Alpha-hemolysin mutants that form narrow channel pores and uses thereof

Abstract

Described herein is an alpha-hemolysin nanopore having a relatively narrow channel and D127G and D128K substitutions relative to SEQ ID NO: 1. The narrow channel reduces the extent to which a nucleic acid template will penetrate the nanopore, while the D127G and D128K substitutions improve the lifetime and arrival rate of the narrow channel pore. Polypeptides for forming such nanopores, systems including such nanopores, and methods of making and using such nanopores are also disclosed herein.

Description

[Technical Field]

[0001] Disclosed are compositions and methods relating to variants of the Staphylococcus aureus alpha-hemolysin polypeptide. The alpha-hemolysin variants are useful, for example, as nanopore components in devices for determining polymer sequence information. [Background technology]

[0002] background Hemolysins are members of a family of protein toxins produced by a wide variety of organisms. Some hemolysins, such as alpha-hemolysin, can disrupt the integrity of cell membranes (e.g., host cell membranes) by forming pores or channels in the membrane. The pores or channels formed in the membrane by pore-forming proteins can be used to transport specific polymers (e.g., polypeptides or polynucleotides) from one side of the membrane to the other.

[0003] Alpha-hemolysin (also called α-hemolysin, α-HL, a-HL, or alpha-HL) is a self-assembling toxin that forms channels in the membranes of host cells. Alpha-hemolysin has become a major component of the nanopore sequencing community. Alpha-hemolysin has many advantageous properties, including high stability, self-assembly, and a pore wide enough to accommodate single-stranded but not double-stranded DNA (Kasianowicz et al., 1996).

[0004] Previous studies on DNA detection within a-HL pores have focused on analyzing the ionic current signature as DNA translocates through the pore (Kasianowicz et al., 1996; Akeson et al., 1999; Meller et al., 2001), a challenging task given the translocation rate (approximately 1 nt / μs at 100 mV) and the inherent noise in the ionic current signal. Higher specificity has been achieved in nanopore-based sensors by incorporating permanently tethered probe molecules inside the pore (Howakka et al., 2001a and Howorka et al., 2001b; Movileanu et al., 2000).

[0005] Wild-type alpha-hemolysin produces a significant number of deletion errors, i.e., does not measure bases. Therefore, many efforts have been made to improve alpha-hemolysin nanopores for use in tag-based sequencing by synthesis (SBS), including, for example, U.S. Patent Application Publication No. 2017-0088588 A1, U.S. Patent Application Publication No. 2017-0088890 A1, U.S. Patent Application Publication No. 2017-0306397 A1, U.S. Patent Application Publication No. 2018-0002750 A1, and U.S. Patent Application Publication No. 2018-0002750 A1. However, there is a need for alpha-hemolysin nanopores with improved properties. Summary of the Invention

[0006] Brief Summary of the Invention Disclosed are variants of Staphylococcus aureus alpha-hemolysin polypeptides containing amino acid mutations useful for generating nanopores that can be used in tag-based sequencing-by-synthesis reactions. The variant polypeptides disclosed herein can be used to prepare heptameric nanopores that have a relatively narrow constriction region and a longer pore lifetime compared to pores formed from the reference alpha-hemolysin polypeptide.

[0007] In one aspect, an alpha-hemolysin (alpha hemolysin) polypeptide is provided that includes at least one narrow channel-alpha hemolysin (alpha hemolysin) subunit, the subunit comprising a D127G substitution and a D128K substitution relative to SEQ ID NO:1. In some embodiments, the amino acid residues corresponding to E111 and / or K147 in SEQ ID NO:1 are selected from the group consisting of glutamic acid, lysine, arginine, and glutamine. In some embodiments, the amino acid residues corresponding to E111 and / or K147 in SEQ ID NO:1 are selected from the group consisting of glutamic acid and lysine. In some embodiments, the narrow channel alpha hemolysin subunit comprises either or both of E111 and K147 (i.e., the wild-type residues at those positions relative to SEQ ID NO:1). In some embodiments, the amino acid residue corresponding to M113 in SEQ ID NO:1 is selected from the group consisting of leucine, isoleucine, valine, and methionine. In some embodiments, the amino acid residue corresponding to M113 in SEQ ID NO:1 is methionine (i.e., the wild-type residue at that position relative to SEQ ID NO:1). In some embodiments, a narrow channel alpha-hemolysin subunit comprises each of E111, M113, and K147 (i.e., the wild-type residues at those positions relative to SEQ ID NO:1). For example, a narrow channel alpha-hemolysin subunit can comprise an amino acid sequence having at least 75%, 80%, 90%, 95%, 98%, or more identity to SEQ ID NO:1, wherein the amino acid sequence comprises a glutamic acid residue at a position corresponding to E111 of SEQ ID NO:1, a methionine residue at a position corresponding to M113 of SEQ ID NO:1, a lysine residue at a position corresponding to K147 of SEQ ID NO:1, a D127G substitution relative to SEQ ID NO:1, and a D128K substitution relative to SEQ ID NO:1. As another example, a narrow channel alpha-hemolysin subunit can comprise an amino acid sequence having at least 75%, 80%, 90%, 95%, 98%, or more identity to SEQ ID NO:2, wherein the amino acid sequence comprises each of G127, K128, E111, M113, and K147 of SEQ ID NO:2.As another example, the narrow channel alpha-hemolysin subunit comprises an amino acid sequence having at least 75%, 80%, 90%, 95%, 98% or more identity to SEQ ID NO:3, wherein the amino acid sequence includes each of G127, K128, E111, M113, and K147 of SEQ ID NO:3. As another example, the narrow channel alpha-hemolysin subunit comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3. As another example, the narrow channel alpha-hemolysin subunit comprises an amino acid sequence having at least 75%, 80%, 90%, 95%, 98% or more identity to SEQ ID NO:4, wherein the amino acid sequence includes N111E, A113M, and N147K substitutions compared to SEQ ID NO:4, and further includes G127 and K128 of SEQ ID NO:4. As another example, a narrow channel alpha-hemolysin subunit comprises an amino acid sequence having at least 75%, 80%, 90%, 95%, 98% or more identity to SEQ ID NO:5, wherein the amino acid sequence comprises an N111E, an A113M, an N147K, and a G128K substitution compared to SEQ ID NO:5, and further comprises G127 of SEQ ID NO:5. As another example, a narrow channel alpha-hemolysin subunit comprises an amino acid sequence having at least 75%, 80%, 90%, 95%, 98% or more identity to SEQ ID NO:6, wherein the amino acid sequence comprises a D127G substitution relative to SEQ ID NO:6, a D128K substitution relative to SEQ ID NO:6, a glutamic acid residue at a position corresponding to E111 of SEQ ID NO:6, a methionine residue at a position corresponding to M113 of SEQ ID NO:6, and a lysine residue at a position corresponding to K147 of SEQ ID NO:6. As another example, the narrow channel alpha hemolysin subunit comprises an amino acid sequence having at least 75%, 80%, 90%, 95%, 98% or more identity to SEQ ID NO:7, wherein the amino acid sequence includes a D127G substitution relative to SEQ ID NO:7, a D128K substitution relative to SEQ ID NO:7, a glutamic acid residue at a position corresponding to E111 in SEQ ID NO:7, a methionine residue at a position corresponding to M113 in SEQ ID NO:7, and a lysine residue at a position corresponding to K147 in SEQ ID NO:7.As another example, the narrow channel alpha hemolysin subunit comprises an amino acid sequence having at least 75%, 80%, 90%, 95%, 98% or more identity to SEQ ID NO:8, wherein the amino acid sequence includes a D127G substitution relative to SEQ ID NO:8, a D128K substitution relative to SEQ ID NO:8, a glutamic acid residue at a position corresponding to E111 in SEQ ID NO:8, a methionine residue at a position corresponding to M113 in SEQ ID NO:8, and a lysine residue at a position corresponding to K147 in SEQ ID NO:8.

[0008] Also provided is a narrow channel alpha-hemolysin nanopore, the nanopore comprising at least six narrow channel alpha-hemolysin subunits comprising D127G and D128K substitutions relative to SEQ ID NO: 1. The nanopore has the following properties: (a) a narrower constriction region than nanopore P-0304; and (b) an extended lifetime compared to nanopore P-0031. In certain embodiments, the narrow channel alpha-hemolysin nanopore described herein is coupled to a DNA polymerase, such as via a covalent bond. In certain exemplary embodiments, the narrow channel alpha-hemolysin nanopore is a 6:1 nanopore, and the DNA polymerase is coupled to the "1" component.

[0009] In certain exemplary embodiments, nucleic acids encoding any of the narrow channel alpha-hemolysin mutant polypeptides described herein are also provided. For example, the nucleic acid sequence can be derived from Staphylococcus aureus αHL (SEQ ID NO: 9). In certain exemplary embodiments, vectors containing any such nucleic acids encoding any one of the hemolysin mutants described herein are also provided. Host cells transformed with the vectors are also provided.

[0010] In certain exemplary aspects, methods are provided for detecting and / or identifying a target nucleic acid molecule using the disclosed narrow-channel alpha-hemolysin nanopore. The methods include, for example, providing a chip comprising a nanopore assembly described herein within a membrane positioned adjacent to or in close proximity to a sensing electrode. The methods then include detecting tagged nucleotides using the nanopore during synthesis of a complementary strand of the target nucleic acid molecule.

[0011] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the scope and spirit of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0012] [Figure 1A] Figure 1 shows two sequencing runs with potential threading issues. (A) Shown is a sequencing run with a clear open channel level 101, tag levels 102a-102d, and a persistent background level 103 likely caused by template threading. [Figure 1B] Figure 1 shows two sequencing runs with potential threading issues. (B) A sequencing run with significant background noise 103 and sequencing removal 104 likely caused by template threading. [Figure 2] FIG. 2 is a graph of attainment velocity (X-axis) versus pore lifetime (Y-axis) for four different pores: P-0031, P-0304, P-0411, and P-0414. [Figure 3] FIG. 3 is a bar graph showing the percentage of wide channel (P-0304) versus narrow channel (P-0411 and P-0414) threaded with alpha-hemolysin nanopores. [Figure 4] FIG. 4 is a sequence alignment between the subunits disclosed in Table 5. DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description The present invention will now be described in detail, by reference only, using the following definitions and examples. All patents and publications mentioned herein, including all sequences disclosed within such patents and publications, are expressly incorporated by reference.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many of the terms used in this invention. Professionals are directed, inter alia, to Sambrook et al., 1989, and Ausubel FM et al., 1993, for definitions and terminology in the art. It is understood that the invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

[0015] Numerical ranges are inclusive of the numbers defining the range. The term about is used herein to mean plus or minus ten percent (10%) of a value. For example, "about 100" refers to any number between 90 and 110.

[0016] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0017] The headings provided herein are not limitations of the various aspects or embodiments of the invention that can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0018] I. Definition Alpha-hemolysin: As used herein, "alpha-hemolysin," "α-hemolysin," "a-HL," and "alpha-hemolysin" are used interchangeably and refer to the polypeptide expressed from the hly gene of Staphylococcus aureus.

[0019] Alpha-hemolysin nanopore: As used herein, "alpha-hemolysin nanopore" refers to a nanopore formed from seven alpha-hemolysin subunits.

[0020] Alpha-hemolysin polypeptide: As used herein, "alpha-hemolysin polypeptide" refers to any polypeptide that includes at least one alpha-hemolysin subunit.

[0021] Alpha-hemolysin subunit: As used herein, "alpha-hemolysin subunit" refers to SEQ ID NO: 1 and variants thereof that are capable of self-assembling into a heptameric nanopore.

