Engineered nanopore with negatively charged polymer threaded through the channel

Abstract

A nanopore is provided having a charged polymer attached thereto. At least one end of the charged polymer is fixed at or near one end of the channel of the nanopore in a position that allows the charged polymer to enter the channel. The charged polymer may optionally be fixed in a threaded configuration. When in a threaded configuration, the charged polymer increases the electrical conductivity of the nanopore while still allowing other polymers (such as nucleic acids or polymer tags of tagged nucleotides) to flow through the channel of the nanopore.

Description

[Technical Field]

[0001] Background of the Invention Technical Field Nanopore constructs and their use for nucleic acid sequencing. [Background technology]

[0002] 2. Description of Related Art The most basic nanopore sequencing systems include a detection electrode positioned near the nanopore, which can detect and record the electrochemical properties of ions flowing through the nanopore. When a relatively large molecule occupies the nanopore, the electrochemical properties detected by the detection electrode change. The identity of the molecule occupying the nanopore can then be determined based on changes in the electrochemical properties, such as a change in the current flowing through the nanopore or a decay in the measured voltage. An overview of nanopore-based sequencing systems can be found in Wang I and Feng.

[0003] The nanopores used in these sequencing systems typically come in one of three flavors: biological nanopores, solid-state nanopores, and hybrid nanopores. Biological nanopores are naturally occurring pore-forming molecules, particularly proteins, such as porins and hemolysins. Commonly used pore-forming proteins include the α-hemolysin (αHL) protein from Staphylococcus aureus, outer membrane protein G (ompG) from Escherichia coli, and porin MspA (MspA) from Mycobacterium smegmatis. In some cases, as in the case of ompG, the pore is formed from a single subunit of the protein. In other cases, as in the case of αHL and MspA, the pore is a multisubunit assembly of the pore-forming protein. For example, αHL forms a heptameric pore structure, and MspA forms an octameric pore structure. Exemplary engineered nanopores based on these proteins are described, for example, in WO 2016 / 069806 (αHL), WO 2017 / 050728 (αHL), WO 2017 / 184866 (αHL), WO 2018 / 002125 (αHL), WO 2012 / 178097 (αHL), Gari (ompG), WO 2017 /

[0004] Solid-state nanopores are pore structures made from synthetic materials, for example, by forming nanometer-sized pores in synthetic membranes. Exemplary materials that can form solid-state nanopores include silicon nitride, silica, alumina, graphene, boron nitride, and molybdenum disulfide. Solid-state nanopores have been reviewed by Chen, Lee, Wasfi, Wang I, and Feng.Hybrid nanopores incorporate both biological and solid-state nanopores. For example, a biological nanopore (such as an αHL nanopore) can be inserted into a solid-state nanopore. Hybrid nanopores are reviewed by Lee, Wasfi, and Feng.

[0004] One approach to nanopore-based nucleic acid sequencing involves threading single-stranded nucleic acids directly through the pore (referred to herein as "direct sequencing"). Each nucleotide (or unique combination of nucleotides) produces a unique change in at least one electrochemical property of the pore. These systems frequently employ means to control the rate at which the nucleic acid moves through the pore, such as tethering enzymes to the pore (including polymerases and helicases), removing negatively charged residues from the pore channel and adding positively charged residues to the pore channel, and adding double-stranded regions to the single-stranded nucleic acid. Exemplary direct sequencing approaches are discussed, for example, by Feng, Manrao, and Wang I.

[0005] Another method involves a sequencing-by-synthesis (SBS) approach, in which a polymerase-catalyzed amplification reaction is performed near the opening of a nanopore with tagged nucleotide polyphosphate molecules. Each tagged nucleotide polyphosphate contains a distinct tag moiety that generates a unique electrochemical signature when it is present in or near the nanopore. When the tagged nucleotide polyphosphate is incorporated into an amplicon, the tag passes into or near the nanopore, and the tag's electrochemical signature is recorded. The sequence of the amplicon is derived from the order in which the tag moieties enter the nanopore. Exemplary tag-based SBS approaches and materials for carrying out such methods are described, for example, in WO 2012-083249, WO 2013 / 154999, U.S. Patent Application Publication No. 2014 / 0309144, U.S. Patent No. 9,017,937, WO 2015 / 148402, WO 2016 / 069806, WO 2016 / 144973, U.S. Patent Application Publication No. 2016 / 022236 No. 3, U.S. Patent Application Publication No. 2016 / 0333327, International Publication No. 2017 / 050728, International Publication No. 2017 / 184866, International Publication No. 2017 / 050722, U.S. Patent Application Publication No. 2017 / 0267983, U.S. Patent Application Publication No. 2018 / 0245147, U.S. Patent Application Publication No. 2018 / 0094249, International Publication No. 2018 / 002125, and Kumar. Various tags have been proposed for use in such systems, including polypeptide (e.g., polylysine tags) and polynucleotide-based tags. See, for example, U.S. Patent No. 8,652,779 and International Publication No. 2017042038. Summary of the Invention

[0006] Summary of the Invention Disclosed herein are engineered nanopores having charged polymers threaded through the nanopore channel, and their use in nanopore-based sequencing systems and methods. It has been discovered that the inclusion of a charged polymer significantly increases the conductance of the pore, thereby aiding in the discrimination of different molecules (such as groups of nucleotides or polymer tags) that occupy the pore during sequencing operations.

[0007] In one embodiment, a charged polymer-linked nanopore (CPL nanopore) is provided, the CPL nanopore comprising a channel having an inlet side and an outlet side, and a charged polymer threaded through the channel, the charged polymer being disposed within the channel and comprising a negatively charged region extending substantially the entire length of the channel. In some embodiments, a first end of the charged polymer is fixed in a fixed position near the inlet side of the channel, and a second end of the charged polymer is fixed in a fixed position near the outlet side of the channel. Exemplary nanopores include those based on α-hemolysin (αHL), outer membrane porin G (OmpG), Mycobacterium smegmatis porin A (MspA), leukocidin nanopore, outer membrane porin F (OmpF) nanopore, cytolysin A (ClyA) nanopore, outer membrane phospholipase A nanopore, Neisseria autotransporter lipoprotein (NalP) nanopore, WZA nanopore, Nocardia farcinica NfpA / NfpB cation-selective channel nanopore, lysenin nanopore, erolysin, and Curlin sigma S-dependent growth subunit G (CsgG) nanopore. In an exemplary embodiment, the biological nanopore is an αHL-based heptameric nanopore, which comprises seven monomeric subunits, each monomeric subunit comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO:1.

[0008] In another embodiment, a system for performing nanopore-based nucleic acid sequencing is provided, which generally includes a CPL-nanopore as disclosed herein and other elements useful for distinguishing between molecules that occupy the nanopore.

[0009] In another embodiment, a method for nanopore-based nucleic acid sequencing using a CPL-nanopore is provided.

[0010] Other details and inventions are described in detail herein. [Brief explanation of the drawings]

