphorylated and non-phosphorylated peptides (see FIG. 13), unmodified and peptides modified with one or two glycans (see FIG. 14), and unmodified and rhamnosylated proteins (FIG. 15).

[0140] Example 7: Mutant proteinaceous nanopores containing beta-barrel pore-forming toxins Examples 1 to 6 concern mutant proteinaceous nanopores containing alpha-helical pore-forming toxins of the actinoporin family and their application as single molecule sensors. To test whether these findings are more broadly applicable to different classes of nanopores with similar dimensions in the sensing region but quite different structural assemblies, we investigated similar mutations and conditions in beta-barrel pores.

[0141] This example shows that beta-barrel pore-forming proteins in which the lumen-facing recognition region of the protein contains one or more mutations to aromatic residues, particularly in combination with nearby acidic mutations, can also be used to provide such nanopore-based sensors.

[0142] We found that we were able to increase the capture (Figure 17A vs. 17B) and resolution (Figure 18A vs. 18B) of a mixture of tryptic peptides using wild-type erolysin pores by lowering the pH of the buffer. However, for the wild-type pore, even at low pH (e.g., pH 3.8), the events we observed were extremely short (Figure 17A / B), and the peptide clusters arising from different peptide populations had a broad distribution and were poorly resolved from each other, making it difficult to distinguish individual peptides from the mixture (Figure 18B).

[0143] We found that substituting lysine at position 238 with phenylalanine (Aer-K238F, Figures 17C and 18C) did not significantly increase peptide residence time (Figure 17C) but slightly improved peptide cluster resolution at pH 3.8 (Figure 18C), and substituting lysine at position 238 with the acidic amino acid aspartic acid (Aer-K238D) significantly increased cluster resolution at low pH over the wild-type pore (Figure 18D). The improved peptide capture and resolution for K238D is due in part to reduced electrostatic repulsion between the nanopore recognition region and the mostly positively charged peptide at low pH and in part to increased cation ion selectivity.

[0144] We further combined the K238D mutation with the introduction of phenylalanine at any of the positions Ala260, Ser264, Gln268, or S272 of aerolysin, and a dramatic improvement in peptide resolution was observed (Figs. 18E–H). The improved resolution between different peptide clusters is due to a combination of improvements that 1) enable more accurate measurement of each single molecule event with longer dwell (retention) times (e.g., Fig. 17D), 2) less spread of the residual current in each cluster that enables more facile resolution of closely spaced clusters from each other, and 3) broader spreading clusters across the full current range. The resolution of the analyte peptide was particularly sharp when the distance between the aspartic acid at position 238 and the introduced aromatic amino acid was less than 4 nm. Thus, the combination of increased negative pores and membrane-spanning aromatic substitutions facing water is important for increasing the capture and resolution of unlabeled peptides. This appears to be particularly important when sampling at acidic pH values (<pH 4.5). Importantly, the combination of mutations in the lumen of the beta-barrel type pore creates a ring of sensing residues similar to that in the constriction of the FraC nanopore when engineered for improved peptide discrimination, which indicates that this combination of mutations can be engineered to be the sensing constriction for a variety of both alpha-helical and beta-barrel type nanopores with similar sensing constriction geometries (e.g., engineered to introduce mutations to non-conserved inward-facing residues using well-known structure modeling and homology modeling tools in the art), an encompassing feature.

[0145] Figure 18I shows the capture and resolution of a tryptic digest peptide mixture using the mutant Aer-K238W pore, illustrating that the aromatic mutation significantly improves peptide detection relative to wild-type aerolysin.

[0146] (Expression and purification of proaerolysin) A plasmid containing the gene encoding proerolysin extended with a 6-histidine tag at the C-terminus was introduced into BL21(DE3) cells using electroporation. Transformants were grown overnight at 37°C on LB agar plates (supplemented with 1% glucose and 100 μg / l ampicillin). The following day, OD 600 Colonies were resuspended in 200 mL of 2YT medium and grown at 37 °C until an A of 0.6-0.8 was reached. At that point, expression was induced by the addition of 0.5 mM IPTG, and the culture was incubated overnight at 25 °C. Cells were then pelleted by centrifugation at 4000 rpm for 15 min, and the cell pellets were stored at -80 °C for at least 30 min. For protein purification, cell pellets from 100 ml of culture were resuspended in 20 ml of lysis buffer (containing 150 mM NaCl, 20 mM imidazole, and 15 mM Tris (buffered to pH 7.5), 1 mM MgCl2, 0.2 units / ml DNase1, and approximately 1 mg of lysozyme). The mixture was incubated at room temperature for 30 min, and then sonicated using a Branson Sonifier 450 (2 min, duty cycle 30%, power control 3) to ensure complete disruption of the cells. Cell debris was pelleted by centrifugation at 6000 rpm for 20 min, and the supernatant was carefully transferred to a fresh Falcon tube. Meanwhile, 200 μl of the Ni-NTA bead solution was washed with wash buffer (150 mM NaCl, 20 mM imidazole, and 15 mM Tris (buffered to pH 7.5)). The beads were added to the supernatant and incubated at room temperature for 5 min. The solution was then loaded onto a Micro Bio-Spin column (Bio-Rad) and then washed with 5 ml of wash buffer. Bound proteins were eluted in a fraction of 200 μl of elution buffer (150 mM NaCl, 300 mM imidazole, 15 mM Tris (buffered to pH 7.5)). The proaerolysin fractions can be stored at 4°C for several weeks.