[0022] Amino acid: As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid, in some embodiments, an amino acid is a D-amino acid, and in some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or obtained from a natural source. As used herein, "synthetic amino acid" or "unnatural amino acid" encompasses chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids, including the carboxy- and / or amino-terminal amino acids in a peptide, can be modified by methylation, amidation, acetylation, and / or substitution with other chemicals without adversely affecting their activity. Amino acids may participate in disulfide bonds. The term "amino acid" is used interchangeably with "amino acid residue" and can refer to free amino acids and / or peptide amino acid residues. Whether the term refers to a free amino acid or a peptide residue will be clear from the context in which the term is used. Note that all amino acid residue sequences are represented herein by formulae in which the left-right orientation is in the conventional direction from amino terminus to carboxy terminus.

[0023] Arrival rate: As used herein, the "arrival rate" of an alpha-hemolysin nanopore is a measure of the frequency with which the alpha-hemolysin nanopore captures the tag of a biotinylated tag molecule. For example, the arrival rate can be determined by obtaining a chip with multiple pores of interest inserted into the bilayer, flowing streptavidin-biotin-TAG through the chip, and measuring the average time between capture events in each of the multiple pores (typically at a very low AC modulation frequency, such as about 50 Hz). The arrival rate is the average time between events across all pores.

[0024] Base pair (bp): As used herein, a base pair refers to the partnering of adenine (A) with thymine (T), adenine (A) with uracil (U), or cytosine (C) with guanine (G) in a double-stranded nucleic acid.

[0025] Complementary: As used herein, the term "complementary" refers to the broad concept of sequence complementarity between regions of two polynucleotide strands or between two nucleotides through base pairing. It is known that adenine nucleotides can form specific hydrogen bonds ("base pairing") with thymine or uracil nucleotides. Similarly, it is known that cytosine nucleotides can base pair with guanine nucleotides.

[0026] Linked alpha-hemolysin polypeptide: An alpha-hemolysin polypeptide comprising multiple alpha-hemolysin subunits separated from one another by one or more flexible linker sequences. Exemplary methods and considerations for producing linked alpha-hemolysin polypeptides are disclosed, for example, by Hammerstein and U.S. Patent Application Publication No. 2017-0088890 A1.

[0027] Expression cassette: An "expression cassette" or "expression vector" is a recombinantly or synthetically produced nucleic acid construct having a series of designated nucleic acid elements that enable transcription of a particular nucleic acid in a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.

[0028] Heterologous: A "heterologous" nucleic acid construct or sequence has a portion of sequence that is not native to the cell in which it is expressed. Heterologous, with respect to a regulatory sequence, refers to a regulatory sequence (i.e., a promoter or enhancer) that does not function in nature to regulate the same gene whose expression it currently regulates. Generally, heterologous nucleic acid sequences are not endogenous to the cell or part of the genome in which they are present, but have been added to the cell by infection, transfection, transformation, microinjection, electroporation, etc. A "heterologous" nucleic acid construct can contain a regulatory sequence / DNA coding sequence combination that is the same as or different from the regulatory sequence / DNA coding sequence combination found in the native cell.

[0029] Host cell: The term "host cell" refers to a cell that contains a vector and supports the replication, and / or transcription or transcription and translation (expression) of an expression construct. Host cells for use in the present invention can be prokaryotic cells, such as E. coli or Bacillus subtilus, or eukaryotic cells, such as yeast, plant, insect, amphibian, or mammalian cells. Generally, host cells are prokaryotic, e.g., E. coli.

[0030] Isolated: An "isolated" molecule is a nucleic acid molecule that is separated from at least one other molecule with which it is normally associated, e.g., in its natural environment. Isolated nucleic acid molecules include nucleic acid molecules contained in cells that normally express the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0031] Lifespan: As used herein, the "lifespan" of an alpha-hemolysin nanopore species is a measure of the percentage of alpha-hemolysin nanopores that remain capable of capturing biotinylated tag molecules for one hour on a nanopore sequencing array. For example, lifetime can be determined by obtaining a chip with multiple pores of interest inserted into a bilayer, flowing streptavidin-biotin-TAG over the chip, and tracking the activity of all individual nanopores on the chip over one hour. The lifetime of a pore species is the percentage of pores that remain active for the entire one-hour period.

[0032] Mutation: As used herein, the term "mutation" refers to a change introduced into a parent sequence, including but not limited to, a substitution, insertion, and / or deletion (including truncation). The result of a mutation includes, but is not limited to, the production of a new characteristic, property, function, phenotype, or trait not found in the protein encoded by the parent sequence.

[0033] Nanopore: As used herein, the term "nanopore" generally refers to a pore, channel, or passageway formed or otherwise provided in a membrane. The membrane may be an organic membrane, such as a lipid bilayer, or a synthetic membrane, such as a membrane made of a polymeric material. The membrane may be a polymeric material. The nanopore may be positioned adjacent to or in close proximity to sensing circuitry, such as a complementary metal-oxide semiconductor (CMOS) or field-effect transistor (FET) circuitry, or an electrode connected to such sensing circuitry. In some examples, the nanopore has a characteristic width or diameter on the order of about 0.1 nanometers (nm) to about 1000 nm. Some nanopores are proteins. Alpha-hemolysin is an example of a nanopore-forming polypeptide.

[0034] Narrow channel alpha-hemolysin nanopore: As used herein, a narrow channel alpha-hemolysin nanopore is an alpha-hemolysin nanopore that comprises at least six narrow channel alpha-hemolysin subunits.

[0035] Narrow channel alpha-hemolysin polypeptide: As used herein, a narrow channel alpha-hemolysin polypeptide is an alpha-hemolysin polypeptide that includes at least one narrow channel alpha-hemolysin subunit.

[0036] Narrow channel alpha-hemolysin subunit: As used herein, a narrow channel alpha-hemolysin subunit is an alpha-hemolysin subunit that, when aligned with SEQ ID NO: 1, has (a) an amino acid (e.g., glutamic acid, lysine, arginine, or glutamine) having a side chain longer than that of asparagine at a position corresponding to E111 of SEQ ID NO: 1, (b) an amino acid (e.g., glutamic acid, lysine, arginine, or glutamine) having a side chain longer than that of asparagine at a position corresponding to K147 of SEQ ID NO: 1, and / or (c) an amino acid (leucine, isoleucine, valine, methionine, etc.) having a side chain longer than that of alanine (at a position corresponding to M113 of SEQ ID NO: 1).

[0037] Nucleic Acid Molecule: The term "nucleic acid molecule" includes RNA, DNA, and cDNA molecules. It is understood that, as a result of the degeneracy of the genetic code, numerous nucleotide sequences encoding a given protein, such as alpha-hemolysin and / or variants thereof, can be produced. The present invention contemplates all possible variant nucleotide sequences encoding variant alpha-hemolysins, all of which are possible given the degeneracy of the genetic code.

[0038] Percent identity: The term "% identity" refers to the level of nucleic acid or amino acid sequence identity between a nucleic acid sequence encoding any one of the polypeptides of the present invention or an amino acid sequence of a polypeptide of the present invention when aligned using a sequence alignment program. For example, as used herein, 80% identity encompasses homologs of a given sequence having greater than 80% identity over the length of the given sequence. Exemplary levels of identity include, but are not limited to, 75%, 80%, 85%, 90%, 95%, 98% or more identity to a given sequence, e.g., the coding sequence of any one of the polypeptides of the present invention described herein. Exemplary computer programs that can be used to determine identity between two sequences include, but are not limited to, BLAST programs publicly available on the Internet, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN. See also Altschul, et al., 1990 and Altschul, et al., 1997. Sequence searches are typically performed using the BLASTN program when evaluating a given nucleic acid sequence against nucleic acid sequences in GenBank DNA sequences and other public databases. The BLASTX program can be used to search nucleic acid sequences translated in all reading frames against amino acid sequences in GenBank protein sequences and other public databases. Both BLASTN and BLASTX are run using default parameters of an open gap penalty of 11.0 and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. (See, e.g., Altschul, S.F., et al., Nucleic Acids Res. 25:3389-3402, 1997.) Alignment of selected sequences to determine "percent identity" between two or more sequences can be performed, for example, using the CLUSTAL-W program in MacVector version 13.0.7, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

[0039] Promoter: As used herein, the term "promoter" refers to a nucleic acid sequence that functions to direct the transcription of a downstream gene. A promoter is generally appropriate for the host cell in which the target gene is being expressed. A promoter, along with other transcriptional and translational regulatory nucleic acid sequences, "control sequences," are necessary to express a given gene. Generally, transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.

[0040] Purified: As used herein, "purified" means that a molecule is present in a sample at a concentration of at least 95% or at least 98% by weight of the sample in which it is contained.

[0041] Tag: As used herein, the term "tag" refers to a nanopore-detectable moiety, which can be an atom or molecule, or a collection of atoms or molecules. The label can provide an optical, electrochemical, magnetic, or electrostatic (e.g., inductive, capacitive) signature that can be detected using the nanopore. Typically, when a nucleotide is attached to a tag, the tag is referred to as a "tagged nucleotide."

[0042] Mutant: As used herein, the term "mutant" refers to a polypeptide that exhibits an altered primary amino acid sequence when compared to the wild-type polypeptide from which it is derived.

[0043] Mutant alpha-hemolysin polypeptide: The term "variant alpha-hemolysin polypeptide" or "variant αHL polypeptide" refers to an alpha-hemolysin polypeptide that includes at least one variant alpha-hemolysin subunit.

[0044] Mutant alpha-hemolysin subunit: The term "mutant alpha-hemolysin" or "mutant αHL" refers to an alpha-hemolysin polypeptide that has one or more substitutions, insertions, or deletions relative to SEQ ID NO:1.

[0045] Mutant narrow channel alpha-hemolysin nanopore: The term "mutant narrow channel alpha-hemolysin nanopore" means a narrow channel alpha-hemolysin nanopore in which at least one of the six narrow channel alpha-hemolysin subunits is a mutant narrow channel alpha-hemolysin subunit.

[0046] Mutant narrow channel alpha-hemolysin polypeptide: The term "mutant narrow channel alpha-hemolysin polypeptide" is an alpha-hemolysin polypeptide that includes at least one mutant narrow channel alpha-hemolysin subunit.

[0047] Mutant narrow channel alpha-hemolysin subunit: The term "mutant narrow channel alpha-hemolysin subunit" refers to a narrow channel alpha-hemolysin subunit that has one or more substitutions, insertions, or deletions compared to SEQ ID NO:1.

[0048] Vector: As used herein, the term "vector" refers to a nucleic acid construct designed for transfer between different host cells. An "expression vector" refers to a vector capable of incorporating and expressing heterologous DNA fragments in foreign cells. Many prokaryotic and eukaryotic expression vectors are commercially available. The selection of an appropriate expression vector is within the knowledge of one of ordinary skill in the art.

[0049] Wild-type alpha-hemolysin: As used herein, the term "wild-type alpha-hemolysin" refers to an alpha-hemolysin subunit comprising SEQ ID NO:1.

[0050] II. Nomenclature In the present specification and claims, the conventional one-letter and three-letter codes for amino acid residues are used.

[0051] For ease of reference, variations in this application are described using the following nomenclature: original amino acid(s); position(s); replacement amino acid(s). According to this nomenclature, for example, the substitution of valine with lysine at position 149 would be: Val149Lys or V149K It is shown as follows.

[0052] Multiple mutations include: Ala1Lys+Asn47Lys+Glu287Arg or A1K+N47K+E287R and represent mutations at positions 1, 47, and 287, substituting alanine for lysine, asparagine for lysine, and glutamic acid for arginine, respectively. Ranges of amino acid substitutions are indicated by dashes, e.g., the range of glycine residues from residues 127 to 131 is 127-131Gly or 127-133G.

