US20180002750A1 - Long lifetime alpha-hemolysin nanopores - Google Patents

Long lifetime alpha-hemolysin nanopores Download PDF

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US20180002750A1
US20180002750A1 US15/638,273 US201715638273A US2018002750A1 US 20180002750 A1 US20180002750 A1 US 20180002750A1 US 201715638273 A US201715638273 A US 201715638273A US 2018002750 A1 US2018002750 A1 US 2018002750A1
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amino acid
seq
hemolysin
variant
acid sequence
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Mark Ambroso
Timothy Craig
Matthew DiPietro
Corissa Harris
Marshall Porter
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Roche Sequencing Solutions Inc
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Roche Sequencing Solutions Inc
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Priority to US15/638,273 priority Critical patent/US20180002750A1/en
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Priority to US16/519,251 priority patent/US10934582B2/en
Priority to US17/157,576 priority patent/US11613778B2/en
Priority to US18/171,969 priority patent/US12091714B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • This application hereby incorporates-by-reference a sequence listing submitted herewith in a computer-readable format, having a file name of 33725_WO_seqlist_ST25, created on Jun. 29, 2017, which is 56,233 bytes in size.
  • Hemolysins are members of a family of protein toxins that are produced by a wide variety of organisms. Some hemolysins, for example alpha hemolysins, can disrupt the integrity of a cell membrane (e.g., a host cell membrane) by forming a pore or channel in the membrane. Pores or channels that are formed in a membrane by pore forming proteins can be used to transport certain polymers (e.g., polypeptides or polynucleotides) from one side of a membrane to the other.
  • a cell membrane e.g., a host cell membrane
  • Pores or channels that are formed in a membrane by pore forming proteins can be used to transport certain polymers (e.g., polypeptides or polynucleotides) from one side of a membrane to the other.
  • Alpha-hemolysin ( ⁇ -HL, a-HL or alpha-HL) is a self-assembling hemolysin toxin that forms a channel in the membrane of a host cell. More particularly, seven alpha-hemolysin monomers assemble into a heptameric, beta-barrel pore in biological membranes. Alpha-hemolysin has many advantageous properties including high stability and self-assembly into a nanopore that is wide enough to accommodate single stranded DNA but not double stranded DNA (Kasianowicz et al., 1996). Based on these properties and other properties, alpha-hemolysin has become a principal component for the nanopore sequencing community.
  • nanopores using wild-type alpha-hemolysins are only able to generate sequence data for a short amount of time.
  • the lifetime of the alpha-hemolysin nanopore during the sequencing reaction often serves as the rate-limiting feature of the sequencing reaction.
  • use of wild-type alpha hemolysin often results in a significant number of deletion errors, i.e., bases that are not measured. Therefore, alpha-hemolysin nanopores with improved properties, including increased sequencing lifetimes, are desired.
  • mutant staphylococcal alpha hemolysin ( ⁇ HL) polypeptides that, when incorporated into a nanopore, improve the lifetime of the nanopore during a DNA sequencing reaction.
  • a nanopore including one or more of the variants described herein lasts longer—and hence provides more sequencing data—than a nanopore that consists of wild-type alpha hemolysin.
  • the ⁇ -hemolysin ( ⁇ -HL) variants comprise a substitution at a position corresponding to any one of E111N, M113A, K147N, or a combination thereof of SEQ ID NO: 14 (the mature, wild-type alpha hemolysin sequence).
  • the ⁇ -hemolysin variant may also include a substitution at H35G or K135G of SEQ ID NO: 14.
  • the ⁇ -hemolysin variant may also, in certain aspects, include one or more one or more glycine residues at residues 126-131 of SEQ ID NO: 14, such as a series of glycine residues that span the entire length of residues 126 through 131 of SEQ ID NO: 14.
  • the variant may also include a poly-G substitution corresponding to amino acids 127-129 of the amino acid sequence set forth as SEQ ID NO: 14, resulting in a span of glycine residues from 126 through 131 of SEQ ID NO: 14.
  • the ⁇ -hemolysin variant includes an amino acid sequence having at least one of the substitutions described herein, while the sequence of the ⁇ -hemolysin variant has at least 80%, 90%, 95%, 98%, or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 14.
  • the ⁇ -hemolysin variant includes an amino acid sequence having at least 80%, 90%, 95%, 98%, or more sequence identity to the amino acid sequence set forth as SEQ ID NOS: 17, 18, 19, 20, or 22.
  • the alpha-hemolysin variant described herein is bound to a DNA polymerase, such as via a covalent bond.
  • the alpha-hemolysin variant is bound to the DNA polymerase via a SpyTag/SpyCatcher linkage.
  • the alpha-hemolysin variant is bound to the DNA polymerase via an isopeptide bond.
  • a heptameric nanopore assembly includes at least one or more of the alpha-hemolysin variants described herein.
  • the heptameric nanopore assembly may include one or more alpha-hemolysin proteins having a substitution at E111N, M113A, K147N, or combinations thereof of SEQ ID NO: 14, such as described herein.
  • the heptameric nanopore assembly may include one or more alpha-hemolysin proteins having a substitution at E111S, M113S, T145S, K147S or L135I or combinations thereof of SEQ ID NO: 14, such as described herein.
  • each of the seven alpha-hemolysin monomers of the heptameric nanopore are alpha-hemolysin variants as described herein.
  • the variant can be the same variant or a combination of different variants described herein.
  • the nanopore assembly includes one or more variants having at least 80%, 90%, 95%, 98%, or more sequence identity to the amino acid sequence set forth as SEQ ID NOS: 17, 18, 19, 20, or 22.
  • the lifetime of the resultant nanopore is increased by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more when compared to a heptameric nanopore assembly consisting of native alpha-hemolysin.
  • nucleic acids encoding any of the alpha hemolysin variants described herein can be derived from Staphylococcus aureus (SEQ ID NO: 1).
  • vectors that include an any such nucleic acids encoding any one of the hemolysin variants described herein.
  • a host cell that is transformed with the vector.
  • a method of producing an alpha-hemolysin variant as descried herein includes, for example, the steps of culturing a host cell including the vector in a suitable culture medium under suitable conditions to produce alpha-hemolysin variant.
  • the variant is then obtained from the culture using methods known in the art.
  • a method of detecting a target molecule includes, for example, providing a chip comprising a nanopore assembly as described herein in a membrane that is disposed adjacent or in proximity to a sensing electrode. The method then includes directing a nucleic acid molecule through the nanopore.
  • the nucleic acid molecule is associated with a reporter molecule and includes an address region and a probe region.
  • the reporter molecule is associated with the nucleic acid molecule at the probe region and is coupled to a target molecule.
  • the method further involves sequencing the address region while said nucleic acid molecule is directed through said nanopore to determine a nucleic acid sequence of said address region.
  • the target molecule is identified, with the aid of a computer processor, based upon a nucleic acid sequence of the determined address region determined.
  • FIG. 1A is histogram showing lifetime assessment for a standard H144A nanopore.