[0011] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] Schematic showing a charged polymer bound to a nanopore. [Figure 1B] Illustrated is a charged polymer bound to a nanopore at only its first end in the released configuration. [Figure 1C] Diagram showing a charged polymer bound to a nanopore at only its first end in an inserted configuration. [Figure 1D] FIG. 1 shows a charged polymer attached to a first end near the entrance side of the nanopore channel and a second end near the exit side of the nanopore in an inserted configuration. [Figure 1E] FIG. 1 shows a charged polymer in an inserted configuration, with a first end near the entrance side of the nanopore channel and a second end attached to an external entity near the exit side of the nanopore. [Figure 1F] 1 shows an exemplary method for anchoring a charged polymer to a nanopore in an inserted configuration, where the first end is covalently attached to the nanopore and the second end is linked to the exterior of the nanopore. [Figure 1G] 1 shows an exemplary method for linking a charged polymer to a nanopore in an intercalated configuration, where the second end of the charged polymer is covalently attached to the nanopore and the first end is linked to the exterior of the nanopore. [Figure 2] Graph of the conductance of a charged polymer-linked nanopore (black squares) versus a nanopore without a charged polymer (gray circles) at various voltage levels. [Figure 3] Cross-section of a heptameric α-hemolysin nanopore, with the various regions indicated. [Figure 4] Cross-section of a heptameric α-hemolysin nanopore with a charged polymer threaded through the channel. The charged polymer is linked at one end to an N17C substitution in the nanopore and at the second end to streptavidin via a biotin moiety. [Figure 5] FIG. 1 shows an exemplary nanopore sequencing complex comprising a charged polymer-linked nanopore as disclosed herein. [Figure 6] FIG. 1 is a top view of an exemplary nanopore sensor chip including a charged polymer-linked nanopore as disclosed herein. [Figure 7] FIG. 1 shows an exemplary nanopore cell containing a nanopore sequencing complex comprising a charged polymer-linked nanopore as disclosed herein. [Figure 8] FIG. 1 shows an exemplary embodiment of an active sequencing complex for performing tag-based SBS nucleic acid sequencing. [Figure 9] FIG. 1 shows an exemplary embodiment of an active sequencing complex for performing direct sequencing. [Figure 10] FIG. 1 illustrates a particular embodiment of a charged polymer. [Figure 11] Figure 1 shows the results of cation exchange chromatography purification of 1:6 α-hemolysin nanopore with an N17C substitution and a SpyCatcher moiety in one monomer. [Figure 12] Image of SDS-PAGE separation of cation exchange chromatography purified 1:6 nanopore shown in Figure 11. Lane 0: Molecular weight standard. Lane 1: Peak P1 from cation exchange in the absence of SpyCatcher-GFP. Lane 2: Peak P1 from cation exchange mixed with SpyCatcher-GFP. Lane 3: Peak P2 from cation exchange in the absence of SpyCatcher-GFP. Lane 4: Peak P2 from cation exchange mixed with SpyCatcher-GFP. [Figure 13]Figure 1 shows the results of a series of capture events with a charged polymer-tethered α-hemolysin nanopore. The charged polymer-tethered α-hemolysin nanopore was first recorded with the charged polymer only tethered to the nanopore at its first end (left of arrow). Streptavidin was then flowed over the chip on the trans side of the barrier, and recording resumed (right of arrow). DETAILED DESCRIPTION OF THE INVENTION

[0012] Detailed Description of the Invention 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.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2nd 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.Experts are particularly directed to Sambrook et al., 1989 and Ausubel FM et al., 1993 for definitions and terms in the art.It should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described, as these may vary.

[0014] 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.

[0015] 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.

[0016] I. Terminology α-Hemolysin: As used herein, "α-hemolysin," "α-hemolysin," and "αHL" are used interchangeably to refer to a monomeric protein that self-assembles into a heptameric, water-filled transmembrane channel (i.e., nanopore). Depending on the context, the term can also refer to the transmembrane channel formed by seven monomeric proteins.

[0017] Base pair (bp): As used herein, 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.

[0018] Capture event: The insertion of a molecule into a nanopore sufficient to cause a change in the characteristics of the ionic current flowing through the nanopore such that the change is detectable by a detection electrode.

[0019] 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.

[0020] Isolated: An "isolated" molecule is a biomolecule that is separated from at least one other molecule with which it is normally associated, eg, in its natural environment.

[0021] Monomer subunit: A structural subunit of a multimeric protein complex. For example, a heptameric α-hemolysin pore contains seven α-hemolysin monomer subunits. A monomer subunit that is not oligomerized into a multimeric subunit is referred to herein as a "non-oligomerized monomer subunit."

[0022] Monomer unit: A structural subunit of a polymer.

[0023] 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.

[0024] Nanopore: As used herein, the term "nanopore" generally refers to a pore, channel, or passageway formed or otherwise provided in an electrically resistive barrier (such as a lipid membrane, a silicon layer, a polymer layer, or a graphene layer) through which an ionic current may pass. Unless otherwise specified, the general term "nanopore" is intended to include biological nanopores, solid-state nanopores, and hybrid nanopores.

[0025] Nanopore sequencing complex: A site at which nanopore-based sequencing methods may be performed, generally comprising at least (a) a nanopore configured to establish current flow through a channel and allow a molecule of interest (such as a nucleic acid or a polymer tag of a tagged nucleotide) to enter the channel, and (b) an electrode or set of electrodes configured to detect characteristics of the nanopore sequencing complex (e.g., resistance, capacitance, voltage decay, and ionic current flow, etc.).

[0026] Native Amino Acid: Any amino acid in an amino acid sequence that, when aligned with a reference amino acid sequence, is the same as the amino acid occupying the corresponding position in the reference sequence.

[0027] Non-native moiety: A component of a nanopore that has a portion not found in a reference structure. For example, if the nanopore comprises a polypeptide, a "non-native amino acid" is any amino acid having a side chain that represents a substitution or insertion at a particular position relative to the reference amino acid sequence, or that represents a chemical modification of the side chain of a native amino acid.

[0028] Nucleic acid molecule: The term "nucleic acid molecule" includes RNA, DNA, and cDNA molecules. It will be understood that, as a result of the degeneracy of the genetic code, numerous nucleotide sequences can be produced that encode a given protein, such as α-hemolysin and / or variants thereof. The present invention contemplates all possible variant nucleotide sequences.

[0029] Peptide: The terms "peptide" and "peptide linkage" are intended to refer to any backbone linkage between two amino acids and / or amino acid analogs resulting from a condensation reaction between the carboxylic acid moiety of one amino acid or amino acid analog and the amino group of a second amino acid or amino acid analog. Unless otherwise clear from the context, these terms are intended to be understood in all cases to include (but not be limited to) linkages between α-amino acids, β-amino acids, γ-amino acids, δ-amino acids, and combinations thereof, as well as linkages between backbone carboxylic acid moieties and side chain amino moieties (e.g., with an ε-linked lysine).

[0030] Peptide chain: The term "peptide chain" is intended to refer to any sequence of two or more amino acids and / or amino acid analogs linked by peptide linkages.

[0031] Peptidomimetic: The terms "peptidomimetic" and "peptidomimetic linkage" refer to a backbone linkage between two amino acid analogs or between an amino acid and an amino acid analog, including, but not limited to, peptoids (amino acids in which the side chain is linked to the amino group), azapeptides (replacement of the α-carbon with a nitrogen), oligoureas (peptide linkages substituted with urea linkages), arylamides, oligohydrazides, and the like.

[0032] Peptidomimetic chain: The term "peptidomimetic chain" is intended to refer to any sequence of two or more amino acids and / or amino acid analogs linked by peptidomimetic backbone linkages.

[0033] Percent homology: The term "% homology" is used interchangeably herein with the term "% identity" and 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% homology is equivalent to 80% sequence identity as determined by a defined algorithm; thus, a homolog of a given sequence has greater than 80% sequence identity over the 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, for example, 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, such as 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.)) Unless otherwise specified, references to an alignment of two amino acid sequences refer to the alignment obtained using the EMBOSS Needle pairwise sequence alignment tool with a BLOSUM 62 matrix, a GAP OPEN setting of 10, a GAP EXTEND setting of 0.5, an END GAP PENALTY setting of "false", an END GAP OPEN setting of 10, and an END GAP EXTEND setting of 0.5 (available from EMBL-EBI).

[0034] Polypeptide: Unless otherwise specified or clear based on the context of this disclosure, the term "polypeptide" shall be understood in its broadest sense and shall encompass any sequence of two or more amino acids and / or amino acid analogs linked by peptidic and / or peptidomimetic linkages.

[0035] 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.

[0036] Tag: As used herein, the term "tag" refers to a nanopore-detectable moiety, which can be an atom or molecule, or an assembly of atoms or molecules. The label can provide an optical, electrochemical, magnetic, or electrostatic (e.g., inductive, capacitive) signature, which can be detected using the nanopore. Typically, when a nucleotide is attached to a tag, the tag is referred to as a "tagged nucleotide."

[0037] Variant: As used herein, the term "variant" of a reference polypeptide or nucleic acid is any such molecule that contains at least one molecular alteration relative to the reference molecule.

[0038] II. Creation of Charged Polymers and Polymer-Linked Nanopores Disclosed herein are biological nanopores having charged polymers attached in a configuration that allows the charged polymer to be threaded through the channel of the nanopore.

[0039] As shown in FIG. 1A , charged polymer 100 includes at least (a) a negatively charged region 101 having a high density of negative charges; and (2) an entity at or near a first end 102 of charged polymer 100 that facilitates binding to the nanopore at or near the entrance to the nanopore channel. In some embodiments, charged polymer 100 further includes an entity at a second end 103 of charged polymer 100 that facilitates binding to either the nanopore or an entity located outside the nanopore. The terms “first end” and “second end” include, but are not limited to, conjugates formed on terminal monomer units of the charged polymer. In some embodiments, the conjugate forming first end 102 can be located on an interior portion of charged polymer 100, as long as negatively charged portion 101 can still thread into the channel and increase the conductance of the channel. In some embodiments, the conjugates forming the first end 102 and the second end 103 can be located in an interior portion of the charged polymer 100 such that the negatively charged portion 101 can still thread into the channel, increasing the conductance of the channel, and the second end 103 can still be anchored at or near the outlet of the channel.

