Methods for nanopore-based analyte detection

Abstract

The invention provides a method for translocating an analyte through a nanopore disposed within a membrane, the analyte comprising a protein, a polypeptide or a peptide, wherein a current or a voltage or a change thereof is detected while the analyte is translocating through the nanopore and one or more characteristics of the analyte are determined.

Description

METHODS FOR NANOPORE-BASED ANALYTE DETECTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of United Kingdom Patent Application No. 2313202.0, filed August 30, 2023, which is herein incorporated by references in its entirety.BACKGROUND

[0002] Nanopore sequencing is an approach to sequencing of nucleic acid molecules. Using nanopore sequencing, a single molecule of DNA or RNA can be sequenced without the need for PCR amplification or chemical labeling of the sample. Nanopore sequencing can offer low-cost genotyping, high mobility for testing, and rapid processing of samples with the ability to display results in real-time. It has been used in the rapid identification of viral pathogens, epidemiological monitoring, environmental monitoring, food safety monitoring, human genome sequencing, plant genome sequencing, monitoring of antibiotic resistance, haplotyping, and other applications.SUMMARY

[0003] Molecules can be detected and characterized by nanopores and nanopore sensors based on capture and modulation of ionic current. Nanopores may identify and characterize many analytes, such as nucleic acid molecules, peptides, polypeptides, or proteins, or fragments thereof, or any combination thereof. There is a need for improved nanopore, nanopore systems, and methods thereof for the detection, capture, and analysis of analytes. Recognized herein are composition, methods, and systems for enhancing proteomic characterization.

[0004] In an aspect, the present disclosure provides a method for identifying a polypeptide characteristic, comprising (a) translocating a polypeptide through a nanopore disposed within a membrane; (b) detecting a current or change thereof while the polypeptide is translocating through the nanopore; and (c) using the current or change thereof detected in (b) to identify a characteristic of the polypeptide with an accuracy of at least 90%.

[0005] In some embodiments, the characteristic comprises a sequence, a length, an identity, a secondary structure, a tertiary structure, a modification to the polypeptide, or combinations thereof.

[0006] In some embodiments, the nanopore is a biological nanopore.

[0007] In some embodiments, the biological nanopore is selected from FraC, a-hemolysin, CytK, Lysenin, MspA, CsgG, Aerolysin, or FhuA. In some embodiments, the biological nanopore comprises an outer membrane protein (OMP) such as OmpG or OmpF.

[0008] In some embodiments, the biological nanopore comprises one or more point mutations.WSGR Docket Number: 64828-710.601

[0009] In some embodiments, the one or more point mutations affects a diameter of the biological nanopore.

[0010] In some embodiments, the one or more mutations create smaller openings on a cis side of the biological nanopore. In some embodiments, the one or more mutations create smaller openings on a trans side of the biological nanopore. In some embodiments, the one or more mutations create smaller openings in the center of the biological nanopore. In some embodiments, the one or more mutations constrict multiple point throughout a channel of the biological nanopore.

[0011] In some embodiments, the diameter of the biological nanopore is from about 0.5 nm to about 2 nm.

[0012] In some embodiments, the one or more point mutations affects a charge of the biological nanopore.

[0013] In some embodiments, the net charge of the biological nanopore is positive.

[0014] In some embodiments, the charge of the channel of the biological nanopore comprises positively charged portions.

[0015] In some embodiments, the net charge of the biological nanopore is negative.

[0016] In some embodiments, the charge of the channel of the biological nanopore comprises negatively charged portions.

[0017] In some embodiments, the one or more point mutations allow for conductance at a set pH.

[0018] In some embodiments, the pH is from about 5 to about 10.

[0019] In some embodiments, the one or more point mutations are one or more lumen facing mutations.

[0020] In some embodiments, the nanopore is an artificial nanopore.

[0021] In some embodiments, the polypeptide is unfolded prior to translocation.

[0022] In some embodiments, the polypeptide is unfolded by one or more unfoldases selected from the group consisting of prokaryotic AAA+ unfoldase, ClpX, PAN unfoldase, and Valosin-containing protein-like ATPase.

[0023] In some embodiments, the one or more of unfoldases are coupled to the nanopore. In some embodiments, the one or more of unfoldases are coupled to the nanopore by covalent or non-covalent forces. In some embodiments, the one or more of unfoldases are coupled to the nanopore by Pi bonding, Pi-Pi bonding, H-bonding, electrostatic interactions, or hydrophobic interfaces, or combinations thereof.

[0024] In some embodiments, the one or more unfoldases are suspended in an electrolyte solution on one side of the membrane.

[0025] In some embodiments, the one or more unfoldases are configured to position proximal to the nanopore upon a binding event with the polypeptide.

[0026] In some embodiments, the nanopore comprises a proteasome and / or a translocase.

[0027] In some embodiments, either the proteasome or the translocase are located on a cis-side of the nanopore. In some embodiments, either the proteasome or the translocase are located on a trans-side of the nanopore. InWSGR Docket Number: 64828-710.601 some embodiments, either the proteasome or the translocase are located on both a cis-side and / or a trans-side of the nanopore.

[0028] In some embodiments, both the proteasome and translocase are located on a cis-side of the nanopore. In some embodiments, both the proteasome and the translocase are located on a trans-side of the nanopore. In some embodiments, both the proteasome and the translocase are located on both a cis-side and a trans-side of the nanopore.

[0029] In some embodiments, the proteasome comprises one or more subunits.

[0030] In some embodiments, the proteasome is fused with the nanopore.

[0031] In some embodiments, the polypeptide is translocated from a cis to a trans side of the membrane. In some embodiments, the polypeptide is translocated from a trans to a cis side of the membrane. In some embodiments, a polypeptide bound to a translocase is partially translocated from a cis to trans side of the membrane, then the translocase pulls the partially translocated portion of the polypeptide back through the pore in a trans to cis direction.

[0032] In some embodiments, the one or more of unfoldases are positioned on the cis side of the membrane.

[0033] In some embodiments, the one or more of unfoldases are positioned on the trans side of the membrane.

[0034] In some embodiments, the polypeptide is fragmented before translocation.

[0035] In some embodiments, a polypeptide fragment comprises a portion comprising a non-natural amino acid, polyethylene glycol, PNA, DNA, or RNA, or combinations thereof.

[0036] In some embodiments, the polypeptide is fragmented by one or more proteases comprising trypsin-type or chymotrypsin-type of activity.

[0037] In some embodiments, the one or more proteases are coupled to the nanopore.

[0038] In some embodiments, the one or more proteases are positioned on a cis side of the membrane

[0039] In some embodiments, the polypeptide is translocated through the nanopore sequentially.

[0040] In some embodiments, translocation is affected by a molecular motor.

[0041] In some embodiments, the step size of translocation is determined by the molecular motor.

[0042] In some embodiments, the molecular motor is ATP driven. In some embodiments, the molecular motor is NTP driven.

[0043] In some embodiments, the step size of translocation is from about 0.2 to about 80 Angstroms. In some embodiments, the step size of translocation is from about 0.5 to about 20 Angstroms.

[0044] In some embodiments, translocation occurs in the absence of a molecular motor. In some embodiments, translocation occurs with a molecular motor that is not being actively driven by ATP or NTP.

[0045] In some embodiments, the rate of translocation is from about 0.1 nm / s to about 300 nm / s.WSGR Docket Number: 64828-710.601

[0046] In some embodiments, the membrane is an insulating membrane.

[0047] In some embodiments, the insulating membrane is a phospholipid bilayer.

[0048] In some embodiments, the insulating membrane is a solid-state membrane.

[0049] In some embodiments, the membrane has a thickness from about 4 nm to about 20 nm.

[0050] In some embodiments, the polypeptide is unlabeled.

[0051] In some embodiments, the polypeptide comprises a tag.

[0052] In some embodiments, the polypeptide comprises an exogenous sequence.

[0053] In some embodiments, the exogenous sequence is about 5 to about 50 amino acids in length.

[0054] In some embodiments, the polypeptide is suspended in an electrolytic solution.

[0055] In some embodiments, the electrolytic solution comprises water, potassium, lithium sodium, calcium, magnesium, phosphate, sulfate, or chloride, or any combination thereof.

[0056] In some embodiments, the concentration of one or more electrolytes in the electrolytic solution is from about 0.1 M to about 1.5 M.

[0057] In some embodiments, the pH of the electrolytic solution is from about 5 to about 10.

[0058] In some embodiments, the pH of the electrolytic solution is different between a trans-side and a cis- side of a membrane.

[0059] In some embodiments, a terminus of the polypeptide is chemically modified with a leader or a tail.

[0060] In some embodiments, a machine learning algorithm is used to identify the characteristic of the polypeptide using the current signal. In some embodiments, the characteristic comprises a sequence.

[0061] In some embodiments, identity of the characteristics is determined with an accuracy from about 90% to about 99.99%.

[0062] In some embodiments, the multi pass accuracy is from about 90% to about 99.99%.

[0063] In some embodiments, the single pass accuracy is from about 90% to about 99.99%.

[0064] In some embodiments, the polypeptide comprises post-translational modifications.

[0065] In some embodiments, the detected change in ionic current is from about 0.1 pA to about 150 pA.

[0066] In some aspects, the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane, wherein the step size of translocation is about 0.2 to about 80 Angstroms; detecting a current or change thereof while the polypeptide is translocating through the nanopore; and using the current or change thereof detected in (b) to characterize a property of the polypeptide or to identify a sequence of the polypeptide.

[0067] In some embodiments, the step size is dependent on the polypeptide structure.WSGR Docket Number: 64828-710.601

[0068] In some aspects, the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane, wherein the rate of translocation is about 0.1 nm / s to about 300 nm / s; detecting a current or change thereof while the polypeptide is translocating through the nanopore; and using the current or change thereof detected in (b) characterize a property of the polypeptide or to identify a sequence of the polypeptide.

[0069] In some aspects, the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane; detecting a current or change thereof while the polypeptide is translocating through the nanopore; and using the current or change thereof detected in (b) to identify a sequence of the polypeptide with a sensing throughput of at least 1 molecule / minute.

[0070] In some aspects, the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane; detecting a current or change thereof while the polypeptide is translocating through the nanopore; and using the current or change thereof detected in (b) to identify a sequence of the polypeptide wherein the average read length is at least about 1 to about 10 amino acids.

[0071] In some aspects, the present disclosure provides a method for proteome analysis, comprising: providing a cell comprising a plurality of polypeptides; translocating a polypeptide of the plurality of polypeptides through a nanopore disposed within a membrane; detecting a current or change thereof while the polypeptide of the plurality of polypeptides is translocating through the nanopore; using the current or change thereof detected in (b) to identify a sequence of the polypeptide of the plurality of polypeptides; and characterizing one or more properties of a proteome using the sequence of the polypeptide identified in (d), wherein proteome coverage is at least 50%.

[0072] In some aspects, the present disclosure provides a method for proteome analysis, comprising: providing a cell comprising a plurality of polypeptides; translocating a polypeptide of the plurality of polypeptides through a nanopore disposed within a membrane; detecting a current or change thereof while the polypeptide of the plurality of polypeptides is translocating through the nanopore; using the current or change thereof detected in (b) to identify a sequence of the polypeptide of the plurality of polypeptides; and characterizing one or more properties of a proteome using the sequence of the polypeptide identified in (d), wherein sequence coverage is at least 10%.

[0073] In some aspects, the present disclosure provides a device for identifying a polypeptide sequence, comprising: a first chamber configured to prepare a biological sample for polypeptide sequencing; a second chamber in fluidic communication with the first chamber, the second chamber comprising a support configured to connect and disconnect with a sensor array; a sensor array connected to the support, wherein the sensor arrayWSGR Docket Number: 64828-710.601 comprises: a plurality of membranes comprising a plurality of pores, the plurality of pores configured to translocate the plurality of polypeptides; and a plurality of electrodes configured to measure a plurality of electrical signals from the plurality of polypeptides translocating through the plurality of pores; and a recording device configured to receive and record a plurality of electrical signals from the plurality of electrodes.

[0074] In some aspects, the present disclosure provides a device for identifying a polypeptide sequence, comprising: a first chamber configured to prepare a biological sample for polypeptide sequencing by binding a plurality of polypeptides in the biological sample with a plurality of biomolecules; a second chamber in fluidic communication with the first chamber, the second chamber comprising a support configured to connect and disconnect with a sensor array; a sensor array connected to the support, wherein the sensor array comprises: a plurality of membranes comprising a plurality of pores, the plurality of pores configured to translocate the plurality of polypeptides, wherein the plurality of biomolecules are configured to facilitate translocating the plurality of polypeptides; and a plurality of electrodes configured to measure a plurality of electrical signals from the plurality of polypeptides translocating through the plurality of pores; and a recording device configured to receive and record a plurality of electrical signals from the plurality of electrodes.

[0075] In some embodiments, the sensor array comprises a plurality of chambers, wherein the plurality of membranes form a plurality of surfaces of the plurality of chambers.

[0076] In some embodiments, the plurality of chambers comprise a volume from about 0.1 μL to about 250 μL.

[0077] In some embodiments, a chamber of the plurality of chambers comprises a volume from about 0.0001 μL to about 1.0 μL.

[0078] In some embodiments, the plurality of chambers comprise a thickness of at most about 3 mm.

[0079] In some embodiments, the plurality of surfaces each comprise an area of at most about 5 mm2.

[0080] In some embodiments, a surface of the plurality of surfaces comprises an area of at most 100 μm2.

[0081] In some embodiments, the device is configured to connect to a recording device.

[0082] In some embodiments, the recording device comprises an analog-to-digital converter.

[0083] In some embodiments, the recording device comprises an amplifier.

[0084] In some embodiments, the plurality of electrodes are disposed on a second plurality of surfaces of the plurality of chambers.

[0085] In some embodiments, the sensor array comprises an adhesive configured to adhere the plurality of membranes to the plurality of chambers.

[0086] In some embodiments, the device comprises a display for displaying the electrical signal.

[0087] In some embodiments, the device comprises a flow cell.WSGR Docket Number: 64828-710.601

[0088] In some aspects, the present disclosure provides a microfluidic device for polypeptide characterization, comprising: one or more microfluidic channels for flowing fluid comprising a polypeptide therethrough; and one or more nanopores disposed within a membrane in fluid communication with the one or more microfluidic channels, the one or more nanopores configured to effect a change in a current applied across the membrane upon translocation of the polypeptide therethrough, wherein the changed effected in the current corresponds to a characteristic of the polypeptide.

[0089] In some embodiments, the membrane comprises from about 10 to about 100,000 pores

[0090] In some embodiments, the surface area of the membrane is at most about 5 mm2.

[0091] In some embodiments, the surface area of the nanopores within the membrane is from about 50 to about 500 nm2.

[0092] In some embodiments, the membrane is disposed within a fluidic chamber comprising an anode and a cathode.

[0093] In some embodiments, each of the anode and cathode is independently positioned on either a cis side or a trans side of the membrane.

[0094] In some embodiments, the device further comprises a potential generator for applying a potential difference across the anode and cathode.

[0095] In some embodiments, the potential across the anode and cathode generated by the potential generator is from about 10 mV to about 1 V.

[0096] In some embodiments, the microfluidic device further comprises a pump for flowing the fluid through the one or more microfluidic channels.

[0097] In some aspects, the present disclosure provides a kit for use with a device for identifying a polypeptide sequence, comprising: a chip comprising a sensor array, the sensor array comprising a plurality of chambers or wells comprising a plurality of lipids and pores; and a biomolecule configured to bind to a polypeptide to facilitate translocation of the polypeptide through a pore.

[0098] In some aspects, the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane; detecting an electrical signal while the polypeptide is translocating through the nanopore; and assigning an identification to the polypeptide based on the electrical signal and a database, the database comprising a plurality of reference signals for the polypeptide, proteoforms thereof, and post-translationally modified variants thereof.

[0099] In some aspects, the present disclosure provides a method for characterizing and / or identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane; detecting an electrical signal while the polypeptide is translocating through the nanopore; andWSGR Docket Number: 64828-710.601 assigning an identification to the polypeptide based on the electrical signal and a machine learning algorithm, the machine learning algorithm configured to distinguish between a plurality of reference signals for the polypeptide, proteoforms thereof, and post-translationally modified variants thereof.

[0100] In some aspects, the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide from a biological sample through a nanopore disposed within a membrane; detecting an electrical signal while the polypeptide is translocating through the nanopore; and assigning an identification to the polypeptide based on the electrical signal and a database, the database comprising a plurality of reference signals for a plurality of polypeptides, wherein the plurality of polypeptides comprise expressible polypeptides, or proteoforms thereof, or post-translationally modified variants thereof, or any combination thereof, based on genomic information of the biological sample.

[0101] In some aspects, the present disclosure provides a method for identifying a polypeptide sequence, comprising: contacting a composition comprising a biological sample with a nanopore, the biological sample comprising an initial volume of at most about 50 μL, wherein the composition comprises a polypeptide and a nucleic acid from the biological sample; translocating the polypeptide through a nanopore disposed within a membrane; detecting an electrical signal while the polypeptide is translocating through the nanopore; and assigning an identification to the polypeptide based on the electrical signal.

[0102] In some aspects, the present disclosure provides a method for generating a sample profile, comprising: receiving an electrical signal, wherein the electrical signal is derived at least from one analyte of a sample translocating across a nanopore; and generating the sample profile based on the electrical signal and a database, wherein the database comprises a plurality of reference electrical signals for a plurality of analytes; wherein the reference electrical signals for the plurality of analytes comprise expressible polypeptides, or proteoforms thereof, or post-translationally modified variants thereof, or combinations thereof.

[0103] In some embodiments, the reference electrical signals further comprise small molecules, metabolites, DNA, or RNA, or combinations thereof.

[0104] In some embodiments, the analyte is derived from a patient.

[0105] In some embodiments, the sample profile comprises an assessment of patient health.

[0106] In some embodiments, the analyte comprises a metabolite, a small molecule, a biopolymer, or a biomolecule.

[0107] In some embodiments, the sample profile comprises an identification of a phenotype.

[0108] In some embodiments, the sample profile comprises an identification of a metabolic state.

[0109] In some embodiments, the sample profile comprises an identification of a disease.WSGR Docket Number: 64828-710.601

[0110] In some embodiments, the electrical signal is derived from a plurality of analytes of a sample translocating across a nanopore.

[0111] In some embodiments, generating a sample profile comprises counting event instances of a subset of the plurality of analytes.

[0112] In some embodiments, the sample profile comprises an environmental profile.

[0113] In some embodiments, assigning an identification comprises determining a degree of similarity with a previously identified phenotypic profile.

[0114] In some embodiments, the degree of similarity is measured by the proteomic coverage.

[0115] In some aspects, the present disclosure provides a method for generating a sample profile, comprising: receiving an electrical signal, wherein the electrical signal is derived from at least one analyte translocating across a nanopore; and generating a sample profile based on the electrical signal and a machine learning algorithm configured to distinguish between a plurality of reference signals for the analyte.

[0116] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0117] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0118] In some aspects, the present disclosure provides a method, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting (1) a current or change thereof; or (2) a voltage or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%.

[0119] In some aspects, the present disclosure provides a method for determining a characteristic of an analyte, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof, wherein (i) an average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second, or (ii) an average rate of translocation is between about 0.1 nm / s to about 10000 nm / s; (b) detecting (1) a current or change thereof; or (2) a voltage or change thereof while the at least the portion of the analyte isWSGR Docket Number: 64828-710.601 translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte.