  • the y-axis is the number of pores which had a lifetime within the bin on the x-axis.
  • the x-axis is the number of 100 s intervals (reps) in which the current passing through the channel corresponded to that of a single Hemolysin nanopore. This experiment was run for 7200 s, or 72 reps.
  • FIG. 1B is a graph showing an analysis of the failure mechanisms of the H144A nanopore for the same run as FIG. 1A .
  • This experiment was run for 7200 s, or 72 reps. represents an analysis of the failure mechanisms of the nanopore for the same run as FIG. 1A .
  • individual pores are displayed as dots in a number of categories. The first category is for those pores which survived until the end of the experiment; their mode of failure was that the instrument was shut off. The second category is for cells that were turned off by the Genia FPGA because the current increased very quickly to a level>10 ⁇ of the current of a single nanopore, which is a general indicator that the lipid bilayer was disrupted.
  • the third category is for when the open channel current increases from that of a single pore to that of a multiple of a single pore, but lower than 10 ⁇ the current. This typically indicates that 2, 3,4, 5, 6, 7, 8, or 9 nanopores inserted into the bilayer that originally only harbored one.
  • the noisy bin contains pores where some unknown mode of failure occurred, which typically results in an unstable level of current is being measured; these may be due to electrode failure.
  • the last category is bilayer, and corresponds to the situation where current is no longer measured passing through a nanopore, but rather the characteristically low conductance of a lipid bilayer is seen.
  • FIG. 2A is a histogram showing lifetime assessment for an N Rectification nanopore.
  • the y-axis is the number of pores which had a lifetime within the bin on the x-axis.
  • the x-axis is the number of 100 s intervals (reps) in which the current passing through the channel corresponded to that of a single N Rectification Hemolysin nanopore. This experiment was run for 3600 s, or 36 reps.
  • the third category is for when the open channel current increases from that of a single pore to that of a multiple of a single pore, but lower than 10 ⁇ the current. This typically indicates that 2, 3, 4, 5, 6, 7, 8, or 9 nanopores inserted into the bilayer that originally only harbored one.
  • the noisy bin contains pores where some unknown mode of failure occurred, which typically results in an unstable level of current is being measured; this may be due to electrode failure.
  • the last category is bilayer, and corresponds to the situation where current is no longer measured passing through a nanopore, but rather the characteristically low conductance of a lipid bilayer is seen.
  • FIG. 3A is a histogram showing lifetime assessment for an N Rectification nanopore.
  • the y-axis is the number of pores which had a lifetime within the bin on the x-axis.
  • the x-axis is the number of 100 s intervals (reps) in which the current passing through the channel corresponded to that of a single N Rectification Hemolysin nanopore. This experiment was run for 18000 s, or 180 reps.
  • FIG. 3B is a graph showing an analysis of the failure mechanisms of the N Rectification nanopore for the same run as FIG. 3A .
  • This experiment was run for 3600 s, or 36 reps. represents an analysis of the failure mechanisms of the nanopore for the same run as FIG. 1A .
  • individual pores are displayed as dots in a number of categories. The first category is for those pores which survived until the end of the experiment; their mode of failure was that the instrument was shut off. The second category is for cells that were turned off by the Genia FPGA because the current increased very quickly to a level>10 ⁇ of the current of a single nanopore, which is a general indicator that the lipid bilayer was disrupted.
  • the third category is for when the open channel current increases from that of a single pore to that of a multiple of a single pore, but lower than 10 ⁇ the current. This typically indicates that 2, 3, 4, 5, 6, 7, 8, or 9 nanopores inserted into the bilayer that originally only harbored one.
  • the noisy bin contains pores where some unknown mode of failure occurred, which typically results in an unstable level of current is being measured; this may be due to electrode failure.
  • the last category is bilayer, and corresponds to the situation where current is no longer measured passing through a nanopore, but rather the characteristically low conductance of a lipid bilayer is seen.
  • FIG. 4A is a graph showing the time-to-thread for control heptameric nanopores as compared to heptameric nanopores including one alpha-hemolysin/phi29 DNA Polymerase conjugate and six alpha-hemolysin variants, each variant having substitutions at H35G+V149K+H144A (i.e., a 1:6 ratio), as set forth in SEQ ID NO: 4.
  • the control nanopores (labeled “WT”) include a 1:6 ratio of alpha-hemolysin/phi29 DNA Polymerase conjugate to six wild-type alpha-hemolysins.
  • 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.
  • Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
  • synthetic amino acid or “non-natural 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 carboxy- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, and/or substitution with other chemical without adversely affecting their activity. Amino acids may participate in a disulfide bond.
  • Base Pair refers to a partnership of adenine (A) with thymine (T), adenine (A) with uracil (U) or of cytosine (C) with guanine (G) in a double stranded nucleic acid.
  • Expression cassette is a nucleic acid construct generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell.
  • the recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
  • the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.
  • Host cell By the term “host cell” is meant a cell that contains a vector and supports the replication, and/or transcription or transcription and translation (expression) of the 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. In general, host cells are prokaryotic, e.g., E. coli.
  • Lifetime As used herein, the term “lifetime” or “nanopore lifetime” is used generally to refer to the overall length of time that a nanopore functions, in a sequencing reaction, to provide useful sequencing data. More particularly, the lifetime of a nanopore can be measured by measuring the time between the start of an experiment and when the nanopore ceases to function properly, as determined by open channel current level.
  • Modified alpha-hemolysin refers to an alpha-hemolysin originated from another (i.e., parental) alpha-hemolysin and contains one or more amino acid alterations (e.g., amino acid substitution, deletion, or insertion) compared to the parental alpha-hemolysin.
  • a modified alpha-hemolysin of the invention is originated or modified from a naturally-occurring or wild-type alpha-hemolysin.
  • Mutation refers to a change introduced into a parental sequence, including, but not limited to, substitutions, insertions, deletions (including truncations).
  • substitutions include, but are not limited to, the creation of a new character, property, function, phenotype or trait not found in the protein encoded by the parental sequence.
  • Nanopore generally refers to a pore, channel, or passage formed or otherwise provided in a membrane.
  • a membrane may be an organic membrane, such as a lipid bilayer, or a synthetic membrane, such as a membrane formed of a polymeric material.
  • the membrane may be a polymeric material.
  • the nanopore may be disposed adjacent or in proximity to a sensing circuit or an electrode coupled to a sensing circuit, such as, for example, a complementary metal-oxide semiconductor (CMOS) or field effect transistor (FET) circuit.
  • CMOS complementary metal-oxide semiconductor
  • FET field effect transistor
  • a nanopore has a characteristic width or diameter on the order of 0.1 nanometers (nm) to about 1000 nm.
  • Some nanopores are proteins. Alpha-hemolysin is an example of a protein nanopore.
  • nucleic acid molecule includes RNA, DNA, and cDNA molecules. It will be understood that, as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding a given protein such as alpha-hemolysin and/or variants thereof may be produced. The present invention contemplates every possible variant nucleotide sequence, encoding variant alpha-hemolysin, all of which are possible given the degeneracy of the genetic code.