[0147] (Multimerization of proerolysin using trypsin) Proerolysin is incubated with trypsin at a mass ratio of 1:1000 for 15 min at room temperature. Trypsin cleaves off the C-terminal peptide, generating proerolysin monomers that can assemble into heptamer pores that can be characterized by electrophysiological experiments.

[0148] (Planar lipid bilayer electrophysiological recordings) The electrophysiology chamber consisted of two compartments separated by a 25 μm thick Teflon (Goodfellow Cambridge Ltd) membrane. The Teflon membrane contained one aperture of approximately 100-200 μm diameter. A lipid membrane was formed by first applying 5 μl of a 5% solution of hexadecane (Sigma Aldrich) in pentane (Sigma Aldrich) to the Teflon membrane near the aperture. The pentane was left to dry and 400 μl of run buffer (1 M KCl, 50 mM citric acid (pH 3.8 or Bis-Tris-propane titrated to pH 3.0), or 1 M KCl, 50 mM Tris (buffered to pH 7.5)) was added to both sides. 20 μl of a 10 mg / ml solution of DPhPC dissolved in pentane was added on top of the buffer on each side of the chamber. The chamber was left to dry for approximately 2 min to allow evaporation of the pentane. A silver / silver chloride electrode was attached to each compartment. The cis compartment was connected to the ground electrode, and the trans compartment was connected to the working electrode. Planar lipid bilayers were fabricated using the Langmuir-Blodgett technique as described by Maglia et al. (Huang et al. Nat. Commun. 2017).

[0149] (trypsin digestion of lysozyme) 100 μg of lysozyme (Carl Roth, from chicken egg white, albumin-free) was dissolved in 150 mM NaCl, 15 mM Tris (buffered to pH 7.5). Prior to digestion, free cysteines were alkylated to prevent the formation of disulfide bonds after digestion. For this, 3 μL of 200 mM DTT was added and the sample was incubated at 37 °C for 15 min, followed by denaturation at 95 °C for 15 min. Then, 7 μL of 200 mM IAA was added and the sample was incubated at room temperature in the dark for 15 min. After alkylation, lysozyme was digested overnight at 37 °C with Trypsin Singles, Proteomics Grade-kit (Sigma Aldrich, Catalog #T7575-1KT) at a mass ratio of lysozyme:trypsin of 50:1.

[0150] (Detection of lysozyme digests using erythrolysinpore) Elorolysin was added to the cis chamber and the bilayer was disrupted and reformed until a single channel was inserted into the bilayer. The orientation of the pores can be detected by a small asymmetry in the pore IV curve. First, a 2 min blank was recorded at an applied potential of +150 mV, after which 4 μl of trypsin-digested lysozyme was added to the cis compartment of the chamber. The analyte was measured for at least 10 min at an applied potential of +150 mV.

[0151] (Data Recording) Ion current recordings were obtained using an Axopatch 200B (Axon Instruments) combined with a Digidata 1550B A / D converter (Axon instruments) similar to a previous study (Huang et al. Nat. Commun. 2019). The sampling frequency was set to 50 kHz for analyte recordings, and the analog Bessel filter was set to 10 kHz. Data were recorded using a Clampex 10 (Molecular Devices).

[0152] (Example 8: Mutant Protein Nanopore Containing Cytolysin K Beta-Barrel Type Pore-Forming Protein) Example 7 relates to single molecule analysis using aerolysin, a modified beta-barrel type pore-forming protein. In this example, aromatic mutations, preferably in combination with nearby acidic mutations, are used to illustrate the ability to capture and discriminate unlabeled peptides for other beta-barrel type pores, preferably when used under low pH conditions (<pH 4). Functionally similar mutations were introduced into cytolysin K (CytK).

[0153] CytK is known to be a nanopore capable of conducting current when inserted into the membrane (Hardy et al, FEMS Microbiol Lett. 2001), but the structure of CytK is unknown. Therefore, a homology model was constructed by mapping the CytK sequence to the sequence and structure of the alpha-hemolysin nanopore from Staphylococcus aureus to identify the beta-barrel region and the putative analyte recognition region (Figure 20A). We identified the beta-barrel region as including the section extending from amino acid E112 to amino acid S134 and from amino acid S137 to amino acid K155, where the even residues in the range of E112 - S130 and the odd residues in the range of S137 - K155 face the inner lumen water (Figure 20A).