[0040] The negatively charged region 101 contains a sufficient density of negatively charged monomer units to increase the conductivity of the nanopore while allowing other polymers (such as nucleic acids or polymer tags of tagged nucleotides) to flow through the nanopore channel. At least a portion of the monomer units in the negatively charged region have a negative charge at neutral pH. Furthermore, the negatively charged region does not have a significant number of monomer units with bulky side chains. Exemplary monomer units include monomer units containing phosphate groups capable of forming phosphodiester bonds (e.g., nucleotides, nucleotide derivatives, and alkyl glycol phosphates (e.g., ethylene glycol phosphate)), negatively charged polypeptides (e.g., polypeptides containing a high concentration of aspartic acid or glutamic acid, explicitly including polyaspartic acid and polyglutamic acid). In one embodiment, the negatively charged region comprises, consists essentially of, or consists of a polymer chain of monomeric units linked by phosphodiester bonds, including, but not limited to, nucleotides, nucleotide derivatives, abasic sites (including polymers formed from alkyl glycol phosphates), and combinations thereof. In a further embodiment, the negatively charged region has the following structure: [ka] (In the formula, R 1 In another embodiment, R 1 is two carbons long. 1 is two carbons long. 1 is three carbons long. 1 is four carbons long. 1 is 5 carbons long. 1 is six carbons long. 1 is 7 carbons long. 1 is 8 carbons long.1 is 9 carbons long. 1 is 10 carbons long. In another embodiment, the charged polymer has the structure [ka] where a is an integer selected such that the length of the charged polymer is at least as long as the length of the channel of the nanopore to which it is connected, and R 1 is an alkyl chain of 2 to 10 carbons in length (including all integers in between), and R 2 and R 3 is a nucleotide, b is 0 to 10, c is 0 to 10, and R 4 and R 5 is the first end, and R 4 and R 5 and the other is the second end). In some embodiments, a is 10 to 100. In some embodiments, a is 10 to 90. In some embodiments, a is 10 to 80. In some embodiments, a is 10 to 70. In some embodiments, a is 10 to 60. In some embodiments, a is 10 to 50. In some embodiments, a is 20 to 100. In some embodiments, a is 20 to 90. In some embodiments, a is 20 to 80. In some embodiments, a is 20 to 70. In some embodiments, a is 20 to 60. In some embodiments, a is 20 to 50.

[0041] Figures 1B, 1C, 1D, and 1E show cross sections of a nanopore 104 with a charged polymer attached. The nanopore 104 includes a channel 105 that runs through the body of the nanopore and forms a pathway through which molecules can pass from one side of the nanopore to the other. When used to sequence nucleic acids or other molecules, the side of the channel through which the molecule to be detected enters is called the channel entrance 105a, and the opposite side of the channel is called the channel exit 105b. The polymer-conjugated pore is formed by fixing the first end 102 in a fixed position near the channel entrance 104. The charged polymer 100 can then be captured by the nanopore, and the negatively charged portion 102 can be threaded through the channel and, if desired, past the channel exit 105b. In configurations where only the first end 102 is fixed in place (Figures 1B and 1C), the charged polymer 100 can occupy two configurations: The nanopore may be configured in two ways: an "exhausted" configuration (FIG. 1B), in which the negatively charged moiety 101 is substantially outside the channel 105; or an "inserted" configuration (FIG. 1C), in which the negatively charged moiety 101 threads through the channel 105 and, optionally, beyond the channel outlet 105b. Any method for creating such a bond may be used. For example, the bond may be formed by a covalent bond between a reactive moiety on the first end 102 and another reactive moiety on the nanopore or on a surface disposed on the outside and near the entrance of the nanopore. For example, the first end 102 may contain N-hydroxysuccinimide (NHS) or sulfo-NHS, which may be reacted with a primary amine disposed on the nanopore or another surface near the entrance of the pore (such as a bead or wall of a well into which the nanopore is inserted). As another example, the first end 102 may include a maleimide, an iodoacetyl group, or a pyridyl disulfide, which may react with a sulfhydryl (such as a cysteine) located on the nanopore or another surface near the entrance to the pore (such as a bead or wall of a well into which the nanopore is inserted).As another example, first end 102 may include a primary amine, which can react (in combination with EDC) with a carboxyl group located on the nanopore or another surface near the entrance to the pore (such as a bead or wall of a well into which the nanopore is inserted). Many other binding chemistries are known in the art and can be used as well. Alternatively, first end 102 may include a first member of a specific binding pair that interacts with a second member of the specific binding pair bound to the nanopore or on a surface located outside and near the entrance to the nanopore. Exemplary specific binding pairs include biotin / avidin, biotin / streptavidin, and antibody / epitope. In one embodiment, first end 102 includes biotin, which is bound by avidin or streptavidin tethered to the nanopore or another surface near the entrance to the pore (such as a bead or wall of a well into which the nanopore is inserted). In another embodiment, first end 102 comprises an epitope tag (such as a hapten, FLAG tag, HA tag, His tag, Myc tag, V5 tag, Xpress tag, Thrombin tag, BAD tag, Factor Xa tag, VSVG tag, SV40 NLS tag, Protein C tag, S tag, OneStrap tag, SB1 tag, etc.) that is bound by an anti-epitope tag antibody or antibody fragment tethered to the nanopore or another surface near the entrance to the pore (such as a bead or wall of a well into which the nanopore is inserted).

[0042] In some configurations, the charged polymer 100 further includes a second end 103 that is anchored to a binding entity 106 (FIG. 1E), such as an antibody or streptavidin molecule, at or near the channel exit 105b (FIG. 1D, indicated by the dashed line between the nanopore 104 and the second end 103), or outside the channel exit 105b. With the second end 103 anchored in place, the charged polymer occupies only the inserted configuration. Any method for creating such a bond can be used. For example, the second end 103 may be formed by a covalent bond between a reactive moiety on the charged polymer and another reactive moiety on the nanopore or on a surface outside the nanopore. For example, N-hydroxysuccinimide (NHS) and sulfo-NHS are commonly used to covalently attach labels to primary amines. Maleimides, iodoacetyl groups, and pyridyl disulfides are commonly used to covalently attach labels to sulfhydryls (such as cysteines). Primary amines in combination with EDC are commonly used to covalently attach labels to carboxyl groups (such as aspartic acid side chains, glutamic acid side chains, or the carboxy terminus). Hydrazine and alkoxyamines are commonly used to covalently attach labels to glycoproteins. As another example, the charged polymer 100 may be attached to a first member of a specific binding pair that interacts with a second member of the specific binding pair located near the outlet side of the channel. Exemplary specific binding pairs include biotin / avidin, biotin / streptavidin, and antibody / epitope. In one embodiment, the second end 103 comprises biotin, which is bound by avidin or streptavidin tethered to a nanopore near the outlet side of the channel or disposed on the exterior surface of the well or the outlet side of the channel.In another embodiment, the second end 103 comprises an epitope tag (such as a hapten, FLAG tag, HA tag, His tag, Myc tag, V5 tag, Xpress tag, Thrombin tag, BAD tag, Factor Xa tag, VSVG tag, SV40 NLS tag, Protein C tag, S tag, OneStrap tag, SB1 tag, etc.) that is bound by an anti-epitope tag antibody or antibody fragment tethered to the nanopore near the outlet side of the channel, or is positioned within the well or on the outer surface of the outlet side of the channel.

[0043] FIG. 1F shows an embodiment in which first end 102 includes a reactive moiety 102a and second end 103 includes a first member of a specific binding pair 103a. Reactive moiety 102a reacts with a reactive moiety on nanopore 104a to form a covalent bond 107 between first end 102 and nanopore 104 (arrow I). The reaction product is fractionated by affinity chromatography using the second member of the specific binding pair as a capture agent to separate the polymer-conjugated nanopore from the unconjugated nanopore (not shown). The purified polymer-conjugated nanopore is then inserted into an electrochemically resistant barrier 108 on a biochip (arrow II). Channel 105 creates a pathway for ion flow through barrier 108, with channel inlet 105a located on a first side of the barrier and channel outlet 105b located on a second side of the barrier. A second member of specific binding pair 106 is positioned on a second side of the barrier adjacent channel outlet 105b. An electric potential is established across the barrier (arrow III), causing second end 103 to thread through channel 105 and exit channel outlet 105b, where the first member of specific binding pair 103a interacts with the second member of specific binding pair 106, thereby locking second end 103 in place. This configuration holds negatively charged moiety 101 in an inserted position within the channel.