[0120] In some aspects, the present disclosure provides a method for characterizing an analyte, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting an electrical signal or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) assigning one or more characteristics to the at least the portion of the analyte based on the electrical signal and a database, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more proteoforms thereof, or one or more variants thereof, or one or more fragments thereof, or a combination thereof.

[0121] In some aspects, the present disclosure provides a method for sample analysis, comprising: (a) providing a sample comprising a plurality of analytes, wherein the plurality of analytes comprises a first analyte and a second analyte; (b) translocating at least a portion of the first analyte through a first nanopore disposed within a first membrane and at least a portion of the second analyte through a second nanopore disposed within a second membrane, wherein the at least a portion of the first analyte comprises at least a portion of a first protein, at least a portion of a first polypeptide, or at least a portion of a first peptide, or first fragments thereof, or a combination thereof, wherein the at least a portion of the second analyte comprises at least a portion of a second protein, at least a portion of a second polypeptide, or at least a portion of a second peptide, or second fragments thereof, or a combination thereof; (c) detecting (i) (1) a first current or change thereof or (2) a first voltage or change thereof while the at least the portion of the first analyte is translocating through the first nanopore, and (ii) (3) a second current or change thereof, or (4) a second voltage or change thereof while the at least the portion of the second analyte is translocating through the second nanopore; (d) using (i) (1) the first current or change thereof or (2) the first voltage or change thereof to determine a first characteristic of the at least the portion of the first analyte and (ii) (3) the second current or change thereof or (4) the second voltage or change thereof to determine a second characteristic of the at least the portion of the second analyte; and (e) characterizing one or more properties of the sample using the first characteristic or the second characteristic determined in (d).

[0122] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all withoutWSGR Docket Number: 64828-710.601 departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCORPORATION BY REFERENCE

[0123] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and the disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0125] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0126] FIGs.1A-1C illustrate electro-osmotic nanopore systems for the translocation and characterization of polymer analytes such as polypeptides through the nanopores, in accordance with some embodiments. FIG.1A illustrates a strong cis-to-trans EOF across the system for capture and translocation of a polymer analyte in the cis to trans direction, in accordance with some embodiments. Arrows through the pore indicate the magnitude of the ion flow in each direction, showing that the EOF can be generated by a large net flow of ions from cis to trans. FIG.1B illustrates a strong cis-to-trans EOF established in a system with positive voltage applied to the trans compartment across the membrane. FIG.1C illustrates a strong cis-to-trans EOF established in a system with negative voltage applied to the trans compartment across the membrane.

[0127] FIG. 2 shows example nanopore-based systems for characterizing and / or translocating polymer analytes. A translocase motor can aid translocation of the analyte through the nanopore, progressing along the polymer analyte in the direction of the subset arrow (moving away from termini PA towards termini PB of polymer analyte.WSGR Docket Number: 64828-710.601

[0128] FIGs.3A-3D show CytK nanopores. FIG.3A illustrates a cross-section of a surface representation of WT-CytK nanopores in 1 M KCl, pH 7.5. The nanopore was made by homology modelling from the alpha- hemolysin nanopore. FIG. 3B shows a cartoon representation of WT-CytK β-barrel region. The N-terminal strand is in dark gray and the C-terminal strand is in light gray. The charged residues are underlined. FIG.3C shows a schematic of the residues in each beta strand of the transmembrane beta-barrel region of wild-type CytK. FIG. 3D shows a cross-section of a surface (left) and cartoon (right) representation of a high ion selectivity mutant, CytK-2E-4D.

[0129] FIG. 4. shows an amino acid sequence and corresponding schematic representation of the three designed unstructured model polypeptide analytes, in accordance with some embodiments (which can be referred to as S1, tzatziki and mujdei). Solid circles indicate negatively charged amino acids, and open circles indicate positively charged amino acids.

[0130] FIGs. 5A-5E illustrate translocation of analyte S1 through wildtype (WT)-CytK nanopores, in accordance with some embodiments. FIG. 5A shows a schematic representation of the translocation of S1 through WT-CytK. The arrow denotes the analyte S1 translocating through the nanopore by an electrophoretic force (EF). FIG.5B shows a voltage dependency of translocation rates for type 1 and type 2 blockades. FIG. 5C shows a voltage dependency of the excluded current (Iex(%)) for type 1 and type 2 blockades. For FIGs.5B and 5C, type 1 blockades are shown as black squares and type 2 blockades are shown as light gray circles. FIG. 5D shows representative traces at –160 mV bias, in accordance with some embodiments. IO denotes the open pore current measurement and IB denotes the blocked pore current measurement. FIG.5E shows dwell time versus current amplitude at –160 mV bias.

[0131] FIGs. 6A-6E illustrate translocation of S1 through 2E-4D-CytK nanopores, in accordance with some embodiments. FIG. 6A shows a schematic representation of the translocation of S1 through 2E-4D-CytK, in accordance with some embodiments. The arrows denote the analyte S1 translocating through the nanopore by an electrophoretic force (EF; solid arrow) and electro-osmotic force (EOF; dotted arrow). The EF and EOF are in the same direction. FIG.6B shows a voltage dependency of translocation rate for analyte S1, in accordance with some embodiments. FIG.6C shows a voltage dependency of the excluded current (Iex%), in accordance with some embodiments. FIG. 6D shows representative traces at –40 mV bias, in accordance with some embodiments. FIG.6E shows a dwell time versus current amplitude at –40 mV bias, in accordance with some embodiments. Experiments were performed at pH 7.5 and under 1 M KCl. Traces were collected at 50 KHz sampling rates and filtered at 10 KHz using a Bessel filter.

[0132] FIGs. 7A-7E illustrate translocation of tzatziki through 2E-4D-CytK nanopores, in accordance with some embodiments. FIG.7A shows a schematic representation of the translocation of S1 through 2E-4D-CytK, in accordance with some embodiments. The arrows denote the analyte tzatziki translocating through theWSGR Docket Number: 64828-710.601 nanopore by an electrophoretic force (EF; solid arrow) and electro-osmotic force (EOF; dotted arrow). The EF and EOF are in opposing directions. FIG.7B shows a voltage dependency of translocation rate, in accordance with some embodiments. FIG.7C shows a voltage dependency of the excluded current (Iex%), in accordance with some embodiments. FIG. 7D shows representative traces at –160 mV bias, in accordance with some embodiments. FIG. 7E shows dwell time versus current amplitude at –160 mV bias, in accordance with some embodiments. Experiments were performed at pH 7.5 and under 1 M KCl. Traces were collected at 50 KHz sampling rates and filtered at 10 KHz using a Bessel filter.

[0133] FIGs. 8A-8E illustrate translocation of mujdei through 2E-4D-CytK nanopores, in accordance with some embodiments. FIG.8A shows a schematic representation of the translocation of S1 through 2E-4D-CytK, in accordance with some embodiments. The arrows denote the analyte tzatziki translocating through the nanopore by an electrophoretic force (EF; solid arrow) and electro-osmotic force (EOF; dotted arrow). The EF and EOF are in opposing directions. FIG.8B shows a voltage dependency of translocation rate, in accordance with some embodiments. FIG.8C shows a voltage dependency of the excluded current (Iex%), in accordance with some embodiments. FIG. 8D shows representative traces at –160 mV bias, in accordance with some embodiments. FIG. 8E shows dwell time versus current amplitude at –160 mV bias, in accordance with some embodiments. Experiments were performed at pH 7.5 and under 1 M KCl. Traces were collected at 50 KHz sampling rates and filtered at 10 KHz using a Bessel filter.

[0134] FIGs.9A-9H illustrate translocation of model substrates through nanopores, in accordance with some embodiments. For each of FIGs. 9A-9H, panel (i) shows the cut-through of a surface representation of the nanopores (left) and a cartoon representation of its β-barrel region (right), panel (ii) indicates the entry or translocation of analyte S1, panel (iii), if present, indicates the entry or translocation of analyte tzatziki and panel (iv), if present, shows the entry or translocation of analyte mudjei. FIG.9A shows representative current traces of substrates through WT-CytK, in accordance with some embodiments. FIG. 9B shows representative current traces of substrates through K128D-CytK, in accordance with some embodiments. FIG. 9C shows representative current traces of substrates through K128-K155Q-CytK, in accordance with some embodiments. FIG.9D shows representative current traces of K128D-K155D-CytK, in accordance with some embodiments. FIG. 9E shows representative current traces of K128D-K155Q-Q122D-CytK, in accordance with some embodiments. FIG. 9F shows representative current traces of K128D-K155D-Q145D-CytK, in accordance with some embodiments. FIG.9G shows representative current traces of substrates through K128D-K155D- T147D-CytK, in accordance with some embodiments. FIG.9H shows representative current traces of substrates through K128D-K155D-Q145D-S151D-CytK, in accordance with some embodiments.

[0135] FIGs. 10A-10G show translocation of model substrates through nanopores in accordance with some embodiments. FIG. 10A shows a cut-through of a surface representation of the WT-CytK nanopore (left) andWSGR Docket Number: 64828-710.601 a cartoon representation of its β-barrel region (right). Plots depict the voltage dependency of the excluded current (Iex%) for type 1 (dark gray circle) and type 2 blockades (light gray circle) (left) and a voltage dependency of translocation rates for type 1 blockade (dark gray square) and type 2 blockades (light gray circle) (right) for the translocation of substrates through WT-CytK. FIG. 10B shows a cut-through of a surface representation of the K128D-K155Q-CytK nanopore (left) and a cartoon representation of its β-barrel region (right). Plots depict voltage dependency of translocation rates (right) and voltage dependency of excluded current (Iex(%)) (left) for the translocation of substrates through K128D-K155Q-CytK. FIG.10C shows a cut- through of a surface representation of the K128D-K155D-CytK nanopore (left) and a cartoon representation of its β-barrel region (right). Plots depict voltage dependency of translocation rates for the two types of events, type 1 blockade (dark gray square) and type 2 blockade (light gray circle), and voltage dependency of excluded current (Iex(%)) of substrates through K128D-K155D-CytK. FIG. 10D shows a cut-through of a surface representation of the K128D-K155Q-Q122D-CytK nanopore (left) and a cartoon representation of its β-barrel region (right). Plots depict voltage dependency of translocation rates (right), and voltage dependency of excluded current (Iex(%)) (left) of substrates through K128D-K155Q-Q122D-CytK. FIG. 10E shows a cut- through of a surface representation of the K128D-K155D-Q145D-CytK nanopore (left) and a cartoon representation of its β-barrel region (right). Plots depict voltage dependency of translocation rates (right), and voltage dependency of excluded current (Iex(%)) (left) of analyte S1 and analyte tzatziki through K128D- K155D-Q145D-CytK. FIG.10F shows a cut-through of a surface representation of the K128D-K155D-T147D- CytK nanopore (left) and a cartoon representation of its β-barrel region (right). Plots depict voltage dependency of translocation rates (right), and voltage dependency of excluded current (Iex%)(left) of analyte S1 and analyte tzatziki through K128D-K155D-T147D-CytK. FIG. 10G shows a cut-through of a surface representation of the K128D-K155D-Q145D-S151D-CytK nanopore (left) and a cartoon representation of its β-barrel region (right). Plots depict voltage dependency of translocation rates (right), and voltage dependency of excluded current (Iex%)(left) of analyte S1, analyte tzatziki, and analyte mujdei through K128D-K155D-Q145D-S151D- CytK.

[0136] FIGs. 11A-11F illustrate translocation of unfolded MalE219a across 2E-4D-CytK nanopores. FIG. 11A shows representative traces of the translocation of MalE219a across 2E-4D-CytK in 2M urea. FIG. 11B shows dwell time versus amplitude of current blockades under –100 mV. FIG. 11C shows a cut-through of a surface representation of the nanopores (left) and a cartoon representation of its β-barrel region (right). FIG. 11D shows a cartoon representation of MalE219a. FIG.11E shows a voltage dependency of the translocation speed. FIG. 11F shows a voltage dependency of the excluded current. Traces were collected at 50 KHz sampling rates and filtered at 10 KHz using a Bessel filter.WSGR Docket Number: 64828-710.601

[0137] FIGs.12A-12F show translocation of unfolded H152A-GBP across 2E-4D-CytK nanopores. FIG.12A shows representative traces of the translocation of H152A-GBP across 2E-4D-CytK in 2.4 M urea showing the two levels, Level 1 (L1) and level 2 (L2), of the translocation blockades. FIG. 12B shows dwell time versus amplitude of current blockades under –100 mV. FIG.12C shows a cut-through of a surface representation of the nanopores (left) and a cartoon representation of its β-barrel region (right). FIG. 12D shows a cartoon representation of MalE219a. FIG.12E shows a voltage dependency of the translocation speed. FIG.12F shows a voltage dependency of the excluded current for L1 and L2 levels as indicated in panel A. Traces were collected at 50 KHz sampling rates and filtered at 10 KHz using a Bessel filter.

[0138] FIGs.13A-13E illustrate malE219a translocation through the 2E-2D CytK mutant in the presence of 1 M and 1.8 M GuHCl. FIG. 13A shows a cut-through of a surface representation of the nanopores (left) and a cartoon representation of its β-barrel region (right), in accordance with some embodiments. FIG.13B shows a voltage dependency of the dwell time of malE219a in 1 M (squares) and 1.8 M (circles) GuHCl. FIG. 13C shows a voltage dependency of the excluded current of malE219a in 1 M (squares) and 1.8 M (circles) GuHCl. FIG. 13D shows a dwell time versus amplitude dependence at various voltages in 1M GuHCl as indicated. FIG.13E shows a dwell time versus amplitude dependence at various voltages in 1M GuHCl as indicated.

[0139] FIGs.14A-14D show characterizations of the 2E-4D-CytK nanopore in the two denaturants. IV curves for 2E-4D-CytK nanopores in the urea is shown in FIG. 14A and GuHCl in FIG. 14B. Numerical values of the asymmetry using the ratio of the ionic current at -100 mV and +100 mV in different concentrations of urea (FIG.14C) and GuHCl (FIG.14D).

[0140] FIGs.15A-15C show MalE219a transport across WT-CytK. FIG. 15A shows translocation events of 100 nM of malE219a-D10 unfolded by 2 M urea, in accordance with some embodiments. FIG.15B shows 100 nM of malE219a added in the cis chamber did not induce events. FIG.15C shows a sequence of malE219a- D10.

[0141] FIGs.16A-16C show actinoporins common sequence alignment and wild-type Fragaceatoxin C. FIG. 16A shows common sequence alignment of some actinoporins, the dots represent the same amino acid as the common sequence, other amino acid differences between the pores are represented by their single-letter code. FIG.16B shows an artistic representation of Fragaceatoxin C (PDB: 4TSY) inserted into a lipid bilayer, across which a voltage is applied. Several non-conserved positions are enlarged. FIG.16C shows representative traces of the octameric (T1) and heptameric (T2) form of wild-type Fragaceatoxin C under an applied potential of -50 mV in 1M KC1 and 50 mM citric acid titrated with bis-tris propane to pH 3.8. Traces were collected at a sampling frequency of 50 kHz, using a 10 kHz Bessel filter and 5 kHz Gaussian filter.

[0142] FIG.17 shows alignment between Fragaceatoxin C homologues. Positions in homologs corresponding to D10 and G13 in Fragaceatoxin C are outlined by black boxes.WSGR Docket Number: 64828-710.601

[0143] FIGs.18A-18D show electrophysiology recordings of (mutant) Fragaceatoxin C with trypsin digested lysozyme, in accordance with some embodiments. FIG. 18A shows representative electrical ionic current traces of (mutant) Fragaceatoxin C combined with equal units of trypsin digested lysozyme added to the first side (e.g., cis side) and under an applied potential of -50 mV. The lowest current level is the open-pore current of the pore (Io), and the step-like upwards events may be the result of captured analytes occluding a portion of the ionic current flowing through the nanopore (event blockades, IB). FIG.18B shows representative trace of octameric Fragaceatoxin C (T1). FIG. 18C shows representative trace of heptameric Fragaceatoxin C (T2). FIG.18D shows representative trace of Fragaceatoxin C mutant (G13F).

[0144] FIGs. 19A-19E show event count and signal correlation of (mutant)’ Fragaceatoxin C with trypsin digested lysozyme. FIGS. 19A-19D show observed excluded current (Iex%) spectra from tryptic digest of lysozyme. FIG.19A shows octameric wild-type Fragaceatoxin C (T1). FIG.19B shows heptameric wild-type Fragaceatoxin C (T2). FIG. 19C shows Fragaceatoxin C mutant G13F. FIG. 19D shows Fragaceatoxin C mutant G13N. FIG.19E shows squared first derivative Euclidean cosine correlation of residual current spectra of (mutant) Fragaceatoxin C combined with equal units of trypsin digested lysozyme. Boxes 1-3 surrounding multiple mutants represent similar signals.

[0145] FIGs.20A-20C show peptide recognition of (mutant) Fragaceatoxin C. FIG.20A shows the location of mutations in the lumen of Fragaceatoxin C (modeled on PDB: 4TSY) marked by arrows. FIG. 20B shows Gaussian fits to histograms of the excluded currents from the clustered event blockade for the capture and detection of Angiotensin IV, Angiotensin III, Angiotensin I and Angiotensinogen recorded under an applied potential of -50 mV. FIG.20C shows excluded current % (IEX%) versus dwell time scatter plots of the single- molecule peptide event blockades detected by the different pore types.

[0146] FIG.21 shows peptide recognition of (mutant) Fragaceatoxin C.

[0147] FIG. 22 shows an electrophysiology setup of an analytical system comprising a nanopore, in accordance with some embodiments.

[0148] FIGs. 23A-23D show bottom-up nanopore-based proteomics. FIG. 23A shows an artistic representation of protease protein digestion to digest a protein into a mixture of peptide fragments. FIG. 23B shows an artistic representation of the experimental setup of a nanopore system. FIG. 23C shows an artistic representation of the resulting ionic current data for detected peptides from a nanopore-based electrophysiology experiment. FIG.23D shows an artistic representation of a resulting residual current versus standard deviation spectrum obtained from analysis of the individual single-molecule event blockades.

[0149] FIGs. 24A-24B show excluded current - mass calibration using peptides and the spectrum obtained from tryptic lysozyme peptides. FIG. 24A shows the mass of the synthetic model peptides (circles) plotted against the average measured excluded current (%) for each peptide when added to the G13F-FraC-T1 nanoporeWSGR Docket Number: 64828-710.601 system. FIG.24B shows excluded current spectrum (histogram of the excluded currents from event blockades) recorded from addition of a mixture of all the model peptides to a G13F-FraC-T1 pore.

[0150] FIGs. 25A-25B show nanopore experiments compared to electrospray ionization mass spectrometry. FIG. 25A shows residual current spectrum as obtained by nanopore electrophysiology using G13F-FraC-T1 and a tryptic digest of Gallus-gallus lysozyme. FIG.25B shows mass spectrometry results from the same tryptic digest, but measured with a mass spectrometer (ESI-MS). The resulting peptide masses were mapped to residual current using the logistic function prediction shown in FIG.24A with a standard deviation of 0.5 Iex%.