  • promoter refers to a nucleic acid sequence that functions to direct transcription of a downstream gene.
  • the promoter will generally be appropriate to the host cell in which the target gene is being expressed.
  • the promoter together with other transcriptional and translational regulatory nucleic acid sequences are necessary to express a given gene.
  • control sequences also termed “control sequences”
  • the 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.
  • purified means that a molecule is present in a sample at a concentration of at least 95% by weight, or at least 98% by weight of the sample in which it is contained.
  • purifying generally refers to subjecting transgenic nucleic acid or protein containing cells to biochemical purification and/or column chromatography.
  • Tag refers to a detectable moiety that may be atoms or molecules, or a collection of atoms or molecules.
  • a tag may provide an optical, electrochemical, magnetic, or electrostatic (e.g., inductive, capacitive) signature, which signature may be detected with the aid of a nanopore.
  • a nucleotide is attached to the tag it is called a “Tagged Nucleotide.”
  • the tag may be attached to the nucleotide via the phosphate moiety.
  • Time-To-Thread means the time it takes the polymerase-tag complex or a nucleic acid strand to thread the tag into the barrel of the nanopore.
  • Variant refers to a modified protein which displays altered characteristics when compared to the parental protein, e.g., altered ionic conductance.
  • variable hemolysin gene or “variant hemolysin” means, respectively, that the nucleic acid sequence of the alpha-hemolysin gene from Staphylococcus aureus has been altered by removing, adding, and/or manipulating the coding sequence or the amino acid sequence of the expressed protein has been modified consistent with the invention described herein.
  • Vector refers to a nucleic acid construct designed for transfer between different host cells.
  • An “expression vector” refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.
  • Wild-type refers to a native gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally-occurring source.
  • % homology is used interchangeably herein with the term “% identity” herein and refers to the level of nucleic acid or amino acid sequence identity between the nucleic acid sequence that encodes any one of the inventive polypeptides or the inventive polypeptide's amino acid sequence, when aligned using a sequence alignment program.
  • 80% homology means the same thing as 80% sequence identity determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence identity over a length of the given sequence.
  • Exemplary levels of sequence identity include, but are not limited to, 80, 85, 90, 95, 98% or more sequence identity to a given sequence, e.g., the coding sequence for any one of the inventive polypeptides, as described herein.
  • Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet. See also, Altschul, et al., 1990 and Altschul, et al., 1997.
  • Sequence searches are typically carried out using the BLASTN program when evaluating a given nucleic acid sequence relative to nucleic acid sequences in the GenBank DNA Sequences and other public databases.
  • the BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the 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.)
  • a preferred alignment of selected sequences in order to determine “% identity” between two or more sequences is performed using for example, 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.
  • Staphylococcus aureus alpha hemolysin wild type sequences are provided herein (SEQ ID NO:1, nucleic acid coding region; SEQ ID NO:14, protein sequence) and available elsewhere (National Center for Bioinformatics or GenBank Accession Numbers M90536 and AAA26598).
  • Point mutations may be introduced by any method known in the art.
  • a point mutation may be made using QuikChange Lightning 2 kit (Stategene/Agilent) following manufacturer's instructions.
  • Primers can be ordered from commercial companies, e.g., IDT DNA.
  • the alpha-hemolysin variants provided herein include specific substitutions—or one or more combination of substitutions—such that nanopores incorporating the variants have improved nanopore lifetime with stable, open channels during a nucleic acid sequencing reaction. By improving nanopore lifetime, sequencing reactions using such long-lifetime nanopores are able to generate more usable sequencing data over the course of a longer sequencing reaction.
  • the variants include a particular mutation or series of mutations.
  • the variant may include an amino acid substitution of any one of E111N/E111S, M113A/M113S, L135I, T145S, K147N/K147S or a combination thereof of SEQ ID NO: 14.
  • the variant may also include a H35G substitution of SEQ ID NO: 14.
  • the variant may include a poly-G substitution at residues 127-129 of SEQ ID NO: 14.
  • the variant may further include a K131G mutation.
  • the E111N/E111S, M113A/M113S, L135I, T145S, K147N/K147S, and/or H35G substitutions described herein are accompanied by a series of poly-G amino acids at residues 126-131 of SEQ ID NO: 14.
  • the alpha-hemolysin variants described herein may additionally include an amino acid substitution at H144A of SEQ ID NO: 14.
  • the alpha-hemolysin variants include specific combinations of substitutions.
  • the alpha hemolysin variant may include an E111N+K147N substitution or an E111S+K147S substitution. Additionally or alternatively, the alpha-hemolysin variant may include an E111N+K147N+M113A substitution or an E111S+K147S+M113S substitution.
  • the alpha hemolysin variants include one of the following combinations of substitutions/residues:
  • the variant includes one or more of the substitutions described herein, while the overall sequence of the variant retains up to 80%, 85%, 90%, 95%, 98% or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 2.
  • the variant includes one or more of the substitutions described herein, while the overall sequence of the variant retains up to 80%, 85%, 90%, 95%, 98% or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 14.
  • the variant has 80%, 85%, 90%, 95%, 98% or more sequence identity to the amino acid sequence set forth SEQ ID NOS: 17, 18, 19, 20, or 22.
  • nanopores including such variants have less net salt movement across the pore when the nanopore is subjected to an alternating current.
  • the resultant nanopore has an improved lifetime compared to, for example, a nanopore consisting of wild-type (native) alpha-hemolysins.
  • the alpha-hemolysin variants described herein that improve nanopore lifetime may also improve Time-To-Thread during a sequencing reaction. That is, when the variant is incorporated into a nanopore, both the lifetime of the nanopore and the Time-To-Thread are improved, thus resulting in a superior nanopore.
  • any of the H35G, E111N, M113A, K147N, or 127-129G substitutions may improve both lifetime of the nanopore and Time-To-Thread.
  • any of the E111S, M113S, T145S, K147S, or L135I mutations may improve both lifetime of the nanopore and Time-To-Thread.
  • additional substitutions may be incorporated into the variants to improve Thread-To-Thread of a resultant nanopore, thereby improving the overall functioning of the nanopore (both the lifetime and Thread-To-Thread).
  • a variant resulting in improved nanopore lifetime and improved time to formed from muting one or more of the amino acids of SEQ ID NO:14 identified in Table 1 has 80%, 85%, 90%, 95%, 98% or more sequence identity to the sequence set forth as SEQ ID NO: 14.
  • the mutation results in the addition of a positive charge.
  • the mutation may result in a substitution of an amino acid residue identified in Table 1 to an arginine, lysine, histidine, asparagine, or other amino acid that can carry a positive charge.
  • the mutation in addition to a H35G, E111N/E111S, M113A/M113S, L135I, T145S, K147N/K147S, and/or 127-129G substitution (or combination thereof) to improve nanopore lifetime, the mutation may include a particular, additional substitution to also improve Time-To-Thread.