[0044] 1G shows an embodiment in which first end 102 includes a first member of a specific binding pair 103a and second end 103 includes a reactive moiety 102a. Reactive moiety 102a reacts with a reactive moiety on nanopore 104a to form a covalent bond 107 between second end 103 and nanopore 104 (arrow I). The reaction product is fractionated by affinity chromatography using the second member of the specific binding pair as a capture agent to separate the polymer-conjugated nanopore from the unconjugated nanopore (not shown). The purified polymer-conjugated nanopore is then inserted into an electrochemically resistant barrier 108 on a biochip (arrow II). Channel 105 creates a pathway for ion flow through barrier 108, with channel inlet 105a located on a first side of the barrier and channel outlet 105b located on a second side of the barrier. The second member of specific binding pair 106 is positioned on a first side of the barrier adjacent to channel entrance 105a. An electric potential is established across the barrier (arrow III), causing first end 102 to thread through channel 105 and out channel entrance 105a, where the first member of specific binding pair 103a interacts with the second member of specific binding pair 106, thereby locking first end 102 in place. This configuration holds negatively charged moiety 101 in an inserted position within the channel.

[0045] As shown in Figure 2, when the charged polymer is in the inserted configuration, the conductance of the pore increases substantially. Without being bound by theory, this enhanced conductance can be attributed to the polymer carrying a large counterion cloud that facilitates the transport of counterions through the channel.

[0046] B. α-hemolysin nanopore In one embodiment, the nanopore comprises a biological nanopore based on α-hemolysin (αHL). The αHL nanopore is a heptameric structure formed from seven monomer subunits of the αHL polypeptide from Staphylococcus aureus. A cross-section of an exemplary αHL nanopore is shown in FIG. 3. The αHL nanopore has a cap region 301 and a beta-barrel region 302, with a channel 303 extending axially through the cap and stem regions. The channel 303 can be divided into an entrance 304, a constriction region 305, a beta-barrel body 306, and a beta-barrel exit 307.

[0047] References herein to the "beta-barrel region" include the constriction region 305, the beta-barrel body 306, and the beta-barrel exit 307, respectively. References herein to the "αHL nanopore" refer to a heptameric pore of seven αHL monomer subunits. The amino acid sequence corresponding to a wild-type αHL monomer subunit can be found in SEQ ID NO: 1. Unless otherwise indicated, all amino acid numbering for an αHL monomer subunit refers to SEQ ID NO: 1. References to an αHL monomer subunit "comprising a substitution at position #" or "comprising substitution X#Y" are understood to mean that the monomer subunit amino acid sequence, when aligned with SEQ ID NO: 1, has a substitution at the position corresponding to the recited position in SEQ ID NO: 1. As used herein, a "non-native amino acid" is an amino acid at a position in the monomer subunit amino acid sequence that represents a substitution or insertion when aligned with SEQ ID NO: 1. In one embodiment, the polypeptide comprises at least one αHL monomer subunit that has 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.

[0048] Table 1 lists the solvent-facing amino acid residues located at the entrance 304, constriction region 305, or beta-barrel 306 when a monomer subunit consisting of SEQ ID NO:1 self-assembles into a homoheptameric αHL nanopore in the presence of DPhPC in an aqueous solution of 20 mM Tris-HCl pH 8.0, 200 mM NaCl at 37° C. "#" 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 αHL nanopore in which the amino acid is located.

[0049] [Table 1]

[0050] These sites can generally be modified (e.g., by substitution, insertion, or deletion) to modify various properties of the nanopore. Exemplary engineered αHL nanopores useful in the present invention can be found, for example, in Ayub, Wang II, WO 2014 / 100481, WO 2016 / 069806, WO 2017 / 050718, WO 2017 / 184866, and WO 2018 / 002125 (each incorporated by reference).

[0051] The αHL monomer subunit of the nanopore can contain modifications that confer specific characteristics to the pore. One example includes substitutions that control the ability of a non-oligomerizing monomer subunit to self-oligomerize. For example, an αHL monomer subunit with a substitution at H35 (e.g., an H35G / L / D / E substitution) does not substantially oligomerize at room temperature or below (e.g., 25°C or below), but stably oligomerizes upon elevation to higher temperatures (e.g., 35°C). In an exemplary embodiment, the αHL monomer subunit further comprises an H35G / L / D / E substitution. 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 substitutions that improve the expression level of the αHL monomer subunit in recombinant cells used to express the monomer subunit. Another example includes substitutions that reduce the coefficient of variation (CV) of the pore arrival rate, such as D227N.

[0052] In some embodiments, the nanopore is a narrow pore. Reference herein to a "narrow" pore is intended to mean that at least six monomer subunits of the pore comprise E111, M113, and K147, or that the nanopore contains substitutions at E111, M113, and / or K147 sufficient to narrow the constriction region compared to a nanopore having six monomer subunits of the pore that contain E111, M113, and K147. In other embodiments, the nanopore contains substitutions that widen the constriction region. These substitutions replace the side chains of amino acids that form the constriction region with amino acids having shorter and / or less bulky side chains. Examples include E111A / S, M113A / S, and K147A / S / N substitutions. In one example, at least three monomer subunits of the αHL nanopore comprise one or more substitutions selected from the group consisting of E111A / S, M113A / S, and K147A / S / N. In one example, at least four monomer subunits of the αHL nanopore comprise one or more substitutions selected from the group consisting of E111A / S, M113A / S, and K147A / S / N. In one example, at least five monomer subunits of the αHL nanopore comprise one or more substitutions selected from the group consisting of E111A / S, M113A / S, and K147A / S / N. In one example, at least six monomer subunits of the αHL nanopore contain one or more substitutions selected from the group consisting of E111A / S, M113A / S, and K147A / S / N (comprising 6:1 monomer subunits, the "6" component having substitutions corresponding to E111A / S, M113A / S, and K147A / S / N).

[0053] Each monomer subunit of an αHL nanopore can have the same primary amino acid sequence (referred to as a "homoheptamer"), or at least one monomer subunit of the heptamer can have an amino acid sequence that differs from the amino acid sequence of the other monomer subunits (referred to as a "heteroheptamer"). A heteroheptameric αHL nanopore may be referred to herein by the ratio of different monomer subunit species used in the nanopore. For example, a "6:1 αHL nanopore" has six monomer subunits with the same amino acid sequence and one monomer subunit with a different amino acid sequence. In such an example, reference to "6" components shall mean each of the six identical monomer subunits, and reference to "1" component shall mean one monomer subunit with a different amino acid sequence. In some embodiments, each monomer subunit of an αHL nanopore is arranged in a polypeptide that does not contain additional monomer subunits (referred to herein as a "non-oligomerized monomer subunit"). An exemplary method for producing homoheptamers and heteroheptamers from non-oligomerized monomer subunits is disclosed in US Patent Application Publication No. 2017-0088890. For example, a 6:1 heteroheptamer can be produced by mixing two different monomer preparations (e.g., one in which the monomer 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, the oligomerization is carried out in the presence of trimethylamine N-oxide (TMAO), such as 0.1-5 M TMAO, 1-4 M TMAO.In one embodiment, αHL monomer subunits having a set of substitutions relative to SEQ ID NO:1, including an H35G substitution, are oligomerized in the presence of an aqueous buffer containing 0.1 to 5 M TMAO at 37° C. In another embodiment, αHL monomer subunits having a set of substitutions relative to SEQ ID NO:1, including an H35G substitution, are oligomerized in the presence of an aqueous buffer containing 0.2 to 4 M TMAO at 37° C. In another embodiment, αHL monomer subunits having a set of substitutions relative to SEQ ID NO:1, including an H35G substitution, are oligomerized in the presence of an aqueous buffer containing about 0.2 M to about 3 M TMAO at 37° C. In other embodiments, the nanopore comprises at least one set of linked monomer subunits. Exemplary methods for generating αHL nanopores from linked monomer subunits of αHL monomer subunits are disclosed, for example, in Hammerstein and U.S. Patent Application Publication No. 2017-0088890. In some embodiments, the αHL nanopore is a 6:1 nanopore, and a charged polymer is attached to the "1" moiety. In a further embodiment, the "1" component comprises an N17C substitution to facilitate attachment of a charged polymer to the "1" subunit. In yet another embodiment, the "1" subunit comprises an N17C substitution, and either the first or second end of the charged polymer comprises a maleimide to facilitate attachment of a charged monomer to the "1" subunit. In yet another embodiment, the "1" subunit comprises an N17C substitution, and either the first or second end of the charged polymer comprises a maleimide to facilitate attachment of a charged monomer to the "1" subunit, and the other of the first and second ends comprises a first member of a specific binding pair (such as biotin or an epitope tag). Figure 4 shows an embodiment comprising one αHL monomer subunit with an N17C substitution, where a charged polymer is attached to the N17C via reaction of a cysteine ​​side chain with a maleimide moiety located at a first end of the charged polymer, and the second end is further anchored in place outside the outlet side of the channel via binding of a biotin molecule located at the second end of the charged polymer to a streptavidin bead.