[0053] III. Development Background A "wide channel" alpha-hemolysin nanopore is one in which one or more amino acids forming the constriction site have been modified to a residue with a shorter side chain compared to wild-type alpha-hemolysin. This provides a wider diameter at the constriction site than a pore with the native residues, allowing the tag to flow more freely through the beta-barrel. Table 1 lists the solvent-facing amino acid residues of SEQ ID NO: 1 that form the channel. "#" indicates the position within SEQ ID NO: 1, "AA" indicates the amino acid at the listed position in SEQ ID NO: 1, and "Position" indicates the subregion of the alpha-hemolysin nanopore in which the amino acid is located. [Table 1]

[0054] As can be seen from the figure, in classical "wide channel" alpha-hemolysin, in which three amino acids: E111, M113, and K147 constitute the constriction site, both E111 and K147 are modified to asparagine (i.e., E111N and K147N substitutions relative to SEQ ID NO: 1), and M113 is modified to alanine (M113A substitution relative to SEQ ID NO: 1).

[0055] Wide-channel alpha-hemolysin pores typically have relatively high threading rates but have several limitations. Figure 1 shows two tag-based sequencing-by-synthesis (SBS) runs using wide-channel alpha-hemolysin nanopores. The dark band at the top is the open channel level 101. Tags occupying the nanopore channel are recorded as signal changes (in this case, conductance levels) relative to the open channel, with different tags resulting in different signal changes 102a–102d. However, persistent background bands are frequently observed (103), and convolution of tag signals can occur, which increases as threading speed increases. Furthermore, as shown in (B), suppression of sequencing activity can also be observed (104). Both issues limit the throughput and accuracy of tag-based SBS. Without wishing to be bound by theory, anomalous patterns may arise, at least in part, from threading of template nucleic acids and / or primers into the nanopore. Background levels are believed to be caused by templates and / or primers that are partially inserted into and ejected from the nanopore, while inhibition is caused by templates or primers that fully thread the nanopore.

[0056] The present disclosure demonstrates that pairing narrow-channel alpha-hemolysin nanopores with D127G and D128K substitutions results in relatively long lifetimes and acceptable arrival rates (Figure 2), while significantly reducing the number of pores exhibiting threading (Figure 3).

[0057] IV. Polypeptides Comprising One or More Mutant Narrow Channel Alpha-Hemolysin Subunit(s) In one aspect, an isolated polypeptide is provided that comprises, consists essentially of, or consists of a mutant narrow channel alpha-hemolysin subunit, the subunit comprising a D127G substitution and a D128K substitution relative to SEQ ID NO: 1. Mutant narrow channel alpha-hemolysin subunits generally have at least the following characteristics: (a) at least 75% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; (b) a D127G substitution relative to SEQ ID NO: 1; (c) a D128K substitution relative to SEQ ID NO: 1, and (d) One or more of the following: (d1) an amino acid at a position corresponding to E111 of SEQ ID NO: 1 having a side chain longer than that of asparagine (e.g., glutamic acid, lysine, arginine, glutamine, etc.); (d2) an amino acid at a position corresponding to K147 in SEQ ID NO: 1 (e.g., glutamic acid, lysine, arginine, or glutamine) having a side chain longer than that of asparagine, and / or (d3) an amino acid at a position corresponding to M113 in SEQ ID NO: 1, which has a side chain longer than that of alanine (e.g., leucine, isoleucine, valine, methionine, etc.) It has.

[0058] The combination of substitutions at D127 and D128 compared to SEQ ID NO: 1 and longer amino acids at the constriction site reduces template threading compared to similar pores with wide channels (e.g., pores containing E111N, M113A and K147N), while improving the lifetime of the resulting pores and resulting in acceptable arrival rates.

[0059] In some embodiments described herein, mutant narrow channel alpha-hemolysin nanopores are characterized according to their "threading ratio." In this context, "threading ratio" refers to the percentage of 6:1 narrow channel alpha-hemolysin nanopores with high quality reads (HQR) indicative of a threaded state, where the "6" component is the mutant narrow channel alpha-hemolysin subunit and the "1" component is subunit G2043. The percentage of pores in a threaded state can be calculated as described in Example 5. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a threading ratio of less than 15%. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a threading ratio of less than 10%. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a threading ratio of less than 5%. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a threading ratio of less than 2%.

[0060] In some embodiments described herein, mutant narrow channel alpha-hemolysin nanopores are characterized according to their "lifetime." In this context, "% lifetime" refers to the percentage of 6:1 narrow channel alpha-hemolysin nanopores that remain active toward T40-tagged streptavidin after 1 hour of exposure to a 350 mV sequencing waveform, where the "6" component is the mutant narrow channel alpha-hemolysin subunit and the "1" component is subunit G2043. % lifetime can be calculated as described in Example 4. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a % lifetime greater than 60%. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a % lifetime greater than 70%. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a % lifetime greater than 75%. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a % lifetime greater than 80%.

[0061] In some embodiments described herein, mutant narrow channel alpha-hemolysin nanopores are characterized according to their "arrival rate." In this context, "arrival rate" shall mean the average arrival rate of T40-tagged streptavidin on a 6:1 narrow channel alpha-hemolysin nanopore during 15 minutes of exposure to a 50 Hz, 150 mV waveform, where the "6" component is the mutant narrow channel alpha-hemolysin subunit and the "1" component is subunit G2043. Arrival rates may be calculated as described in Example 4. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have arrival rates of less than 25 ms. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have arrival rates of less than 20 ms. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have arrival rates of less than 15 ms.

[0062] In certain exemplary embodiments, the mutant narrow channel alpha-hemolysin subunit provided herein has 80%, 85%, 90%, 95% or more identity to the sequence set forth as SEQ ID NO: 1, except that the amino acid sequence (a) comprises either or both of a D127G and a D128K substitution relative to SEQ ID NO: 1, (b) further comprises an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, compared to SEQ ID NO: 1, and / or (c) an amino acid at M113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, compared to SEQ ID NO: 1. The amino acids at E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading rate that is less than the threading rate of pore P-0304. In one embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 15%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 10%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 5%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 2%. In yet another embodiment, the mutant narrow channel alpha-hemolysin subunit comprises each of E111, M113, and K147.

[0063] In another embodiment, the mutant narrow channel alpha-hemolysin subunit comprises an amino acid sequence having at least 75%, 80%, 90%, 95%, 98% or more identity to SEQ ID NO:2, wherein the amino acid sequence comprises (a) each of G127 and K128, and (b) an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, relative to SEQ ID NO:2, and / or (c) an amino acid at M113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, relative to SEQ ID NO:2. The amino acids at E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading rate that is less than the threading rate of pore P-0304. In one embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 15%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 10%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 5%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 2%. In yet another embodiment, the mutant narrow channel alpha-hemolysin subunit comprises each of E111, M113, and K147. In yet another embodiment, the mutant narrow channel alpha-hemolysin subunit comprises or consists of SEQ ID NO:2.

[0064] In another embodiment, the mutant narrow channel alpha-hemolysin subunit comprises an amino acid sequence having at least 75%, 80%, 90%, 95%, 98% or more identity to SEQ ID NO:3, wherein the amino acid sequence comprises (a) each of G127 and K128, and (b) an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, relative to SEQ ID NO:3, and / or (c) an amino acid at M113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, relative to SEQ ID NO:3. The amino acids at E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading rate that is less than the threading rate of pore P-0304. In one embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 15%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 10%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 5%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 2%. In yet another embodiment, the mutant narrow channel alpha-hemolysin subunit comprises each of E111, M113, and K147. In yet another embodiment, the mutant narrow channel alpha-hemolysin subunit comprises or consists of SEQ ID NO:3.

[0065] In certain exemplary embodiments, the mutant narrow channel alpha-hemolysin subunit has 75%, 80%, 85%, 90%, 95% or more identity to the sequence set forth as SEQ ID NO:4, except that the amino acid sequence includes (a) each of G127 and K128 of SEQ ID NO:4, (b) an amino acid at either or both of N111 and N147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, compared to SEQ ID NO:4, and / or (c) an amino acid at A113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, compared to SEQ ID NO:4. The amino acids at N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio that is less than the threading ratio of pore P-0304. In one embodiment, the amino acids N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 15%. In another embodiment, the amino acids N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 10%. In another embodiment, the amino acids N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 5%. In another embodiment, the amino acids N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 2%. In yet another embodiment, the mutant narrow channel alpha-hemolysin subunit comprises each of an N111E substitution, an A113M substitution, and an N147K substitution relative to SEQ ID NO:4. In another embodiment, the amino acids N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 2%. In yet another embodiment, the mutant narrow channel alpha-hemolysin subunit comprises each of an N111E substitution, an A113M substitution, and an N147K substitution relative to SEQ ID NO:4.

[0066] In certain exemplary embodiments, the mutant narrow channel alpha-hemolysin subunit has 75%, 80%, 85%, 90%, 95% or more identity to the sequence set forth as SEQ ID NO:5, except that the amino acid sequence includes either or both of (a1) G127 of SEQ ID NO:5, and (a2) a G128K substitution relative to SEQ ID NO:5, (b) an amino acid at either or both of N111 and N147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, relative to SEQ ID NO:5, and / or (c) an amino acid at A113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, relative to SEQ ID NO:5. The amino acids at N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio that is less than the threading ratio of pore P-0304. In one embodiment, the amino acids N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 15%. In another embodiment, the amino acids N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 10%. In another embodiment, the amino acids N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 5%. In another embodiment, the amino acids N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 2%. In yet another embodiment, the mutant narrow channel alpha-hemolysin subunit comprises each of an N111E substitution, an A113M substitution, and an N147K substitution relative to SEQ ID NO:5. In another embodiment, the amino acids N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 2%. In yet another embodiment, the mutant narrow channel alpha-hemolysin subunit comprises each of an N111E substitution, an A113M substitution, and an N147K substitution relative to SEQ ID NO:5.

[0067] In certain exemplary embodiments, the mutant narrow channel alpha-hemolysin subunit has 75%, 80%, 85%, 90%, 95% or more identity to the sequence set forth as SEQ ID NO:6, except that the amino acid sequence includes (a) either or both of a D127G and a D128K substitution relative to SEQ ID NO:6, and (b) an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, relative to SEQ ID NO:6, and / or (c) an amino acid at M113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, relative to SEQ ID NO:6. The amino acids at E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading rate that is less than the threading rate of pore P-0304. In one embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 15%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 10%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 5%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 2%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 2%. In yet another embodiment, the mutant narrow channel alpha-hemolysin subunit comprises each of E111, K147, and M113 relative to SEQ ID NO:6.

[0068] In certain exemplary embodiments, the mutant narrow channel alpha-hemolysin subunit has 75%, 80%, 85%, 90%, 95% or more identity to the sequence set forth as SEQ ID NO:7, except that the amino acid sequence includes (a) either or both of a D127G and a D128K substitution relative to SEQ ID NO:7, and (b) an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, relative to SEQ ID NO:7, and / or (c) an amino acid at M113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, relative to SEQ ID NO:7. The amino acids at E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading rate that is less than the threading rate of pore P-0304. In one embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 15%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 10%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 5%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 2%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 2%. In yet another embodiment, the mutant narrow channel alpha-hemolysin subunit comprises each of E111, K147, and M113 relative to SEQ ID NO:7.

[0069] In certain exemplary embodiments, the mutant narrow channel alpha-hemolysin subunit has 75%, 80%, 85%, 90%, 95% or more identity to the sequence set forth as SEQ ID NO: 8, except that the amino acid sequence includes (a) either or both of a D127G and a D128K substitution relative to SEQ ID NO: 8, and (b) an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, relative to SEQ ID NO: 8, and / or (c) an amino acid at M113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, relative to SEQ ID NO: 8. The amino acids at E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading rate that is less than the threading rate of pore P-0304. In one embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 15%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 10%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 5%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 2%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin subunit has a threading ratio of less than 2%. In yet another embodiment, the mutant narrow channel alpha-hemolysin subunit comprises each of E111, K147, and M113 relative to SEQ ID NO:8.