[0151] FIGs.26A-26C show the reproducibility of nanopore protein spectra, using three independent repeats of the sensing of proteolytic digestions. FIG.26A shows results from bovine serum albumin (BSA). FIG.26B shows results from dihydrofolate reductase (DHFR). FIG. 26C shows results from elongation factor P (EFP). For each figure, the left-side panel shows the excluded current histograms with a normalized area of 100%, which were obtained from the excluded current versus dwell time scatters of all event blockades shown in the respective right-side panels.

[0152] FIGs. 27A-27B shows spectral matching using squared first difference correlation coefficient. FIG. 27A shows example representative baseline corrected residual current spectra of the measurement of peptide fragment mixtures from 9 tryptic digested proteins. Unique spectra were observed for each protein type. For all digested proteins of FIG. 27A, the right-side panel shows the excluded current histograms with a normalized area of 100%, which were obtained from the excluded current versus dwell time scatters of all event blockades shown in the respective left-side panel. FIG. 27B shows leave-one-out spectral matching of the baseline corrected residual current spectra using Euclidean cosine cross-correlation.

[0153] FIG. 28 shows the detection of proteins kemptide (LRRASLG) and phosphorylated kemptide (LRRA{pS}LG). The graph shows that the peptides can be detected as two distinct clusters, plotting residual current (Ires = blockade current / open-pore current) versus dwell time.

[0154] FIG.29 shows the detection of glycopeptides. The peptides were unmodified peptide (ANVTLNTAG), peptide with one glycan (ANVT(Glc)LNTAG and peptide with two glycans (ANVT(Glc)LNTT(Glc)G).

[0155] FIGs.30A-30B show the detection of rhamnosylated proteins. FIG.30A shows unmodified Elongation Factor P (EF-P), with residual current (IRes) plotted against dwell time. FIG. 30B shows and rhamnosylated EF-P, with residual current (IRes) plotted against dwell time.

[0156] FIGs.31A-31C show discrimination between single amino changes. FIG.31A shows detection of two forms of enkephalin with sequences added to the cis-chamber of G13F-FraC-T1 pores, in accordance with some embodiments: YGGFL, and YdAGFdL, wherein d represents a D-amino acid; all other amino acids are L- amino acid. FIGS. 31B-31C show differences in nanopore signal due to the presence of D-amino acids, with analytes added to the cis compartment (FIG.31B) or trans compartment (FIG.31C).WSGR Docket Number: 64828-710.601

[0157] FIGs.32A-32D show detection of trypsinated lysozyme in Aerolysin nanopores, including WT-Aer at pH 7.5 (FIG. 32A), WT-Aer at pH 3.8 (FIG.32B), Aer-K238F at pH 3.8 (FIG.32C) and Aer-K238D-S264F at pH 3.0 (FIG. 32D). The open-pore current (Io) and exemplary step-like current blockades (IB) from peptide captures are marked.

[0158] FIGs. 33A-33I show detection of trypsinated lysozyme in Aerolysin nanopores. The pores included WT-Aerolysin at pH 7.5 (FIG. 33A), WT-Aerolysin at pH 3.8 (FIG. 33B), K238F aerolysin at pH 3.8 (FIG. 33C), K238D aerolysin at pH 3.0 (FIG. 33D), K238D-A260F aerolysin at pH 3.0 (FIG.33E), K238D-S264F aerolysin at pH 3.0 (FIG.33F), K238D-Q268F aerolysin at pH 3.0 (FIG.33G), K238D-S272F aerolysin at pH 3.0 (FIG.33H). FIG.33I shows measurement of 4μg trypsinated lysozyme added to the cis compartment (final concentration l0ng / μl) of nanopore system comprising Aer-K238W.

[0159] FIGs.34A-34C show detection of trypsinated lysozyme in Cytolysin K (CytK) nanopores. The current traces show representative sections of ionic current data for selected pores, comprising either WT-CytK at pH 3.8 (FIG.34A), CytK-K128F at pH 3.8 (FIG.34B), or CytK-S126F-K128D at pH 3.8 (FIG.34C). The open- pore current (Io) and exemplary step-like current blockades (IB) from peptide captures are marked in each plot.

[0160] FIGs.35A-35H show detection of trypsinated lysozyme in Cytolysin K (CytK) nanopores. FIG. 35A shows a homology model of CytK (left) mapped onto the structure of the alpha-hemolysin nanopore from Staphylococcus aureus, and predicted beta-strand showing inward water-facing amino acids for the beta-barrel lumen of the nanopore (right). FIGS.35B-35G shows residual current versus dwell time scatter of individual peptide blockades provoked by 4μg of trypsinated lysozyme added to the trans-chamber of a system comprising either (FIG.35B) wild type (WT-CytK) at pH 3.8, (FIG.35C) K128F CytK nanopore at pH 3.8, (FIG.35D) S126F- K128D CytK nanopore at pH 3.8, (FIG. 35E) S120F - K128D CytK nanopore at pH 3.0 (FIG. 35F) Q122F - K128D CytK nanopore at pH 3.0, (FIG.35G) G124F - K128D CytK nanopore at pH 3.0. FIG.35H shows measurement of two peptides (10 μM Lys4 and 10μM Lys7) added to the trans compartment a system comprising K128W CytK nanopore.

[0161] FIGs.36A-36B show detection of Lys-C digested lysozyme in Lysenin nanopores. FIG.36A shows a nanopore system comprising wildtype lysenin (Lys-WT). FIG. 36B shows a nanopore system comprising mutant lysenin (Lys-E76F).

[0162] FIGs. 37A-37F show detection of non-proteinaceous small molecules. The system comprised heptameric wild-type FraC (FIG. 37A), mutant FraC_G13F nanopores with Thioflavin (FIG. 37B and FIG. 37C), octameric wild-type FraC (FIG. 37D), or mutant FraC_G13F nanopores with Vitamin B12 (FIG. 37E and FIG.37F).

[0163] FIG.38 shows the design of a transmembrane protein device for single-molecule protein analysis.WSGR Docket Number: 64828-710.601

[0164] FIGs. 39A-39D show the fabrication and electrical optimization of a nanopore. FIG.39A shows the effects of linker length on the nanopore expression in E. coli cells, insertion efficiency and nanopore stability. FIG. 39B shows the electrical properties of ^4 mutant. The left schematic of FIG. 39B shows the linker sequence of ^4 mutant. The middle current representation of FIG.39B shows electrical recordings of a single nanopore at ±35 mV. The right plot of FIG. 39B shows a histogram of the unitary conductance values of 59 nanopores at-35 mV. FIG.39C shows the electrical properties of ^2 mutant. The left schematic of FIG.39C shows the linker sequence of ^2 mutant. The middle current representation of FIG.39C shows a current trace and the current histogram corresponding the insertion of individual pore into a lipid membrane at +35 mV. The right plot of FIG. 39C shows a histogram of the unitary conductance values of 59 artificial nanopores at-35 mV. FIG. 39D shows interaction of DPhPC with the artificial transmembrane pore generated by molecular dynamics simulations.

[0165] FIGs.40A-40H show the electrical properties of optimized artificial pore ( ^2) and discrimination of substrates. FIG. 40A shows the schematic of an ion-current measurement setup. FIG. 40B shows a current trace recorded through an efficient single pore after optimization at ±35 mV. FIG. 40C shows averaged current—voltage (I— V) characteristics of three different nanopores. The error bars represent a standard deviation from the mean curve of the ion selectivity of the nanopore (FIG.40D). Determination of the reversal potential shows that the pore is cation-selective, as expected from the electrostatic potentials at their constrictions. FIG.40E shows the chemical structure of beta-cyclodextrin (β-CD), scatter plots of Ires% versus dwell time, and representative trace. FIG. 40F shows the chemical structure of gamma-cyclodextrin (γ-CD), scatter plots of Ires % versus dwell time, and representative trace. FIG. 40G shows peptide sequences of angiotensin I, scatter plots of Ires % versus dwell time, and representative trace. FIG. 40H shows peptide sequences of dynorphin A, scatter plots of Ires % versus dwell time, and representative trace.

[0166] FIGs.41A-41E show the design of the artificial proteasome-nanopore. FIG.41A shows the structure of T. acidophilum proteasome-PA26. The C-terminal of PA26 (S231) is near L21 of the α subunit. FIG. 41B shows the reconstitution of artificial proteasome-nanopore. To obtain subcomplex 3, two separate vectors were used to express the four proteins. FIG.41C shows SDS-PAGE (left) and native PAGE (right) analyses of the purified complex 3. SDS-PAGE revealed the presence of three unique bands of PAuA20 (top), α∆12 (middle), and β (bottom) with molecular weights of 52.7, 25.8, and 22.3 kDa, respectively. FIG.41D shows behavior of a single pore at ±35 mV in 1 M NaCl, 15 mM Tris, pH 7.5. FIG. 41E depicts a cut-through of a surface representation of artificial transmembrane proteasome.

[0167] FIGs.42A-42C show SDS-PAGE analysis the hydrolyzing activity of subcomplex 3. FIG.42A shows β-casein (1 mg / mL) that was incubated with subcomplex 3 at 53°C in buffer A (50 mM Tris, pH 7.5, 150 mM NaCl). FIG. 42B shows β-casein (1 mg / mL) that was incubated with subcomplex 3 for 2 hours in buffer A.WSGR Docket Number: 64828-710.601 FIG.42C shows β-casein (1 mg / mL) that was incubated with subcomplex 3 at 53°C for 0.5 hour in buffer B (50 mM Tris, pH 7.5, 0.3-1.0 M NaCl). The β-casein / subcomplex 3 concentration ratio was 42.

[0168] FIGs.43A-43F show the discrimination of substrates with the proteasomal nanopore. FIG.43A shows typical current trace provoked by the analyte substrate 1 (S1) using an inactive proteasome-nanopore. FIG.43B shows translocation of S1 (20 μM) through an inactive proteasome-nanopore mediated by VAT (20.0 μM) and ATP (2.0 mM). FIG.43C shows when an inactive proteasome is used in the presence of ATP and VAT, GFP- ssrA is unfolded and translocated intact through the proteasome chamber and nanopore. FIG.43D shows typical current traces provoked by S1 using an active proteasome-nanopore. FIG. 43E shows when an active proteasome is used, in the presence of VAT and ATP, only rare and fast events may be observed suggesting that the active proteasome-nanopore can cleave S1 efficiently, producing small fragments. As shown in FIG. 43F, when an active proteasome is used in the presence of ATP and VAT, unfolded GFP-ssrA is cleaved in the proteasomal chamber and the degraded peptides may be too short to be detected by the nanopore.

[0169] FIGs. 44A-44B show discrimination of substrates with proteasomal nanopore. FIG. 44A shows sequence comparison of substrate 1 (S1) and substrate 2 (S2). FIG.44B shows scatter plots of fraction blockade versus time and representative blockades induced by cleaved S1 and S2.

[0170] FIGs.45A-45D show the design and membrane insertion of PA26 artificial nanopore. FIG.45A shows a ribbon diagram of the structure of anthrax protective antigen (PDB ID: 3J9C). FIG.45B shows a structure of PA26 (PDB ID: 1YA7). FIG.45C shows the structure of artificial PA26-nanopore. FIG.45D shows a current trace shows insertion of individual pore.

[0171] FIGs. 46A-46D show the design and insertion of ATPase artificial nanopores. FIG. 46A shows a ribbon diagram of the structure of anthrax protective antigen (PDB ID: 3J9C). The transmembrane region is highlighted in blue. FIG. 46B shows the structure of Aquifex aeolicus ATPase (PDB ID: 3M0E). FIG. 46C shows the structure of artificial ATPase transmembrane pore. FIG. 46D shows a current trace shows insertion and ATP hydrolysis of individual pore.

[0172] FIG.47 shows the design of a ClpP-artificial nanopore for single-molecule protein analysis.

[0173] FIG. 48 shows the current-voltage (I-V) characteristics of three different nanopores: opened ClpP- nanopore, closed ClpP-nanopore, and PA-nanopore.

[0174] FIG.49 shows the controlled translocation through the ClpP-nanopore.

[0175] FIGs. 50A-50G show the preparation and characterization of type I, type II, and type III FraC nanopores. FIG. 50A shows a cut through of a surface representation of WT-FraC oligomer (PDB: 4TSY) colored according to the vacuum electrostatic potential as calculated by PyMOLPyMOL. FIG.50B shows the percentage of the distribution of type I, type II, and type III for WT-FraC, W112S-FraC, W116S-FraC and W112S-W116S-FraC at pH 7.5 and 4.5. FIG.50C shows IV curves of type II nanopores formed by WT-FraC,WSGR Docket Number: 64828-710.601 W116S-FraC and W112S-W116S-FraC at pH 7.5 (15 mM Tris-HCl, 1 M KCl). FIG. 50D shows the single nanopore conductance of W116S-FraC in 1 M KC1 (0.1 M citric acid and 180 mM Tris base) at pH 4.5. FIG. 50E shows typical current traces for the three nanopore types of W116S-FraC in 1 M KCl at pH 4.5 under -50 mV applied potential. FIG.50F shows reversal potentials measured under asymmetric condition of KCl (1960 mM cis, 467 mM trans) at pH 4.5 for the three W116S-FraC nanopore types. FIG.50G shows molecular models of three types of FraC nanopores constructed from the FraC crystals structure using the symmetrical docking function of Rosetta.

[0176] FIGs. 51A-51F show single channel conductance distributions of FraC nanopores at pH 7.5 and 4.5. FIG. 51A shows a table reporting the average conductance values obtained by fitting Gaussian functions to conductance histograms, in accordance with some embodiments. S.D. represents the standard deviation of all single channels (number given as n). In FIG. 51B-51F, each panel represents a different batch of FraC nanopores as indicated, in accordance with some embodiments.

[0177] FIGs. 52A-52B show discrimination of angiotensin peptides in mixture with type II W116S-FraC nanopores. FIG.52A shows (i) sequences of angiotensin I (DRVYIHPFHL), II (DRVYIHPF), III (RVYIHPF) and IV (VYIHPF) with corresponding Ires% measured at -30 mV; (ii) blockades provoked by the four angiotensin peptides; (iii) density plot of the Ires% versus the standard deviation of the current amplitude for angiotensin I added to the cis compartment; and (iv) density plot after further addition of angiotensin II, angiotensin III, and angiotensin IV to the cis chamber. FIG. 52B shows discrimination of angiotensin II and angiotensin A (ARVYIHPF), showing (i) table showing the sequences, the molecular weights and the Ires% of the peptides; (ii) representative traces of the peptide blockades; and color density plot of the Ires% versus the standard deviation of the current amplitude for angiotensin II blockades prior (iii) and after (iv) the further addition of angiotensin A to the cis chamber.

[0178] FIGs. 53A-53C show an evaluation of biological peptides having different chemical compositions. Relation between the molecular weight and Ires% of peptide using: type I WT-FraC nanopores (FIG. 53A), type II W116S-FraC nanopores (FIG. 53B), and type III W112S-W116S-FraC nanopores (FIG. 53C) at pH 4.5. The solid line represents a second order polynomial fitting.

[0179] FIGs. 54A-54D show a nanopore peptide mass spectrometer at pH 3.8. FIG. 54A shows amino acid sequences of four different peptides and their overall charge at different pH. The chargeable amino acids are underlined. FIG.54B shows pH dependence of the Ires% for the four peptides (cis) shown in FIG.54A using type II W116S-FraC nanopores under -30 mV applied potential. FIG. 54C shows comparison of the Ires% versus the mass of peptides at pH 4.5 and 3.8. FIG.54D shows voltage dependence of c-Myc dwell times at different pHs.WSGR Docket Number: 64828-710.601

[0180] FIGs.55A-55C show discrimination of short peptide mixture with type III FraC nanopores comprising mutant W112S-W116S-FraC. FIG. 55A shows sequence, Ires% (-50 mV) and molecular weight (M.W) of angiotensin IV (VYIHPF), angiotensin 4-8 (YIHPF), endomorphin I (YPWF), and leucine enkephalin (Leu- enkephalin; YGGFL). FIG.55B shows blockades provoked by the different peptides. FIG.55C shows density plot showing the Ires% versus the standard deviation of the current blockade for the mixture of angiotensin IV, angiotensin 4-8, endomorphin I and leucine-enkephalin.

[0181] FIGs. 56A-56B show characterization of type II FraC nanopores comprising an oxidized cysteine at position 10. Difference between the DOC / W116S type II pore (FIG.56A) and the oxidized DIOC / W116S type II pore (FIG.56B).

[0182] FIGs. 57A-57D show wild type FraC (WtFraC) and D10R-K159E FraC (ReFraC) nanopores. FIG. 57A shows a cross-section through octameric WtFraC showing coulombic surface coloring. Aspartate residue 10 (D10), located in the constriction zone of WtFraC, is indicated. FIG.57B shows a top view on WtFraC (top) and ReFraC (bottom). FIG. 57C shows single channel conductance histogram for ReFraC (left) and WtFraC (right) at +50 mV in IM NaCl, 15 mM Tris-HCl pH 7.5. FIG.57D shows raw trace of WtFraC (top) and ReFraC (bottom).

[0183] FIGs.58A-58F show DNA discrimination with ReFraC. FIG.58A shows representative blockades of a homopolymeric DNA strand in complex with NA using ReFraC. The cartoon representations on the right of each current trace shows the interpretation of the current blockades. FIG. 58B shows representative distributions of residual currents obtained for A20, C20, T20 homopolymeric strands with ReFraC nanopores. FIG.58C shows current blockades of a continuous trace induced by homopolymeric C20 and A20 nucleotides to the same ReFraC pore. Traces shown were digitally filtered with 100 Hz cut-off. FIG. 58D shows a distribution of residual currents imposed by mixtures of C20 and A20 homopolymeric strands. FIG.58E shows continuous trace of an experiment to resolve mixtures of homopolymeric C20and T20nucleotides and FIG.58F shows the distribution of residual currents imposed by mixtures of C20 and T20 homopolymeric strands.

[0184] FIGs.59A-59B show unzipping / translocation of dsDNA by ReFraC. FIG.59A shows a representative trace of ReFraC capturing a NA:A(dsDNA)C complex at +50 mV. The open pore current is denoted as “1” and for comparison indicated after capture of the complex. States 2 and 3 are indicative of the block. Upon reversal of potential (“4”) the block is immediately released indicating that the double-stranded region NA:A(dsDNA)C complex was peeled off. FIG. 59B shows at + 100 mV, in more than half of the cases (insert) a single block (“2”) is observed after the dsDNA part is pushed through (deformation, brackets) and upon application of - 30 mV the block cannot be released immediately (“3”). At higher negative potentials the block can be released, indicating a rotaxane was formed.WSGR Docket Number: 64828-710.601

[0185] FIGs. 60A-60B show unitary channel conductance distribution and voltage current dependence determined for WtFraC and ReFraC nanopores. FIG. 60A shows unitary channel conductance distribution measured for WtFraC (top) and ReFraC (bottom) pre-oligomerized pores reconstituted in planar lipid bilayers. FIG.60B shows voltage current dependence measured for WtFraC and ReFraC nanopores.

[0186] FIG. 61 shows hemolytic activity of the WtFraC, D10R FraC and ReFraC. Hemolysis rate was calculated as inverse of the time elapsed till 50% decrease in turbidity (measured as optical density at 650 nm wavelength) observed in 1% of horse erythrocytes suspension in 15 mM Tris-HCl pH 7.5150 mM NaCl.