  • the variant may additionally include an amino acid substitution of any one of V149K, E287R, T109K, P151K, or combinations thereof of SEQ ID NO: 14.
  • the variant may include one or more these same substitutions, while the overall sequence can have up to 80%, 85%, 90%, 95%, 98% or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 14.
  • one or more of the first 17 amino acids of SEQ ID NO: 14 mutated to either an A, N, K, or combinations thereof. Additionally or alternatively, any of the variants may include a series of glycine residue substitutions spanning from residue 127 to residue 131 of the sequence set forth as SEQ ID NO: 14, as described herein.
  • an ⁇ -hemolysin variant can include various combinations of substitutions as described herein, in certain example embodiments the ⁇ -hemolysin variant includes particular combinations of substitutions to improve Time-to-Thread.
  • an ⁇ -hemolysin variant may include the following combinations of amino acid substitutions of the sequence set forth as SEQ ID NO: 14:
  • the amino acid substitution described herein allows the addition of heterologous molecules, such as polyethylene glycol (PEG).
  • the a-HL variant has one or more post-translational modifications.
  • the substitution is a non-native amino acid that is basic or positively charged at a pH from about 5 to about 8.5.
  • the amino acids forming all or a part of the variants described herein may be stereoisomers. Additionally or alternatively, the amino acids forming all or a part of the variants described herein may be modifications of naturally occurring amino acids, non-naturally occurring amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, and the like.
  • the amino acids forming the variants described herein may be one or more of the 20 common amino acids found in naturally occurring proteins, or one or more of the modified and unusual amino acids. In certain example embodiments, the amino acids may be D- or L-amino acids.
  • the variants may also include one or more modified amino acids.
  • the modified amino acid may be a derivatized amino acid or a modified and unusual amino acid.
  • modified and unusual amino acids include but are not limited to, 2-Aminoadipic acid (Aad), 3-Aminoadipic acid (Baad), ⁇ -Amino-propionic acid (Bala, ⁇ -alanine), 2-Aminobutyric acid (Abu, piperidinic acid), 4-Aminobutyric acid (4Abu), 6-Aminocaproic acid (Acp), 2-Aminoheptanoic acid (Ahe), 2-Aminoisobutyric acid (Aib), 3-Aminoisobutyric acid (Baib), 2-Aminopimelic acid (Apm), 2,4-Diaminobutyric acid (Dbu), Desmosine (Des), 2,2′-Diaminopimelic acid (Dpm), 2,3-Diaminopropionic acid (Dpr),
  • the amino acid sequence of the variant is sequential, without any modified and unusual amino acids interrupting the sequence of D- or L-amino acids.
  • the sequence may include one or more modified and unusual amino acids as noted above.
  • the sequence of the variant may be interrupted by one or more modified and unusual amino acids. Accordingly, provided are pseudopeptides and peptidomimetics, including structures that have a non-peptidic backbone.
  • the variants include dimers or multimers of peptides.
  • any of the amino acid sequences described herein may also include a linker sequences or affinity tags, and further may include sequences for removing such tags (e.g., protease cleavage sites).
  • the sequences may include a linker/TEV/HisTAG sequence at the C-terminal end having the sequence GLSA ENLYFQG HHHHHH (SEQ ID NO: 16, where the TEV sequence is underlined).
  • such a sequence allows for the purification of the variant.
  • the alpha-hemolysin peptides described herein can be assembled into a multimeric protein assembly (i.e., a nanopore).
  • the resultant nanopore will include multiple, alpha-hemolysin subunits.
  • a heptameric alpha-hemolysin nanopore includes seven subunits.
  • any of the alpha-hemolysin variants described herein can be used in nanopore assembly.
  • the subunits of a given nanopore for example, can be identical copies of the same polypeptide or they can be different polypeptides.
  • each of the seven subunits of a heptameric assembly may have an amino acid sequence corresponding to the amino acid sequence set forth as SEQ ID NOS: 17, 18, 19, 20, or 22.
  • the subunits may include the substitutions identified in SEQ ID NOS: 17, 18, 19, 20, or 22, but may only have 80%, 85%, 90%, 95%, 98% or more sequence identity to the amino acid sequence set forth as SEQ ID NOS: 17, 18, 19, 20, or 22, respectively.
  • each of the subunits may include the same series of substitutions, with one or more of each of subunits of the nanopore having a different overall amino acid sequence. That is, while a particular substitution or combination of substitutions may be conserved among all subunits in a given nanopore, the overall amino acid sequences of the various subunits may be different.
  • a nanopore including variant alpha-hemolysin subunits that are the same or substantially the same provide an improved lifetime as compared to a nanopore having a mixture of different alpha-hemolysin subunits.
  • a nanopore including only alpha-hemolysin variant subunits having 80%, 85%, 90%, 95%, 98% or more sequence identity to one of the amino acid sequence set forth as SEQ ID NOS: 17, 18, 19, 20, or 22 may have a greater lifetime than a nanopore that includes variant subunits along with wild-type (native) subunits.
  • one or more of the subunits may additionally include substitutions that improve Time-To-Thread as described herein.
  • resultant nanopore has both improved lifetime and increased Time-to-Thread.
  • the nanopore may include a mixture of the variants described herein and other alpha-hemolysin polypeptides, such as wild-type (native) alpha-hemolysins.
  • the nanopores have a defined ratio of modified subunits (e.g., a-HL variants) to un-modified subunits (e.g., a-HL).
  • the nanopore has a polymerase attached to one of the subunits to form a nanopore assembly.
  • a method for assembling a protein having a plurality of subunits includes providing a plurality of first subunits 2705 and providing a plurality of second subunits 2710, where the second subunits are modified when compared with the first subunits.
  • the first subunits are wild-type (e.g., purified from native sources or produced recombinantly).
  • the second subunits can be modified in any suitable way.
  • the second subunits have a protein (e.g., a polymerase) attached (e.g., as a fusion protein).
  • the modified subunits can comprise a chemically reactive moiety (e.g., an azide or an alkyne group suitable for forming a linkage).
  • the method further comprises performing a reaction (e.g., a Click chemistry cycloaddition) to attach an entity (e.g., a polymerase) to the chemically reactive moiety.
  • the method can further include contacting the first subunits with the second subunits 2715 in a first ratio to form a plurality of proteins 2720 having the first subunits and the second subunits.
  • one part modified aHL subunits having a reactive group suitable for attaching a polymerase can be mixed with six parts wild-type aHL subunits (i.e., with the first ratio being 1:6).
  • the plurality of proteins can have a plurality of ratios of the first subunits to the second subunits.
  • the mixed subunits can form several nanopores having a distribution of stoichiometries of modified to un-modified subunits (e.g., 1:6, 2:5, 3:4).
  • the proteins are formed by simply mixing the subunits.