[0070] The mutant narrow-channel alpha-hemolysin subunits disclosed herein can include additional modifications compared to any of SEQ ID NOS: 1-8 that alter or improve the characteristics of the resulting nanopore. Numerous schemes and mutations for generating alpha-hemolysin mutants useful for nanopore-based sequencing have been described in the art, including, for example, Noskov, Bhattacharya, and Stoddart, PCT / US2015 / 57902, U.S. Pat. No. 10,301,31, PCT / EP2016 / 072220, U.S. Pat. No. 10,227,645, PCT / US2017 / 028636, U.S. Pat. No. 10,351,908, PCT / EP2017 / 065972, U.S. Pat. No. 10,934,582, PCT / EP2019 / 054792, and U.S. Patent Application Publication No. 2020-0385433, each of which is incorporated herein by reference. As one non-limiting example, the mutant narrow channel alpha-hemolysin subunit can contain substitutions that control the ability of a non-oligomerizing alpha-hemolysin subunit to self-oligomerize. For example, an alpha-hemolysin subunit with a substitution at H35 (e.g., an H35G / L / D / E substitution) does not substantially oligomerize at or below room temperature (e.g., 25°C or below), but stably oligomerizes when heated to higher temperatures (e.g., 35°C). Other examples of substitution strategies for controlling self-oligomerization and / or directing specific patterns of oligomerization are disclosed, for example, in WO 2017 / 050718. Another example includes a substitution that reduces the coefficient of variation (CV) of the pore arrival rate, such as D227N. In some embodiments, the mutant narrow channel alpha-hemolysin subunit has a set of modifications to any of SEQ ID NOs: 1-8 that result in a lifespan of 80% or greater. In some embodiments, the mutant narrow channel alpha-hemolysin subunit has a set of modifications to any of SEQ ID NOs: 1-8 that result in an arrival rate of 15 ms or less. In some embodiments, the mutant narrow channel alpha-hemolysin subunit has a set of modifications to any of SEQ ID NOs: 1-8 that result in an 80% or greater lifetime and an arrival rate of 15 ms or less.In yet other embodiments, the mutant narrow channel alpha hemolysin subunit has a set of modifications to any of SEQ ID NOs: 1-8 that result in a lifetime of 80% or greater, an arrival speed of 15 ms or less, and a threading rate of less than 2%.

[0071] The polypeptide can comprise one to seven mutant narrow channel alpha-hemolysin subunits. In one embodiment, the polypeptide disclosed herein comprises a single α mutant narrow channel alpha-hemolysin subunit. In another embodiment, the polypeptide is a linked alpha-hemolysin polypeptide comprising two to seven mutant narrow channel alpha-hemolysin subunits, expressly including a polypeptide comprising two narrow channel alpha-hemolysin subunits, a polypeptide comprising narrow channel alpha-hemolysin subunits, a polypeptide comprising four narrow channel alpha-hemolysin subunits, a polypeptide comprising five narrow channel alpha-hemolysin subunits, a polypeptide comprising six narrow channel alpha-hemolysin subunits, and a polypeptide comprising seven narrow channel alpha-hemolysin subunits. Exemplary methods and considerations for producing linked alpha-hemolysin polypeptides are disclosed, for example, by Hammerstein and U.S. Patent Application Publication No. 2017-0088890 A1. In one embodiment, each narrow channel alpha-hemolysin subunit of the linked narrow channel alpha-hemolysin polypeptide is separated from the other narrow channel alpha-hemolysin subunit(s) by a linker sequence. In one embodiment, the linker sequence is a flexible linker. Exemplary flexible linkers are disclosed, for example, by Hammerstein and Chen.

[0072] The polypeptide may also contain moieties useful for purifying the polypeptide, such as epitope tags, protease cleavage sites, and the like.

[0073] The polypeptide may also include an entity (herein referred to as a "binding moiety") useful for binding other active agents (such as a polymerase) to the polypeptide. Exemplary conjugation moieties include, for example, the SpyTag / SpyCatcher peptide system (Zakeri et al. PNAS 109:E690-E697 2012), native chemical ligation systems (Thapa et al., Molecules 19:14461-14483 2014), sortase systems (Wu and Guo, J Carbohydr Chem 31:48-66 2012; Heck et al., Appl Microbiol Biotechnol 97:461-475 2013), transglutaminase systems (Dennler et al., Bioconjug Chem 25:569 578 2014), and formylglycine conjugation systems (Rashidian et al., Bioconjug Chem 24:1277-1294). 2013), click chemistry attachment systems, or components of other chemical ligation techniques known in the art.

[0074] V. Nucleic Acids, Expression Cassettes, Expression Vectors, Recombinant Cells, and Methods for Producing Polypeptides In another aspect of the disclosure, an isolated polynucleotide is provided, the isolated polynucleotide comprising a nucleotide sequence encoding an isolated polypeptide described in Section IV. In one embodiment, the nucleic acid is an expression cassette comprising a nucleotide sequence encoding the polypeptide linked to a set of nucleic acid transcription elements (e.g., promoter, enhancer, start and stop codons, ribosome binding site, etc.) sufficient for transcription of the nucleotide sequence encoding the polypeptide in a prokaryotic or eukaryotic cell or a cell-free expression system.

[0075] In another aspect, a vector containing a nucleotide encoding a polypeptide is provided. The vector can be, for example, a cloning vector or an expression vector. Suitable vector backbones include those routinely used in the art, such as plasmids, artificial chromosomes, BACs, or PACs. Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, Wisconsin), Clonetech (Palo Alto, California), Stratagene (La Jolla, California), and Invitrogen / Life Technologies (Carlsbad, California). Vectors typically contain one or more regulatory regions. Regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription initiation sites, termination sequences, polyadenylation sequences, and the like.

[0076] In another embodiment, a host cell comprising the expression vector is provided. For example, a host cell useful for producing a polypeptide is transformed or transiently or stably transfected with the expression vector. In another aspect of the disclosure, a method for preparing a mutant alpha-hemolysin polypeptide described herein is provided, comprising: (a) culturing a host cell comprising an expression vector disclosed herein under conditions sufficient to induce expression of the polypeptide; and (b) purifying the polypeptide from the host cell. Such methods are well known in the art, and many systems therefor are commercially available.

[0077] VI. Mutant narrow-channel alpha-hemolysin nanopore In one embodiment, a mutant narrow channel alpha-hemolysin nanopore or hybrid nanopore is provided that comprises a mutant narrow channel alpha-hemolysin nanopore as a biological component, the mutant narrow channel alpha-hemolysin nanopore having the following properties: (a) a lower threading rate than nanopore P-0304; (b) an extended lifetime compared to nanopore P-0031 (see Table 2). [Table 2]

[0078] In some embodiments, the mutant narrow-channel alpha-hemolysin nanopore further has an attainment rate that is comparable to or better than the attainment rate of pore P-0411 or P-0414. [Table 3] [Table 4]

[0079] Each subunit of a mutant narrow-channel alpha-hemolysin nanopore can be identical (referred to as a "homoheptamer"), or at least one subunit of the heptamer can have a different primary amino acid sequence and / or a modification, such as a modification to facilitate polypeptide binding, compared to the other subunits (referred to as a "heteroheptamer"). A heteroheptameric alpha-hemolysin nanopore may be referred to herein by the ratio of different subunit species used in the nanopore. For example, a "6:1 alpha-hemolysin nanopore" has six identical subunits and one different subunit. In such an example, reference to "6" components shall mean each of the six identical subunits, and reference to "1" component shall mean one different subunit. In some embodiments, each subunit of the alpha-hemolysin nanopore is arranged in a polypeptide that does not include additional subunits (referred to herein as "non-oligomerizing subunits"). An exemplary method for producing homoheptamers and heteroheptamers from non-oligomerized alpha-hemolysin subunits is disclosed in U.S. Patent Application Publication No. 2017-0088890 A1. For example, a 6:1 heteroheptamer can be produced by mixing two different subunit preparations (e.g., one in which the subunit is modified with an entity that can be used to bind to a polymerase and another entity that does not contain such a modification). The entity intended to produce the resulting heptamer is provided in molar excess over the other heptamer in the presence of a membrane, and the mixture is incubated overnight at 37°C in an aqueous solution (e.g., 20 mM Tris-HCl pH 8.0, 200 mM NaCl or 20 mM sodium citrate pH 3, 400 mM NaCl, 0.1% TWEEN 20 + 0.2 M TMAO). The resulting heptamer is then purified by cation exchange chromatography. In some embodiments, oligomerization is carried out in the presence of trimethylamine N-oxide (TMAO), such as 0.1-5 M TMAO, 1-4 M TMAO, etc. In other embodiments, the nanopore comprises at least one set of linked subunits.Exemplary methods for generating alpha-hemolysin nanopores from tethered alpha-hemolysin subunits are disclosed, for example, in Hammerstein and US Patent Application Publication No. 2017-0088890 A1.

[0080] The mutant narrow-channel alpha-hemolysin nanopore described herein may also include a polymerase bound thereto. In one embodiment, a single polymerase is bound to the mutant narrow-channel alpha-hemolysin nanopore. Exemplary polymerases include those derived from Clostridium phage phiCPV4 DNA polymerase (described by GenBank accession number YP_00648862 and referred to herein as "Pol6"), phi29 DNA polymerase, T7 DNA pol, T4 DNA pol, E. coli DNA pol 1, Klenow fragment, T7 RNA polymerase, and E. coli RNA polymerase, as well as associated subunits and cofactors. In one embodiment, the polymerase is a Pol6-derived DNA polymerase. Exemplary Pol6 derivatives useful for nanopore-based sequencing are disclosed, for example, in U.S. Patent Application Publication Nos. 2016 / 0222363, 2016 / 0333327, 2017 / 0267983, 2018 / 0094249, and 2018 / 0245147. Exemplary methods for attaching polymerases to alpha-hemolysin nanopores include the SpyTag / SpyCatcher peptide system (Zakeri et al. PNAS 109:E690-E697 2012), native chemical ligation systems (Thapa et al., Molecules 19:14461-14483 2014), sortase systems (Wu and Guo, J Carbohydr Chem 31:48-66 2012; Heck et al., Appl Microbiol Biotechnol 97:461-475 2013), transglutaminase systems (Dennler et al., Bioconjug Chem 25:569-578 2014), and formylglycine ligation systems (Rashidian et al., Bioconjug Chem 24:1277-1294). 2013), click chemistry linkage systems, or other chemical ligation techniques known in the art.In one embodiment, the polymerase is bound to one amino acid side chain of the alpha-hemolysin subunit. In one embodiment, the alpha-hemolysin nanopore is a 6:1 nanopore and the polymerase is bound to the "1" component. In one embodiment, the alpha-hemolysin nanopore is a 6:1 nanopore and the polymerase is bound to the "1" component and the polymerase is a DNA polymerase. In another embodiment, the alpha-hemolysin nanopore is a 6:1 nanopore and the polymerase is bound to the "1" component and the polymerase is a Pol6-derived DNA polymerase.

[0081] In some embodiments described herein, mutant narrow channel alpha-hemolysin nanopores are characterized according to their "threading ratio." In this context, "threading ratio" refers to the percentage of mutant narrow channel alpha-hemolysin nanopores that have high quality reads (HQRs) indicative of a threaded state. The percentage of pores in a threaded state can be calculated as described in Example 5. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a threading ratio of less than 15%. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a threading ratio of less than 10%. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a threading ratio of less than 5%. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a threading ratio of less than 2%.

[0082] In some embodiments described herein, mutant narrow channel alpha-hemolysin nanopores are characterized according to their "lifetime." In this context, "% lifetime" shall mean the percentage of mutant narrow channel alpha-hemolysin nanopores that remain active toward T40-tagged streptavidin after 1 hour of exposure to a 350 mV sequencing waveform. % lifetime may be calculated as described in Example 4. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a % lifetime greater than 60%. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a % lifetime greater than 70%. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a % lifetime greater than 75%. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have a % lifetime greater than 80%.