[0187] FIGs.62A-62B show translocation and immobilization of A(dsDNA)C DNA substrate recorded with ReFraC nanopore. A(dsDNA)C substrate (depicted above the trace) was made by annealing of ohgo I (5’ biotinylated AAAAAAAAAAAAAAAAAAAAGTGCTACGACTCTCTGTGTGCCCCCCCCCCCCCCCCCCCC) and oligo II (CACACAGAGAGTCGTAGCAC). FIG.62A shows blockades provoked on ReFraC nanopore by 1 µM of A(dsDNA)C alone (left) and in complex with 0.25 µM of neutravidin (right), substrates were added in cis under +50 mV applied potential. FIG. 62B shows blockades provoked on ReFraC nanopore by 1 µM of A(dsDNA)C alone (left) and in complex with 0.25 µM of neutravidin (right), substrates were added in cis under at + 70 mV.

[0188] FIG. 63 shows representative traces showing stepwise enhancements of the residual current within A(dsDNA)C-neutravidin blockades provoked on ReFraC nanopore. 1 µM of A(dsDNA)C and 0.25 µM of neutravidin were present in cis at +50 mV.

[0189] FIG. 64 shows traces depicting rotaxane formation by A(dsDNA)C-neutravidin driven into ReFraC nanopore at +100 mV applied potential.1 µM of A(dsDNA)C and 0.250 µM of neutravidin were added in cis. Voltage stepping protocols are shown with the lines below each current trace diagram. Rotaxanes were dismantled by switching the applied potential to -40 mV.

[0190] FIGs. 65A-65B show representative traces showing pseudorotaxane and rotaxane formation by oligonucleotide I - neutravidin immobilized within the ReFraC nanopore. FIG. 65A shows pseudorotaxane formation provoked by 1 µM of oligo I and 0.25 µM of neutravidin present in cis. FIG. 65B shows rotaxane formation by 1 µM of oligonucleotide I and 0.25 µM neutravidin present in cis while 1 µM of oligonucleotide II was added in trans. Rotaxanes were dismantled by switching the applied potential to -40 mV (two arrows above the trace indicate the dismantling of rotaxane).

[0191] FIGs.66A-66C show capture of an oligopeptide (Endothelin 1) and a protein (Chymotrypsin) with two FraC variants at two different pH conditions, in accordance with some embodiments. FIG. 66A shows cross- sections of wild type FraC (WtFraC, PDB: 4TSY) and D10R-K159E-FraC (ReFraC). FIGs. 66B-66C show representative traces induced by 1 µM endothelin 1 (FIG. 66B) and 200 nM chymotrypsin (FIG. 66C) toWSGR Docket Number: 64828-710.601 WtFraC (left) and ReFraC (right). Chymotrypsin (PDB: 5CHA) and human endothelin 1 (PDB: 1EDN) are shown as surface representations. The coloring represents the electrostatic potential of the molecular surface as calculated by APBS(13) (pH 7.5 in 1 M KC1) with light gray and dark gray corresponding to negative and positive potentials (range -4 to +4 kbT / ec), respectively.

[0192] FIGs. 67A-67B show electrostatic distribution and ion-selectivity of WtFraC and ReFraC. FIG. 67A shows the monomer averaged simulated electrostatic potentials reveal the negatively and positively charged constrictions of WtFraC and ReFrac, respectively. FIG. 67B shows determination of the reversal potential shows that WtFraC and ReFrac may be respectively cation- and anion-selective, as expected from the electrostatic potentials at their constrictions.

[0193] FIGs. 68A-68E show biomarker characterization with WtFraC at pH 4.5. FIG. 68A shows, from top to bottom: (i) surface representation with molecular surface and cartoon representations (PyMOL) of chymotrypsin (25 kDa, PDB: 5CHA), (ii) a representative trace obtained under -150 mV applied potential, (iii) a heatplot depicting the dwell time distribution versus Ires% at -150 mV, and (iv) the voltage dependence of Ires%, the voltage dependence of the dwell times, and the capture frequency. FIGs. 68B-68E show the same information for β2- macroglobulin (FIG. 68B; 11.6 kD, PDB: 1LDS), human EGF (FIG.68C; 6.2 kD, PDB: 1JL9), endothelin 1 (FIG. 68D; 2.5 kD, PDB: 1EDN) and angiotensin I (FIG. 68E; 1.3 kD), respectively. Angiotensin I is depicted as a random structure drawn with PyMOL.

[0194] FIGs. 69A-69E show discrimination of endothelin 1 and 2 with WtFraC at pH 4.5. FIG. 69A shows molecular surface representation of endothelin 1 and endothelin 2 using electrostatic coloring (PyMOL). FIG. 69B shows amino acid sequences of endothelin 1 and 2 (top), and Ires% and dwell time for endothelin 1 and endothelin 2 blockades at -50 mV in pH 4.5 buffer (1 M KCl, 0.1 M citric acid, 180 mM Tris-Base (bottom). Lines (6901) indicate the disulfide bridges in each oligopeptide. FIG.69C shows representative endothelin 1 and endothelin 2 blockades to the same FraC nanopore under -50 mV applied potential. FIG. 69D shows histogram (left) of residual currents provoked by 2 µM endothelin 1 and corresponding heatplot depicting the standard deviation of the current amplitude versus Ires% (right). FIG. 69E shows the same as FIG. 69D but after addition of 8 µM endothelin 2 to the same pore revealing a second population.

[0195] FIG. 70 shows a computer system for implementing a method, a system, or a device of the present disclosure, in accordance with some embodiments.

[0196] FIGS.71A-71B show a device comprising a nanopore as described herein.

[0197] FIG. 72 shows a method using devices, nanopores, systems, and sample preparation as described herein.

[0198] FIG.73 shows a kit as described herein.WSGR Docket Number: 64828-710.601

[0199] FIG.74 shows an exemplary system for characterizing and translocating polymer analytes, for example mixed amino-acid composition protein analytes, through a nanopore in a membrane.

[0200] FIGs.75A-75B illustrate an example leader with potential components. FIG.75A shows components of a (peptide based) “leader construct” (6) for attaching to a target protein substrate of interest, which can assist loading / binding of protein translocase motor(s) for unfolding and controlling translocation of the target protein substrate through a nanopore. The construct comprises a number of possible elements: 1. Recognition motif, 2. Capture motif, 3. Stall motif, 4. Block motif, 5. Coupling motif. FIG.75B shows an illustrative schematic of a leader construct (6) that is attached to a target protein substrate(s) of interest (7), e.g. a folded or structured protein.

[0201] FIG. 76 shows an exemplary process of a method of loading a protein translocase (8) onto a leader construct (6). A translocase first binds to a leader construct (B) at or near the recognition motif, and then proceeds to translocate along the construct (C) in the direction of the subset arrow via NTP hydrolysis until encountering the stall and / or blocking motifs that stall / pause the progression of the translocase (D).

[0202] FIGs. 77A-77B show an exemplary process of loading multiple protein translocases onto a leader construct. FIG. 77A shows a schematic of a substrate designed to load and stall one protein translocase. The capture (2) and / or stall (3) motifs in combination have a footprint long enough to accommodate a single translocase. FIG.77B shows a schematic of a substrate designed to load and stall n multiple protein translocase, comprising a longer combination of capture (2) and / or stall (3) motifs that can effectively stall and accommodate the binding footprints of the n multiple translocases, such that the trailing translocase motor(s) cannot push the leading translocase(s) through the stall / block motifs.

[0203] FIGs. 78A-78B show exemplary methods of loading a leader construct (6) with protein translocase(s) (8) and attaching the leader construct to a protein of interest (7). FIG.78A shows leader constructs can first be coupled to target protein analytes, then loaded with translocases. FIG.78B shows leader substrates can be pre- loaded with translocases, and then coupled to target protein analytes.

[0204] FIGs.79A-79D show electrical recordings of the capture of a Maltose Binding Protein substrate (MBP- 1) in wild-type alpha-hemolysin nanopores (WT αHL). Figures show selected representative regions of recording at -80 mV (FIG.79A), -120 mV (FIG.79B), -160 mV (FIG.79C) and -180 mV (FIG. 79D) (trans electrode).

[0205] FIGs. 80A-80D show the same as detailed in FIGs. 79A-79D, but using wild-type CytK nanopores (WT CytK).

[0206] FIGs. 81A-81C show a representative example of electrical current vs time traces for testing of a Maltose Binding Protein substrate:ClpX translocase complexes (MBP-1:ClpX) in weak EOF wild-type alpha-WSGR Docket Number: 64828-710.601 hemolysin (FIGs. 81A-81B) or wild-type CytK (WT CytK) (FIG. 81C). Measurements were carried out in a system similar to that described in FIG.1 (except with low or zero EOF nanopores).

[0207] FIGs. 82A-82B show a representative examples of electrical current vs time traces for testing of a Maltose Binding Protein substrate:ClpX translocase complexes (MBP-1:ClpX) in strong EOF CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores according to the system described in FIG.74. FIGs.82A-82B show representative sections at -80 mV from separate experiments. The characteristic ClpX controlled MBP-1 translocations are marked by numbered arrows.

[0208] FIG.83 shows a representative zoomed single example event of ClpX controlled MBP-1 translocation through CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores. Events start with a blockade (S1) from the open pore level (state i) to an almost 0 pA level (state ii), and terminate (S3) with a return to open pore current levels (state iv) when the ClpX reaches the end of the MBP-1.

[0209] FIGs.84A-84B show representative zoomed examples of ClpX controlled MBP-1 translocation events through CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores. The spectra illustrate MBP-1 polypeptide translocations through CytK 4D2E.

[0210] FIGs.85A-85D show examples of ClpX controlled MBP-1 translocation events for selected high EOF nanopore systems using high ion-selectivity nanopores. Events acquired from a CytK_4D2E nanopore (CytK K128D K155D S120D Q122D) system at -80 mV (FIG. 85A), a CytK_3D1F2E nanopore (CytK K128F_S120D_Q122D_K155D) system -80 mV (FIG. 85B), a CytK_4D2E_Alt nanopore (CytK K128D K155D S120D S151D) system at -80mV (FIG.85C), a CytK_2D1F2E nanopore (CytK K128F S120D Q122D) system at -120 mV (FIG.85D), all in cis and trans solutions of 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5, with preloaded MBP-1:ClpX in the cis compartment (to a final concentration 0.2 µM ClpX, 0.1 µM MBP-1 and 2.5 mM ATP).

[0211] FIGs. 86A-86B show representative zoomed exemplary events of ClpX controlled translocation of MBP-1 substrates (FIG.86A) compared to GFP-1 substrates (FIG.86B) through CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores at -80 mV.

[0212] FIG. 87 shows exemplary electrical current vs. time traces for testing of a Maltose Binding Protein:ClpX translocase complexes (MBP-1:ClpX) in strong EOF CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores with non-hydrolyzable Gamma-S-ATP.

[0213] FIG. 88 shows a histogram of the translocation duration for 35 full-length ClpX controlled MBP-1 translocations through a CytK 4D2E nanopore at -80 mV (cis: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5 containing 0.2 µM ClpX:0.1 µM MBP-1, 2.5 mM ATP; trans: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5).WSGR Docket Number: 64828-710.601

[0214] FIGs.89A-89B show a comparison of ClpX controlled MBP-1 translocations at -80 mV through CytK 4D2E nanopore systems (cis: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5, 2.5 mM ATP; trans: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5) without (FIG.89A) and with (FIG.89B) pre-loading incubation to form the MBP-1:ClpX complexes.

[0215] FIG. 90 shows exemplary capture and ClpX controlled trans-to-cis translocation of MBP-1 through CytK 4D2E nanopores that were inserted from the cis compartment (cis and trans solutions of 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2). Preloaded MBP-1:ClpX complexes were added to the trans compartment (to a final concentration 0.2 µM ClpX, 0.1 µM MBP-1 and 2.5 mM ATP) and captured into the trans entrance of the nanopores at +80 mV, and translocated under ClpX control as a result of the strong trans- to-cis EOF created at positive applied voltage.

[0216] FIGs. 91A-91B show gel results of ClpX activity assays. FIG. 91A shows a gel showing the results of a ClpX / ClpP degradation assay of a GFP-ssrA substrate (GFP-0) under varying concentrations of KCl. FIG. 91B shows gel showing the results of a degradation assay of GFP-0 under varying concentration of potassium glutamate (KGlu). (65 nM ClpX, 65nM ClpP, 2800 nM GFP)

[0217] FIG.92 shows representative ClpX controlled translocation of MBP-1 through a CytK 4D2E nanopore at -120 mV in a nanopore system.

[0218] FIGs. 93A-93E show a schematic showing an exemplary “Out mode” method for characterizing a target protein by capturing it from the first side (e.g., cis side) into the nanopore of a system setup with high net cis-to-trans EOF, in conjunction with a protein translocase orientated on the target protein such that it then pulls the polypeptide back out through the same nanopore to the first side (e.g., cis side).

[0219] FIG. 94 shows a schematic of current vs time for a translocation event resulting from translocase controlled polypeptide translocation through a nanopore as described by the scheme in FIGs.93A-93E.

[0220] FIGs.95A-95D show structural models of CytK. FIG.95A shows a structural model of the wild-type CytK nanopore from homology mapping onto the structure of the alpha-hemolysin nanopore. FIG.95B shows a schematic of the residues in each beta strand of the transmembrane beta-barrel region of wild-type CytK, marking water-facing residues of the down- and up- strands most suitable for mutagenesis. FIG.95C shows a model of the CytK 4D2E nanopore (CytK K128D K155D S120D Q122D), showing very high net negative internal charge due to mutations. FIG.95D shows a schematic location of mutations to negative residues in the barrel region of the CytK 4D2E nanopore.

[0221] FIGs. 96A-96E show recordings of different substrates in CytK nanopores. FIG. 96A shows a schematic of the substrate design. FIGs.96B-96E show electrophysiology reads acquired from {GFP}-{MBP- 1}, {LIVBP}-{MBP-1}, {SpuE}-{MBP-1}, and {GBP}-{MBP-1} substrates. Each substrate shows a similar ionic current pattern in the region corresponding to MBP as marked by the underlying arrows, and a uniqueWSGR Docket Number: 64828-710.601 ionic current signature specific to the attached protein in the second sections as marked by the indicated arrows below.

[0222] FIG.97 shows a single-molecule read of a 88 kDa MBP-MBP fusion protein.

[0223] FIGs.98A-98D show stalling of ClpX at 37°C using blocking domains. FIG.98A shows unfolding of GFP-1 with (+ClpX) and without ClpX (-ClpX); FIG.98B shows mNG with an alpha-helical blocking domain; FIG. 98C shows mNG with a helix-turn-helix blocking domain, and FIG. 98D shows mNG with a hairpin blocking domain.

[0224] FIGs. 99A-99B show depictions of maltose-binding protein (MBP) monomers (FIG. 99A) and maltose-binding protein dimers (FIG.99B) for analysis.

[0225] FIG.100 shows an electrophysiology recording of a MBP protein lacking the ssrA recognition motif, that was tagged on the C-terminus to allow binding of ClpX. ClpX controlled MBP translocations (marked by arrows) are evident from the characteristic pattern of changing ionic current signals.

[0226] FIGs.101A-101D show attachment of a single-stranded DNA to ClyA nanopore. FIG.101A depicts a side view (left) and top view (right) of ClyA structure (PDB: 6mrt). Serine at position 110 (S110) was genetically mutated to cysteine to enable site-specific chemical modification. FIG. 101B shows a schematic model showing the conjugation strategy of attaching ssDNA to ClyA nanopore. FIG. 101C shows a SDS- PAGE analysis of the conjugation efficiency. Lane 1: protein ladder, lane 2: ClyA-S110C monomer, lane 3: after reaction of ClyA-S110C with maleimide-PEG4-DBCO (ClyA-DBCO), lane 4: after reaction of purified ClyA-DBCO with f-azide (ClyA-f). FIG. 101D shows a native polyacrylamide gel analysis of the oligomerization of ClyA-f. Lane 5: ClyA-f after oligomerization, lane 6: S110C mutated ClyA after oligomerization.

[0227] FIGs.102A-102F depict functionalization of ClyA nanopore with Spike nanobody Ty1 and electrical characterization of the nanopore. FIG.102A shows a schematic model showing the strategy of functionalizing ClyA nanopore with Ty1 nanobody. FIG. 102B shows I-V curves of ClyA-S110C (triangle), ClyA-f (square) and ClyA-f-Ty1 (circle) at applied potential ranging from -90 to 90 mV (three independent experiments). FIG. 102C shows a histogram showing conductance distribution of ClyA-f nanopore with (white) and without (black) Ty1 nanobody. FIG.102D depicts representative current traces of ClyA-f-Ty1 under an applied potential of - 20 mV. The terms “in” and “out” represented the nanobodies locating inside (blocked pore) and outside (open pore) of the nanopore respectively. Io is the open pore current and Ib is the blocked pore current. FIG. 102E shows an all-point histogram of the current traces shown in FIG. 102D, which demonstrated well-defined distribution of the blockade signals. FIG. 102F shows a schematic model interpreting the reversible conformation change between blocked (left) and open (right) states of ClyA-f-Ty1 at applied potential of -20 mV, which corresponded to the movement of one of the Ty1 nanobodies in and out of the vestibule of the pore.WSGR Docket Number: 64828-710.601

[0228] FIGs. 103A-103F show single channel recording traces of ClyA-f-Ty1 and the analysis of residual current Ib / Io, tin and tout under different applied potentials. FIG.103A shows representative current traces of ClyA-f-Ty1 under applied potentials ranging from -10 to -40 mV. FIG. 103B shows an all-point histogram of current traces depicted in FIG.103A, showing Ty1 nanobodies tend to reside in ClyA nanopore with increasing applied potentials. FIGs.103C-103D show a histograms of logarithmic time of Ty1 locating inside and outside of ClyA, respectively. FIGs. 103E-103F show the influence of applied potentials on the average logarithmic time of Ty1 locating inside and outside of ClyA.

[0229] FIGs. 104A-104B depict nanobody attachment to ClyA through DNA oligo hybridization verified using DNase I. FIG.104A shows current traces of ClyA-f-Ty1 before and after the addition of 5 U DNase I in the presence of 2.5 mM MgCl2 at an applied potential of -20 mV. FIG. 104B shows enlarged representative current traces from FIG.104A, showing that the nanobodies attached to the ClyA nanopore were removed after the addition of DNase I after about 30 mins. All-point histograms were displayed on the top of the panel showing the current distribution before and after the addition of DNaseI. The schematic model shown above depicts how the nanobodies were removed from ClyA nanopore.

[0230] FIG.105 depicts detection of Spike protein by nanobody-functionalized nanopores. The upper current trace shows ClyA-f-Ty1 before and after the sequential addition of 6 µM BSA and 2.3 nM Spike protein. Below the upper current trace are enlarged representative current traces and all-point histograms of the current distribution. From left to right are: (i) representative traces before addition of BSA and spike protein; (ii) representative traces after addition of BSA and prior to spike protein; and (iii) after addition of BSA and spike protein. showed before and after the addition BSA and Spike proteins.