  • a detergent e.g., deoxycholic acid
  • the nanopores can also be formed, for example, using a lipid (e.g., 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) or 1,2-di-0-phytanyl-sn-glycero-3-phosphocholine (DoPhPC)) and moderate temperature (e.g., less than about 100° C.).
  • DPhPC 1,2-diphytanoyl-sn-glycero-3-phosphocholine
  • DoPhPC 1,2-di-0-phytanyl-sn-glycero-3-phosphocholine
  • mixing DPhPC with a buffer solution creates large multi-lamellar vesicles (LMV), and adding aHL subunits to this solution and incubating the mixture at 40° C. for 30 minutes results in pore formation.
  • LMV multi-
  • the method can further comprise fractionating the plurality of proteins to enrich proteins that have a second ratio of the first subunits to the second subunits 2725.
  • nanopore proteins can be isolated that have one and only one modified subunit (e.g., a second ratio of 1:6).
  • any second ratio is suitable.
  • a distribution of second ratios can also be fractionated such as enriching proteins that have either one or two modified subunits.
  • the total number of subunits forming the protein is not always 7 (e.g., a different nanopore can be used or an alpha-hemolysin nanopore can form having six subunits) as depicted in FIG. 27 of WO2014/074727.
  • proteins having only one modified subunit are enriched.
  • the second ratio is 1 second subunit per (n ⁇ 1) first subunits where n is the number of subunits comprising the protein.
  • the first ratio can be the same as the second ratio, however this is not required. In some embodiments, proteins having mutated monomers can form less efficiently than those not having mutated subunits. If this is the case, the first ratio can be greater than the second ratio (e.g., if a second ratio of 1 mutated to 6 non-mutated subunits are desired in a nanopore, forming a suitable number of 1:6 proteins may require mixing the subunits at a ratio greater than 1:6).
  • Proteins having different second ratios of subunits can behave differently (e.g., have different retention times) in a separation.
  • the proteins are fractionated using chromatography, such as ion exchange chromatography or affinity chromatography. Since the first and second subunits can be identical apart from the modification, the number of modifications on the protein can serve as a basis for separation.
  • either the first or second subunits have a purification tag (e.g., in addition to the modification) to allow or improve the efficiency of the fractionation.
  • a poly-histidine tag His-tag
  • streptavidin tag streptavidin tag
  • other peptide tag is used.
  • the first and second subunits each comprise different tags and the fractionation step fractionates on the basis of each tag.
  • a charge is created on the tag at low pH (Histidine residues become positively charged below the pKa of the side chain).
  • ion exchange chromatography can be used to separate the oligomers which have 0, 1, 2, 3, 4, 5, 6, or 7 of the “charge-tagged” aHL subunits.
  • this charge tag can be a string of any amino acids which carry a uniform charge.
  • FIG. 28 and FIG. 29 show examples of fractionation of nanopores based on a His-tag.
  • FIG. 28 shows a plot of ultraviolet absorbance at 280 nanometers, ultraviolet absorbance at 260 nanometers, and conductivity. The peaks correspond to nanopores with various ratios of modified and unmodified subunits.
  • FIG. 29 of WO2014/074727 shows fractionation of aHL nanopores and mutants thereof using both His-tag and Strep-tags.
  • an entity e.g., a polymerase
  • the protein can be a nanopore protein and the entity can be a polymerase.
  • the method further comprises inserting the proteins having the second ratio subunits into a bilayer.
  • a nanopore can comprise a plurality of subunits.
  • a polymerase can be attached to one of the subunits and at least one and less than all of the subunits comprise a first purification tag.
  • all of the subunits comprise a first purification tag or a second purification tag.
  • the first purification tag can, for example, be a poly-histidine tag (e.g., on the subunit having the polymerase attached).
  • a polymerase e.g., DNA polymerase
  • Any DNA polymerase capable of synthesizing DNA during a DNA synthesis reaction may be used in accordance with the methods and compositions described herein.
  • Example DNA polymerases include, but are not limited to, phi29 (Bacillus bacteriophage ⁇ 29), pol6 (Clostridium phage phiCPV4; GenBank: AFH27113.1) or po17 (Actinomyces phage Av-1; GenBank: ABR67671.1).
  • a DNA-manipulating or modifying enzyme such as a ligase, nuclease, phosphatase, kinase, transferase, or topoisomerase.
  • the polymerase is a polymerase variant.
  • the polymerase variant may include any of the polymerase variants identified in U.S. patent application Ser. No. 15/012,317 (published as the US 2016/0222363 A1, also referred to herein as the “317 application”).
  • Such variants include, for example, one or more amino acid substitutions at H223A, N224Y/L, Y225L/T/I/F/A, H227P, 1295 W/F/M/E, Y342L/F, T343N/F, 1357G/L/Q/H/W/M/A/E/Y/P, S360G, L361M/W/V, 1363V, S365Q/W/M/A/G, S366A/L, Y367L/E/M/P/N, P368G, D417P, E475D, Y476V, F478L, K518Q, H527 W/R/L, T529M/F, M531H/Y/A/K/R/W/T/L/V, N535L/Y/M/K/I, G539Y/F, P542E/S, N545K/D/S/L/R, Q546 W
  • the polymerase includes one or more such substitutions and has 80%, 90%, 95%, 98% or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 15.
  • the polymerase variant may have altered enzyme activity, fidelity, processivity, elongation rate, sequencing accuracy, long continuous read capability, stability, or solubility relative to the parental polymerase.
  • the polymerase can be attached to the nanopore assembly in any suitable way known in the art. See, for example, PCT/US2013/068967 (published as WO2014/074727; Genia Technologies), PCT/US2005/009702 (published as WO2006/028508), and PCT/US2011/065640 (published as WO2012/083249; Columbia Univ).
  • the polymerase is attached to the nanopore (e.g., hemolysin) protein monomer and then the full nanopore heptamer is assembled (e.g., in a ratio of one monomer with an attached polymerase to 6 nanopore (e.g., hemolysin) monomers without an attached polymerase).
  • the nanopore heptamer can then be inserted into the membrane.
  • Another method for attaching a polymerase to a nanopore involves attaching a linker molecule to a hemolysin monomer or mutating a hemolysin monomer to have an attachment site and then assembling the full nanopore heptamer (e.g., at a ratio of one monomer with linker and/or attachment site to 6 hemolysin monomers with no linker and/or attachment site).
  • a polymerase can then be attached to the attachment site or attachment linker (e.g., in bulk, before inserting into the membrane).
  • the polymerase can also be attached to the attachment site or attachment linker after the (e.g., heptamer) nanopore is formed in the membrane.
  • a plurality of nanopore-polymerase pairs are inserted into a plurality of membranes (e.g., disposed over the wells and/or electrodes) of the biochip.
  • the attachment of the polymerase to the nanopore complex occurs on the biochip above each electrode.
  • the polymerase can be attached to the nanopore with any suitable chemistry (e.g., covalent bond and/or linker).
  • the polymerase is attached to the nanopore with molecular staples.
  • molecular staples comprise three amino acid sequences (denoted linkers A, B and C).