[0154] (Expression and Purification of (Mutant) CytK) A plasmid containing the gene encoding CytK extended by a 6-histidine residue at the C-terminus was introduced into BL21(DE3) cells by electroporation. The transformed cells were grown overnight at 37 °C on an LB agar plate (1% glucose, 100 μg / l ampicillin). OD 600Colonies were resuspended and grown in 200 mL of 2YT medium at 37°C until the ΔH was 0.6–0.8, after which expression was induced by the addition of 0.5 mM IPTG and the cultures were incubated overnight at 25°C. Cells were pelleted by centrifugation and stored at -80°C for at least 30 min. Cell pellets were lysed by resuspension in lysis buffer (150 mM NaCl, 20 mM imidazole, 15 mM Tris, pH 7.5, 1 mM MgCl2, 0.2 units / ml DNase1, approximately 1 mg lysozyme), incubated at room temperature for 30 min, and then sonicated (Branson Sonifier 450, 2 min). Cell debris was pelleted by centrifugation and the supernatant containing CytK was collected. CytK was extracted from the supernatant and purified using Ni-NTA beads, and the final eluate was aliquoted into 200 μl portions (150 mM NaCl, 300 mM imidazole, 15 mM Tris (buffered to pH 7.5)) and then stored at 4°C.

[0155] (Planar lipid bilayer electrophysiological recordings) Electrophysiological measurements were performed as described in Example 7. CytK was added to the cis chamber, disrupting and reforming the DPhPC bilayer in the nanopore system until a single nanopore was inserted into the bilayer. The orientation of the pore can be detected by the asymmetry in the pore's IV curve. All recordings were made in the presence of 1 M KCl in both the cis and trans compartments at either pH 3.8 (50 mM citric acid (titrated with bis-tris propane to pH 3.8)) or pH 7.5 (50 mM Tris (buffered to pH 7.5)). First, a 2-minute blank open pore current was recorded at an applied potential of +100 mV, after which 4 μl of trypsin-digested lysozyme was added to either the cis or trans compartment of the chamber. Analytes were measured for at least 10 minutes at applied potentials of -100 mV to +100 mV as indicated. Ion current was recorded using a Digidata 1440A (Molecular Devices) connected to an Axopatch 200B amplifier (Molecular Devices). The sampling frequency was set to 50 kHz for analyte recording, and the analog Bessel filter was set to 10 kHz. Data was recorded using a Clampex 10 (Molecular Devices). Event blockage data was analyzed as described herein to measure event blockage resulting from peptide capture and extract metrics including mean open pore current, mean blockage current, duration of blockage (residence time), standard deviation of blockage current, etc.

[0156] (result) As in Example 7, a nanopore sensing system including a CytK nanopore was tested using a digested peptide mixture generated from trypsinized lysozyme. At either pH 7.5 or pH 3.8, under either positive or negative applied potentials across a wide range of voltages, wild-type CytK shows little to no capture of peptides from the trypsinized lysozyme sample, including when the sample was added to either the cis or trans compartment. For example, Figures 19A and 20B show that a low number of events were detected using the wild-type CytK nanopore when a trypsinized lysozyme sample was added to the trans compartment with a positive applied potential on the trans electrode to drive electrophoretic capture of mostly positively charged peptides (+100 mV, 1 M KCl, pH 3.8).

[0157] According to our predicted structure, the lysine residue at position 128 and the glutamic acid residue at position 139 are predicted to be inward-facing residues in the recognition region. In accordance with previous findings described herein, a phenylalanine was introduced by substitution into K128 of the CytK monomer close to the acidic E139, for reasons previously mentioned, to both reduce the net positive charge in the nanopore and introduce aromaticity for improved peptide detection. The K128F mutation caused a dramatic improvement in the ability to both capture (Figure 19B) and discriminate (Figure 20C) different peptides at low pH relative to the wild-type nanopore. Extremely good results were also obtained with the K128W mutation (Figure 20H).

[0158] In another implementation, an aromatic amino acid was introduced near the additional negative mutations by substituting lysine at position 238 with aspartic acid and serine at position 126 with phenylalanine (CytK-S126F-K128D), similar to the strategy employed in Example 7. Similar to what was observed for the erolysin nanopore system, this combination of aromatic amino acid substitutions near acidic amino acid substitutions further improved the resolution of distinct peptides through a combination of improved metrics including better capture (FIG. 19C), longer residence time of peptide blockade (FIG. 19C), tighter clusters with less spread in residual current (FIG. 20D), and clusters that are more widely spread across the entire current range from minimum to maximum (FIG. 20D).