[0231] FIGs. 106A-106G show the effect of BSA on nanobody internalization. FIGs. 106A-106C show histograms distribution of tout before and after the addition of 3 µM BSA or 6 µM BSA to the first side (e.g., a cis side) of a ClyA-f-Ty1 nanopore system. The histograms were fitted with single exponential function. FIGs. 106D-106G show the change of blockade percentage, open percentage, average time of the coupled Ty1 staying inside the ClyA nanopore (tin), average time of Ty1 staying outside the ClyA nanopore (tout) with increasing concentration of BSA, respectively. (n=4, each experiment was conducted with independent nanopores. Error bars represent standard deviations).

[0232] FIGs.107A-107E show the effect of adding Spike protein to the ClyA-f-Ty1 pore. FIG.107A depicts current traces showing the transition of the pore from a dynamic state (alternating between Ty1 in the pore and out the pore) to a fully open state (with the Ty1 trapped outside the pore through binding to Spike protein) in the period immediately following the addition of 2.3 nM Spike protein. FIG. 107B shows current traces of ClyA-f-Ty1 in the period approximately 25 minutes post addition of 2.3 nM Spike protein. FIG. 107C shows an all-point histogram of the current traces presented in FIG.107B. FIGs.107D-107E show histograms of theWSGR Docket Number: 64828-710.601 logarithm of tin (FIG. 107D) and tout (FIG.107E) after the addition of 2.3 nM Spike protein in of the period about 25 minutes post addition of Spike protein. The histograms were fitted with the Gauss distribution function.

[0233] FIG.108A-108D show open probability of ClyA-f-Ty1 correlates positively with Spike trimer protein concentration. FIG. 108A shows representative current traces of ClyA-f-Ty1 before and after the addition of increasing concentration of Spike trimer protein. FIG. 108B shows an all-point histograms were displayed to show the current distribution before and after the addition of increasing concentration of Spike protein. FIG. 108C shows a curve regression of the open probability in the function of spike concentrations. The curve was fitted by using Hill-Langmuir equation (n=1.31, Kd=760.6 pM). FIG.108D shows a schematic model depicting the dynamics of the interaction between ClyA-f-Ty1 and Spike protein. Ty1 nanobodies dynamically move in and out of ClyA nanopore under applied potential.

[0234] FIGs.109A-109J show the influence of Spike proteins concentration on binding kinetics to the ClyA- f-Ty1 pore. FIGs. 109A-109D show histograms of log10(tout) at Spike concentration of 115 pM, 230 pM, 345 pM, 460 pM, respectively. The data were fitted with Guass distribution. FIGs.109E-109H show histograms of log10(tin) at Spike concentration of 115 pM, 230 pM, 345 pM, 460 pM, respectively. The data were fitted with Guass distribution. FIGs.109I-109J show the concentration dependency of the logarithm of tout and tin.

[0235] FIGs. 110A-110H show the behavior of ClyA-f-Ty1 in the presence of blood. FIG. 110A shows a schematic model showing electrical measurement of ClyA-f-Ty1 in the presence of blood. FIG. 110B shows current traces showing the current change before and after addition of 1 µL of blood to the ClyA-f-Ty1 nanopore presenting in 500 µL electrolyte buffer. FIGs.110C and FIG. 110E show representative current traces in the presence of 6 µM BSA (FIG. 110C) and after addition of 1 µL of blood (FIG.110E). FIGs.110D and FIG. 110F show all-point histograms of the current traces before (FIG.110D) and after (FIG.110F) addition of 1 µL of blood. FIG.110G shows a histogram of the logarithm of dwell time in level0 before and after the addition of 1 µL of blood. FIG. 110H shows a histogram of the logarithm of dwell time in level1 before and after the addition of 1 µL of blood.

[0236] FIGs. 111A-111B show detection of spike trimer in the presence of blood. FIGs. 111A-111B show representative current traces before (FIG.111A) and after (FIG.111B) the addition of 2.3 nM Spike protein in the presence of 1 µL blood at a bias of -20 mV.

[0237] FIGs. 112A-112B show detection of Her2 with functionalized nanopores. FIG. 112A shows representative current traces of ClyA attached by 2Rs15d nanobody (ClyA-f-15d) before and after the addition of 32.8 nM Her2 protein under an applied potential of -20 mV. FIG.112B shows representative current traces of ClyA attached by 2Rb17c nanobody (ClyA-f-17c) before and after the addition of 20.8 nM Her2 protein at the same applied potential.WSGR Docket Number: 64828-710.601

[0238] FIGs.113A-113E show functionalized ClyA nanopore for the detection of muPA. FIG. 113A shows the crystal structure of muPA in complex with nb22 nanobody (PDB: 5LHR). Reported binding affinity of nb22 to muPA56: kon = (4.6 ± 0.8) x 105 M-1 s-1, koff = (7.8 ± 2.2) x 10-5 s-1, KD = 0.2 ± 0.03 nM. FIG. 113B shows representative current traces of ClyA-f-nb22 before and after adding 3 nM muPA under -15 mV applied potential. FIG. 113C shows enlarged representative current traces after adding 3 nM muPA at -15 mV. FIG. 113D shows a heatmap of the blockade events observed after the addition of 3 nM muPA with the logarithm of the dwell time against current blockade percentage. FIG. 113E shows the schematic model showing the conformation changes of ClyA-f-nb22 in response to muPA proteins.

[0239] FIGs.114A-114C shows schematic illustrations of some of the options for coupling targeting moieties R to nanopores N via the hybridization of duplexed oligonucleotide (e.g. dsDNA) linkers L (where one oligonucleotide strand of the duplex linker L is coupled to the nanopore N, and the other complementary strand is coupled to the binding moiety R). FIG. 114A-114C illustrates 3 possible options for coupling the components. FIG.114A shows the nanopore N and binding moiety R components are located at opposite ends of the duplex linker L. FIG. 114B shows the nanopore N and binding moiety R components are located at the same end of the hybridized duplex linker L. FIG.114C shows one or both the nanopore N and binding moiety R components are coupled to the oligonucleotide strands of the hybridized duplex linker L at an internal position along the strand.

[0240] FIGs. 115A-115D show schematic illustrations of a nanopore N with a linker L that is initially in a protected state (FIG.115A), comprising a hybridized protecting polynucleotide strand (i) that can be removed by applying voltage to the nanopore in a membrane system to capture and strip the protecting strand from the linker L (FIG.115B). The deprotected nanopore (FIG.115C) can then be combined with a selected binding moiety R which will hybridize to the linker L to create the functional N-L-R nanopore system (FIG.115D).

[0241] FIG. 116 shows a depiction of a computer system that is programmed or otherwise configured to implement the methods provided herein.

[0242] FIGs. 117A-117B show example workflow methods for signal extraction and processing with representative current signals (e.g., electrophysiology traces or waveforms). FIG. 117A shows a schematic illustrating a method for extracting a signal or portion thereof or change thereof and processing the signal. FIG. 117B shows the representative signals for each step shown in the method schematic of FIG.117A, beginning with raw signal (step 1), extracted and denoised portions of signal (reads, putative reads and other event types, step 2), filtering (e.g., based on read metrics) to remove events that do not meet the criteria of good reads (step 3), segmenting the reads and performing merging and other resegmentation steps to produce a segmented read (squiggle) (steps 4 and 5), and finally performing further filtering to remove reads based on the metrics of the segmented reads.WSGR Docket Number: 64828-710.601

[0243] FIG.118 shows a representative schematic of a model architecture of a model described herein.

[0244] FIG.119 shows a representative workflow for scaling a reference squiggle to read as described herein.

[0245] FIG. 120 shows a representative workflow for polypeptide identification. The polypeptide identification can be for a single molecule.

[0246] FIG.121 shows a representative workflow for polypeptide quantification.

[0247] FIG.122 shows a representative workflow for alignment and consensus of signal reads.

[0248] FIGs.123A-123D show alignment and generation of consensus sequence from data. FIG.123A shows representative current signal reads for protein translocation events of data produced by a nanopore system described herein. FIG.123B shows representative reference signals (e.g., generated by machine learning from trained models as described herein). FIG.123C shows an example of an alignment schematic mapping of one of the signal reads of the detected current signals of FIG.123A and the reference signals of FIG.123B. FIG. 123D shows an example of the output consensus of multiple signal reads of FIG.123A that all aligned to one of the reference signals of FIG. 123B all piled up into a consensus plot with shared x-axis sequence position (also called index position) and scaled and normalized current (performed during scaling as described herein).

[0249] FIGs. 124A-124E show representative raw current signal (specifically portions of signal reads corresponding to protein translocations through a nanopore)showing how point mutations and other small amino acid motif 2-mer or 3-mer changes in the analytes alters the ionic current signal where indicated by the dotted box. FIG. 124A shows the current signal from an analyte with the KNK motif, with point mutations relative to KWK and ENK motifs. FIG.124B shows the current signal from an analyte with the WWW motif, with 2-mer and 3-mer changes relative to analytes with the other motifs. FIG. 124C shows the current signal from the ENE motif, with a single point mutation relative to the ENK motif. FIG. 124D shows the current signal from an analyte with the KWK motif, with a point mutation relative to the KNK motif. FIG.124E shows the current signal from an analyte with the ENK motif, with a single point mutation relative to the KNK and ENE motifs.

[0250] FIGs. 125A-125B show the consensus analysis of point mutation sequencing of maltose protein containing analyte MBP-1. Circles represent wildtype MBP (MBP-MBP1), triangles represent MBP1 with ENK mutation, squares represent MBP1 with KWK mutation, and downward-facing triangles represent MBP1 with WWW mutation. FIGs. 125A-125B show the consensus squiggle along index positions of the analyte (determined as described herein by aligning many reads to a reference squiggle of MBP-1 and adjusted the reference squiggle based on the deviations in the reads) as the lines and markers. Overlaid over the consensus lines and markers are the points corresponding to the average current for the given segments aligned the sequence index position for all the reads used in the consensus analysis. The spread of points are tightly clustered around the mean of the consensus squiggle at each index position, showing that all the single moleculeWSGR Docket Number: 64828-710.601 reads closely match the consensus. FIG.125B shows the zoom region of FIG.125A containing the mutation, illustrating the clear deviations in current that are observed in the reads and the consensus from the point mutations.

[0251] FIGs.126A-126D show consensus squiggles (overlaid with their corresponding aligned reads, plotting the 1-standard deviation spread of the difference in the current of the corresponding segment at each index position as the width of the lines) from samples containing protein analytes with and without post-translational modifications (PTMs). FIG. 126A shows detection of phosphorylation, specifically a phosphorylated serine residue, from the sequence index. FIG. 126B shows detection of glycosylation, specifically a glycosylated cysteine residue, from the sequence index. FIG.126C shows detection of acetylation, specifically an acetylated lysine residue, from the sequence index. FIG.126D shows detection of deamidation from the sequence index. Resides Q255, N203, N175, and Q174 were deaminated.

[0252] FIGs.127A-127B show a poly-PTM measurements from an analyte. FIG.127A shows the consensus squiggle plots for both treated (phosphorylated) and untreated samples, obtained from alignment of the reads for the samples as described herein. The figure plots current vs index position for the two consensus squiggles obtained (lines), overlaying points corresponding to the average current for the given segments aligned the sequence index position for all the reads used in the consensus. Also overlaid is a transparent thicker line showing the 1-standard deviation spread in the points across index positions of the analyte. FIG. 127B shows the zoom of FIG.127A in the region containing two deviations that arise from two phosphorylation sites along the proteins. Both Site 1 and Site 2 showed clear deviations in current for the modified reads and consensus squiggle vs the unmodified.

[0253] FIGs. 128A-128C show detection of post-translational modifications and variants of analytes in a mixed population. FIG. 128A shows a multiple aligned reads (aligned as described herein to a reference squiggle to align the segments in sequence index space) overlaid into a pile-up of reads for the sample measured at 0 hours of mixing (e.g., treatment). FIG. 128B shows the similar multiple aligned reads pile-up from the sample measured at 16 hours after mixing (e.g., treatment). The degraded sub-population can be viewed as a new population of reads containing a clear downward deflection in current at the position indicated by the arrow. FIG. 128C shows quantification of the percentage of reads aligning to the degraded sub-population in the mixture versus the sample time points. The percent of modified analyte is shown on the y-axis over time on the x-axis. Over 0 hours to 72 hours post-treatment, the amount of modified analyte increased from 0% to 57%.

[0254] FIGs.129A-129B show bi-directional current signal reads of protein analyte nanopore translocations. FIG. 129A shows reading of the MBP1 analyte in C-terminus to N-terminus direction when fed through a nanopore cis-to-trans as described herein. FIG.129B shows reading of the MBP1 analyte in N-terminus to C- terminus direction when fed through a nanopore cis-to-trans as described herein.WSGR Docket Number: 64828-710.601

[0255] FIG.130 shows a representative current waveform for a MBP-1 analyte translocated through an MspA nanopore with VAT (VAT-ΔN unfoldase). S. and E. mark the start and end of the reads respectively, and IO the open-pore current. Measurements obtained with a MspA_D90N nanopore in 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5. The applied voltage is -80 mV. The cis compartment contained a final concentration of 0.2 µM VAT-ΔN, 0.1 µM substrate and 2.5 mM ATP.

[0256] FIG. 131 shows a representative portion of the electrophysiology current signal of a substrate with truncation of a C-terminal peptide tag. The substrate had no poly-glycine region . Measurements obtained with a MspA_D90N nanopore in 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5. The applied voltage is -80 mV. The cis compartment contained a final concentration of 0.2 µM ClpX, 0.1 µM MBP and 2.5 mM ATP.

[0257] FIG.132 shows translocation read of a C-terminus to C-terminus linked protein created by chemical conjugation of a tag to the substrate. The location of recognizable and repeatable features in the reads are indicated by arrows. Measurements obtained with a MspA_D90N nanopore in 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5. The applied voltage is -80 mV. The cis compartment contained a final concentration of 0.2 µM ClpX, 0.1 µM MBP-C and 2.5 mM ATP.

[0258] FIGs. 133A-133F show representative portions of current signal reads for different proteins. FIG. 133A shows the current signal for translocation of alpha-synuclein through a nanopore, displayed as current (pA) versus time (s). FIG.133B shows the current signal for translocation of p53 through a nanopore, displayed as current (pA) versus time (s). FIG. 133C shows the current signal for translocation of TauF through a nanopore, displayed as current (pA) versus time (s). FIG.133D shows the current signal for translocation of a nanobody through a nanopore, displayed as current (pA) versus time (s). FIG. 133E shows the current signal for translocation of a light chain of an antibody through a nanopore, displayed as current (pA) versus time (s). FIG.133F shows the current signal for translocation of Titin I27 through a nanopore, displayed as current (pA) versus time (s).

[0259] FIGs. 134A-134C show representative current signal reads for the translocation of long peptides (peptides with a length of 900 amino acids or more). FIG. 134A shows a representative current signal for translocation of analyte DNAK-E.coli-MBP1 through a nanopore, displayed as current (pA) versus time (s). FIG. 134B shows a representative current signal for translocation of analyte CH60-E.coli-MBP1 through a nanopore, displayed as current (pA) versus time (s). FIG. 134C shows a representative current signal for translocation of analyte ODP2-E.coli-MBP1 through a nanopore, displayed as current (pA) versus time (s).

[0260] FIG. 135 shows a schematic for isoform characterization that involves comparing multiple signals reads for protein isoform translocations against multiple predicted reference squiggles to score and then identify the isoform identify of each read.WSGR Docket Number: 64828-710.601

[0261] FIGs.136A-136D show schematic current signal reads of current versus time that illustrate differences in the kinetics or speed of translocation as a function of the position through the read / sequence. FIG. 136A illustrates longer lived segments or pauses in the middle of translocations (marked by arrows), after which the read continues to translocate at the normal average speed. FIG 136B illustrates stalls (marked by arrows) that terminate the translocation of the reads (e.g. requiring the read to be ejected). FIG.136C shows representative current signal reads for CH60 protein translocations, marking pauses and changes in kinetics by arrows. FIG. 136D shows representative current signal reads for antibody protein translocations, marking pauses and changes in kinetics by arrows. Pauses and other changes in kinetics can be used for determination of polypeptide characteristics.

[0262] FIGs. 137A-137E show schematic representations of electrophysiology systems as described herein. FIG. 137A shows an enclosed trans compartment fluidically connected to an open cis compartment by a membrane. FIG. 137B shows an enclosed cis compartment directly connected to an enclosed trans compartment, via a membrane, both compartments within a solid substrate. FIG. 137C shows an aqueous cis droplet connected to an aqueous trans droplet. The droplets can have electrodes in or connected to the aqueous droplet. FIG. 137D shows an aqueous droplet connected to a continuous aqueous phase. FIG.137E shows an aqueous droplet connected to an enclosed compartment (trans compartment).

[0263] FIGs. 138A-138E show a schematic of a system for translocating a protein substrate through a nanopore which is initially captured from the trans, then translocated with a motor protein trans-to-cis against a net cis-to-trans EOF (after a reversal of voltage and EOF in step D and E).

[0264] FIG.139 shows a schematic of the electrophysiology signal obtained from implementing the system as shown in FIGs.138A-138E.

[0265] FIGs.140A-140E show a schematic of a system for translocating a protein substrate, initially captured from the trans, then translocated with a motor protein cis-to-trans with a net cis-to-trans EOF, (after a reversal of voltage and EOF in step D and E).

[0266] FIG. 141 shows shows a schematic of the electrophysiology signal obtained from implementing the system as shown in FIGs.140A-140E.

[0267] FIGs. 142A-142E show a schematic of a system for translocating a protein substrate through a nanopore, which is initially captured from the trans, then translocated with a motor protein trans-to-cis against a net cis-to-trans EOF.

[0268] FIG.143 shows a schematic of the electrophysiology signal obtained from implementing the system as shown in FIGs.142A-142E.WSGR Docket Number: 64828-710.601

[0269] FIG. 144A-144D show a schematic of a system for translocating a protein substrate through a nanopore, which is initially captured from the cis after binding to translocases on the cis, then translocated with a motor protein cis-to-trans with the net cis-to-trans EOF.

[0270] FIG.145 shows a schematic of the electrophysiology signal obtained from implementing the system as shown in FIGs.144A-144D.

[0271] FIG. 146 shows representative electrophysiology reads of ClpX controlled MBP-1 translocations through MspA nanopores, obtained from multiple different single nanopores in multiple membranes (formed on separate trans compartments with a common cis compartment) on a array chip where the nanopores are individually electrically addressed and measured in parallel. DETAILED DESCRIPTION

[0272] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed.

[0273] Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure.

[0274] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The use of the words “a” or “an” when used in conjunction with the term “comprising” herein may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0275] It should also be noted that the term “or” can be generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0276] The term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, can be meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. As used herein, “about” and “approximately” may mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given range of values.WSGR Docket Number: 64828-710.601

[0277] The compositions and methods of the present invention encompass polypeptides and nucleic acids having the sequences specified, or sequences substantially identical or similar thereto, e.g., sequences at least 80%, 85%, 90%, 95% identical or higher to the sequence specified. In the context of an amino acid sequence, the term “substantially identical” can be used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are (i) identical to, or (ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and / or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 80%, 85%, 90%.91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.5%, 99.9%, or 100% sequence identity to a reference sequence, e.g., a sequence provided herein. In the context of nucleotide sequence, the term “substantially identical” can be used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.5%, 99.9%, or 100% sequence identity to a reference sequence, e.g., a sequence provided herein.

[0278] The term “variant” can refer to a polypeptide and / or at least a portion of a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or can be encoded by a substantially identical nucleotide sequence. In some embodiments, the variant can be a functional variant.