[0054] The αHL nanopore described herein can also include a polymerase bound thereto. Such an embodiment is particularly useful for performing tag-based SBS methods. In one embodiment, a single polymerase is bound to the αHL nanopore. Exemplary polymerases include those derived from the DNA polymerase Clostridium phage phiCPV4 (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 DNA polymerase derived from Pol6. 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 the αHL nanopore 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 linking systems, or other chemical ligation techniques known in the art.In yet other embodiments, one αHL monomer subunit is expressed as a fusion protein with a polymerase. In one embodiment, the polymerase is attached to an amino acid side chain of one monomer subunit. In one embodiment, the αHL nanopore is a 5:1:1 nanopore, in which the polymerase is attached to one "1" component and a charged polymer is attached to the other "1" component. In certain embodiments, a 5:1:1 nanopore is provided, in which one "1" component comprises a member of the SpyCatcher / SpyTag binding system and the other "1" component comprises an N17C substitution. In another embodiment, the αHL nanopore is a 6:1 nanopore, in which the polymerase and charged polymer are attached to the "1" component and the polymerase is a DNA polymerase.

[0055] C. Alternative Nanopores In one embodiment, the nanopore comprises a biological nanopore that is not an αHL. Exemplary non-αHL biological nanopores include the outer membrane porin G (OmpG) nanopore from Escherichia coli (the standard full-length unprocessed sequence disclosed in Uniprot accession number P76045-1), Mycobacterium smegmatis porin A (MspA) (the standard full-length unprocessed sequence disclosed in Uniprot accession number A0QR29-1), the dodecamer connector channel from the bacteriophage phi29 DNA packaging motor (Phi29), Bacillus anthracis protective antigen, PA 63(PA63), leukocidin nanopore, outer membrane porin F (OmpF) nanopore, ferric hydroxamate uptake component A (FhuA) from Escherichia coli (E. coli), cytolysin A (ClyA) nanopore, outer membrane phospholipase A nanopore, Neisseria self-transport lipoprotein (NalP) nanopore, WZA nanopore, Nocardia farcinica NfpA / NfpB cation-selective channel nanopore, lysenin nanopore, erolysin, the DNA packaging motor of bacteriophage SPP1 (SPP1), and Curlin sigma S-dependent multiplication subunit G (CsgG) nanopore. Reviews of the use of various nanopore proteins can be found, for example, in Gari (OmpG), Haque et al. (MspA and Phi29), and Wang II (Phi29, MspA, CsgG, PA63, ClyA, FhuA, SPP1).

[0056] III. Nucleic Acid Sequencing Systems and Methods Systems and methods are provided for performing nucleic acid sequencing using the disclosed nanopores. Systems for nanopore-based nucleic acid sequencing generally include a chip having a plurality of nanopore sequencing complexes comprising the charged polymer-linked nanopores disclosed herein, and a computing system adapted to record changes in one or more electrical properties of the nanopore sequencing complexes.

[0057] FIG. 5 illustrates an exemplary nanopore sequencing complex 500. An electrochemically resistant barrier 501 separates a first electrolyte solution 502 from a second electrolyte solution 503. The side of the barrier on which the first electrolyte solution is disposed is referred to as the cis side of the barrier, and the side on which the second electrolyte solution is disposed is referred to as the trans side. A nanopore 504 having a charged polymer attached thereto, as described herein, is inserted into the barrier 501, and a channel 505 is formed to allow ion exchange between the first and second electrolyte solutions, with the inlet side of the channel and the first end of the charged polymer facing the cis side of the barrier, and the outlet side of the channel and the second end of the charged polymer facing the trans side of the barrier. The working electrode 506 and the counter electrode 507 are operably connected to a signal source 508. The signal source 508 applies a voltage signal between the working electrode 506 and the counter electrode 507. The nanopore 504 is positioned relative to the electrodes such that a change in at least one electrical property of the nanopore can be detected and transmitted to a computing system. When the system is used in a sequencing-by-synthesis method, the system further includes (f) a nucleic acid polymerase 507 associated with the nanopore on the cis side of the barrier, and (g) a set of polymer-tagged nucleoside-5'-oligophosphates (N5OPs) 510 disposed in the first electrolyte solution.

[0058] Any semipermeable membrane, limited to those that allow the transmembrane flow of water but are impermeable to the flow of ions or other osmolytes, can be used as an electrochemically resistant barrier, so long as a nanopore described herein can be inserted therein. For example, the disclosed methods and systems can be used with membranes that are polymers. In some embodiments, the membrane is a copolymer. In some embodiments, the membrane is a triblock copolymer. In an exemplary embodiment, the membrane is an ABA triblock copolymer, where "A" is poly-b-(methyloxazoline) and "B" is poly(dimethylsiloxane)-poly-b-(methyloxazoline) (Pmoxa-PDMS-Pmoxa membrane). In other embodiments, the electrochemically resistant barrier may be a lipid bilayer.Exemplary materials used to form lipid bilayers include, for example, diphytanoyl-phosphatidylcholine (DPhPC), 1,2-diphytanoyl-sn-glycero-3-phosphocholine, 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine (DOPhPC), palmitoyl-oleoyl-phosphatidylcholine (POPC), dioleoyl-phosphatidyl-methyl ester (DOPME), dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, 1,2-di-O-phytanyl-sn-glycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy( 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-7000], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl, GM1 ganglioside, lysophosphatidylcholine (LPC), or any combination thereof.

[0059] The electrochemically resistant barrier 501 separates a second electrolyte 503 on the trans side of the barrier from a first electrolyte 502 on the cis side of the barrier. The first electrolyte 502 and the second electrolyte 503 are aqueous solutions buffered to optimal ion concentrations and maintained at an optimal pH to open the nanopore and keep the barrier as intact as possible. The first electrolyte can contain the free nanopore (prior to insertion into the barrier), the nucleic acid of interest, and any auxiliary reagents necessary to sequence the nucleic acid of interest (such as primer nucleic acid and N5OP for SBS sequencing). The first and second electrolytes can further contain one or more of lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, calcium chloride (CaCl), strontium chloride (SrCl), manganese chloride (MnCl), and magnesium chloride (MgCl).

[0060] A single free nanopore (not shown) can be inserted into barrier 501 by an electroporation process triggered by a voltage signal, thereby forming nanopore 504 in barrier 501. Channel 505 traverses barrier 501 and provides the only path for ion flow from first electrolyte 502 to working electrode 506.

[0061] In some embodiments, the working electrode 506 is a metal electrode. For non-faradaic conduction, the working electrode 506 can be made of a metal or other material that is resistant to corrosion and oxidation, such as, for example, platinum, gold, titanium nitride, and graphite. For example, the working electrode 506 can be a platinum electrode with electroplated platinum. In another example, the working electrode 506 can be a titanium nitride (TiN) working electrode. The working electrode 506 can be porous, thereby increasing its surface area and providing a capacitance associated with the working electrode 506. Because the working electrode of a nanopore sequencing complex can be independent from the working electrode of another nanopore sequencing complex, the working electrode can be referred to as a cell electrode in this disclosure.

[0062] The counter electrode (CE) 507 can be an electrochemical potential sensor. In some embodiments, the counter electrode 507 is shared among multiple nanopore sequencing complexes and can therefore be referred to as a common electrode. The common electrode can be configured to apply a common potential to the first electrolyte 502 in contact with the nanopore 504. The counter electrode 507 and the working electrode 506 can be connected to a signal source 508 to provide an electrical stimulus (e.g., a voltage bias) across the barrier 501, which can be used to sense electrical properties (e.g., resistance, capacitance, voltage decay, and ionic current flow) of the barrier 501. The signal source 508 can apply a voltage signal between the working electrode 506 and the counter electrode 507.