[0159] Aromatic mutations placed much higher in the barrel of erolysin (S120, Q122, or G124) combined with K128D also resulted in better resolution of tryptic lysozyme peptides. See Figures 20E, F, and G.

[0160] Thus, the data demonstrate that aromatic substitutions, preferably close to acidic amino acid substitutions, result in sensing regions that improve the ability to capture and discriminate unlabeled peptides, especially at low pH conditions.

[0161] Of note, in Examples 7 and 8, we have demonstrated two different dominant mechanisms for controlling peptide capture in CytK and the erolysin nanopore. For example, we have demonstrated that the erolysin nanopore can efficiently capture and discriminate peptides at a positive applied potential when the analyte is in the cis compartment. Thus, the analyte is mostly positively charged at pH 3.8 or pH 3.0, but is captured against the electrophoretic direction due to the dominant electroosmotic capture conditions. In contrast, we have demonstrated that CytK can efficiently capture and discriminate peptides at a positive applied potential when the analyte is in the trans compartment. Thus, the analyte is mainly captured by electrophoretic forces under pH 3.8 conditions, and the electroosmotic component was adjusted to near zero by the substitution of additional acidic residues (see Table 4). Our results indicate that the introduction of aromatic residues in beta-barrel pore-forming toxins works regardless of the analyte capture mechanism, and that the introduction of acidic residues under low pH conditions is an important tool for adjusting and controlling cation selectivity and electroosmotic capture. Table 4: Ion selectivity of FraC, elolysin, and CytK nanopores. Reversal potentials were measured from IV curves between -100 mV and +100 mV under asymmetric salt conditions (2 M KCl in trans and 0.5 M KCl in cis), buffered to the indicated pH with 50 mM Tris (for pH 7.5) or 50 mM citric acid (titrated to pH 3.8 with bis-tris propane). Reversal potentials (applied voltage at zero net current) were determined by linear regression of the IV curves between -20 mV and +20 mV. [Table 4]

[0162] Example 9: Mutant protein nanopore containing lysenin beta barrel pore-forming protein This example shows that a further exemplary beta-barrel pore-forming protein, lysenin, was successfully mutated to demonstrate that aromatic substitution of non-aromatic lumen-facing residues improves the ability to capture and discriminate unlabeled peptides.

[0163] The plasmid was introduced into BL21(DE3) E. coli competent cells by electroporation with lysenin from Eisenia fetida. The cells were then grown overnight at 37°C on lysogeny broth (LB) agar plates (containing 100 μL / mL ampicillin). The LB plates were then picked and inoculated into 400 mL of 2xYT medium. The culture was then grown at 37°C with shaking at 200 rpm until the absorbance at 600 nm of the cell culture was 0.8. After this, 0.5 mM isopropyl-D-thiogalactopyranoside (IPTG) was added to the medium and the culture was grown overnight at 25°C with shaking at 200 rpm. The next day, the cells were harvested by centrifugation (4000 rpm, 15 min) and the resulting pellet was frozen at -80°C for 30 min.

[0164] Cells were resuspended in 40 mL of lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.02% DDM (10 mM imidazole, 1 mM MgCl2 with 0.2 mg / mL lysozyme and 10 μL DNase I)) and mixed for 30 minutes. The lysate was sonicated (at 40% power) for 2 minutes and centrifuged (4000 rpm) for 15 minutes at 4°C. The supernatant was then incubated with 150 μL of washed Ni-NTA beads at 20 rpm for 15 minutes. The Ni-NTA beads were loaded onto a gravity flow column and washed * After washing with buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM imidazole, and 0.02% DDM), the protein was eluted in three elution steps with 150 μL of elution buffer. * (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 300 mM imidazole, and 0.02% DDM). Lysenin monomer was stored at 4°C.

[0165] Lysenin was allowed to polymerize by incubation with liposomes (with 1:1 sphingomyelin:DPHPC lipid composition) at a protein:liposome ratio of 1:10 for 1 h at 37°C. Liposomes were then disrupted by addition of 0.6% LDAO. The solution was diluted 20-fold with wash buffer and mixed with 150 μl of washed Ni-NTA beads. The solution was then loaded onto a gravity-flow column and washed with wash buffer. Multimers were eluted in 150 μL fractions with elution buffer (containing 1 M imidazole, 150 mM NaCl, 15 mM Tris (buffered to pH 7.5)). Multimers were stored at 4°C.

[0166] Figure 21 shows the results obtained with 0.5 μg of Lys-C digested lysozyme (final concentration 1.25 ng / μl) added to the trans compartment of assay systems containing either wild-type Lys (panel A) or Lys-E76F (panel B). The introduction of aromatic residues in the lumen led to more pronounced peptide clusters for larger peptides.