  • Linker A can extend from a hemolysin monomer
  • Linker B can extend from the polymerase
  • Linker C then can bind Linkers A and B (e.g., by wrapping around both Linkers A and B) and thus the polymerase to the nanopore.
  • Linker C can also be constructed to be part of Linker A or Linker B, thus reducing the number of linker molecules.
  • the polymerase is linked to the nanopore using SolulinkTM chemistry.
  • SolulinkTM can be a reaction between HyNic (6-hydrazino-nicotinic acid, an aromatic hydrazine) and 4FB (4-formylbenzoate, an aromatic aldehyde).
  • the polymerase is linked to the nanopore using Click chemistry (available from LifeTechnologies for example).
  • Click chemistry available from LifeTechnologies for example.
  • zinc finger mutations are introduced into the hemolysin molecule and then a molecule is used (e.g., a DNA intermediate molecule) to link the polymerase to the zinc finger sites on the hemolysin.
  • the SpyTag/SpyCatcher system which spontaneously forms covalent isopeptide linkages under physiological conditions, may be used to join an alpha-hemolysin monomer to the polymerase. See, for example, Li et al, J Mol Biol. 2014 Jan. 23; 426(2):309-17.
  • an alpha-hemolysin protein can be expressed having a SpyTag domain.
  • the DNA polymerase to be joined to the alpha-hemolysin may be separately expressed as fusion protein having a SpyCatcher domain.
  • the SpyTag and SpyCatcher proteins interact to form the alpha-hemolysin monomer that is linked to a DNA polymerase via a covalent isopeptide linkage.
  • the SpyTag domain is attached to the alpha-hemolysin via a linker sequence.
  • the linker-SpyTag protein may include the sequence GGSSGGSSGG AHIVMVDAYKPTK (SEQ ID NO: 21), with the underlined portion being the linker sequence and the bolded portion being the SpyTag sequence.
  • a HisTag is attached to the SpyTag sequence of the SpyTag sequence.
  • the HisTag may be linked to the SpyTag via a KG linker.
  • the polymerase may be attached to a nanopore monomer before the nanopore monomer is incorporated into a nanopore assembly.
  • the purified alpha-hemolysin/SpyTag fusion protein is mixed with purified polymerase/SpyCatcher fusion protein, thus allowing the SpyTag and SpyCatcher proteins bind each other to form an alpha-hemolysin/polymerase monomer.
  • the monomer can then be incorporated into the nanopore assembly as described herein to form a heptameric assembly.
  • the polymerase is attached to the nanopore assembly after formation of the nanopore assembly.
  • the fusion protein is incorporated into the nanopore assembly to form the heptameric nanopore assembly.
  • the polymerase/SpyCatcher fusion protein is then mixed with the heptameric assembly, thus allowing the SpyTag and SpyCatcher proteins bind each other, which in turn results in binding of the polymerase to the nanopore assembly.
  • the nanopore assembly may be configured, for example, to have only a single SpyTag, which therefore allows the attachment of a single polymerase/SpyCatcher.
  • alpha-hemolysin for example, mixing the alpha-hemolysin/SpyTag proteins with additional alpha-hemolysin proteins results in heptamers having 0, 1, 2, 3, 4, 5, 6, or 7 alpha-hemolysin/SpyTag subunits. Yet because of the different number of SpyTag sequences (0, 1, 2, 3, 4, 5, 6, or 7) associated with each heptamer, the heptamers have different charges.
  • the heptamers can be separated by methods known in the art, such as via elution with cation exchange chromatography. The eluted fractions can then be examined to determine which fraction includes an assembly with a single SpyTag. The fraction with a single SpyTag can then be used to attach a single polymerase to each assembly, thereby creating a nanopore assemblies with a single polymerase attached thereto.
  • the different heptamer fraction can be separated based on molecular weight, such as via SDS-PAGE.
  • a reagent can then be used to confirm the presence of SpyTag associated with each fraction.
  • a SpyCatcher-GFP green fluorescent protein
  • the fraction with a single SpyTag can be identified, as evidenced by the furthest band migration and the presence of GFP fluorescence in the SDS-PAGE gel corresponding to the band. For example, a fraction containing seven alpha-hemolysin monomers and zero SpyTag fusion proteins will migrate the furthest, but will not fluoresce when mixed with SpyCatcher-GFP because of the absence of the SpyTag bound to the heptamers.
  • the fraction containing a single SpyTag will both migrate the next furthest (compared to other fluorescent bands) and will fluoresce, thereby allowing identification of the fraction with a single SpyTag bound to the heptamer.
  • the polymerase/SpyCatcher fusion protein can then be added to this fraction, thereby linking the polymerase to the nanopore assembly.
  • a heptameric nanopore may include seven variant subunits, with each subunit having a sequence corresponding to one of the amino acid sequence set forth in SEQ ID NOS: 17, 18, 19, 20, or 22 or to an amino acid sequence that is, respectively, 80%, 85%, 90%, 95%, 98% or more identical thereto, with a DNA polymerase attached to one of the subunits.
  • Pore based sensors can be used for electro-interrogation of single molecules.
  • a pore based sensor can include a nanopore of the present disclosure formed in a membrane that is disposed adjacent or in proximity to a sensing electrode.
  • the sensor can include a counter electrode.
  • the membrane includes a trans side (i.e., side facing the sensing electrode) and a cis side (i.e., side facing the counter electrode).
  • a nanopore including one or more of the alpha-hemolysin variants described herein will have an improved nanopore lifetime relative to a nanopore including wild-type alpha-hemolysin (i.e., a nanopore without any of the substitutions described herein).
  • the greater the number of variants included in the nanopore corresponds to a greater level of improvement in nanopore lifetime.
  • a heptameric nanopore where each of the seven alpha-hemolysin subunits corresponds to one of the sequences set forth as SEQ ID NOS: 17, 18, 19, 20, or 22 or a sequence that is, respectively, 80%, 85%, 90%, 95%, 98% or more identical thereto, may have a longer lifetime than a nanopore including only wild-type (native) alpha-hemolysin or fewer than seven variants. That is, in certain example embodiments, the greater of number of variants included in the nanopore corresponds to a longer nanopore lifetime.
  • all seven of the subunits of a heptameric nanopore include substitutions, such as the same substitution or overlapping substitutions that improve nanopore lifetime.
  • the variants in a given nanopore may be the same variant or a combination of different variants.
  • the lifetime of a nanopore including one or more alpha-hemolysin variants as described herein, such as those provided in SEQ ID NOS: 17, 18, 19, 20, and 22, is increased by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more as compared to a nanopore including only wild-type (native) alpha-hemolysin.
  • the lifetime of a nanopore including one or more alpha-hemolysin variants as described herein is doubled or tripled as compared to a nanopore including only wild-type (native) alpha-hemolysin.
  • the time for a tag to thread through the pore may be decreased.
  • the TTT for a nanopore comprising one or more of the variants described herein may be decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more as compared to a heptameric nanopore assembly consisting of wild-type (native) alpha-hemolysin.