[0083] In some embodiments described herein, mutant narrow channel alpha-hemolysin nanopores are characterized according to their "arrival rate." In this context, "arrival rate" shall mean the average arrival rate of T40-tagged streptavidin on a mutant narrow channel alpha-hemolysin nanopore during 15 minutes of exposure to a 50 Hz, 150 mV waveform. Arrival rates may be calculated as described in Example 4. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have an arrival rate of less than 25 ms. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have an arrival rate of less than 20 ms. In some embodiments, mutant narrow channel alpha-hemolysin nanopores have an arrival rate of less than 15 ms.

[0084] In one embodiment, the mutant narrow channel alpha-hemolysin nanopore comprises 1, 2, 3, 4, 5, 6, or 7 narrow channel alpha-hemolysin subunits having the following characteristics: (a) at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:1; (b) a D127G substitution relative to SEQ ID NO:1; (c) a D128K substitution relative to SEQ ID NO:1, and (d) one or more of: (d1) an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, compared to SEQ ID NO:1, and / or (d2) an amino acid at M113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, compared to SEQ ID NO:1. The amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio that is less than the threading ratio of pore P-0304. In one embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 15%. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 10%. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 5%. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 2%.In yet another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least the "6" components include: (a1) either or both of a D127G substitution and a D128K substitution relative to SEQ ID NO: 1; (a2) an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, relative to SEQ ID NO: 1; and / or (a3) ​​an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, relative to SEQ ID NO: 1; and / or (a4) an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, relative to SEQ ID NO: 1; , valine, or methionine, and (b) the component "1" comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO: 1, and is further bound to or adapted to bind to a polymerase. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least "6" components comprise an amino acid sequence having (a1) a D127G substitution relative to SEQ ID NO:1, (a2) a D128K substitution relative to SEQ ID NO:1, and (a3) ​​each of E111, M113, and K147 of SEQ ID NO:1; and (b) "1" component comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:1, and is further bound to or adapted to bind to a polymerase.

[0085] In one embodiment, the mutant narrow channel alpha-hemolysin nanopore comprises 1, 2, 3, 4, 5, 6, or 7 narrow channel alpha-hemolysin subunits having the following characteristics: (a) at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:2; (b) comprises each of G127 and K128 of SEQ ID NO:2; (c) further comprises (c1) an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, compared to SEQ ID NO:2, and / or (c2) an amino acid at M113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, compared to SEQ ID NO:2. The amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio that is less than the threading ratio of pore P-0304. In one embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 15%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 10%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 5%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 2%. In yet another embodiment, the narrow channel alpha hemolysin subunit(s) comprises each of E111, M113, and K147.In yet another embodiment, the narrow channel alpha hemolysin subunit comprises or consists of SEQ ID NO:2.In yet another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least the "6" components (a1) comprise each of G127 and K128 relative to SEQ ID NO:2, (a2) further comprise an amino acid at either or both of E111 and K147 having a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, relative to SEQ ID NO:2, and / or (a3) ​​an amino acid at M113 having a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, relative to SEQ ID NO:2, and (b) the "1" component comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:2, and is further bound to or adapted to bind to a polymerase. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least "6" components comprise each of G127, K128, E111, M113, and K147 of SEQ ID NO:2, and (b) "1" component comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:2, and is further bound to or adapted to bind to a polymerase.

[0086] In one embodiment, the mutant narrow channel alpha-hemolysin nanopore comprises 1, 2, 3, 4, 5, 6, or 7 narrow channel alpha-hemolysin subunits having the following characteristics: (a) at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:3; (b) comprises each of G127 and K128 of SEQ ID NO:3; (c) further comprises (c1) an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, compared to SEQ ID NO:3, and / or (c2) an amino acid at M113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, compared to SEQ ID NO:3. The amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio that is less than the threading ratio of pore P-0304. In one embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 15%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 10%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 5%. In another embodiment, the amino acids E111, K147, and / or M113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 2%. In yet another embodiment, the narrow channel alpha hemolysin subunit(s) comprises each of E111, M113, and K147.In yet another embodiment, the narrow channel alpha hemolysin subunit(s) comprises or consists of SEQ ID NO:3.In yet another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least the "6" components (a1) comprise each of G127 and K128 relative to SEQ ID NO:3, (a2) further comprise an amino acid at either or both of E111 and K147 having a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, relative to SEQ ID NO:3, and / or (a3) ​​an amino acid at M113 having a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, relative to SEQ ID NO:3, and (b) the "1" component comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:3, and is further bound to or adapted to bind to a polymerase. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least "6" components comprise each of G127, K128, E111, M113, and K147 of SEQ ID NO:3, and (b) "1" component comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:3, and is further bound to or adapted to bind to a polymerase.

[0087] In one embodiment, the mutant narrow channel alpha-hemolysin nanopore comprises 1, 2, 3, 4, 5, 6, or 7 narrow channel alpha-hemolysin subunits having the following characteristics: (a) at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO: 4; (b) each of G127 and K128 of SEQ ID NO: 4; (c1) further comprising an amino acid at either or both of N111 and N147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, compared to SEQ ID NO: 4; and / or (c2) further comprising an amino acid at A113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, compared to SEQ ID NO: 4. The amino acids N111, N147, and / or A113 are selected so that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio that is less than that of pore P-0304. In one embodiment, the amino acids N111, N147, and / or A113 are selected so that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 15%. In another embodiment, the amino acids N111, N147, and / or A113 are selected so that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 10%. In another embodiment, the amino acids N111, N147, and / or A113 are selected so that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 5%. In another embodiment, the amino acids N111, N147, and / or A113 are selected so that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 2%. In yet another embodiment, the polypeptide comprises each of an N111E substitution, an A113M substitution, and an N147K substitution relative to SEQ ID NO: 4. In yet another embodiment, the polypeptide comprises each of G127 and K128 relative to SEQ ID NO: 4, and further comprises each of an N111E, A113M, and N147K substitution relative to SEQ ID NO: 4.In yet another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least the "6" components (a1) comprise each of G127 and K128 relative to SEQ ID NO:4, (a2) further comprise an amino acid at either or both of N111 and N147 having a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, relative to SEQ ID NO:4, and / or (a3) ​​an amino acid at A113 having a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, relative to SEQ ID NO:4, and (b) the "1" component comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:4, and is further bound to or adapted to bind to a polymerase. In yet another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least the "6" components comprise each of G127 and K128 relative to SEQ ID NO:4, and further comprise each of an N111E substitution, an N147K substitution, and an A113M substitution relative to SEQ ID NO:4; and (b) the "1" component comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:4, and is further bound to or adapted to bind to a polymerase.

[0088] In one embodiment, the mutant narrow channel alpha-hemolysin nanopore comprises 1, 2, 3, 4, 5, 6, or 7 narrow channel alpha-hemolysin subunits having the following characteristics: (a) at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO: 5; and (b) ( The mutant narrow channel alpha-hemolysin nanopore comprises (b1) G127 of SEQ ID NO:5, and (b2) a G128K substitution relative to SEQ ID NO:5, and (c) further comprises (c1) an amino acid at either or both N111 and N147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, relative to SEQ ID NO:5, and / or (c2) an amino acid at A113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, relative to SEQ ID NO:5. The amino acids at N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio that is less than that of pore P-0304. In one embodiment, the amino acids at N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 15%. In another embodiment, the amino acids N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 10%. In another embodiment, the amino acids N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 5%. In another embodiment, the amino acids N111, N147, and / or A113 are selected such that the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 2%. In yet another embodiment, the polypeptide comprises each of an N111E substitution, an A113M substitution, and an N147K substitution relative to SEQ ID NO:5. In yet another embodiment, the polypeptide comprises G127 of SEQ ID NO:5, and a G128K substitution, an N111E substitution, an A113M substitution, and an N147K substitution relative to SEQ ID NO:5.In yet another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least the "6" components comprise: (a1) G127 of SEQ ID NO:5; (a2) a G128K substitution relative to SEQ ID NO:5; (a3) ​​an amino acid at either or both of N111 and N147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, relative to SEQ ID NO:5; and (a4) an amino acid at A113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, relative to SEQ ID NO:5; and (b) the "1" component comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:5, and is further bound to or adapted to bind to a polymerase. In yet another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least "6" components comprise G127 of SEQ ID NO:5 and each of a G128K substitution, an N111E substitution, an N147K substitution, and an A113M substitution relative to SEQ ID NO:5; and (b) "1" component comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:5, and is further bound to or adapted to bind to a polymerase.

[0089] In one embodiment, the mutant narrow channel alpha-hemolysin nanopore comprises 1, 2, 3, 4, 5, 6, or 7 narrow channel alpha-hemolysin subunits having the following characteristics: (a) at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:6; (b) either or both of a D127G and a D128K substitution relative to SEQ ID NO:6; (c) further comprising (c1) an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, compared to SEQ ID NO:6; and / or (c2) an amino acid at M113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, compared to SEQ ID NO:6. The amino acids E111, K147 and / or M113 are selected such that the percentage of nanopores exhibiting a threaded state is reduced compared to pore P-0304. In one embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 15%. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 10%. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 5%. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 2%.In yet another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least the "6" component comprises: (a1) either or both of a D127G and a D128K substitution relative to SEQ ID NO: 6; (a2) an amino acid having a side chain longer than asparagine, e.g., glutamic acid, lysine, arginine, or glutamine, at either or both of E111 and K147 relative to SEQ ID NO: 6; and / or (a3) ​​an amino acid having a side chain longer than alanine, e.g., glutamic acid, lysine, arginine, or glutamine, at M113 relative to SEQ ID NO: 6. (b) component "1" comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:6, and is further bound to or adapted to bind to a polymerase. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least "6" components comprise an amino acid sequence having (a1) a D127G substitution relative to SEQ ID NO:6, (a2) a D128K substitution relative to SEQ ID NO:6, and (a3) ​​each of E111, M113, and K147 of SEQ ID NO:6; and (b) "1" component comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:6, and is further bound to or adapted to bind to a polymerase.

[0090] In one embodiment, the mutant narrow channel alpha-hemolysin nanopore comprises 1, 2, 3, 4, 5, 6, or 7 narrow channel alpha-hemolysin subunits having the following characteristics: (a) at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO: 7; (b) one or both of a D127G and a D128K substitution relative to SEQ ID NO: 7; (c) further comprising: (c1) an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, compared to SEQ ID NO: 7; and / or (c2) an amino acid at M113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, compared to SEQ ID NO: 7. The amino acids E111, K147 and / or M113 are selected such that the percentage of nanopores exhibiting a threaded state is reduced compared to pore P-0304. In one embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 15%. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 10%. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 5%. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 2%.In yet another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least the "6" component comprises: (a1) either or both of a D127G and a D128K substitution relative to SEQ ID NO: 7; (a2) an amino acid having a side chain longer than asparagine, e.g., glutamic acid, lysine, arginine, or glutamine, at either or both of E111 and K147 relative to SEQ ID NO: 7; and / or (a3) ​​an amino acid having a side chain longer than alanine, e.g., glutamic acid, lysine, arginine, or glutamine, at M113 relative to SEQ ID NO: 7. (b) component "1" comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:7, and is further bound to or adapted to bind to a polymerase. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least "6" components comprise an amino acid sequence having (a1) a D127G substitution relative to SEQ ID NO:7, (a2) a D128K substitution relative to SEQ ID NO:7, and (a3) ​​each of E111, M113, and K147 of SEQ ID NO:7; and (b) "1" component comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:7, and is further bound to or adapted to bind to a polymerase.