[0167] (References) (1)Robertson,JWF;Rodrigues,CG;Stanford,VM;Rubinson,KA;Krasilnikov,OV;Kasianowicz,JJSingle-Molecule Mass Spectrometry in Solution Using a Solitary Nanopore.Proc.Natl.Acad.Sci.2007,104(20),8207-8211. (2)Huang, G.;Voet,A.;Maglia,G.FraC Nanopores with Adjustable Diameter Identify the Mass of Opposite-Charge Peptides with 44 Dalton Resolution.Nat.Commun.2019,10(1),835. (3)Chavis,AE;Brady,KT;Hatmaker,GA;Angevine,CE;Kothalawala,N.;Dass,A.;Robertson,JWF;Reiner,JESingle Molecule Nanopore Spectrometry for Peptide Detection.ACS Sensors 2017,2(9),1319-1328. (4)Anderluh,G.;Macek,P.Cytolytic Peptide and Protein Toxins from Sea Anemones(Anthozoa: Actiniaria).Toxicon 2002,40(2),111-124. (5) Garcia-Ortega, L.; Alegre-Cebollada, J.; Garcia-Linares, S.; Bruix, M.; Martinez-del-Pozo, A.; Gavilanes, JG. Biomembr.2011,1808(9),2275–2288. (6)Ros,U.;Rodriguez-Vera,W.;Pedrera,L.;Valiente,PA;Cabezas,S.;Lanio,ME;Garcia-Saez,AJ;Alvarez,C Rev. 2015,116,70–78. (7)Huang,G.;Willems,K.;Soskine,M.;Wloka,C.;Maglia,G.Electro-Osmotic Capture and Ionic Discrimination of Peptide and Protein Biomarkers with FraC Nanopores.Nat.Commun.2017,8(1),935. (8)Tanaka,K.;Caaveiro,J.M.M.;Morante,K.;Gonza´lez-Man~as,J.M.;Tsumoto,K.Structural Basis for Self-Assembly of a Cytolytic Pore Lined by Protein and Lipid.Nat Commun 2015,6. (9)Li,J.;Hibbert,D.B.;Fuller,S.;Vaughn,G.A Comparative Study of Point-to-Point Algorithms for Matching Spectra.Chemom.Intell.Lab.Syst.2006,82(1),50-58. (10)Mu¨llner,D.Fastcluster: Fast Hierarchical,Agglomerative Clustering Routines for R and Python.J.Stat.Software;Vol 1,Issue 9 2013. (11)Thapa,P et al.;Native chemical ligation: a boon to peptide chemistry.Molecules.2014 Sep 12;19(9):14461-83. (12)Hardy,S..P,Granum,E., CytK toxin of Bacillus cereus forms pores in planar lipid bilayers and is cytotoxic to intestinal epithelia.FEMS Microbiol Lett.2001;197(1):47-51 (13)Dal Peraro,van der Goot,Pore-forming toxins: ancient,but never really out of fashion.Nat Rev Microbiol.2016 Feb;14(2):77-92. (14)Shengli Zhang,Gang Huang,Roderick Versloot,Bart Marlon Herwig,Paulo Cesar Telles de Souza,Siewert-Jan Marrink,Giovanni Maglia,Bottom-up fabrication of a multi-component nanopore sensor that unfolds,processes and recognizes single proteins.BioRxiv,2020. (15)Scott et al.Constructing ion channels from water-soluble α-helical barrels.Nat Chem.2021 May 10. (16)Vorobieva et al.De novo design of transmembrane β barrels.Science.2021 Feb 19;371(6531) (17)Spruijt,Tusk,Bayley.DNA scaffolds support stable and uniform peptide nanopores.Nat Nanotechnol.2018 Aug;13(8):739-745. (18)Kristan,K.C.,Viero,G.,Dalla Serra,M.,Macek,P.& Anderluh,G.Molecular mechanism of pore formation by actinoporins.Toxicon,2009 1125-1134. (19)Heron et al,Simultaneous measurement of ionic current and fluorescence from single protein pores.J Am Chem Soc.2009;131(5):1652-3. (20)Spaan, van Strijp, Torres, Leukocidins: staphylococcal bi-component pore-forming toxins find their receptors.Nat Rev Microbiol.2017 Jul;15(7):435-447. (21)Hammerstein, Jayasinghe, Bayley.Subunit dimers of alpha-hemolysin expand the engineering toolbox for protein nanopores.J Biol Chem.2011 Apr 22;286(16):14324-34. (22)Gouaux et al.Subunit stoichiometry of staphylococcal alpha-hemolysin in crystals and on membranes: a heptameric transmembrane pore.Proc Natl Acad Sci US A.1994 Dec 20;91(26):12828-31. (23)Crnkovic´,Srnko,Anderluh.Biological Nanopores: Engineering on Demand.Life(Basel).2021 Jan 5;11(1):27.