[0279] The term “functional variant” can refer to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or can be encoded by a substantially identical nucleotide sequence, and can be capable of having one or more activities of the reference amino acid sequence.

[0280] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) can be performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In some embodiments, the length of a reference sequence aligned for comparison purposes can be at least about 30%, at least about 40%, at least about 50%, at least about 60%, and at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions can then be compared. When a position in the first sequence can be occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” can beWSGR Docket Number: 64828-710.601 equivalent to amino acid or nucleic acid “homology”). A nanopore described herein may comprise one or more components. The one or more components may be of a family of binary toxin, or a mutant thereof, or a functional homolog thereof, or a functional ortholog thereof, or a functional paralog thereof. “Homologs” can refer to proteins, peptides, oligopeptides, polypeptides having amino acid substitutions, deletions, or insertions, or any combination thereof relative to an unmodified (e.g., wild-type) protein and having similar biological and / or functional activity as the unmodified protein from which they are derived. “Ortholog” can refer to a gene or protein from different organisms (e.g., different species) that are derived from a common ancestral gene. “Paralog” can refer to a gene or protein from the same organism (e.g., same species) that can be a product of gene duplication of a common ancestral gene.

[0281] The percent identity between the two sequences may be a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol.48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A set of parameters (and the one that should be used unless otherwise specified) can be a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0282] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res.WSGR Docket Number: 64828-710.601 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0283] The term “amino acid” can embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Amino acids can include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing. As used herein the term “amino acid” can comprise both the D- or L- optical isomers and peptidomimetics.

[0284] A “conservative amino acid substitution” can be one in which the amino acid residue can be replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains can include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), or aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), or any combination thereof.

[0285] As used herein, the term “mutation” can refer to an alteration in the nucleotide sequence of the genome of an organism, virus, or extrachromosomal DNA. In some embodiments, the mutation may be a large-scale mutation, such as amplifications (or gene duplications) or repetitions of a chromosomal segment, deletions of large chromosomal regions, chromosomal rearrangements (e.g., chromosomal translocations, chromosomal inversions, non-homologous chromosomal crossover, and interstitial deletions), and loss of heterozygosity. In some embodiments, the mutation may be a small-scale mutation, such as insertions, deletions, and substitution mutations. As used herein, the term “substitution mutation” can refer to the transition that exchange a single nucleotide for another. A mutation herein may comprise a chemical conjugation to a non-natural amino acid.

[0286] In one approach to single-molecule analyte sequencing, analytes may be unfolded and translocated through a nanopore. In some cases, analyte domains may be recognized, a complex and inconsistent current signal obtained from measurement of an analyte-translocase complex and arising from the unfolding process that the analyte undergoes in the system may prevent the recognition of protein, polypeptides, or peptide sequences or subtle characteristics thereof. In another approach, analytes might be cleaved at specific sites and nanopore currents may be used to identify the released polypeptide or peptides. Therefore, the present disclosure may be aimed at designing and engineering new, analyte-based (e.g., protein-based) nanopores and nanopore systems that may be capable (for example, as part of a multi-protein sensor complex) of unfolding proteins, controlling processive and unidirectional transit across the nanopore, and producing highly consistent and reproducible signals that enable the analytes to be accurately characterized. Thus, the properties of a sampleWSGR Docket Number: 64828-710.601 containing a plurality of proteins (including complex mixtures of different protein types) may be accurately determined.

[0287] The detection of analytes and the sequencing of DNA using biological nanopores has seen major advances over recent years. There remains a long-felt need for the detection and sequencing of proteins with nanopores. However, this may be complicated by the complex physio-chemical structure of polypeptides or proteins, and / or the lack of understanding of the mechanism of capture and recognition of polypeptides by nanopores. In fact, it can be challenging to measure and / or analyze one or more analytes from a real complex sample or small sample volumes, and to do so with high speed, a high degree of accuracy or sensitivity, and / or capability for real time analysis. To achieve this, requires a combination of capable measurement and data processing methods and systems disclosed herein. As an example, the alignment methods disclosed herein can work incredibly well because the movement control is achieved to yield consistent high quality data signals.

[0288] Importantly, the ability to accurately characterize peptides / polypeptides / proteins (or the properties of the samples from which they are derived) can be highly dependent on the consistency (e.g., between runs, systems, nanopores, membranes) and quality of the signal, which in turn depends on the translocation properties as the molecules move through the nanopore. The methods and systems disclosed herein allow for translocation movement of single reads of non-nucleic acid based polymer analytes (e.g., one or more peptides, polypeptides, or proteins) through nanopores with high quality movement characteristics from which the step size can be obtained from reads.

[0289] One of the goals of the present invention is to improve the accuracy of characterizing individually captured peptides by a nanopore sensor. To that end, engineered proteinaceous nanopores can be developed to improve the capture of unlabeled peptides and / or labeled analytes (e.g., unlabeled or labeled non-nucleic acid based polymer analytes), to increase a residence (dwell time) of analytes in a nanopore sensor, and to improve the discrimination between analyte species. The present disclosure provides engineered nanopores to improve analyte sensing under about physiological conditions as well as at low pH conditions that may be optimized for analyte detection.

[0290] It was previously not thought possible to push / feed analytes into pores from, for example, the cis side in their native form (e.g. without attaching or conjugating to DNA leaders or adding other (e.g. polyanion) tags to create electrophoretic capture motifs) due to their complex composition. The diverse charge can result in the unfolded peptides being sometimes attracted and / or repelled from a nanopore depending on charge and applied voltage. Thus, it was not possible to translocate a diverse repertoire of complex peptides or polypeptides through nanopores by electrophoretic mechanisms alone. Indeed, previous studies demonstrated translocation of either very short peptides with a contour length shorter than the length of the nanopore channel or of very carefully selected (model) analytes, whose charge, structure or added electrophoretic tags favor capture and translocationWSGR Docket Number: 64828-710.601 through nanopores by electrophoresis. However, in no way is this representative for the broad amino acid composition of proteins that are found in nature.

[0291] The present disclosure provides novel approaches that may be simple and / or provide robust mechanisms of feeding non-nucleic acid based polymer analyte (e.g., polypeptides, peptides, full-length proteins, or any combination thereof) through nanopores (e.g., for the purpose of sequencing and / or characterizing them). It was found that these goals can be achieved by using a large and dominant cis-to-trans electro-osmotic flow (EOF), generated by a large cis-to-trans excess of ions flowing through the nanopore, in conjunction with a translocase on the cis side of a nanopore that can controllably feed and pass a wide range of analytes from cis to trans through the nanopore against the direction of the electrophoretic forces (EPFs).

[0292] The present disclosure provides novel approaches that can yield highly reproducible signals from peptide / polypeptide / protein analytes. The present disclosure further demonstrates that the portions of the signals that result from translocation of peptide / polypeptide / protein analytes may be highly consistent between different measurements of the same type of analyte. The present disclosure further demonstrates that the high signal consistency and reproducibility between different measurements of single analyte molecules (e.g. from different instruments, different systems, different membranes and nanopores, different times, different samples, etc.) can enable signals from analyte molecules to be combined and compared in analytical bioinformatics methods disclosed herein. For example, the high reproducibility may enable forming databases that are generated from a plurality of the same type of molecule (where multiple reads of the same molecule improves the precision of the database information). The high reproducibility can mean molecules can be compared with high accuracy on a single molecule basis, either to each other or to an artificial reference signal that is generated by training from a plurality of training molecules. For example, the high reproducibility enables characteristics (e.g. identification, variants, modifications, length, speed, etc.) to be determined with high accuracy by comparison to reference information. High reproducibility may mean molecules can be combined into sets and analysed in aggregate (e.g. to achieve higher accuracy by averaging or to determine properties of a sample that require a plurality of molecules). The high reproducibility can mean that cleaner (less noisy and variable) signal data may be measured on a molecule by molecule basis, which can provide more detail of the analytes. For example, very small changes in the analytes (e.g. relative to an unchanged reference signal) can be detected with higher accuracy (e.g. the ability to measure many types of single point variants and larger motifs at high accuracy).

[0293] Furthermore, the present disclosure in combination with the analytical methods shown herein (including comparisons to references and databases) also can demonstrate that the EOF exerts a powerful force that stretches and / or pulls on the portions of the peptide / polypeptide / protein analyte within or near the nanopore while the analyte resides in the nanopore. An advantage of the EOF may be that this force also keeps the motorWSGR Docket Number: 64828-710.601 bound to the analyte held against the nanopore entrance during motor controlled analyte translocation. Another advantage of the EOF force acting on the portions of the peptide / polypeptide / protein in the nanopore may be improved signal quality, for example, resulting from more consistent signal molecule-to-molecule, and / or by limiting variable changes in current signal that can result from the random stochastic movements of the portion of the peptide / polypeptide / protein in the nanopore (e.g. crumpling, folding, wobbling, shifting up and down of portions of the peptide / polypeptide / protein in the nanopore). The present disclosure demonstrates that the EOF in the systems and methods of the invention can further improve the consistency and reproducibility of portions of the signal, enabling signals obtained from multiple sources (e.g. from different instruments, different systems, different membranes and nanopores, different times, different samples, or any combination thereof) to be combined and measured in aggregated analyses, and / or to be compared (e.g. to each other or to signals in databases generated from other analytes) to accurately determine characteristics of the analytes or samples of a plurality of analytes (including complex mixtures of different analytes).

[0294] Further, the EOF exerts a stretching force on the peptide / polypeptide / protein that can reduce the length of the portion of the peptide / polypeptide / protein that is in the “reader” region of the nanopore (for example the one or more constrictions of the nanopore that give rise to the majority of the changes in current). The reduction in the portion within the reader can have several benefits, including simplifying the signal, increasing the magnitude of current ranges as the analyte moves, provides higher resolution of closely separated features along the analyte, and / or simplifies the analysis of the sequence-to-signal (due to reduced sequence contributing to the signal).

[0295] The present disclosure provides novel approaches that yield highly reproducible signals from one or more analytes (e.g., one or more peptides, polypeptides, proteins, or any combinations thereof). The present disclosure further demonstrates that the portions of the signals that can result from translocation of non-nucleic acid based polymer analytes can be highly consistent between different measurements of the same type of analyte. The present disclosure demonstrates that the high signal consistency and reproducibility between different measurements of single analyte molecules (e.g. from different instruments, different systems, different membranes and nanopores, different times, different samples, or any combinations thereof) can enable signals from analyte molecules to be combined and / or compared in analytical bioinformatics methods disclosed herein. For example, the high reproducibility enables forming databases that can be generated from a plurality of the same type of molecule (e.g., where multiple reads of the same molecule can improve the precision of the database information). The high reproducibility can demonstrate that molecules can be compared with high accuracy on a single molecule basis, to each other and / or to an artificial reference signal that can be generated by training from a plurality of training molecules. For example, the high reproducibility can enable one or more characteristics (e.g. identification, variants, modifications, length, speed, or any combinations thereof) to beWSGR Docket Number: 64828-710.601 determined with high accuracy by comparison to reference information. High reproducibility means molecules can be combined into sets and analyzed in aggregate. For example, one or more analytes may be combined and / or analyzed in aggregate to achieve higher accuracy by averaging and / or to determine properties of a sample that require a plurality of analytes. The high reproducibility can mean that cleaner (e.g., less noisy and variable) signal data may be measured on a molecule by molecule basis, which can provide more detail of the analytes. For example, enabling very small changes in the analytes (e.g. relative to an unchanged reference signal) may be detected with higher accuracy (e.g. the ability to measure many types of single point variants and larger motifs at high accuracy).

[0296] Furthermore, the present disclosure in combination with the analytical methods shown herein (for example, including comparisons to references and databases) can also demonstrate that the electro-osmotic force (EOF) can exert a powerful force that stretches and pulls on the portions of the one or more analytes (e.g., one or more non-nucleic acid based polymer analytes) within or near a nanopore while the one or more analytes reside in the nanopore. An advantage of the EOF is that this force can also keep a motor protein as described herein bound to the analyte held against the nanopore entrance during motor controlled analyte translocation. An advantage of the EOF force acting on the portions of the one or more analytes (e.g., one or more non-nucleic acid based polymer analytes) in the nanopore can be improved signal quality. The improved signal quality may result from more consistent signal molecule-to-molecule, for example by limiting variable changes in current signal that can result from random stochastic movements of the portion of the one or more non-nucleic acid based polymer analytes in the nanopore. Random stochastic movements of the one or more analytes may comprise crumpling, folding, wobbling, shifting up and down, or any combination thereof of portions of the one or more analytes in the nanopore. The present disclosure demonstrates that the EOF in the systems and methods of the invention can further improve the consistency and reproducibility of portions of the signal. This may enable signals obtained from multiple sources (e.g. from different instruments, different systems, different membranes and nanopores, different times, different samples, or any combinations thereof) to be combined and / or measured in aggregated analyses, and may be compared to each other and / or to signals in one or more databases generated from other analytes. These comparisons may accurately determine characteristics of the analytes and / or samples of a plurality of analytes (e.g. including complex mixtures of different analytes).

[0297] Further, the EOF can exert a stretching force on the one or more non-nucleic acid based polymer analytes that may reduce the length of the portion of the one or more non-nucleic acid based polymer analytes that may be in the “reader” region of the nanopore. The “reader” region can comprise one or more constrictions (e.g., constriction regions) of a nanopore that may give rise to one or more changes in current. The reduction in the portion within the constriction regions can have several benefits, including, but not limited to: (i) simplifying a signal, (ii) increasing a magnitude of current ranges as one or more analytes move, (iii) providing higherWSGR Docket Number: 64828-710.601 resolution of closely separated features along the one or more analytes, (iv) simplifying an analysis of the sequence-to-signal (e.g., due to reduced sequence contributing to the signal), or (v) any combination thereof.

[0298] In some aspects, the present disclosure provides a membrane comprising a pore. A pore can be inserted into a membrane such as a lipid bilayer. The pore can be a biological pore. A pore can be engineered to bind with specific analytes. In some embodiments, a pore can be engineered to bind with a class of analytes (e.g., a peptide). In some embodiments, a pore can be engineered to not to bind with a class of analytes. In some embodiments, a pore can be engineered to permit certain ionic species to traverse through the pore. In some embodiments, a pore can be engineered to prevent certain ionic species from traversing through the pore. Various design aspects of pore and systems and methods for achieving those aspects are disclosed herein.

[0299] In some aspects, the present disclosure provides a sensor array. The array of sensors can comprise two or more sensors. The array of sensors can comprise at least about 1, 2, 3, 4, 8, 16, 32, 64, 96, 100, 500, 1000, 2000, or greater than about 2000 sensors. The array of sensors can comprise, at most about 2000, 1000, 500, 100, 96, 64, 32, 16, 8, 4, 3, 2, 1, or less than about 1 sensor. In some embodiments, an array of sensors can comprise between about 1 sensor to about 1,000 sensors. In some embodiments, an array of sensors can comprise between about 1 sensor to about 2 sensors, about 1 sensor to about 3 sensors, about 1 sensor to about 4 sensors, about 1 sensor to about 8 sensors, about 1 sensor to about 16 sensors, about 1 sensor to about 32 sensors, about 1 sensor to about 64 sensors, about 1 sensor to about 96 sensors, about 1 sensor to about 400 sensors, about 1 sensor to about 800 sensors, about 1 sensor to about 1,000 sensors, about 2 sensors to about 3 sensors, about 2 sensors to about 4 sensors, about 2 sensors to about 8 sensors, about 2 sensors to about 16 sensors, about 2 sensors to about 32 sensors, about 2 sensors to about 64 sensors, about 2 sensors to about 96 sensors, about 2 sensors to about 400 sensors, about 2 sensors to about 800 sensors, about 2 sensors to about 1,000 sensors, about 3 sensors to about 4 sensors, about 3 sensors to about 8 sensors, about 3 sensors to about 16 sensors, about 3 sensors to about 32 sensors, about 3 sensors to about 64 sensors, about 3 sensors to about 96 sensors, about 3 sensors to about 400 sensors, about 3 sensors to about 800 sensors, about 3 sensors to about 1,000 sensors, about 4 sensors to about 8 sensors, about 4 sensors to about 16 sensors, about 4 sensors to about 32 sensors, about 4 sensors to about 64 sensors, about 4 sensors to about 96 sensors, about 4 sensors to about 400 sensors, about 4 sensors to about 800 sensors, about 4 sensors to about 1,000 sensors, about 8 sensors to about 16 sensors, about 8 sensors to about 32 sensors, about 8 sensors to about 64 sensors, about 8 sensors to about 96 sensors, about 8 sensors to about 400 sensors, about 8 sensors to about 800 sensors, about 8 sensors to about 1,000 sensors, about 16 sensors to about 32 sensors, about 16 sensors to about 64 sensors, about 16 sensors to about 96 sensors, about 16 sensors to about 400 sensors, about 16 sensors to about 800 sensors, about 16 sensors to about 1,000 sensors, about 32 sensors to about 64 sensors, about 32 sensors to about 96 sensors, about 32 sensors to about 400 sensors, about 32 sensors to about 800 sensors, about 32 sensors to about 1,000WSGR Docket Number: 64828-710.601 sensors, about 64 sensors to about 96 sensors, about 64 sensors to about 400 sensors, about 64 sensors to about 800 sensors, about 64 sensors to about 1,000 sensors, about 96 sensors to about 400 sensors, about 96 sensors to about 800 sensors, about 96 sensors to about 1,000 sensors, about 400 sensors to about 800 sensors, about 400 sensors to about 1,000 sensors, or about 800 sensors to about 1,000 sensors. The array of sensors can comprise the same membrane, the same pore, and the same electrolyte conditions. At least two sensors in the array of sensors can comprise a different membrane, a different pore, and / or different electrolyte conditions. The array of sensors can provide signals in parallel, which can increase the throughput of analyte detection and / or identification. Sensors in the array may each analyze the same sample. In some embodiments, one fraction of sensors in the array can analyze one sample, and another fraction of sensors in the array can analyze a different sample. Various configurations and embodiments for arrays of sensors are provided herein.

[0300] In some aspects, the present disclosure provides a device. The device can comprise a sensor or an array of sensors. The device can comprise an electrical energy source and two electrodes. One of the two electrodes may be disposed on one side of the membrane of a sensor, and another electrode may be disposed on the other side. The electrical energy source can apply a potential between the two electrodes, which can cause ions in an electrolyte to conduct through the fluid, and through the pore of the sensor. The potential can also cause an analyte, if charged, to translocate through the pore. The potential can create an electrophoretic force (EPF), described further in detail elsewhere in this application, which can provide a driving force for an analyte to translocate through the pore in order to generate a change in signal. The device may further comprise two or more additional electrodes. For example, these electrodes can be configured to measure the electrical potential across the nanopore and / or membrane that changes when an analyte translocates through a pore. These electrodes can be configured to measure the current across a membrane as an analyte translocates through a pore. The device can be in electrical communication with a recording device to record measured signals. The device can be in electrical communication with a computer or a processor (e.g., a circuit, or an integrated circuit, or any combination thereof), which can receive a signal from the sensor or the array of sensors, store the signals in digital form, and / or process the signal. The device can comprise a flow cell. The flow cell can comprise or be fluidically coupled to the sensor or the sensor array. The sensor or the sensor array can be integrated with the flow cell into a single piece, or they can be separate. The device can comprise or be fluidically coupled to a fluidic control system (e.g., pumps, or controllers, or any combination thereof). In some embodiments, the fluidic control system may comprise a pump, a pressure based flow controller, a pressurized reservoir, a pressure sensor, a vacuum control system, one or more valves, a bubble trap, or fluidic channels that can generate a capillary-force, or combinations thereof. In some embodiments, a pump may be a syringe pump, a peristaltic pump, or piezoelectric pump. The device can be a handheld device or a tabletop device. The device can be configured to detect a single analytes (e.g., chemical species) (e.g., detecting the presence of a particularWSGR Docket Number: 64828-710.601 pathogen like coronavirus). The device can be configured to detect a variety of analytes (e.g., chemical species). The device can be configured to identify any analytes (e.g., chemical species) in a sample. Various forms of devices and methods of using devices are disclosed herein.