[0091] In one embodiment, the mutant narrow channel alpha-hemolysin nanopore comprises 1, 2, 3, 4, 5, 6, or 7 narrow channel alpha-hemolysin subunits having the following characteristics: (a) at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:8; (b) one or both of a D127G and a D128K substitution relative to SEQ ID NO:8; (c) further comprising: (c1) an amino acid at either or both of E111 and K147 that has a longer side chain than asparagine, such as glutamic acid, lysine, arginine, or glutamine, compared to SEQ ID NO:8; and / or (c2) an amino acid at M113 that has a longer side chain than alanine, such as leucine, isoleucine, valine, or methionine, compared to SEQ ID NO:8. The amino acids E111, K147 and / or M113 are selected such that the percentage of nanopores exhibiting a threaded state is reduced compared to pore P-0304. In one embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 15%. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 10%. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 5%. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore has a threading ratio of less than 2%.In yet another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least the "6" component comprises: (a1) either or both of a D127G and a D128K substitution relative to SEQ ID NO:8; (a2) an amino acid having a side chain longer than asparagine, e.g., glutamic acid, lysine, arginine, or glutamine, at either or both of E111 and K147 relative to SEQ ID NO:8; and / or (a3) ​​an amino acid having a side chain longer than alanine, e.g., glutamic acid, lysine, arginine, or glutamine, at M113 relative to SEQ ID NO:8. (b) component "1" comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:8, and is further bound to or adapted to bind to a polymerase. In another embodiment, the mutant narrow channel alpha-hemolysin nanopore is a 6:1 heteroheptamer, wherein (a) at least "6" components comprise an amino acid sequence having (a1) a D127G substitution relative to SEQ ID NO:8, (a2) a D128K substitution relative to SEQ ID NO:8, and (a3) ​​each of E111, M113, and K147 of SEQ ID NO:8; and (b) "1" component comprises an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity to SEQ ID NO:8, and is further bound to or adapted to bind to a polymerase.

[0092] VII. SBS Sequencing Systems and Methods In one embodiment, a system for performing nucleic acid sequencing by synthesis (SBS) is provided, the system comprising: (a) a mutant narrow-channel alpha-hemolysin nanopore as disclosed in Section VI; (b) a nucleic acid polymerase associated with the nanopore; (c) a set of nucleotide oligophosphates disposed in an electrolyte solution, the nucleotide oligophosphates comprising positively charged tags capable of threading the nanopore of a); and (d) at least one electrode configured to record characteristics of the current flowing through the channel.

[0093] FIG. 4 shows an exemplary embodiment of a nanopore sequencing complex 500 for performing tag-based SBS nucleotide sequencing. An electrically resistive barrier 501 separates a bulk electrolyte 502 from a second electrolyte 503. A heptameric alpha-hemolysin nanopore 504 as disclosed herein is disposed within the electrically resistive barrier 501, and a channel of the nanopore 505 provides a pathway through which ions can flow between the bulk electrolyte 502 and the second electrolyte 503. A working electrode 506 is disposed on the side of the electrically resistive barrier 501 containing the second electrolyte 503 (referred to as the "trans side" of the electrically resistive barrier) and is positioned near the heptameric alpha-hemolysin nanopore 504. A counter electrode 507 is disposed on the side of the electrically resistive barrier 501 containing the bulk electrolyte 502 (referred to as the "cis side" of the electrically resistive barrier). A signal source 508 is adapted to apply a voltage signal between the working electrode 506 and the counter electrode 507. A polymerase 509 associates with a heptameric alpha-hemolysin nanopore 504, and a primed template nucleic acid 510 associates with the polymerase. Bulk electrolyte 502 contains four different polymer-tagged nucleoside oligophosphates 511 (tags shown as 511a). Polymerase 509 catalyzes the incorporation of polymer-tagged nucleotides 511 into the template amplicon. Once polymer-tagged nucleoside oligophosphates 511 are properly complexed with polymerase 509, tag 511a can be drawn (e.g., loaded) into the nanopore by an electrical force, such as the force generated in the presence of an electric field generated by an applied voltage across electrically resistive barrier 501 and / or nanopore 504. Tag 511a occupies the channel of nanopore 504 but affects the flow of ions through nanopore 504, thereby generating an ion-blocking signal 512. Each nucleotide 511 has a unique polymer tag 511a that generates a unique ion-blocking signal due to the different chemical structure and / or size of the tag 511a. By identifying the unique ion-blocking signal 512, the identity of the unique tag 511a (and therefore the nucleotide 510 with which it is associated) can be identified. This process is repeated iteratively as each nucleotide 510 is incorporated into the amplicon. [Example]

[0094] VIII. Working Examples Example 1: Generation and Expression of Mutant Alpha-Hemolysin Polypeptides DNA encoding wild-type alpha-hemolysin having the amino acid sequence of SEQ ID NO:1 was purchased from a commercial source. Sequence modifications were performed by site-directed mutagenesis using the QuikChange Multi Site-Directed Mutagenesis Kit (Agilent, La Jolla, CA, USA) to generate nucleic acids encoding SEQ ID NOs:2-8, each with a C-terminal linker / TEV / HisTag. SEQ ID NOs:5, 7, and 8 were also expressed with a C-terminal SpyTag. Escherichia coli (E. coli) BL21 DE3 cells (ThermoFisher, Waltham, MA, USA) were transformed with the pET-26b(+) vector, and the transformed cells were cultured for protein expression according to the manufacturer's instructions. Cultured cells were harvested by centrifugation and then lysed by sonication. Polypeptides bearing cleavable epitope tags were purified from the lysate by affinity column chromatography (TALON® metal affinity resin, Takara Bio USA). The epitope tag was cleaved, and the mutant alpha-hemolysin polypeptides were separated from the cleaved tag and uncleaved polypeptide by affinity column chromatography (TALON® metal affinity resin, Takara Bio USA). Proteins were stored at 4°C if used within 5 days, otherwise 8% trehalose was added and stored at -80°C. The amino acid sequences of the mutant alpha-hemolysin polypeptides thus produced and their alignment with SEQ ID NO: 1 are shown in Figure 4. The sequences shown include the alpha-hemolysin subunit sequences, excluding the associated SpyTag sequence.

[0095] Example 2: Nanopore assembly Using approximately 10 mg of total protein, the following combinations of alpha-hemolysin / SpyTag and desired alpha-hemolysin mutant proteins were mixed together in a 9:1 ratio (w / w) of subunit 1 to subunit 2 to form a heptameric mixture: [Table 5]

[0096] Diphytanoylphosphatidylcholine (DPhPC) lipid was solubilized in either 50 mM Tris, 200 mM NaCl, pH 8 or 150 mM KCl, 30 mM HEPES, pH 7.5 to a final concentration of 50 mg / ml and added to the α-HL subunit mixture to a final concentration of 5 mg / ml. The α-hemolysin subunit mixture was incubated at 37°C for at least 60 minutes. The resulting lipid-protein mixture was then solubilized by adding n-octyl-β-D-glucopyranoside (βOG) to a final concentration of 5% (wt / vol). The sample was centrifuged to remove protein aggregates, leaving behind the lipid complexes, and the supernatant was collected for further purification. The heptameric mixture was then subjected to cation exchange purification, and the elution fraction corresponding to a 6:1 ratio of subunit 1 to subunit 2 was collected.

[0097] Example 3: Pore arrival rate and lifespan To measure the lifetime of the generated nanopores, the 6:1 pores generated in Example 2 were inserted into a sequencing array described in PCT / US14 / 61853. Streptavidin beads conjugated to polydeoxythymidine 40-mers (T40 tags) were flowed through the array, and a sequencing waveform at 350 mV was applied to the system for 1 hour. As the charge polarity changed, tags were inserted (leading to an "inserted state") and ejected from the pore (leading to an "open channel"), as observed by monitoring the change in conductance of individual pores on the array. Pores were considered "active" as long as they continued to exhibit distinct conductance levels correlating with the inserted state and open channel. The "lifetime" of a pore species was determined by calculating the percentage of single pores that remained active throughout the entire 1-hour run.

[0098] To measure pore arrival rates, the same setup as in the lifetime experiments was used, except that the arrays were subjected to a 50 Hz, 150 mV waveform for 15 minutes. The "arrival rate" of a pore species was determined by: (a) determining the average time between pore insertions for each individual pore on the array, and (b) calculating the average of all the averages determined in (a).

[0099] Each experiment was performed for all pores listed in Table 5. The results are reported in Figure 2, where the lifetime (Y-axis) is plotted against the average arrival rate (X-axis) for each pore type. As can be seen, the two narrow-channel alpha-hemolysin nanopores with the D127G+D128K substitution compared to SEQ ID NO: 1 (P-0411 and P-0414) had relatively high lifetimes (>80%) and acceptable arrival rates (<15 ms) comparable to the wide-channel alpha-hemolysin nanopore (P-0304). The narrow-channel alpha-hemolysin nanopore without the D127G+D128K substitution had a much lower lifetime (<10%). This indicates that the D127G+D128K substitution significantly improves the lifetime of narrow-channel alpha-hemolysin nanopores while maintaining acceptable arrival rates.

[0100] Example 5: Mitigation of threading using narrow channel alpha-hemolysin nanopores To assess the effect of narrow channel alpha-hemolysin nanopores on the extent of template threading, standard sequencing experiments were performed with each of the pores from Example 2.

[0101] Escherichia coli (E. coli) BL21 DE3 cells (ThermoFisher, Waltham, Massachusetts, USA) were transformed with the pPR-IBA2 plasmid (IBA Life Sciences, Germany), which contains an expression cassette encoding the Pol6 DNA polymerase-SpyCatcher fusion protein. Transformed cells were cultured for protein expression according to the manufacturer's instructions, and the fusion protein was purified using a cobalt affinity column. The SpyCatcher-polymerase fusion was incubated with the 6:1 nanopore from Example 2 at a 1:1 molar ratio in 3 mM SrCl2 at 4°C overnight. The polymerase-alpha hemolysin heptameric complex was then purified using size-exclusion chromatography.

[0102] Polymerase-pore template complexes were generated from purified polymerase-alpha hemolysin heptameric complexes as described in U.S. Patent Application Publication No. 2017-0268052 and inserted into sequencing arrays as described in PCT / US14 / 61853. Negatively charged tagged nucleotides were inserted into a 20 mM HEPES pH 8, 300 mM KGlu, 3 mM Mg 2+ The system was run in the presence of a buffer containing ATP and a standard sequencing run was performed. The aggregated data from the sequencing run was filtered for only those pores that produced high-quality reads (HQRs), and the percentage of HQRs that showed evidence of template threading was calculated.

[0103] This experiment was repeated for a wide-channel alpha-hemolysin nanopore (pore P-0304) and two narrow-channel alpha-hemolysin nanopores with D127G+D128K substitutions (pores P-0411 and P-0414). As shown in Figure 3, P-0304 had over 15% of its pores indicative of a threaded state, while both P-0411 and P-0414 had less than 2% of their pores indicative of a threaded state.