[0063] FIG. 6 is a top view of an exemplary embodiment of a nanopore sensor chip 600 having an array 640 of nanopore cells 650, each containing a single nanopore sequencing complex 500. Each nanopore cell 650 may include control circuitry integrated on the silicon substrate of the nanopore sensor chip 600. In some embodiments, sidewalls 636 are included in the array 640 to separate groups of nanopore cells 650 so that each group can receive a different sample for characterization. Each nanopore cell can be used to sequence nucleic acids. In some embodiments, the nanopore sensor chip 600 includes a cover plate 630. In some embodiments, the nanopore sensor chip 600 also includes a plurality of pins 610 for interfacing with other circuitry, such as a computer processor.

[0064] In some embodiments, the nanopore sensor chip 600 includes multiple chips in the same package, for example, forming a multi-chip module (MCM) or a system in a package (SiP). The chips can include, for example, memory, a processor, a field programmable gate array (FPGA), an application specific integrated circuit (ASIC), data converters, high-speed I / O interfaces, etc.

[0065] In some embodiments, the nanopore sensor chip 600 is connected (e.g., docked) to a nanochip workstation 620, which can include various components for performing (e.g., automatically performing) various embodiments of the processes disclosed herein. These processes can include, for example, an analyte delivery mechanism, such as a pipette, for delivering a lipid suspension or other membrane structure suspension, an analyte solution, and / or other liquids, suspensions, or solids. Components of the nanochip workstation can further include a robotic arm, one or more computer processors, and / or memory. Multiple polynucleotides can be detected on an array 640 of nanopore cells 650. In some embodiments, each nanopore cell 650 is individually addressable.

[0066] FIG. 7 shows an exemplary embodiment of a nanopore cell containing a nanopore sequencing complex. The nanopore cell 700 can include a well 705 formed from dielectric layers 701 and 704, a barrier 714 formed over the well 705, and a sample chamber 715 separated from the well 705 by the barrier 714. The well 705 can contain a volume of a second electrolyte 706, and the sample chamber 715 can hold a first electrolyte 708 containing a nanopore and an analyte of interest (e.g., a nucleic acid molecule to be sequenced). The nanopore cell 700 can include a working electrode 702 at the bottom of the well 705 and a counter electrode 710 disposed within the sample chamber 715. A signal source 728 can apply a voltage signal between the working electrode 702 and the counter electrode 710. A single nanopore can be inserted into the barrier 714 by an electroporation process triggered by the voltage signal, thereby forming a nanopore 716 within the barrier 714. The barriers (e.g., lipid bilayers 714 or other membrane structures) within the array can be chemically and electrically unconnected to one another. Thus, each nanopore cell in the array can be an independent sequencing machine, generating data specific to a single polymer molecule associated with a nanopore that acts on an analyte of interest and modulates ionic current through an otherwise impermeable barrier.

[0067] As shown in FIG. 7 , the nanopore cell 700 can be formed on a substrate 730, such as a silicon substrate. A dielectric layer 701 can be formed on the substrate 730. Dielectric materials used to form the dielectric layer 701 can include, for example, glass, oxide, nitride, etc. An electrical circuit 722 for controlling electrical stimuli and processing signals detected from the nanopore cell 700 can be formed on the substrate 730 and / or in the dielectric layer 701. For example, multiple patterned metal layers (e.g., metal 1 through metal 6) can be formed in the dielectric layer 701, and multiple active devices (e.g., transistors) can be fabricated on the substrate 730. In some embodiments, a signal source 728 is included as part of the electrical circuit 722. The electrical circuit 722 can include, for example, an amplifier, an integrator, an analog-to-digital converter, a noise filter, feedback control logic, and / or various other components. The electrical circuitry 722 can further be connected to a processor 724 connected to a memory 726, where the processor 724 can analyze the sequencing data and determine the sequences of the polymer molecules sequenced in the array.

[0068] A working electrode 702 may be formed on the dielectric layer 701 and may form at least a portion of the bottom of the well 705 .

[0069] A dielectric layer 704 can be formed on the dielectric layer 701. The dielectric layer 704 forms walls surrounding the well 705. Dielectric materials used to form the dielectric layer 704 can include, for example, glass, oxide, silicon mononitride (SiN), polyimide, or other suitable hydrophobic insulating materials. The top surface of the dielectric layer 704 can be silanized. The silanization can form a hydrophobic layer 720 above the top surface of the dielectric layer 704. In some embodiments, the hydrophobic layer 720 has a thickness of about 1.5 nanometers (nm).

[0070] A well 705 formed by the dielectric layer walls 704 contains a second electrolyte 706 in contact with the working electrode 702. In some embodiments, the second electrolyte 706 has a thickness of about 3 microns (μm).

[0071] A barrier 714 may be formed on the dielectric layer 704 and span the well 705. The barrier 714 is embedded with a single nanopore 716 having a charged polymer attached thereto, as disclosed herein. The nanopore 716 may contain at least a portion of the analyte of interest, the charged polymer, and / or small ions (e.g., Na + , K. + , Ca 2+ , Cl - ) can be large enough to pass between the two sides of the barrier 714. A sample chamber 715 is located on the cis side of the barrier 714 and can hold a solution of an analyte to be characterized.

[0072] In some embodiments, various checks are performed during production of the nanopore cell as part of the calibration. Once the nanopore cell is produced, further calibration steps can be performed, for example, to identify a nanopore cell that is operating as desired (e.g., one nanopore in the cell). Such calibration checks can include physical checks, voltage calibration, open channel calibration, and identification of cells with a single nanopore.

[0073] In use, a plurality of nanopore sequencing complexes generate an active sequencing complex, and a molecule enters the channel of the nanopore, causing a change in one or more electrical properties of the nanopore sequencing complex, which is detected, transmitted to, and correlated by a computing system. In SBS sequencing, the molecule entering the channel is the tagged N5OP polymer tag. In direct sequencing, the molecule entering the channel is a nucleic acid of interest.

[0074] FIG. 8 shows an exemplary embodiment of an active sequencing complex 800 for performing tag-based SBS nucleic acid sequencing. An electrically resistive barrier 801 separates a first electrolyte 802 from a second electrolyte 803. A nanopore 804 is disposed within the electrically resistive barrier 801, and a channel of the nanopore 805 provides a path through which ions can flow between the first electrolyte 802 and the second electrolyte 803. A working electrode 806 is disposed on the side of the electrically resistive barrier 801 containing the second electrolyte 803 (referred to as the "trans side" of the electrically resistive barrier) and is positioned near the nanopore 804. A counter electrode 807 is disposed on the side of the electrically resistive barrier 801 containing the first electrolyte 802 (referred to as the "cis side" of the electrically resistive barrier). A signal source 808 is adapted to apply a voltage signal between the working electrode 806 and the counter electrode 807. A polymerase 809 associates with nanopore 804, and a primed template nucleic acid 810 associates with polymerase 809. First electrolyte 802 contains four different polymer-tagged nucleoside oligophosphates 811 (tags shown as 811a). Polymerase 809 catalyzes the incorporation of polymer-tagged nucleotides 811 into the template amplicon. When polymer-tagged nucleoside oligophosphates 811 are correctly complexed with polymerase 809, tag 811a can be drawn (e.g., loaded) into the nanopore by an electrical force, such as a force generated in the presence of an electric field generated by an applied voltage across electrically resistive barrier 801 and / or nanopore 804. Tag 811a occupies the channel of nanopore 804 but affects the flow of ions through nanopore 804, thereby generating an ion-blocking signal 812. Each nucleotide 811 has a unique polymer tag 811a that generates a unique ion-blocking signal due to the different chemical structure and / or size of the tag 811a. By identifying the unique ion-blocking signal 812, the identity of the unique tag 811a (and therefore the nucleotide 810 with which it is associated) can be identified. This process is repeated iteratively as each nucleotide 811 is incorporated into the amplicon.Exemplary tag-based SBS approaches and materials for carrying out such methods are described in, e.g., WO 2012-083249, WO 2013 / 154999, U.S. Patent Application Publication No. 2014 / 0309144, U.S. Pat. No. 9,017,937, WO 2015 / 148402, WO 2016 / 069806, WO 2016 / 144973, U.S. Patent Application Publication No. 2016 / 0222363, U.S. Pat. The tags are described in U.S. Patent Application Publication No. 2016 / 0333327, WO 2017 / 050728, WO 2017 / 184866, WO 2017 / 050722, U.S. Patent Application Publication No. 2017 / 0267983, U.S. Patent Application Publication No. 2018 / 0245147, U.S. Patent Application Publication No. 2018 / 0094249, WO 2018 / 002125, and Kumar (each of which is incorporated herein by reference). Various tags have been proposed for use in such systems, including polypeptide (such as polylysine tags), polynucleotide, and polyethylene glycol-based tags. See, for example, U.S. Patent No. 8,652,779 and WO 2017042038 (each of which is incorporated herein by reference).