[0301] In some aspects, the nanopores, methods, and system provided herein comprise detecting and / or characterizing one or more characteristics of an analyte. Characteristics of the analyte (e.g., the non-nucleic acid based polymer analyte) may comprise a shape of the non-nucleic acid based polymer analyte, a structure of the non-nucleic acid based polymer analyte, one or more mutations of the non-nucleic acid based polymer analyte, a sequence of the non-nucleic acid polymer analyte, a surface charge of the non-nucleic acid based polymer analyte, one or more post-translation modifications of the non-nucleic acid based polymer analyte, or one or more ligands coupled to the non-nucleic acid based polymer analyte, or any combination thereof.

[0302] In some aspects, the present disclosure provides methods for processing signals. The methods can be implemented on a computer. The methods can be written as a set of instructions, which can be stored in a non- transitory storage medium. The methods can be executable by a computer processor. The methods and algorithms can be configured to store or process one or more signals and determine one or more identifications and / or characteristics of analytes associated with the one or more signals. A computer or processor implementing the methods can be in electrical (wireless or wired) communication with the device. Various methods for processing signals to identify analytes are disclosed herein.

[0303] In some aspects, provided herein are methods for translocating an analyte (e.g., a non-nucleic acid based polymer analyte). In some embodiments, the methods may comprise translocating one or more analytes (e.g., a plurality of analytes, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more analytes). The analyte may be translocated through a pore described herein. The pore (e.g., nanopore) may be disposed within a membrane. The pore may be part of a nanopore system described herein. In some embodiments, at least a portion of an analyte (e.g., a non-nucleic acid based polymer analyte) may be translocated through a pore. The at least a portion of the analyte may comprise at least a portion of a protein, at least a portion of a polypeptide, at least a portion of a peptide, or any combination thereof. The terms “polypeptide” and “peptide” generally to refer to a polymer of amino acids in which an amino acid may be linked to another amino acid by a peptide bond. In some examples, a polypeptide is a protein. The amino acid may be a naturally occurring amino acid or a non-naturally occurring amino acid (i.e., amino acid analogue). The polymer can be linear or branched and can include modified amino acids, and / or may be interrupted by non-amino acids. Polypeptides can occur as single chains or associated chains. The polymer may include a plurality of amino acids and may have a secondary and tertiary structure (i.e., protein). In some examples, the polymer comprises at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1000, at least aboutWSGR Docket Number: 64828-710.601 10,000, or more amino acids. In some embodiments, the at least a portion of the analyte translocated through a pore may comprise at least a portion of an analyte described herein.

[0304] A method may comprise detecting a current or change thereof. A method may comprise detecting a voltage or change thereof. In some embodiments, a signal or change thereof can comprise a measure of an ionic current, voltage, or any combination thereof. The method may comprise detecting a signal or change thereof. In some embodiments, the method may comprise detecting a current or change thereof while there is no analyte in a pore described herein (e.g., the pore can comprise an open pore). In some embodiments, the method may comprise detecting a current or change thereof while at least a portion of an analyte translocates through a pore. In some embodiments, the method may comprise detecting a current or change thereof while at least a portion of an analyte (e.g., one or more analytes) resides in a pore. The method may comprise using a current or change thereof. The method may comprise using a voltage or change thereof. In some embodiments, the method may comprise using a current or change thereof to identify one or more characteristics of an analyte (e.g., a non- nucleic acid based polymer analyte). In some embodiments, the method may comprise using a current or change thereof to determine one or more characteristics of an analyte (e.g., a non-nucleic acid based polymer analyte). Determining a characteristic can comprise measuring a characteristic of an analyte, or quantitating a characteristic of an analyte, or any combination thereof. In some embodiments, the current or change thereof and / or voltage or change thereof may be used to identify a plurality of characteristics (e.g., at least about 1, 2, 3, 4, 5, 10, or more characteristics). In some embodiments, the current or change thereof and / or voltage or change thereof may be used to determine a plurality of characteristics (e.g., at least about 1, 2, 3, 4, 5, 10, or more characteristics). The characteristics of at least a portion of the analyte may comprise characteristics of an analyte described herein (e.g., a shape of the analyte, a structure of the analyte, one or more mutations of the analyte, a sequence of the non-nucleic acid polymer analyte, a surface charge of the analyte, one or more post- translation modifications of the analyte, or one or more ligands coupled to the analyte, or any combination thereof). In some embodiments, the current or change thereof may be used to identify one or more characteristics of an analyte with an accuracy. The accuracy of identifying the one or more characteristics of the analyte may be at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. The accuracy of identifying the one or more characteristics of the analyte may be at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, or less than about 20%. In some embodiments, the accuracy of identifying the one or more characteristics of the analyte may be between about 20% to about 100%. In some embodiments, the accuracy of identifying the one or more characteristics of the analyte may be between about 20% to about 30%, about 20% to about 40%, about 20% toWSGR Docket Number: 64828-710.601 about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 85%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 85%, about 30% to about 90%, about 30% to about 95%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 40% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 100%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 85% to about 90%, about 85% to about 95%, about 85% to about 100%, about 90% to about 95%, about 90% to about 100%, or about 95% to about 100%.

[0305] As an example, a method provided herein can comprise: (a) translocating at least a portion of an analyte through a nanopore disposed within a mem-brane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting (1) a current or change thereof; or (2) voltage or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%.

[0306] In some embodiments, translocating of at least a portion of an analyte can comprise translocating in a C-terminus to N-terminus (C-to-N) direction, or a N-terminus to C-terminus (N-to-C) direction, or any combination thereof. The C-to-N and / or N-to-C directions can be relative to at least a portion of the analyte sequence. Determining one or more characteristics can comprise using the current or change thereof and / or the voltage or change thereof associated with a C-to-N direction, N-to-C direction, or any combination thereof. Determining one or more characteristics can comprise using an electrical signal or change thereof associated with a C-to-N direction, or N-to-C direction, or any combination thereof.

[0307] In some aspects, provided herein are methods for determining one or more characteristics of an analyte (e.g., a non-nucleic acid based polymer analyte). The method may comprise translocating at least a portion of an analyte. The method may comprise translocating one or more analytes (e.g., a plurality of analytes, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more analytes). The analyte may be translocated through a pore described herein (e.g., a nanopore disposed within a membrane). The pore may be part of a nanopore system described herein. In some embodiments, at least a portion of analyte (e.g., at least a portion of a protein, at least a portionWSGR Docket Number: 64828-710.601 of a polypeptide, at least a portion of a peptide, or a combination thereof) may be translocated through a pore at a rate. The rate can comprise an average rate of translocation through the pore. The average rate of translocation may comprise an average of an analyte (e.g., a non-nucleic acid based polymer analyte) translocating through a pore one or more times. The average rate of translocation may comprise an average of two or more analytes translocating through a pore one or more times. A rate of translocation of an analyte through a pore may be expressed as amino acids per second (amino acids / sec) and / or nanometers / sec (nm / s). In some embodiments, a rate of translocation (e.g., an average rate of translocation) may comprise translocation of an analyte with a motor protein (e.g., a translocase) or without a motor protein.

[0308] In some embodiments, an average rate of translocation can comprise at least about 0.1 amino acids / sec, at least about 0.5 amino acids / sec, at least about 0.6 amino acids / sec, at least about 0.7 amino acids / sec, at least about 0.8 amino acids / sec, at least about 0.9 amino acids / sec, at least about 1 amino acid / sec, at least about 2 amino acids / sec, at least about 3 amino acids / sec, at least about 4 amino acids / sec, at least about 5 amino acids / sec, at least about 6 amino acids / sec, at least about 7 amino acids / sec, at least about 8 amino acids / sec, at least about 9 amino acids / sec, at least about 10 amino acids / sec, at least about 11 amino acids / sec, at least about 12 amino acids / sec, at least about 13 amino acids / sec, at least about 14 amino acids / sec, at least about 15 amino acids / sec, at least about 16 amino acids / sec, at least about 17 amino acids / sec, at least about 18 amino acids / sec, at least about 19 amino acids / sec, at least about 20 amino acids / sec, at least about 30 amino acids / sec, at least about 40 amino acids / sec, at least about 50 amino acids / sec, at least about 60 amino acids / sec, at least about 70 amino acids / sec, at least about 80 amino acids / sec, at least about 90 amino acids / sec, at least about 100 amino acids / sec, at least about 200 amino acids / sec, at least about 300 amino acids / sec, at least about 400 amino acids / sec, at least about 500 amino acids / sec, at least about 600 amino acids / sec, at least about 700 amino acids / sec, at least about 800 amino acids / sec, at least about 900 amino acids / sec, at least about 1000 amino acids / sec, at least about 5000 amino acids / sec, at least about 10000 amino acids / sec, at least about 15000 amino acids / sec, at least about 20000 amino acids / sec, at least about 25000 amino acids / sec, at least about 30000 amino acids / sec, at least about 35000 amino acids / sec, at least about 40000 amino acids / sec, at least about 45000 amino acids / sec, at least about 50000 amino acids / sec, or greater than about 50000 amino acids / sec. In some embodiments, an average rate of translocation can comprise at most about 50000 amino acids / sec, at most about 45000 amino acids / sec, at most about 40000 amino acids / sec, at most about 35000 amino acids / sec, at most about 30000 amino acids / sec, at most about 25000 amino acids / sec, at most about 20000 amino acids / sec, at most about 15000 amino acids / sec, at most about 10000 amino acids / sec, at most about 5000 amino acids / sec, at most about 1000 amino acids / sec, at most about 900 amino acids / sec, at most about 800 amino acids / sec, at most about 700 amino acids / sec, at most about 600 amino acids / sec, at most about 500 amino acids / sec, at most about 400 amino acids / sec, at most about 300 amino acids / sec, at most about 200 amino acids / sec, at most aboutWSGR Docket Number: 64828-710.601 100 amino acids / sec, at most about 90 amino acids / sec, at most about 80 amino acids / sec, at most about 70 amino acids / sec, at most about 60 amino acids / sec, at most about 50 amino acids / sec, at most about 40 amino acids / sec, at most about 30 amino acids / sec, at most about 20 amino acids / sec, at most about 19 amino acids / sec, at most about 18 amino acids / sec, at most about 17 amino acids / sec, at most about 16 amino acids / sec, at most about 15 amino acids / sec, at most about 14 amino acids / sec, at most about 13 amino acids / sec, at most about 12 amino acids / sec, at most about 11 amino acids / sec, at most about 10 amino acids / sec, at most about 9 amino acids / sec, at most about 8 amino acids / sec, at most about 7 amino acids / sec, at most about 6 amino acids / sec, at most about 5 amino acids / sec, at most about 4 amino acids / sec, at most about 3 amino acids / sec, at most about 2 amino acids / sec, at most about 1 amino acid / sec, at most about 0.9 amino acids / sec, at most about 0.8 amino acids / sec, at most about 0.7 amino acids / sec, at most about 0.6 amino acids / sec, at most about 0.5 amino acids / sec, at most about 0.1 amino acids / sec, or less than about 0.1 amino acids / sec.

[0309] In some embodiments, an average rate of translocation can be between about 0.1 amino acids / sec to about 100 amino acids / sec. In some embodiments, an average rate of translocation can comprise between about 0.1 amino acids / sec to about 0.5 amino acids / sec, about 0.1 amino acids / sec to about 1 amino acid / sec, about 0.1 amino acids / sec to about 10 amino acids / sec, about 0.1 amino acids / sec to about 20 amino acids / sec, about 0.1 amino acids / sec to about 30 amino acids / sec, about 0.1 amino acids / sec to about 40 amino acids / sec, about 0.1 amino acids / sec to about 50 amino acids / sec, about 0.1 amino acids / sec to about 60 amino acids / sec, about 0.1 amino acids / sec to about 70 amino acids / sec, about 0.1 amino acids / sec to about 80 amino acids / sec, about 0.1 amino acids / sec to about 100 amino acids / sec, about 0.5 amino acids / sec to about 1 amino acid / sec, about 0.5 amino acids / sec to about 10 amino acids / sec, about 0.5 amino acids / sec to about 20 amino acids / sec, about 0.5 amino acids / sec to about 30 amino acids / sec, about 0.5 amino acids / sec to about 40 amino acids / sec, about 0.5 amino acids / sec to about 50 amino acids / sec, about 0.5 amino acids / sec to about 60 amino acids / sec, about 0.5 amino acids / sec to about 70 amino acids / sec, about 0.5 amino acids / sec to about 80 amino acids / sec, about 0.5 amino acids / sec to about 100 amino acids / sec, about 1 amino acid / sec to about 10 amino acids / sec, about 1 amino acid / sec to about 20 amino acids / sec, about 1 amino acid / sec to about 30 amino acids / sec, about 1 amino acid / sec to about 40 amino acids / sec, about 1 amino acid / sec to about 50 amino acids / sec, about 1 amino acid / sec to about 60 amino acids / sec, about 1 amino acid / sec to about 70 amino acids / sec, about 1 amino acid / sec to about 80 amino acids / sec, about 1 amino acid / sec to about 100 amino acids / sec, about 10 amino acids / sec to about 20 amino acids / sec, about 10 amino acids / sec to about 30 amino acids / sec, about 10 amino acids / sec to about 40 amino acids / sec, about 10 amino acids / sec to about 50 amino acids / sec, about 10 amino acids / sec to about 60 amino acids / sec, about 10 amino acids / sec to about 70 amino acids / sec, about 10 amino acids / sec to about 80 amino acids / sec, about 10 amino acids / sec to about 100 amino acids / sec, about 20 amino acids / sec to about 30 amino acids / sec, about 20 amino acids / sec to about 40 amino acids / sec, about 20 aminoWSGR Docket Number: 64828-710.601 acids / sec to about 50 amino acids / sec, about 20 amino acids / sec to about 60 amino acids / sec, about 20 amino acids / sec to about 70 amino acids / sec, about 20 amino acids / sec to about 80 amino acids / sec, about 20 amino acids / sec to about 100 amino acids / sec, about 30 amino acids / sec to about 40 amino acids / sec, about 30 amino acids / sec to about 50 amino acids / sec, about 30 amino acids / sec to about 60 amino acids / sec, about 30 amino acids / sec to about 70 amino acids / sec, about 30 amino acids / sec to about 80 amino acids / sec, about 30 amino acids / sec to about 100 amino acids / sec, about 40 amino acids / sec to about 50 amino acids / sec, about 40 amino acids / sec to about 60 amino acids / sec, about 40 amino acids / sec to about 70 amino acids / sec, about 40 amino acids / sec to about 80 amino acids / sec, about 40 amino acids / sec to about 100 amino acids / sec, about 50 amino acids / sec to about 60 amino acids / sec, about 50 amino acids / sec to about 70 amino acids / sec, about 50 amino acids / sec to about 80 amino acids / sec, about 50 amino acids / sec to about 100 amino acids / sec, about 60 amino acids / sec to about 70 amino acids / sec, about 60 amino acids / sec to about 80 amino acids / sec, about 60 amino acids / sec to about 100 amino acids / sec, about 70 amino acids / sec to about 80 amino acids / sec, about 70 amino acids / sec to about 100 amino acids / sec, or about 80 amino acids / sec to about 100 amino acids / sec.

[0310] In some embodiments, an average rate of translocation can be between about 100 amino acids / sec to about 50,000 amino acids / sec. In some embodiments, an average rate of translocation can comprise between about 100 amino acids / sec to about 500 amino acids / sec, about 100 amino acids / sec to about 1,000 amino acids / sec, about 100 amino acids / sec to about 5,000 amino acids / sec, about 100 amino acids / sec to about 10,000 amino acids / sec, about 100 amino acids / sec to about 15,000 amino acids / sec, about 100 amino acids / sec to about 20,000 amino acids / sec, about 100 amino acids / sec to about 25,000 amino acids / sec, about 100 amino acids / sec to about 30,000 amino acids / sec, about 100 amino acids / sec to about 40,000 amino acids / sec, about 100 amino acids / sec to about 50,000 amino acids / sec, about 500 amino acids / sec to about 1,000 amino acids / sec, about 500 amino acids / sec to about 5,000 amino acids / sec, about 500 amino acids / sec to about 10,000 amino acids / sec, about 500 amino acids / sec to about 15,000 amino acids / sec, about 500 amino acids / sec to about 20,000 amino acids / sec, about 500 amino acids / sec to about 25,000 amino acids / sec, about 500 amino acids / sec to about 30,000 amino acids / sec, about 500 amino acids / sec to about 40,000 amino acids / sec, about 500 amino acids / sec to about 50,000 amino acids / sec, about 1,000 amino acids / sec to about 5,000 amino acids / sec, about 1,000 amino acids / sec to about 10,000 amino acids / sec, about 1,000 amino acids / sec to about 15,000 amino acids / sec, about 1,000 amino acids / sec to about 20,000 amino acids / sec, about 1,000 amino acids / sec to about 25,000 amino acids / sec, about 1,000 amino acids / sec to about 30,000 amino acids / sec, about 1,000 amino acids / sec to about 40,000 amino acids / sec, about 1,000 amino acids / sec to about 50,000 amino acids / sec, about 5,000 amino acids / sec to about 10,000 amino acids / sec, about 5,000 amino acids / sec to about 15,000 amino acids / sec, about 5,000 amino acids / sec to about 20,000 amino acids / sec, about 5,000 amino acids / sec to about 25,000 amino acids / sec, about 5,000 amino acids / sec to about 30,000 amino acids / sec, about 5,000 aminoWSGR Docket Number: 64828-710.601 acids / sec to about 40,000 amino acids / sec, about 5,000 amino acids / sec to about 50,000 amino acids / sec, about 10,000 amino acids / sec to about 15,000 amino acids / sec, about 10,000 amino acids / sec to about 20,000 amino acids / sec, about 10,000 amino acids / sec to about 25,000 amino acids / sec, about 10,000 amino acids / sec to about 30,000 amino acids / sec, about 10,000 amino acids / sec to about 40,000 amino acids / sec, about 10,000 amino acids / sec to about 50,000 amino acids / sec, about 15,000 amino acids / sec to about 20,000 amino acids / sec, about 15,000 amino acids / sec to about 25,000 amino acids / sec, about 15,000 amino acids / sec to about 30,000 amino acids / sec, about 15,000 amino acids / sec to about 40,000 amino acids / sec, about 15,000 amino acids / sec to about 50,000 amino acids / sec, about 20,000 amino acids / sec to about 25,000 amino acids / sec, about 20,000 amino acids / sec to about 30,000 amino acids / sec, about 20,000 amino acids / sec to about 40,000 amino acids / sec, about 20,000 amino acids / sec to about 50,000 amino acids / sec, about 25,000 amino acids / sec to about 30,000 amino acids / sec, about 25,000 amino acids / sec to about 40,000 amino acids / sec, about 25,000 amino acids / sec to about 50,000 amino acids / sec, about 30,000 amino acids / sec to about 40,000 amino acids / sec, about 30,000 amino acids / sec to about 50,000 amino acids / sec, or about 40,000 amino acids / sec to about 50,000 amino acids / sec.