[0104] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0105] Sequence Listing Free Text SEQ ID NO: 1 (mature WT aHL; AAA26598) ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK 50 KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100 YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGHTLKYVQ 150 PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200 NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250 RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293

[0106] SEQ ID NO: 2 (aHL mutant G2055; D13A + H35G + D127G + D128K + H144A + V149K) ADSDINIKTG TT A IGSNTTV KTGDLVTYDK ENGM G KKVFY SFIDDKNHNK 50 KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100 YYPRNSIDTK EYMSTLTYGF NGNVTG Goalkeeper TG KIGGLIGANV SIG A TLKY K Q 150 PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200 NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250 RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293

[0107] SEQ ID NO: 3 (aHL mutant G2097; H35G+N47K+D127G+D128K+H144A+V149K) ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGM G KKVFY SFIDDK K HNK 50 KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100 YYPRNSIDTK EYMSTLTYGF NGNVTG Goalkeeper TG KIGGLIGANV SIG A TLKY K Q 150 PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200 NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250 RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293

[0108] Sequence number 4 (aHL variant G1742; H35G+N47K+E111N+M113A+D127G+D128K+T129G+K131G+H144A+K147N+V149K) ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGM G KKVFY SFIDDK K HNK<000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ SEQ ID NO: 5 (aHL mutant G1678; H35G+E111N+M113A+D127G+D128G+T129G+K131G+K147N) ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGM G KKVFY SFIDDKNHNK 50 KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100 YYPRNSIDTK N Y A STLTYGF NGNVTG GGG G G IGGLIGANV SIG A TL N YVQ 150 PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200 NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250 RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293

[0110] SEQ ID NO: 6 (aHL mutant G639; H35G+N47K+H144A+V149K) ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGM G KKVFY SFIDDK K HNK 50 KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100 YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIG A TLKY K Q 150 PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200 NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250 RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293

[0111] SEQ ID NO: 7 (aHL mutant G1032;K8D) ADSDINI D TG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK 50 KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100 YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV SIGHTLKYVQ 150 PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200 NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250 RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN 293

[0112] SEQ ID NO: 8 (aHL mutant G2043; D128K+V149K) ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK 50 KLLVIRTKGT IAGQYRVYSE EGANKSGLAW PSAFKVQLQL PDNEVAQISD 100 YYPRNSIDTK EYMSTLTYGF NGNVTGD K TG KIGGLIGANV SIGHTLKY K Q 150 PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR 200 NGSMKAADNF LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE 250 RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE RYKIDWEKEE MTN<000053^{8}>293

[0113] [[ID=^{25}]] Sequence number 9 (WT aHL DNA) ATGGCAGATC TCGATCCCGC GAAATTAATA CGACTCACTA TAGGGAGGCC 50 ACAACGGTTT CCCTCTAGAA ATAATTTTGT TTAACTTTAA GAAGGAGATA 100 TACAAATGGA TTCAGATATT AATATTAAAA CAGGTACAAC AGATATTGGT 150 TCAAATACAA CAGTAAAAAC TGGTGATTTA GTAACTTATG ATAAAGAAAA 200 TGGTATGCAT AAAAAAGTAT TTTATTCTTT TATTGATGAT AAAAATCATA 250 ATAAAAAATT GTTAGTTATT CGTACAAAAG GTACTATTGC AGGTCAATAT 300 Note: There seems to be a formatting issue with the tag in the original text which was translated as <000053^{8}> here. It should be corrected to in the actual translation. Also, etc. are likely some kind of specific identifiers in the patent context and are kept as they are without further semantic translation as per the instructions.AGAGTATATA GTGAAGAAGG TGCTAATAAA AGTGGTTTAG CATGGCCATC 350 TGCTTTTAAA GTTCAATTAC AATTACCTGA TAATGAAGTA GCACAAATTT 400 CAGATTATTA TCCACGTAAT AGTATTGATA CAAAAGAATA TATGTCAACA 450 TTAACTTATG GTTTTAATGG TAATGTAACA GGTGATGATA CTGGTAAAAT 500 TGGTGGTTTA ATTGGTGCTA ATGTTTCAAT TGGTCATACA TTAAAATATG 550 TACAACCAGA TTTTAAAACA ATTTTAGAAA GTCCTACTGA TAAAAAAGTT 600 GGTTGGAAAG TAATTTTTAA TAATATGGTT AATCAAAATT GGGGTCCTTA 650 TGATCGTGAT AGTTGGAATC CTGTATATGG TAATCAATTA TTTATGAAAA 700 CAAGAAATGG TTCTATGAAA GCAGCTGATA ATTTCTTAGA TCCAAATAAA 750 GCATCAAGTT TATTATCTTC AGGTTTTTCT CCTGATTTTG CAACAGTTAT 800 TACTATGGAT AGAAAAGCAT CAAAACAACA AACAAATATT GATGTTATTT 850 ATGAACGTGT AAGAGATGAT TATCAATTAC ATTGGACATC AACTAATTGG 900 AAAGGTACAA ATACTAAAGA TAAATGGACA GATAGAAGTT CAGAAAGATA 950 TAAAATTGAT TGGGAAAAAG AAGAAATGAC AAATGGTCTC AGCGCTTGGA 1000 GCCACCCGCA GTTCGAAAAA TAA 1023

[0114] List of Cited References Akeson et al.,Microsecond timescale discrimination among polycytidylic acid,polyadenylic acid,and polyuridylic acid as homopolymers or as segments within single RNA molecules,Biophys.J.(1999)77:3227-3233. Aksimentiev and Schulten,Imaging a-Hemolysin with Molecular Dynamics:Ionic Conductance,Osmotic Permeability,and the Electrostatic Potential Map,Biophysical Journal(2005)88:3745-3761. Bhattacharya et al.,Rectification of the Current in α-Hemolysin Pore Depends on the Cation Type:The Alkali Series Probed by Molecular Dynamics Simulations and Experiments,The Journal of Physical Chemistry(2011),Vol.115,Issue 10,pp.4255-4264. Butler et al.,Single-molecule DNA detection with an engineered MspA protein nanopore,PNAS(2008)105(52):20647-20652. Chen et al.,Fusion Protein Linkers:Property,Design and Functionality,Advanced Drug Delivery Reviews,15 October 2013,Vol.65,Issue 10,pp.1357-1369. Hammerstein et al.,Subunit dimers of α-hemolysin expand the engineering toolbox for protein Nanopores,Journal of Biological Chemistry,Vol.286,Issue 16,pp.14324-34. Howorka et al.,Sequence-specific detection of individual DNA strands using engineered nanopores,Nat.Biotechnol.,19(2001a),pp.636-639. Howorka et al.,Kinetics of duplex formation for individual DNA strands within a single protein nanopore,Proc.Natl.Acad.Sci.USA,98(2001b),pp.12996-13001. Kasianowicz et al.,Nanometer-scale pores:potential applications for analyte detection and DNA characterization,Proc.Natl.Acad.Sci.USA(1996)93:13770-13773. Korchev et al.,Low Conductance States of a Single Ion Channel are not ‘Closed’,J.Membrane Biol.(1995)147:233-239. Krasilnikov and Sabirov,Ion Transport Through Channels Formed in Lipid Bilayers by Staphylococcus aureus Alpha-Toxin,Gen.Physiol.Biophys.(1989)8:213-222. Meller et al.,Voltage-driven DNA translocations through a nanopore,Phys.Rev.Lett.,86(2001),pp.3435-3438. Movileanu et al.,Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore,Nat.Biotechnol.,18(2000),pp.1091-1095. Nakane et al.,A Nanosensor for Transmembrane Capture and Identification of Single Nucleic Acid Molecules,Biophys.J.(2004)87:615-621. Noskov et al.,Ion Permeation through the α-Hemolysin Channel:Theoretical Studies Based on Brownian Dynamics and Poisson-Nernst-Plank Electrodiffusion Theory,Biophysical Journal(2004),Vol.87,Issue 4,pp.2299-2309 Rhee and Burns, Nanopore sequencing technology: nanopore preparations, TRENDS in Biotech. (2007) 25(4):174-181. Song et al., Structure of Staphylococcal α-HemoIysin, a Heptameric Transmembrane Pore, Science (1996) 274:1859-1866. Stoddart et al., Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore, Proceedings of the National Academy of Sciences of the United States of America (2009), Vol. 106, Issue 19, pp. 7702-7707.

[0115] The entirety of each patent, patent application, publication, document, GENBANK sequence, website, and other published material referenced herein, including all tables, drawings, and figures, is incorporated herein by reference. All patent applications and publications are incorporated herein by reference to the same extent as if each were specifically and individually indicated to be incorporated by reference. Citation of the above patents, patent applications, publications, and documents is not an admission that any of the above is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. All patents and publications mentioned herein are indicative of the level of skill of those skilled in the art to which this invention pertains. Some aspects of the invention are described below. 1. A polypeptide comprising a mutant narrow channel alpha-hemolysin subunit, wherein said mutant narrow channel alpha-hemolysin subunit has at least the following characteristics: (a) at least 75% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; (b) a D127G substitution relative to SEQ ID NO: 1; (c) a D128K substitution relative to SEQ ID NO: 1, and (d) One or more of the following: (d1) an amino acid at a position corresponding to E111 of SEQ ID NO: 1, which has a side chain longer than the side chain of asparagine; (d2) an amino acid at a position corresponding to K147 in SEQ ID NO: 1, which has a side chain longer than the side chain of asparagine, and / or (d3) an amino acid at a position corresponding to M113 in SEQ ID NO: 1, having a side chain longer than that of alanine A polypeptide having the formula: 2. The polypeptide of item 1, wherein the mutant narrow channel alpha-hemolysin subunit has at least 80%, at least 85%, at least 90%, at least 95% or more identity to at least one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. 3. The polypeptide according to item 1 or 2, wherein the amino acid at the position corresponding to E111 is selected from the group consisting of glutamic acid, lysine, arginine, and glutamine. 4. The polypeptide according to item 1 or 2, wherein the amino acid at the position corresponding to E111 is selected from the group consisting of glutamic acid and lysine. 5. The polypeptide according to item 1 or 2, wherein the amino acid residue corresponding to E111 is glutamic acid. 6. The polypeptide according to any one of items 1 to 5, wherein the amino acid at the position corresponding to K147 is selected from the group consisting of glutamic acid, lysine, arginine, and glutamine. 7. The polypeptide according to any one of items 1 to 5, wherein the amino acid at the position corresponding to K147 is selected from the group consisting of glutamic acid and lysine. 8. The polypeptide according to any one of items 1 to 5, wherein the amino acid at the position corresponding to K147 is lysine. 9. The polypeptide according to any one of items 1 to 8, wherein the amino acid at the position corresponding to M113 is selected from the group consisting of leucine, isoleucine, valine, and methionine. 10. The polypeptide according to any one of items 1 to 8, wherein the amino acid at the position corresponding to M113 is methionine. 11. (a) an amino acid sequence having at least 75% identity to SEQ ID NO: 1, (a1) D127G and D128K substitutions relative to SEQ ID NO: 1, and (a2) each of E111, M113, and K147 of SEQ ID NO: 1 an amino acid sequence comprising (b) an amino acid sequence having at least 75% identity to SEQ ID NO: 2, the amino acid sequence including each of G127, K128, E111, M113, and K147 of SEQ ID NO: 2; (c) an amino acid sequence having at least 75% identity to SEQ ID NO: 3, the amino acid sequence including each of G127, K128, E111, M113, and K147 of SEQ ID NO: 3; (d) an amino acid sequence having at least 75% identity to SEQ ID NO:4, (d1) each of G127 and K128 of SEQ ID NO: 4; (d2) N111E substitution relative to SEQ ID NO: 4; (d3) N147K substitution relative to SEQ ID NO: 4, and (d4) A113M substitution relative to SEQ ID NO: 4 an amino acid sequence comprising (e) an amino acid sequence having at least 75% identity to SEQ ID NO:5, (e1) G127 of SEQ ID NO: 5, (e2) a G128K substitution relative to SEQ ID NO: 5; (e3) N111E substitution relative to SEQ ID NO: 5; (e4) N147K substitution relative to SEQ ID NO: 5, and (e5) A113M substitution relative to SEQ ID NO: 5 an amino acid sequence comprising (f) an amino acid sequence having at least 75% identity to SEQ ID NO:6, (f1) D127G and D128K substitutions relative to SEQ ID NO: 6; (f2) each of E111, K147, and M113 of SEQ ID NO: 6 an amino acid sequence comprising (g) an amino acid sequence having at least 75% identity to SEQ ID NO: 7, (g1) D127G and D128K substitutions relative to SEQ ID NO: 7, and (g2) each of E111, M113, and K147 of SEQ ID NO: 7 an amino acid sequence comprising: (h) an amino acid sequence having at least 75% identity to SEQ ID NO: 8, (h1) D127G and D128K substitutions relative to SEQ ID NO: 8, and (h2) each of E111, M113, and K147 of SEQ ID NO: 8 an amino acid sequence comprising A polypeptide comprising an amino acid sequence selected from the group consisting of: 12. The polypeptide according to item 11, wherein the amino acid sequence has at least 80%, at least 85%, at least 90%, at least 95% or more identity to at least one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. 13. A narrow-channel alpha-hemolysin nanopore comprising at least one polypeptide according to any of items 1 to 12. 14. The narrow channel alpha-hemolysin nanopore of any of items 13, wherein the nanopore comprises at least six mutant narrow channel alpha-hemolysin subunits comprising D127G and D128K substitutions relative to SEQ ID NO:1. 15. The narrow channel alpha-hemolysin nanopore of item 14, wherein the narrow channel alpha-hemolysin nanopore is a 6:1 nanopore, and the "1" component is bound to a DNA polymerase. 16. A system for performing nucleic acid sequencing by synthesis (SBS), said system comprising: (a) a chip having a plurality of sensing electrodes; (b) an electrochemically resistive barrier disposed on a surface of the chip, the barrier having a cis side and a trans side; (c) a first electrolyte on the cis side of the barrier; (d) a second electrolyte on the transformer side of the barrier; (e) a plurality of narrow channel alpha-hemolysin nanopores according to any one of items 13 to 15, wherein the narrow channel alpha-hemolysin nanopores are disposed within the barrier such that the channel of the narrow channel alpha-hemolysin nanopore allows ion exchange between the first electrolyte solution and the second electrolyte solution, and at least a portion of the narrow channel alpha-hemolysin nanopore is sufficiently close to one of the sensing electrodes such that the sensing electrode can detect at least one characteristic of a current flowing through the channel of the nanopore; (f) a computer system in electronic communication with the sensing electrode, the computer system adapted to record characteristics of the current flowing through the nanopore detected by the sensing electrode; (g) a nucleic acid polymerase associated with the nanopore on the cis side of the barrier, the nucleic acid polymerase capable of catalyzing a template-dependent nucleic acid amplification reaction in the first electrolyte solution; and (f) a set of nucleoside-5'-oligophosphates disposed in the first electrolyte solution, the set including at least a polymer-tagged adenosine nucleoside-5'-oligophosphate, a polymer-tagged guanine nucleoside-5'-oligophosphate, a polymer-tagged cytosine nucleoside-5'-oligophosphate, and either a polymer-tagged thymidine nucleoside-5'-oligophosphate or a polymer-tagged uracil nucleoside-5'-oligophosphate, each of the polymer-tagged nucleoside-5'-oligophosphates being the nucleoside-5'-oligophosphate; A system comprising: 17. A sequencing-by-synthesis (SBS) method for sequencing a template nucleic acid, said method comprising: 17. The system of claim 16, having a plurality of active nanopore sequencing complexes, wherein each active nanopore sequencing complex comprises: at least one of said sensing electrodes; one of the nanopores inserted into the barrier proximate to the sensing electrode, wherein a current flows through the nanopore and a characteristic of the current is detected by the sensing electrode; and a nucleic acid polymerase associated with the nanopore; and the template nucleic acid complexed with the nucleic acid polymerase; 17. The system of claim 16 having a plurality of active nanopore sequencing complexes, incorporating the tagged nucleoside-5'-oligophosphates into the complementary nucleic acid of the template nucleic acid in the active nanopore sequencing complex by a template-dependent nucleic acid amplification reaction catalyzed by the nucleic acid polymerase, wherein the polymer tag of the tagged nucleoside-5'-oligophosphate moves within or near the channel of the nanopore as the tagged nucleoside-5'-oligophosphate is incorporated into the complementary nucleic acid, and wherein movement of the polymer tag within or near the channel changes the characteristics of the current flowing through the nanopore; detecting a change in a characteristic of the current through the nanopore caused by the polymer tag with the sensing electrode and recording the change in the computer system; Correlating each recorded change with one of the tagged nucleoside-5'-oligophosphates, thereby generating a sequence of complementary nucleic acid generated at that electrode. A method comprising:

Claims

1. 1. A polypeptide comprising a mutant narrow channel alpha-hemolysin subunit, which functions as a constituent polypeptide of a nanopore, wherein the mutant narrow channel alpha-hemolysin subunit has at least the following characteristics: (a) at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; (b) a D127G substitution relative to SEQ ID NOs: 1-8; (c) a D128K substitution relative to SEQ ID NOs: 1-8; and (d) one or more of the following: (d1) an amino acid at a position corresponding to E111 of SEQ ID NO: 1, having a side chain longer than that of asparagine; (d2) an amino acid at a position corresponding to K147 in SEQ ID NO: 1, having a side chain longer than that of asparagine, and / or (d3) an amino acid at a position corresponding to M113 in SEQ ID NO: 1, having a side chain longer than that of alanine A polypeptide having the formula:

2. The polypeptide of claim 1, wherein the mutant narrow channel alpha hemolysin subunit has at least 90%, at least 95% or more identity to at least one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:

8.

3. 2. The polypeptide of claim 1, wherein the amino acid at the position corresponding to E111 is selected from the group consisting of glutamic acid, lysine, arginine, and glutamine.

4. 2. The polypeptide of claim 1, wherein the amino acid at the position corresponding to E111 is selected from the group consisting of glutamic acid and lysine.

5. The polypeptide of claim 1 , wherein the amino acid residue corresponding to E111 is glutamic acid.

6. 2. The polypeptide of claim 1, wherein the amino acid at the position corresponding to K147 is selected from the group consisting of glutamic acid, lysine, arginine, and glutamine.

7. 2. The polypeptide of claim 1, wherein the amino acid at the position corresponding to K147 is selected from the group consisting of glutamic acid and lysine.

8. The polypeptide of claim 1 , wherein the amino acid at the position corresponding to K147 is lysine.

9. 2. The polypeptide of claim 1, wherein the amino acid at the position corresponding to M113 is selected from the group consisting of leucine, isoleucine, valine, or methionine.

10. The polypeptide of claim 1, wherein the amino acid at the position corresponding to M113 is methionine.

11. (a) an amino acid sequence having at least 90% identity to SEQ ID NO:1, (a1) a D127G substitution and a D128K substitution relative to SEQ ID NO: 1; and (a2) each of E111, M113, and K147 of SEQ ID NO: 1 an amino acid sequence comprising (b) an amino acid sequence having at least 90% identity to SEQ ID NO: 2, comprising each of G127, K128, E111, M113, and K147 of SEQ ID NO: 2; (c) an amino acid sequence having at least 90% identity to SEQ ID NO: 3, comprising each of G127, K128, E111, M113, and K147 of SEQ ID NO: 3; (d) an amino acid sequence having at least 90% identity to SEQ ID NO:4, (d1) each of G127 and K128 of SEQ ID NO: 4; (d2) an N111E substitution relative to SEQ ID NO: 4; (d3) an N147K substitution relative to SEQ ID NO: 4; and (d4) A113M substitution relative to SEQ ID NO: 4 an amino acid sequence comprising (e) an amino acid sequence having at least 90% identity to SEQ ID NO:5, (e1) G127 of SEQ ID NO: 5, (e2) a G128K substitution relative to SEQ ID NO: 5; (e3) an N111E substitution relative to SEQ ID NO: 5; (e4) an N147K substitution relative to SEQ ID NO: 5, and (e5) A113M substitution relative to SEQ ID NO: 5 an amino acid sequence comprising (f) an amino acid sequence having at least 90% identity to SEQ ID NO:6, (f1) D127G and D128K substitutions relative to SEQ ID NO: 6; (f2) each of E111, K147, and M113 of SEQ ID NO: 6 an amino acid sequence comprising (g) an amino acid sequence having at least 90% identity to SEQ ID NO:7, (g1) a D127G substitution and a D128K substitution relative to SEQ ID NO: 7; and (g2) each of E111, M113, and K147 of SEQ ID NO: 7 an amino acid sequence comprising: (h) an amino acid sequence having at least 90% identity to SEQ ID NO: 8, (h1) a D127G substitution and a D128K substitution relative to SEQ ID NO: 8; and (h2) each of E111, M113, and K147 of SEQ ID NO: 8 an amino acid sequence comprising A polypeptide comprising an amino acid sequence selected from the group consisting of:

12. 12. The polypeptide of claim 11, wherein the amino acid sequence has at least 90%, at least 95% or more identity to at least one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8.

13. 13. A narrow channel alpha-hemolysin nanopore comprising at least one polypeptide according to claim 12.

14. 14. The narrow channel alpha-hemolysin nanopore of claim 13, wherein said nanopore comprises at least six mutant narrow channel alpha-hemolysin subunits comprising D127G and D128K substitutions relative to SEQ ID NO:

1.

15. 15. The narrow channel alpha-hemolysin nanopore of claim 14, wherein said narrow channel alpha-hemolysin nanopore is a 6:1 nanopore, said "1" component binding to a DNA polymerase.

16. 1. A system for performing nucleic acid sequencing by synthesis (SBS), said system comprising: (a) a chip comprising a plurality of sensing electrodes; (b) an electrochemically resistive barrier disposed on a surface of the chip, the barrier having a cis side and a trans side; (c) a first electrolyte on the cis side of the barrier; (d) a second electrolyte on the transformer side of the barrier; and (e) a plurality of narrow-channel alpha-hemolysin nanopores according to any of claims 13-15, wherein the narrow-channel alpha-hemolysin nanopores are disposed within the barrier such that the channel of the narrow-channel alpha-hemolysin nanopore allows ion exchange between the first electrolyte and the second electrolyte, and at least a portion of the narrow-channel alpha-hemolysin nanopore is sufficiently close to one of the sensing electrodes such that the sensing electrode can detect at least one characteristic of a current flowing through the channel of the nanopore; (f) a computer system in electronic communication with the sensing electrode, the computer system adapted to record characteristics of the current flowing through the nanopore detected by the sensing electrode; (g) a nucleic acid polymerase associated with the nanopore on the cis side of the barrier, the nucleic acid polymerase capable of catalyzing a template-dependent nucleic acid amplification reaction in the first electrolyte solution; and (f) a set of nucleoside-5'-oligophosphates disposed in the first electrolyte solution, the set including at least a polymer-tagged adenosine nucleoside-5'-oligophosphate, a polymer-tagged guanine nucleoside-5'-oligophosphate, a polymer-tagged cytosine nucleoside-5'-oligophosphate, and either a polymer-tagged thymidine nucleoside-5'-oligophosphate or a polymer-tagged uracil nucleoside-5'-oligophosphate, each of the polymer-tagged nucleoside-5'-oligophosphates being the nucleoside-5'-oligophosphate; A system comprising:

17. 1. A sequencing-by-synthesis (SBS) method for sequencing a template nucleic acid, the method comprising:

17. The system of claim 16, comprising a plurality of active nanopore sequencing complexes, each active nanopore sequencing complex comprising: at least one of said sensing electrodes; one of the nanopores inserted into the barrier adjacent to the sensing electrode, wherein a current flows through the nanopore and a characteristic of the current is detected by the sensing electrode; a nucleic acid polymerase associated with the nanopore; and the template nucleic acid complexed with the nucleic acid polymerase; providing the system of claim 16 having a plurality of active nanopore sequencing complexes comprising: incorporating the tagged nucleoside-5'-oligophosphate into the complementary nucleic acid of the template nucleic acid in the active nanopore sequencing complex by a template-dependent nucleic acid amplification reaction catalyzed by the nucleic acid polymerase, wherein the polymer tag of the tagged nucleoside-5'-oligophosphate moves within or near the channel of the nanopore as the tagged nucleoside-5'-oligophosphate is incorporated into the complementary nucleic acid, and wherein movement of the polymer tag within or near the channel changes the characteristics of the current flowing through the nanopore; detecting a change in a characteristic of the current through the nanopore caused by the polymer tag with the sensing electrode and recording the change in the computer system; Correlating each recorded change with one of the tagged nucleoside-5'-oligophosphates, thereby generating the sequence of the complementary nucleic acid generated at that electrode. A method comprising:

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