[0075] FIG. 9 illustrates an exemplary embodiment of an active sequencing complex 900 for performing direct sequencing. An electrically resistive barrier 901 separates a first electrolyte 902 from a second electrolyte 903. A nanopore 904 is disposed within the electrically resistive barrier 901, and a channel of the nanopore 905 provides a path through which ions can flow between the first electrolyte 902 and the second electrolyte 903. A working electrode 906 is disposed on the side of the electrically resistive barrier 901 containing the second electrolyte 903 (referred to as the "trans-side" of the electrically resistive barrier) and is positioned near the nanopore 904. A counter electrode 907 is disposed on the side of the electrically resistive barrier 901 containing the first electrolyte 902 (referred to as the "cis-side" of the electrically resistive barrier). A signal source 908 is adapted to apply a voltage signal between the working electrode 906 and the counter electrode 907. The first electrolyte 902 contains a nucleic acid 910 of interest. The nucleic acid of interest 910 can be drawn (e.g., loaded) into the nanopore by an electrical force, such as a force generated in the presence of an electric field generated by a voltage applied across the electrically resistive barrier 901 and / or the nanopore 904. When a nucleotide or sequence of nucleotides occupies the channel of the nanopore 904, they affect the flow of ions through the nanopore 904, thereby generating an ion-blocking signal. Each nucleotide or sequence of nucleotides that occupies the channel 905 can generate a different ion-blocking signal. This process is repeated iteratively as the nucleic acid of interest 910 passes through the channel, and the sequence of the nucleic acid of interest 910 is extrapolated based on the unique sequence of the ion-blocking signals recorded by Feng, Manrao, and Wang I.

[0076] IV. Working Examples Tethering synthetic polymers to the interior of protein nanopores produced α-HL pores with high conductance and narrower ion passageways. When nanopores entrap thin, highly negatively charged polymers, the nanopores exhibit higher conductance than when the entrapped polymer is absent. Without being bound by theory, the mechanism for the observed conductance enhancement may be due to the polymers carrying a large counterion cloud. The entrapment of these polymers within the nanopore facilitates the transport of these counterions, resulting in the observed enhanced conductance regime despite the narrower passageway.

[0077] A. Synthesis of Charged Polymers A charged polymer with the structure disclosed in Figure 10 was synthesized on an ABI 3900 DNA Synthesizer using standard solid-phase phosphoramidite chemistry protocols. The resulting polymer was cleaved from the resin by treatment with concentrated ammonium hydroxide. The product was concentrated in a SPEEDVAC vacuum concentrator (Thermo Fisher Scientific) and then purified by reverse-phase high-performance liquid chromatography (RP-HPLC) to obtain the pure compound. The protected maleimide was removed by heating in dry toluene at 90°C for 3 hours. The product was used without further purification. The maleimide group is highlighted in blue, and the biotin group is highlighted in green.

[0078] B. Expression and Purification of αHL Nanopore Both wild-type α-HL-6X-His (SEQ ID NO: 2) and α-HL-N17C_6X-His (SEQ ID NO: 3) were expressed in BL21 DE3 Star pLys-S E. coli cells grown overnight at 25°C in MAGIC MEDIA E. coli expression medium (Invitrogen). SEQ ID NO: 3 contains a SpyTag near the C-terminus. Each was dissolved by sonication in 25 mM Tris, pH 8.0, 300 mM NaCl, 10 mM imidazole. Both were purified on a TALON column and eluted with the same buffer containing 150 mM imidazole. The His tag of WT α-HL-6X-His was cleaved with TEV protease to generate WT α-HL (SEQ ID NO: 1).

[0079] C. Pore Assembly and Purification For the 1:6 oligomer containing one α-HL-N17C-SpyTag-6X-His (SEQ ID NO: 3) and six WT α-HL (SEQ ID NO: 1), purified α-HL-N17C-SpyTag-6X-His (G124) monomer and His-tagged truncated WT α-HL (G1471) monomer were mixed at a 1:8 (w / w) ratio. The lipid 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was added to a final lipid concentration of 5 mg / mL. The mixture was incubated overnight at 37°C. Lipid vesicles were solubilized in a buffer containing 5% (w / v) n-octyl-β-D-glucoside (β-OG). The oligomers were purified by cation exchange chromatography on a RESOURCE S column in 20 mM sodium acetate (NaAc) buffer, pH 4.8, 30 mM NaCl, 0.1% Tween 20, 1 mM tris(2-carboxyethyl)phosphine (TCEP). The bound protein was eluted with a linear gradient of 20 mM NaAc buffer, pH 4.8, 2 M NaCl, 0.1% Tween 20, 1 mM tris(2-carboxyethyl)phosphine (TCEP). As can be seen in Figure 11, two major peaks, Peak 1 (P1) and Peak 2 (P2), were obtained. Peak 1 corresponded to the 0:7 oligomer, and Peak 2 corresponded to the 1:6 oligomer.

[0080] The 1:6 oligomer (P2) was confirmed by adding SpyCatcher-GFP protein and running an SDS-polyacrylamide gel. The results are shown in Figure 12. Peak P1 did not show an electrophoretic mobility shift when incubated with SpyCatcher-GFP, indicating that it is a 0:7 oligomer. Peak P2 showed an electrophoretic mobility shift when incubated with SpyCatcher-GFP, indicating that it is a 1:6 oligomer.

[0081] D. Chemical conjugation of 1:6 pores with charged polymers The 1:6 oligomer was mixed with the polymer tag at a 1:10 (mol:mol) ratio in 20 mM HEPES pH 7.5, 100 mM NaCl, 0.01% Tween 20, followed by incubation at room temperature for 4 hours. The conjugated pore was bound to MagStrep "Type 3" XT beads and eluted with elution buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 0.001% Tween 20, 8% (w / v) trehalose, 2 mM d-biotin).

[0082] E. Single channel recording Planar bilayers were formed across approximately 100 μm openings in polytetrafluoroethylene membranes using the "Montal-Muller" approach. In this approach, the openings were first treated with a droplet of 10% hexadecane / pentane. A droplet (approximately 5 μL) of 10 mg / mL DPhPC (1,2-diphytanoyl-sn-glycerophosphocholine) in pentane was applied to the top surface of each chamber in a buffer solution (200 mM KGlu, 0.5 mM EDTA, 20 mM HEPES pH 7.5). A lipid monolayer was generated as soon as the solvent evaporated. Bilayers were then fabricated by raising the lipid monolayer on the buffer solution on both sides of the opening.

[0083] A pair of Ag / AgGlu electrodes was prepared. The ground electrode was connected to the cis compartment, and the working electrode was connected to the trans compartment. Purified 1:6 oligomer was applied to the cis compartment. Currents were detected using the pair of Ag / AgGlu electrodes, amplified with a patch-clamp amplifier equipped with HUMMSILENCER technology (AXON AXOPATCH 200B Microelectrode Amplifier, Axon Instruments), filtered with a low-pass Bessel filter (80 dB / decade) with a 1 kHz break frequency, and then digitized with a DIGIDATA 1200 A / D converter (Axon Instruments) at a sampling frequency of 5 kHz. Data samples were stored on the hard disk of a PC computer.

[0084] The first set of capture events was recorded using a charged polymer tethered only at the first end. Streptavidin was then flowed through the trans side of the bilayer, and an additional set of recordings was generated. The results are shown in Figure 13. A fluctuating current was observed at two levels, suggesting that the tethered polymer occupies two states: 1) inserted into the channel and 2) ejected from the channel. Adding streptavidin to the trans side resulted in a higher conductance between the two levels. This indicates that the biotin at the end of the polymer is fixed by streptavidin, thus causing the polymer to remain in a permanently threaded position (Figure 4).