[0168] [Note] [Appendix 1] A proteinaceous nanopore comprising a mutant alpha-helical pore-forming toxin of the actinoporin family or a pore-forming fragment thereof, the lumen-facing recognition region of the pore-forming protein or fragment thereof comprises one or more substitutions of lumen-facing amino acids in the recognition region corresponding to amino acids 10 to 20 of Fragaceatoxin C (FraC; UniProtKB / Swiss-Prot:B9W5G6) with neutral or non-neutral aromatic amino acid residues; Proteinaceous nanopores.

[0169] [Appendix 2] A mutant actinoporin or its alpha-helical transmembrane domain (aa1-27) containing one or more substitutions of the lumen-facing amino acids with Trp, Tyr, or Phe, 2. A proteinaceous nanopore as described in appendix 1.

[0170] [Appendix 3] Fragaceatoxin A(FraA;Swiss-Prot P0DUW8)、Fragaceatoxin B(FraB;Swiss-Prot A0A515MEN7)、Fragaceatoxin C(FraC;Swiss-Prot B9W5G6)、Fragaceatoxin D(FraD;Swiss-Prot P0DUW9)、Fragaceatoxin E(FraE;Swiss-Prot A0A515MEM9)、Equinatoxin II(Eqt-II;Swiss-Prot P61914)、Equinatoxin IV(Eqt-IV;Swiss-Prot P61914)、Equinatoxin V(Eqt-V;Swiss-Prot Q93109)、Urticinatoxin(UcI;Swiss-Prot C9EIC7)、Actitoxin-Oor1b(Or-G;Swiss-Prot Q5I2B1)、Actitoxin-Oor1a(Or-A Swiss-Prot Q5I4B8)、Gigantoxin-4(Gigt 4;Swiss-Prot H9CNF5)、Heteractis magnifica cytolysin III(HmgIII;Swiss-Prot Q9U6X1)、Bandaporin(bp-1;Swiss-Prot C5NSL2)、Cribinopsis japonica toxin I(CJTOX I;Swiss-Prot A0A2Z5Z9X0)、Cribinopsis japonica toxin II(CJTOX II;Swiss-Prot A0A2Z5Z9H5)、Sticholysin I(StI;Swiss-Prot P81662)、Sticholysin II(StII;Swiss-Prot P07845)、Stichotoxin Hcr4a(RTX-A;Swiss-Prot P58691)、Stichotoxin Hcr4b(RTX-SII;Swiss-Prot P0C1F8)、Sagatoxin I(Src I;Swiss-Prot Q86FQ0)、Cytolysin Avt-I(AvtI;Swiss-Prot Q5R231)、Cytolysin PsTX-20A(PsTX20A;mutated alpha-helical pore-forming toxins of the actinoporin family or pore-forming fragments thereof selected from the group consisting of Nigrelysin (Swiss-Prot P0DL55), Nigrelysin (Swiss-Prot A0A345GPN1), and their pore-forming toxin homologs exhibiting at least 90%, preferably at least 95%, sequence identity thereto; 3. A proteinaceous nanopore according to claim 1 or 2.

[0171] [Appendix 4] containing an aromatic substitution of one or more residues corresponding to Asp10, Gly13, Asp17, Lys20, preferably Gly13, of the amino acids of FraC; A proteinaceous nanopore according to any one of claims 1 to 3.

[0172] [Appendix 5] comprising a mutated actinoporin selected from Table 1, A proteinaceous nanopore as described in appendix 4.

[0173] [Appendix 6] one or more further mutations that increase the net negative charge of the pore are introduced into amino acids of the recognition region facing the lumen; 6. A proteinaceous nanopore according to any one of claims 1 to 5.

[0174] [Appendix 7] comprising one or more mutations to Glu and / or Asp residues, 7. A proteinaceous nanopore as described in appendix 6.