  • a nanopore including one or more of the variants described herein may having an increased lifetime of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more—as well as a decreased TTT of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more—as compared to a heptameric nanopore assembly consisting of wild-type (native) alpha-hemolysin.
  • This example illustrates the expression and recovery of protein from bacterial host cells, e.g., E. coli.
  • DNA encoding the wild-type a-HL was purchased from a commercial source. The sequence was verified by sequencing.
  • the gene encoding either a wild-type or variant ⁇ -hemolysin was inserted into a pPR-IBA2 plasmid (IBA Life Sciences, Germany) under the control of T7 promoter.
  • E. coli BL21 DE3 (from Life Technologies) cells were transformed with the expression vector comprising the DNA encoding the wild-type or variant ⁇ -hemolysin using techniques well-known in the art. Briefly, the cells were thawed on ice (if frozen). Next, the desired DNA (in a suitable vector/plasmid) was added directly into the competent cells (should not exceed 5% of that of the competent cells) and mixed by flicking the tube. The tubes were placed on ice for 20 minutes. Next, the cells were placed in a 42° C. water bath for 45 seconds without mixing, followed by placing the tubes on ice for 2 min.
  • the cells were then transferred to a 15 ml sterilized culture tube containing 0.9 ml of SOC medium (pre-warmed at room temperature) and cultured at 37° C. for 1 hr in a shaker. Finally, an aliquot of the cells were spread onto a LB agar plate containing the appropriate antibiotic and the plates incubated at 37° C. overnight.
  • colonies were picked and inoculated into a small volume (e.g., 3 ml) of growth medium (e.g., LB broth) containing the appropriate antibiotic with shaking at 37° C., overnight.
  • growth medium e.g., LB broth
  • Site-directed mutagenesis is carried out using a QuikChange Multi Site-Directed Mutagenesis kit (Stratagene, La Jolla, Calif.) to prepare the example H35G+V149K+H144A (SEQ ID NO: 4) and H35G+E111N+M113A+126 ⁇ 131G+H144A+K147N (SEQ ID NO: 19), with the sequences including a C-terminal linker/TEV/HisTag for purification.
  • QuikChange Multi Site-Directed Mutagenesis kit (Stratagene, La Jolla, Calif.) to prepare the example H35G+V149K+H144A (SEQ ID NO: 4) and H35G+E111N+M113A+126 ⁇ 131G+H144A+K147N (SEQ ID NO: 19), with the sequences including a C-terminal linker/TEV/HisTag for purification.
  • QuikChange Multi Site-Directed Mutagenesis kit (Stratagene, La Jolla, Calif.) is also carried out to prepare a variant (E111N+M113A+126 ⁇ 131G+K147N, SEQ ID NO: 20) for Polymerase attachment, with the variant including a C-terminal SpyTag, KG linker, and HisTag.
  • the variants were expressed and purified as in Example 1.
  • This example describes the assembly of a 1:6 heptameric nanopore including one subunit having a SpyTag sequence for subsequent polymerase attachment (the “ ⁇ -HL-variant-SpyTag” subunit) and six ⁇ -HL-variant subunits with no SpyTag (the “ ⁇ -HL-variant” subunits).
  • the ⁇ -HL-variant-SpyTag (E111N+M113A+126 ⁇ 131G+K147N, SEQ ID NO: 20)) was prepared and expressed as described in Examples 1 and 2 with a C-terminal SpyTag, KG linker, and HisTag.
  • the ⁇ -HL-variant-SpyTag protein was then then purified on a cobalt affinity column using a cobalt elution buffer (200 mM NaCl, 300 mM imidazole, 50 mM tris, pH 8). The protein was stored at 4° C. if used within 5 days, otherwise 8% trehalose was added and stored at ⁇ 80° C.
  • variants of H35G+E111N+M113A+126-131G+H144A+K147N were prepared and expressed as described in Examples 1 and 2 with a linker/TEV/HisTag and purified on a cobalt affinity column using a cobalt elution buffer (200 mM NaCl, 300 mM imidazole, 50 mM tris, pH 8).
  • the ⁇ -HL-variant protein was then incubated with 1 mg of TEV protease for every 5 mg of protein at 4 C for 4 hours.
  • the mixture is purified on a cobalt affinity column to remove TEV protease and undigested protein.
  • the proteins were stored at 4° C. if used within 5 days, otherwise 8% trehalose was added and stored at ⁇ 80° C.
  • the ⁇ -HL-variant-SpyTag to desired ⁇ -HL-variant protein solutions were mixed together at a 1:9 ratio to ultimately facilitate the formation of a mixture of heptamers at the desired ratio. It is expected that such a mixture heptamers will result in various fractions that include varying ratios of ⁇ -HL-variant-SpyTag to ⁇ -HL-variant protein (0:7; 1:6, 2:5, 3:4, etc.), where the ⁇ -HL-variant-SpyTag component is present as 0, 1, 2, 3, 4, 5, 6, or seven monomeric subunits of the heptamer.
  • 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 mixture of ⁇ -HL monomers to a final concentration of 5 mg/ml.
  • the mixture of the ⁇ -HL monomers was incubated at 37° C. for at least 60 min. Thereafter, n-Octyl- ⁇ -D-Glucopyranoside (130G) was added to a final concentration of 5% (weight/volume) to solubilize the resulting lipid-protein mixture.
  • the sample was centrifuged to clear protein aggregates and left over lipid complexes and the supernatant was collected for further purification.
  • the mixture of ⁇ -HL heptamers was then subjected to cation exchange purification and the elution fractions collected. For each fraction, two samples were prepared for SDS-PAGE. The first sample included 15 uL of ⁇ -HL eluate alone and the second sample was combined with 3 ug of SpyCatcher-GFP. The samples were then incubated and sheltered from light and at room temperature for 1-16 hours. Following incubation, 5 uL of 4 ⁇ Laemmli SDS-PAGE buffer (Bio-RadTM) was added to each sample. The samples and a PrecisionPlusTM Stain-Free protein ladder were then loaded onto a 4-20% Mini-PROTEAN Stain-Free protein precast gel (Bio-Rad). The gels were ran at 200 mV for 30 minutes. The gels were then imaged using a Stain-Free filter.
  • the conjugation of SpyCatcher-GFP to heptameric ⁇ -HL/SpyTag can be observed through molecular weight band shifts during SDS-PAGE.
  • Heptamers containing a single SpyTag will bind a single SpyCatcher-GFP molecular and will thus have a shift that corresponds to the molecular weight of the heptameric pore plus the molecular weight of a single SpyCatcher-GFP, while heptamers with two or more SpyTags should have correspondingly larger molecular weight shifts.
  • the peaks eluted off of the cation exchange column during heptameric ⁇ -HL purification above can be analyzed for the ratio of ⁇ -HL/SpyTag to ⁇ -HL-variant.