[0085] The conductance was then measured for the charged polymer-conjugated / streptavidin-trapped nanopore at various voltages and compared to the same pore without the charged polymer. The results are shown in Figure 2. As can be seen, the charged polymer-conjugated pore (line with square hatch marks) had consistently greater conductance levels than the same pore without the charged polymer.

[0086] V. References 1. Ayub et al., Nucleobase Recognition by Truncated α-Hemolysin Pores, ACS Nano, 2015, Vol. 9, No. 8, pp. 7895-7903. 2. Chen & Liu, Fabrication and Applications of Solid-State Nanopores, 2019, Sensors, Vol. 19, No. 8, E1886. 3. Feng et al., Nanopore-based Fourth-generation DNA Sequencing Technology, 2015, Genomics, Proteomics & Bioinformatics, Vol. 13, No. 1, pp. 4-16. 4. Gari et al., Quiet Outer Membrane Protein G (OmpG) Nanopore for Biosensing, ACS Sensors, April 14, 2019, Vol. 4, pp. 1230-35. 5. Hammerstein et al., Subunit dimers of α-hemolysin expands the engineering toolbox for protein nanopores, Journal of Biological Chemistry, Vol. 286, No. 16, pp. 14324-34. 6. Haque et al., Solid-State and Biological Nanopore for Real-Time Sensing of Single Chemical and Sequencing of DNA, Nano Today (February 2013), Vol. 8, No. 1, pp. 56-74. 7. Kumar et al., PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis, 2012, Scientific Reports, Vol. 2, Art. 684. 8. Lee et al., Recent proof in solid-state nanopores, 2018, Advanced Materials, Vol. 30, No. 42. 9. Manrao et al., Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase, 2012, Nature Biotechnology, Vol. 30, pp. 349-53. 10. Pavlenok and Niederweis, Hetero-oligomeric MspA pores in Mycobacterium smegmatis, 2016, FEMS Microbiology Letters, Volume 363, Issue 7, fnw046. 11. Wang et al., The evolution of nanopore sequencing, 2015, Frontiers in Genetics, Vol. 5, Art. 449 (“Wang I”). 12. Wang et al., Engineering of Protein Nanopores for Sequencing, Chemical or Protein Sensing and Disease Diagnosis, 2018, Current Opinions in Biotechnology, Vol. 51, pp. 80-89 ("Wang II"). 13. Wasfi et al., Graphene-based nanopore approaches for DNA sequencing: A literature review, 2018, Biosensors and Bioelectronics, Vol. 119, pp. 191-203.

Claims

1. 1. A nanopore-forming protein comprising: a channel having an inlet side and an outlet side; a charged polymer threaded through the channel; wherein the charged polymer comprises: a first end fixed in position on the inlet side of the channel; a second end located on the outlet side of the channel, optionally fixed in position; a negatively charged region disposed between the first end and the second end and extending substantially the entire length of the channel; where: the nanopore is a heptameric α-hemolysin nanopore; and The charged polymer comprises at least: (i) a negatively charged region 101 having a high density of negative charges; (ii) an entity at or near the first end 102 of the charged polymer 100 that facilitates binding to the nanopore at or near the entrance to the nanopore channel; where: (a) the second end of the charged polymer comprises biotin or a biotin derivative, and the biotin or biotin derivative is immobilized on avidin, streptavidin, or deglycosylated avidin disposed on the outlet side of the channel; or (b) a nanopore-forming protein, wherein the second end of the charged polymer comprises an antibody epitope, the antibody epitope being anchored to an antibody disposed on the outlet side of the channel.

2. 2. The nanopore-forming protein of claim 1, wherein the channel is formed by seven monomer subunits, each monomer subunit having at least 75% sequence identity with SEQ ID NO:1, and the first end is covalently bound to one of the seven monomer subunits.

3. 3. The nanopore-forming protein of claim 1, wherein the negatively charged region comprises at least 10 phosphodiester bonds.

4. 4. The nanopore-forming protein of claim 3, wherein the phosphodiester bond links nucleotides and / or abasic sites.

5. The nanopore-forming protein of claim 4 , wherein the negatively charged region comprises at least 10 abasic sites.

6. The nanopore-forming protein of claim 4 , wherein the negatively charged region comprises at least 20 abasic sites.

7. The abasic site has the following structure: 【Chemistry 1】 where R1 is an alkyl chain 2 to 10 carbons in length. The nanopore-forming protein of claim 4 , wherein

8. 8. The nanopore-forming protein of claim 2, wherein at least six of the seven subunits, when aligned with SEQ ID NO:1, include E111, M113 and K147.

9. 1. A system for performing nanopore-based sequencing, comprising: a chip comprising a plurality of nanopore sequencing complexes; and a computing system adapted to record changes in one or more electrical properties of the nanopore sequencing complexes, wherein each nanopore sequencing complex: (a) an electrochemically resistive barrier disposed on a surface of the chip, the electrochemically resistive barrier having a cis side and a trans side; (b) a first electrolyte on the cis side of the barrier; (c) a second electrolyte on the transformer side of the barrier; (d) the nanopore-forming protein of any one of claims 1 to 7, wherein the inlet side of the channel is on the cis side of the barrier and the outlet side of the channel is on the trans side of the barrier, such that the channel allows ion exchange between the first electrolyte solution and the second electrolyte solution; (e) at least one electrode in electronic communication with the computing system, the at least one electrode being configured to detect a change in at least one electrical property of the nanopore sequencing complex associated with occupancy of the nanopore by a molecule and to transmit the detected change to the computing system; Including, the system.

10. 10. A method for sequencing a template nucleic acid using the system of claim 9, comprising: generating a plurality of active nanopore sequencing complexes, each active nanopore sequencing complex comprising a single-stranded nucleic acid template inserted into the channel of the nanopore; - applying a force to the single-stranded nucleic acid template at each active sequencing complex, the force causing the single-stranded nucleic acid to move through the channel from the entry side to the exit side, and each nucleotide or sequence of nucleotides in the nucleic acid causing a unique change in the electrical properties of the nanopore; - detecting a change in the electrical property of the nanopore caused by the nucleotide or sequence of nucleotides occupying the channel and recording the change in the computer system; - correlating each recorded change with the nucleotide or sequence of nucleotides occupying the channel, thereby generating a sequence of the single-stranded template nucleic acid at that electrode; A method comprising:

11. 1. A system for performing sequencing-by-synthesis (SBS) nucleic acid sequencing, comprising: a chip comprising a plurality of nanopore sequencing complexes; and a computing system adapted to record changes in one or more electrical properties of the nanopore sequencing complexes, wherein each nanopore sequencing complex: (a) an electrochemically resistive barrier disposed on a surface of the chip, the electrochemically resistive barrier having a cis side and a trans side; (b) a first electrolyte on the cis side of the barrier; (c) a second electrolyte on the transformer side of the barrier; (d) the nanopore-forming protein of any one of claims 1 to 7, wherein an inlet side of the channel is on the cis side of the barrier and an outlet side of the channel is on the trans side of the barrier, such that the channel allows ion exchange between the first electrolyte solution and the second electrolyte solution; (e) at least one electrode in electronic communication with the computing system, the at least one electrode being configured to detect a change in at least one electrical property of the nanopore sequencing complex associated with occupancy of the nanopore by a molecule and to transmit the detected change to the computing system; (f) a nucleic acid polymerase associated with the nanopore on the cis side of the barrier; (g) a set of polymer-tagged nucleoside-5′-oligophosphates (N5OPs) disposed in the first electrolyte solution; Including, the system.

12. 12. A method for sequencing a template nucleic acid using the system of claim 11, comprising: generating a plurality of active nanopore sequencing complexes, each active nanopore sequencing complex comprising: a single-stranded nucleic acid template complexed with said nucleic acid polymerase; a primer hybridized to said template nucleic acid; - said set of at least one tagged N5OP associated with said polymerase; generating the image data, - in each active sequencing complex, iteratively ligating the tagged N5OP to the primer by a template-dependent nucleic acid amplification reaction catalyzed by the nucleic acid polymerase, wherein the polymer tag of the tagged N5OP moves within or proximate to the channel of the nanopore when the tagged N5OP is ligated to a complementary nucleic acid, and wherein movement of the polymer tag within or proximate to the channel changes an electrical property of the nanopore; - detecting a change in the electrical property of the nanopore caused by the polymer tag and recording said change in the computer system; - correlating each recorded change with one of said tagged N5OPs, thereby generating the sequence of said complementary nucleic acid generated at that electrode; A method comprising:

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