[0175] [Appendix 8] (i) FraC, FraE, or a functional homologue thereof that has at least 90% sequence identity thereto, which contains an aromatic residue at the position corresponding to Gly13 of FraC; (ii) FraB, Ten-C, Eqt-II, Gigt4, HmgIII, RTX-SII, Hmt, or a functional homologue thereof exhibiting at least 90% sequence identity, comprising an aromatic residue at the position corresponding to Ser13 and, optionally, an acidic residue at position 10; (iii) bp-1, or a functional homologue thereof that has at least 90% sequence identity, containing an aromatic residue at the position corresponding to Asn13; (iv) CJTOX I, or a functional homologue thereof exhibiting at least 90% sequence identity, containing an aromatic residue at the position corresponding to Thr36 and, optionally, an acidic residue at the position corresponding to Gln33; (v) CJTOX II, or a functional homologue thereof exhibiting at least 90% sequence identity, comprising an aromatic residue at the position corresponding to Ala36 and, optionally, an acidic residue at the position corresponding to Gln33; (vi) Cytolysin Avt-I, Cytolysin PsTX-20A, or a functional homologue thereof exhibiting at least 90% sequence identity, comprising an aromatic residue at the position corresponding to Glu13 and, optionally, an acidic residue at the position corresponding to Ala10; (vii) Eqt-IV, or a functional homologue thereof that has at least 90% sequence identity thereto, comprising an aromatic residue at the position corresponding to Ala13 and, optionally, an acidic residue at the position corresponding to Lys10; (viii) Eqt-V, or a functional homologue thereof that has at least 90% sequence identity, containing an aromatic residue at the position corresponding to Thr13; (ix) Nigrelysin or a functional homologue thereof that has at least 90% sequence identity, which contains an aromatic residue at the position corresponding to Asn27; (x) StII, RTX-A, or a functional homologue thereof exhibiting at least 90% sequence identity, comprising an aromatic residue at the position corresponding to Ser11 and, optionally, an acidic residue at the position corresponding to Ala8; (xi) Src I, or a functional homologue thereof that has at least 90% sequence identity, containing an aromatic residue at the position corresponding to Arg12 and, optionally, an acidic residue at the position corresponding to Ala9; (xii) StI, or a functional homologue thereof having at least 90% sequence identity, containing an aromatic residue at the position corresponding to Ser12; (xiii) UcI, or a functional homologue thereof that has at least 90% sequence identity, containing an aromatic residue at the position corresponding to Lys13; or (xiv) Or-G, or a functional homologue thereof having at least 90% sequence identity, comprising an aromatic residue at the position corresponding to Ala8 and, optionally, an acidic residue at the position corresponding to Ala5; or the alpha-helical transmembrane domain (aa1-27) of said polypeptide containing the aforementioned mutations; The mutant actinoporin is selected from the group consisting of: 8. A proteinaceous nanopore according to any one of claims 1 to 7.

[0176] [Appendix 9] A mutated FraC or a pore-forming fragment thereof comprising a mutation Gly13Tyr, Gly13Trp, or Gly13Phe, preferably Gly13Phe; 9. A proteinaceous nanopore as described in appendix 8.

[0177] [Appendix 10] An analytical system comprising a hydrophobic membrane separating a fluid chamber into a cis side and a trans side, the hydrophobic membrane comprising a mutant proteinaceous nanopore described in any one of appendixes 1 to 9.

[0178] [Appendix 11] providing a recombinant monomer of said mutant pore-forming toxin or pore-forming fragment thereof; contacting the monomers with liposomes and / or surfactants to assemble them into oligomers; recovering the oligomer from the liposomes and / or surfactant; and contacting the oligomer with a membrane that may contain sphingomyelin to allow formation of a nanopore; Including, 11. A method for providing a system as described in claim 10.

[0179] [Appendix 12] adding an analyte of interest to a chamber of the analytical system of claim 10; allowing the analyte to contact the nanopore; and Detecting / characterizing at least one property of said analyte; Including, A method for single molecule analysis, preferably for identification and / or sequencing of an analyte of interest.

[0180] [Appendix 13] applying an electric field to the nanopore such that the analyte is electrophoretically and / or electroosmotically trapped in the nanopore. 13. The method according to claim 12.

[0181] [Appendix 14] The analyte of interest has a mass in the range of 200 to 5000 Da, preferably in the range of 200 to 500 Da or 500 to 1700 Da; 14. The method according to claim 12 or 13.

[0182] [Appendix 15] The analyte of interest is a biopolymer, preferably a biopolymer selected from the group consisting of proteins, polypeptides, and oligopeptides; 15. The method according to any one of claims 12 to 14.

[0183] [Appendix 16] The analyte of interest is a proteinaceous material, preferably a peptide, more preferably a peptide of about 30, 20, 15, 10, 5, 3, or 2 amino acids or less in length. 16. The method according to claim 15.

[0184] [Appendix 17] Detecting mutations and / or post-translational modifications of the analyte, including, for example, detecting peptide fragments that differ in single amino acid residues, amino acid chirality, degree of phosphorylation, and / or degree of glycosylation; 14. The method according to claim 12 or 13.

[0185] [Appendix 18] The detection is carried out at a pH ≦ 4.5, preferably below pH 4.0; 18. The method according to any one of claims 12 to 17.

[0186] [Appendix 19] 1. A method for decreasing the translocation rate of a peptide analyte through an alpha-helical or beta-barrel transmembrane pore, comprising: (a) increasing the net aromaticity of the lumen of the pore by substituting one or more lumen-facing non-aromatic amino acids with one or more aromatic amino acids; and (b) passing the polypeptide through the pore, increasing the net aromaticity thereby decreasing the translocation rate of the polypeptide through the pore; A method comprising:

[0187] [Appendix 20] Step (a) comprises providing a proteinaceous nanopore according to any one of claims 1 to 9; 19. The method according to claim 19.