  • the presence of SpyCatcher-GFP attachment can be observed using a GFP-fluorescence filter when imaging the SDS-PAGE gels.
  • the fraction whose molecular weight shift corresponded to a single addition of SpyCatcher-GFP was determined using a molecular weight standard protein ladder. Bio-Rad's stain-free imaging system was used to determine the molecular weight shift. The presence of GFP fluorescence was determined using a blue filter. The presence of fluorescence was used to confirm the presence of the SpyTag protein. The elution fraction corresponding to the 1:6 ratio, i.e., one ⁇ -HL-variant-SpyTag to six ⁇ -HL-variants, was then used for further experiments.
  • a 1:6 heptamer was also produced having a V149K substitution added to each of the H35G+E111N+M113A+126 ⁇ 131G+H144A+K147N (SEQ ID NO: 19) ⁇ -HL-variants. That is, the “six” component of the 1:6 heptamer included a V149K substitution, with the ⁇ -HL-variant-SpyTag “one” component being E111N+M113A+126 ⁇ 131G+K147N denoted as N rectification in Table 1 (SEQ ID NO: 20).
  • 1:6 heptamers In addition to the 1:6 heptamers described above, these same or similar procedures were used to create a 1:6 heptamer having six H35G+V149K+H144A (SEQ ID NO: 4) ⁇ -HL-variants. More particularly, such 1:6 heptamers have a wild-type ⁇ -HL-SpyTag as the “one” component of the 1:6 ratio and a “six” component including six H35G+V149K+H144A (SEQ ID NO: 4) ⁇ -HL-variants.
  • This example provides for the attachment of a polymerase to a nanopore.
  • the polymerase may be coupled to the nanopore by any suitable means. See, for example, PCT/US2013/068967 (published as WO2014/074727; Genia Technologies), PCT/US2005/009702 (published as WO2006/028508), and PCT/US2011/065640 (published as WO2012/083249; Columbia Univ).
  • the polymerase e.g., phi29 DNA Polymerase
  • phi29 DNA Polymerase was coupled to a protein nanopore (e.g. alpha-hemolysin), through a linker molecule.
  • a protein nanopore e.g. alpha-hemolysin
  • the SpyTag and SpyCatcher system which spontaneously forms covalent isopeptide linkages under physiological conditions, was used. See, for example, Li et al, J Mol Biol. 2014 Jan. 23; 426(2):309-17.
  • the Sticky phi29 SpyCatcher HisTag was expressed according to Example 1 and purified using a cobalt affinity column.
  • the SpyCatcher polymerase and the SpyTag oligomerized protein were incubated at a 1:1 molar ratio overnight at 4° C. in 3 mM SrCl 2 .
  • the 1:6-polymerase-template complex is then purified using size-exclusion chromatography.
  • This example shows the activity of the nanopores as provided by Examples 1-4 (nanopores with an attached polymerase).
  • the variant nanopores were assayed to determine the effect of the substitutions. More particularly, the 1:6 ratio nanopores with the “one” component of the nanopore including the ⁇ -HL-variant-SpyTag (with the polymerase attached) and the “six” component including the ⁇ -HL-variants were assayed to determine the effect of the substitutions on nanopore lifetime. Nanopores also including the V149K substitution were similarly assayed to determined nanopore lifetime (see Table 1, below).
  • nanopores having the 1:6 wild-type ⁇ -HL-SpyTag (with polymerase attached) as the “one” component and six H35G+V149K+H144A (SEQ ID NO: 4) ⁇ -HL-variants as the “six” component were analyzed for the effect of the substitutions on Time-To-Thread.
  • the bilayers were formed and pores were inserted as described in PCT/US14/61853 filed 23 Oct. 2014.
  • the nanopore device (or sensor) used to detect a molecule (and/or sequence a nucleic acid) was set-up as described in WO2013123450.
  • This assay was designed to measure the time a nanopore is able to function properly in a lipid bilayer under the effect of alternating voltages, i.e., squarewaves.
  • Nanopore lifetime uses alternating positive and negative voltages (squarewaves) to pore lifetime.
  • Our sequencing complex is comprised of a protein nanopore ( ⁇ HL) which is attached to a single DNA polymerase (see Example 4). Current passes differently through the pore in the positive and negative applied voltages. Nanopores may pass ions differently under positive compared to negative voltages. This leads to a pumping of salt and water ions out of the well. Over the lifetime of the pore, this process gradually deforms the bilayer, and causes the nanopore to no longer conduct ions across the bilayer. When this happens, it marks the end of the pore lifetime. These times are then recorded and plotted as a histogram, as shown in FIG. 1A , FIG. 2A , and FIG. 3A .
  • the Genia Sequencing device is used with a Genia Sequencing Chip.
  • the electrodes are conditioned and phospholipid bilayers are established on the chip as explained in PCT/US2013/026514.
  • Genia's sequencing complex is inserted to the bilayers following the protocol described in PCT/US2013/026514 (published as WO2013/123450).
  • the pore lifetime data was collected using a buffer system comprised of 20 mM HEPES pH 8, 300 mM KGlu, 3 uM tagged nucleotide, 3 mM Mg 2+ , with a voltage applied of 235 mV peak to peak with a modulation rate of 80 Hz.
  • nanopores including 1:6 ratios of ⁇ -HL-variant-SpyTag (E111N+M113A+126 ⁇ 131G+K147N, SEQ ID NO: 20) and ⁇ -HL-variant subunits (H35G+E111N+M113A+126 ⁇ 131G+H144A+K147N (SEQ ID NO: 19)) showed significantly improved lifetimes.
  • addition V149K substitution did reduce the overall level of improved lifetime of the nanopores, the lifetime was nevertheless improved by 5.4% to ⁇ 26% of pores lasting at least 1 hour (as compared to controls) (see Table 1).
  • This assay was designed to measure the time it takes to capture a tagged molecule by a DNA polymerase attached to the nanopore using alternating voltages, i.e., squarewaves.
  • the Genia Sequencing device is used with a Genia Sequencing Chip.
  • the electrodes are conditioned and phospholipid bilayers are established on the chip as explained in PCT/US2013/026514.
  • Genia's sequencing complex is inserted to the bilayers following the protocol described in PCT/US2013/026514 (published as WO2013/123450).
  • the time-to-thread data was collected using a buffer system comprised of 20 mM HEPES pH 8, 300 mM KGlu, 3 uM tagged nucleotide, 3 mM Mg 2+ , with a voltage applied of 235 mV peak to peak with a duty cycle of 80 Hz.
  • time-to-thread was measured by determining how long the second squarewave reported unobstructed open channel current. As an example, if 10 consecutive squarewaves showed tagged nucleotide captures that lasted to the end of the positive portion of the squarewave then the time-to-thread parameter would be calculated from squarewaves 2-10 (the first squarewave does not factor into the calculation because the polymerase did not have a tag bound to it in the previous squarewave). These time-to-thread numbers were then collected for all of the pores in the experiment and statistical parameters extracted from them (such as a mean, median, standard deviation etc.).

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