[0188] [Appendix 21] 11. A device comprising a plurality of the analytical systems of claim 10, preferably wherein said analytical systems comprise distinct pore types.

[0189] [Appendix 22] (i) a mutant proteinaceous nanopore according to any one of claims 1 to 9, an analytical system according to claim 10, or an apparatus according to claim 21, and (ii) an analyte-manipulating enzyme, preferably a protease; Including, A kit of parts for characterizing an analyte of interest.

[0190] [Appendix 23] Use of the analytical system according to claim 10, the device according to claim 21 or the kit according to claim 22 for single molecule analysis, preferably for identification and / or sequencing of biopolymers, more preferably for label-free protein fingerprinting.

Claims

1. A nanopore comprising (i) an alpha-helical pore-forming protein or a fragment thereof, or (ii) a beta-barrel pore-forming protein or a fragment thereof, the beta-barrel pore-forming protein or fragment thereof is not alpha-hemolysin or aerolysin; the alpha-helical pore-forming protein or fragment thereof, or the beta-barrel pore-forming protein or fragment thereof, comprises modifications with one or more natural or unnatural aromatic amino acids; Nanopore.

2. The nanopore comprising the alpha-helical pore-forming protein or a fragment thereof. The nanopore of claim 1 .

3. The alpha-helical pore-forming protein or fragment thereof comprises an actinoporin. The nanopore of claim 1 .

4. The nanopore comprises the beta-barrel pore-forming protein or a fragment thereof. The nanopore of claim 1 .

5. The beta-barrel pore-forming protein or fragment thereof comprises a cytolysin, a leukocidin, a bacterial outer membrane protein porin, or a newly designed pore-forming peptide. The nanopore of claim 1 .

6. The modification is within or adjacent to a constriction region of the nanopore. The nanopore of claim 1 .

7. The modification comprises an amino acid sequence insertion, an amino acid sequence substitution, a chemical modification, a chemical ligation, a chemical functionalization, or a combination thereof. The nanopore of claim 1 .

8. The alpha-helical pore-forming protein or fragment thereof, or the beta-barrel pore-forming protein or fragment thereof, comprises another modification with one or more basic amino acids, one or more acidic amino acids, or a combination thereof. The nanopore of claim 1 .

9. The nanopore comprising a recognition region or constriction region having a diameter of at most 2 nanometers. The nanopore of claim 1 .

10. The modification is configured to increase the net negative charge of the nanopore or increase the net aromaticity of the interior surface of the nanopore. The nanopore of claim 1 .

11. (a) providing a system comprising: (i) a fluid chamber; (ii) a membrane separating a first side from a second side; and (iii) a nanopore disposed within said membrane, said nanopore comprising an alpha-helical pore-forming protein or a fragment thereof, or a beta-barrel pore-forming protein or a fragment thereof, said beta-barrel pore-forming protein or fragment thereof being neither alpha-hemolysin nor erolysin, and said alpha-helical pore-forming protein or fragment thereof, or said beta-barrel pore-forming protein or fragment thereof comprising modification with one or more natural or non-natural aromatic amino acids; (b) contacting the nanopore with an analyte; A method comprising:

12. The method of claim 1, further comprising translocating the analyte through the nanopore. The method of claim 11.

13. The method of claim 1, further comprising measuring a signal produced by translocating the analyte through the nanopore. The method of claim 11.

14. The nanopore comprising the alpha helix pore-forming protein or a fragment thereof. The method of claim 11.

15. The alpha-helical pore-forming protein or fragment thereof comprises an actinoporin. The method of claim 11.

16. The nanopore comprising the beta-barrel pore-forming protein or a fragment thereof. The method of claim 11.

17. The beta-barrel pore-forming protein or fragment thereof comprises a cytolysin, a leukocidin, a bacterial outer membrane protein porin, or a newly designed pore-forming peptide. The method of claim 11.

18. The analyte comprises a peptide, a polypeptide, a protein, or any combination thereof. The method of claim 11.

19. The alpha-helical pore-forming protein or fragment thereof, or the beta-barrel pore-forming protein or fragment thereof, comprises another modification with one or more basic amino acids, one or more acidic amino acids, or a combination thereof. The method of claim 11.

20. The nanopore comprising a recognition region or constriction region of at most 2 nanometers in diameter. The method of claim 11.

21. The method of claim 20, further comprising determining one or more properties of the analyte. The method of claim 11.

22. The one or more properties are selected from the group consisting of the length of the analyte, the volume of the analyte, the mass of the analyte, the shape of the analyte, the charge distribution of the analyte, the identity of the analyte, the sequence of the analyte, and one or more chemical modifications of the analyte.

22. The method of claim 21.

23. A system comprising: (i) a fluid chamber; (ii) a membrane separating a first side from a second side; and (iii) a nanopore described in any one of claims 1 to 10 disposed within the membrane.

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