[0311] In some embodiments, an average rate of translocation can comprise at least about 0.01 nm / s, at least about 0.1 nm / s, at least about 0.5 nm / s, at least about 0.6 nm / s, at least about 0.7 nm / s, at least about 0.8 nm / s, at least about 0.9 nm / s, at least about 1 amino acid / sec, at least about 2 nm / s, at least about 3 nm / s, at least about 4 nm / s, at least about 5 nm / s, at least about 6 nm / s, at least about 7 nm / s, at least about 8 nm / s, at least about 9 nm / s, at least about 10 nm / s, at least about 11 nm / s, at least about 12 nm / s, at least about 13 nm / s, at least about 14 nm / s, at least about 15 nm / s, at least about 16 nm / s, at least about 17 nm / s, at least about 18 nm / s, at least about 19 nm / s, at least about 20 nm / s, at least about 30 nm / s, at least about 40 nm / s, at least about 50 nm / s, at least about 60 nm / s, at least about 70 nm / s, at least about 80 nm / s, at least about 90 nm / s, at least about 100 nm / s, at least about 200 nm / s, at least about 300 nm / s, at least about 400 nm / s, at least about 500 nm / s, at least about 600 nm / s, at least about 700 nm / s, at least about 800 nm / s, at least about 900 nm / s, at least about 1000 nm / s, at least about 5000 nm / s, at least about 10000 nm / s, at least about 15000 nm / s, at least about 20000 nm / s, or greater than about 20000 nm / s. In some embodiments, an average rate of translocation can comprise at most about 20000 nm / s, at most about 15000 nm / s, at most about 10000 nm / s, at most about 5000 nm / s, at most about 1000 nm / s, at most about 900 nm / s, at most about 800 nm / s, at most about 700 nm / s, at most about 600 nm / s, at most about 500 nm / s, at most about 400 nm / s, at most about 300 nm / s, at most about 200 nm / s, at most about 100 nm / s, at most about 90 nm / s, at most about 80 nm / s, at most about 70 nm / s, at most about 60 nm / s, at most about 50 nm / s, at most about 40 nm / s, at most about 30 nm / s, at most about 20 nm / s, at most about 19 nm / s, at most about 18 nm / s, at most about 17 nm / s, at most about 16 nm / s, at most about 15 nm / s, at most about 14 nm / s, at most about 13 nm / s, at most about 12 nm / s, at most about 11 nm / s, at most about 10 nm / s, at most about 9 nm / s, at most about 8 nm / s, at most about 7 nm / s, at most about 6 nm / s, at most about 5 nm / s, atWSGR Docket Number: 64828-710.601 most about 4 nm / s, at most about 3 nm / s, at most about 2 nm / s, at most about 1 amino acid / sec, at most about 0.9 nm / s, at most about 0.8 nm / s, at most about 0.7 nm / s, at most about 0.6 nm / s, at most about 0.5 nm / s, at most about 0.1 nm / s, at most about 0.01 nm / s, or less than about 0.01 nm / s.

[0312] In some embodiments, an average rate of translocation can be between about 0.1 nm / s to about 10000 nm / s. In some embodiments, an average rate of translocation can be between about 0.1 nm / s to about 100 nm / s. In some embodiments, an average rate of translocation can comprise between about 0.1 nm / s to about 0.5 nm / s, about 0.1 nm / s to about 1 amino acid / sec, about 0.1 nm / s to about 10 nm / s, about 0.1 nm / s to about 20 nm / s, about 0.1 nm / s to about 30 nm / s, about 0.1 nm / s to about 40 nm / s, about 0.1 nm / s to about 50 nm / s, about 0.1 nm / s to about 60 nm / s, about 0.1 nm / s to about 70 nm / s, about 0.1 nm / s to about 80 nm / s, about 0.1 nm / s to about 100 nm / s, about 0.5 nm / s to about 1 amino acid / sec, about 0.5 nm / s to about 10 nm / s, about 0.5 nm / s to about 20 nm / s, about 0.5 nm / s to about 30 nm / s, about 0.5 nm / s to about 40 nm / s, about 0.5 nm / s to about 50 nm / s, about 0.5 nm / s to about 60 nm / s, about 0.5 nm / s to about 70 nm / s, about 0.5 nm / s to about 80 nm / s, about 0.5 nm / s to about 100 nm / s, about 1 amino acid / sec to about 10 nm / s, about 1 amino acid / sec to about 20 nm / s, about 1 amino acid / sec to about 30 nm / s, about 1 amino acid / sec to about 40 nm / s, about 1 amino acid / sec to about 50 nm / s, about 1 amino acid / sec to about 60 nm / s, about 1 amino acid / sec to about 70 nm / s, about 1 amino acid / sec to about 80 nm / s, about 1 amino acid / sec to about 100 nm / s, about 10 nm / s to about 20 nm / s, about 10 nm / s to about 30 nm / s, about 10 nm / s to about 40 nm / s, about 10 nm / s to about 50 nm / s, about 10 nm / s to about 60 nm / s, about 10 nm / s to about 70 nm / s, about 10 nm / s to about 80 nm / s, about 10 nm / s to about 100 nm / s, about 20 nm / s to about 30 nm / s, about 20 nm / s to about 40 nm / s, about 20 nm / s to about 50 nm / s, about 20 nm / s to about 60 nm / s, about 20 nm / s to about 70 nm / s, about 20 nm / s to about 80 nm / s, about 20 nm / s to about 100 nm / s, about 30 nm / s to about 40 nm / s, about 30 nm / s to about 50 nm / s, about 30 nm / s to about 60 nm / s, about 30 nm / s to about 70 nm / s, about 30 nm / s to about 80 nm / s, about 30 nm / s to about 100 nm / s, about 40 nm / s to about 50 nm / s, about 40 nm / s to about 60 nm / s, about 40 nm / s to about 70 nm / s, about 40 nm / s to about 80 nm / s, about 40 nm / s to about 100 nm / s, about 50 nm / s to about 60 nm / s, about 50 nm / s to about 70 nm / s, about 50 nm / s to about 80 nm / s, about 50 nm / s to about 100 nm / s, about 60 nm / s to about 70 nm / s, about 60 nm / s to about 80 nm / s, about 60 nm / s to about 100 nm / s, about 70 nm / s to about 80 nm / s, about 70 nm / s to about 100 nm / s, or about 80 nm / s to about 100 nm / s.

[0313] In some embodiments, an average rate of translocation can comprise between about 100 nm / sec to about 20,000 nm / sec. In some embodiments, an average rate of translocation can comprise between about 100 nm / sec to about 200 nm / sec, about 100 nm / sec to about 300 nm / sec, about 100 nm / sec to about 400 nm / sec, about 100 nm / sec to about 500 nm / sec, about 100 nm / sec to about 1,000 nm / sec, about 100 nm / sec to about 2,500 nm / sec, about 100 nm / sec to about 5,000 nm / sec, about 100 nm / sec to about 7,500 nm / sec, about 100 nm / sec to about 10,000 nm / sec, about 100 nm / sec to about 15,000 nm / sec, about 100 nm / sec to about 20,000WSGR Docket Number: 64828-710.601 nm / sec, about 200 nm / sec to about 300 nm / sec, about 200 nm / sec to about 400 nm / sec, about 200 nm / sec to about 500 nm / sec, about 200 nm / sec to about 1,000 nm / sec, about 200 nm / sec to about 2,500 nm / sec, about 200 nm / sec to about 5,000 nm / sec, about 200 nm / sec to about 7,500 nm / sec, about 200 nm / sec to about 10,000 nm / sec, about 200 nm / sec to about 15,000 nm / sec, about 200 nm / sec to about 20,000 nm / sec, about 300 nm / sec to about 400 nm / sec, about 300 nm / sec to about 500 nm / sec, about 300 nm / sec to about 1,000 nm / sec, about 300 nm / sec to about 2,500 nm / sec, about 300 nm / sec to about 5,000 nm / sec, about 300 nm / sec to about 7,500 nm / sec, about 300 nm / sec to about 10,000 nm / sec, about 300 nm / sec to about 15,000 nm / sec, about 300 nm / sec to about 20,000 nm / sec, about 400 nm / sec to about 500 nm / sec, about 400 nm / sec to about 1,000 nm / sec, about 400 nm / sec to about 2,500 nm / sec, about 400 nm / sec to about 5,000 nm / sec, about 400 nm / sec to about 7,500 nm / sec, about 400 nm / sec to about 10,000 nm / sec, about 400 nm / sec to about 15,000 nm / sec, about 400 nm / sec to about 20,000 nm / sec, about 500 nm / sec to about 1,000 nm / sec, about 500 nm / sec to about 2,500 nm / sec, about 500 nm / sec to about 5,000 nm / sec, about 500 nm / sec to about 7,500 nm / sec, about 500 nm / sec to about 10,000 nm / sec, about 500 nm / sec to about 15,000 nm / sec, about 500 nm / sec to about 20,000 nm / sec, about 1,000 nm / sec to about 2,500 nm / sec, about 1,000 nm / sec to about 5,000 nm / sec, about 1,000 nm / sec to about 7,500 nm / sec, about 1,000 nm / sec to about 10,000 nm / sec, about 1,000 nm / sec to about 15,000 nm / sec, about 1,000 nm / sec to about 20,000 nm / sec, about 2,500 nm / sec to about 5,000 nm / sec, about 2,500 nm / sec to about 7,500 nm / sec, about 2,500 nm / sec to about 10,000 nm / sec, about 2,500 nm / sec to about 15,000 nm / sec, about 2,500 nm / sec to about 20,000 nm / sec, about 5,000 nm / sec to about 7,500 nm / sec, about 5,000 nm / sec to about 10,000 nm / sec, about 5,000 nm / sec to about 15,000 nm / sec, about 5,000 nm / sec to about 20,000 nm / sec, about 7,500 nm / sec to about 10,000 nm / sec, about 7,500 nm / sec to about 15,000 nm / sec, about 7,500 nm / sec to about 20,000 nm / sec, about 10,000 nm / sec to about 15,000 nm / sec, about 10,000 nm / sec to about 20,000 nm / sec, or about 15,000 nm / sec to about 20,000 nm / sec.

[0314] A method may comprise detecting a current or change thereof. In some embodiments, a current or change thereof may be detected while there is no analyte in a pore described herein (e.g., the pore can comprise an open pore). In some embodiments, a current or change thereof may be detected while at least a portion of an analyte translocates through a pore. In some embodiments, the method may comprise using a current or change thereof to determine one or more characteristics of an analyte (e.g., a non-nucleic acid based polymer analyte) and / or at least a portion of an analyte. In some embodiments, the current or change thereof may be used to determine a plurality of characteristics (e.g., at least about 1, 2, 3, 4, 5, 10, or more characteristics). The characteristics of at least a portion of the analyte may comprise characteristics of an analyte described herein.

[0315] As an example, provided herein is a method for determining a characteristic of an analyte comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a mem-brane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, atWSGR Docket Number: 64828-710.601 least a portion of a peptide, or a combination thereof, wherein (i) an average rate of translocation is between about 0.1 amino ac-ids per second to about 35000 amino acids per second or (ii) an average rate of translocation is between about 0.1 nm / s to about 10000 nm / s; (b) detecting (1) a current or change thereof, or (2) voltage or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte.

[0316] In some embodiments, the methods described herein may comprise translocating an additional analyte (e.g., an additional non-nucleic acid based polymer analyte) through a pore (e.g., a nanopore). The additional analyte may be translocated through a same pore as a first analyte. The additional analyte may be translocated through a different pore (e.g., an additional pore) as a first analyte. In some embodiments, an additional analyte comprises at least a portion of the additional analyte. The at least a portion of the additional analyte can comprise a non-nucleic acid based polymer analyte (e.g., at least a portion of an additional protein, at least a portion of an additional polypeptide, at least a portion of an additional peptide, or a combination thereof). In some embodiments, at least a portion of the additional analyte may translocate through a pore described herein. The additional analyte may translocate through a pore described herein with an average rate of translocation described herein. In some embodiments, an additional current or change thereof and / or an additional voltage or change thereof may be detected. In some embodiments, the additional current or change thereof and / or the additional voltage or change thereof may be detected while at least a portion of an additional analyte translocates through a pore. In some embodiments, the additional current or change thereof and / or the additional voltage or change thereof may be detected while at least a portion of an additional analyte resides in a pore (e.g., the additional analyte enters a pore from a first side and may not exit a pore on a second side). In some embodiments, the additional current or change thereof and / or the additional voltage or change thereof may be used to identify and / or determine one or more characteristics of at least a portion of the additional analyte. A characteristic of the additional analyte can comprise a shape of the additional analyte, a structure of the additional analyte, one or more mutations of the additional analyte, a sequence of the additional analyte, a surface charge of the additional analyte, one or more post-translation modifications of the additional analyte, or one or more ligands coupled to the additional analyte, or any combination thereof.

[0317] In some aspects, provided herein is a method for characterizing an analyte (e.g., a non-nucleic acid based polymer analyte). The analyte may be translocated through a pore described herein. The pore (e.g., nanopore) may be disposed within a membrane. The pore may be part of a nanopore system described herein. In some embodiments, at least a portion of an analyte (e.g., a non-nucleic acid based polymer analyte) may be translocated through a pore. The at least a portion of the analyte may comprise at least a portion of a protein, at least a portion of a polypeptide, at least a portion of a peptide, or any combination thereof. A signal or changeWSGR Docket Number: 64828-710.601 thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof) may be detected. In some embodiments, a signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof) may be detected while there is no analyte in a pore described herein (e.g., the pore can comprise an open pore). In some embodiments, a signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof) may be detected while at least a portion of an analyte translocates through a pore. In some embodiments, a signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof) may be detected while at least a portion of an analyte resides in a pore.

[0318] One or more characteristics may be assigned to at least a portion of an analyte (e.g., at least a portion of a non-nucleic acid based polymer analyte). The one or more characteristics may be assigned based on the signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof). The one or more characteristics may be assigned based on an electrical signal or change thereof, a database, or any combination thereof. The database may comprise one or more signals (e.g., reference signals). The reference signal can comprise a signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof) identified and / or determined for an analyte. In some embodiments, the reference signal may be for one or more analytes (e.g., one or more polypeptides, one or more proteins, or one or more peptides, or one or more fragments thereof, or one or more proteoforms thereof, or one or more variants thereof, or any combination thereof). In some embodiments, variants of the analyte (e.g., non-nucleic acid based polymer analyte) may comprise one or more post-translational modifications (PTMs) and / or one or more conjugations to the analyte (e.g., drug conjugate, barcode, polynucleotide, or leader construct, or any combination thereof). The one or more post-translational modifications may comprise naturally occurring PTMs, or non-naturally occurring PTMs, or any combination thereof.

[0319] As an example, provided herein is a method for characterizing an analyte comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting an electrical signal or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) assigning one or more characteristics to the at least the portion of the analyte based on the electrical signal and a database, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more fragments thereof, or one or more proteoforms thereof, or one or more variants thereof, or a combination thereof.

[0320] In some embodiments, assigning can comprise measuring one or more characteristics, or quantitating one or more characteristics, or any combination thereof. In some embodiments, the one or more characteristicsWSGR Docket Number: 64828-710.601 may be assigned by scoring a signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof). The signal or change thereof may be scored against the one or more reference signals and / or at least a portion of the one or more reference signals. In some embodiments, scoring may comprise aligning at least a portion of the signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof). In some embodiments, at least a portion of the signal or change thereof may be aligned with the one or more reference signals and / or at least a portion of the one or more reference signals. In some embodiments, the alignment may comprise soft alignment.

[0321] In some embodiments, the methods provided herein may be repeated to generate a plurality of signals or changes thereof (e.g., electrical signals or changes thereof, currents or changes thereof, or any combination thereof). For example, a plurality of analytes may translocate through a plurality of nanopores. The plurality of analytes may translocate through a plurality of nanopores disposed in membranes. As the plurality of analytes translocates through the plurality of nanopores, a plurality of signals (e.g., electrical signals, current signals, or any combination thereof) may be generated. The plurality of signals (e.g., electrical signals, current signals, or any combination thereof) may be detected. One or more characteristics may be assigned to the plurality of analytes (e.g., the plurality of non-nucleic acid based polymer analytes). The one or more characteristics may be assigned based on the plurality of signals (e.g., electrical signals, current signals, or any combination thereof). The one or more characteristics may be assigned based on the plurality of signals, a database, or any combination thereof.

[0322] In some aspects, provided herein is a method for sample analysis. A sample may be provided. In some embodiments, the sample can comprise an analyte. In some embodiments, the sample can comprise a plurality of analytes (e.g., a plurality of non-nucleic acid based polymer analytes). At least a subset of the plurality of analytes may undergo any of the methods and / or system disclosed herein. The plurality of analytes can comprise a first analyte and a second analyte. In some embodiments, the plurality of analytes may be translocated. The plurality of analytes may be translocated through a pore described herein (e.g., a pore disposed within a membrane). In some embodiments, at least a portion of the analyte (e.g., a first analyte and / or a second analyte) may be translocated through a pore. The at least a portion of the analyte may comprise at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or any combination thereof. In some embodiments, translocation of the analyte (e.g., the first analyte and / or the second analyte) through a pore may generate a signal or change thereof (e.g., a first current or change thereof and / or a second current or change thereof). The first current or change thereof and / or the second current or change thereof may be detected. In some embodiments, the first current or change thereof and / or the second current or change thereof may be used to determine a characteristic (e.g., a first characteristic and / or a second characteristic). In some embodiments, the first current or change thereof and / or the second current or change thereof may be used to determine aWSGR Docket Number: 64828-710.601 plurality of characteristics. At least a plurality of characteristics may be determined using the methods and / or system disclosed herein. In some embodiments, a sample may be analyzed by using the first characteristic and / or a second characteristic. For example, one or more properties of the sample may be characterized by using the first characteristic and / or a second characteristic.

[0323] As an example, a method for sample analysis can comprise: (a) providing a sample comprising a plurality of analytes, wherein the plurality of analytes comprises a first analyte and a second analyte; (b) translocating at least a portion of the first analyte through a first nanopore disposed within a first membrane and at least a portion of the second analyte through a second nanopore disposed within a second membrane, wherein the at least a portion of the first analyte comprises at least a portion of a first protein, at least a portion of a first polypeptide, at least a portion of a first peptide, or a combination thereof, wherein the at least a portion of the second analyte comprises at least a portion of a second protein, at least a portion of a second polypeptide, at least a portion of a second peptide, or a combination thereof; (c) detecting (i) (1) a first current or change thereof, or (2) a first voltage or change thereof while the at least the portion of the first analyte is translocating through the first nanopore, and (ii) (3) a second current or change thereof, or (4) a second voltage or change thereof while the at least the portion of the second analyte is translocating through the second nanopore; (d) using (i) (1) the first current or change thereof, or (2) the first voltage or change thereof to determine a first characteristic of the at least the portion of the first analyte and (ii) (3) the second current or change thereof, or (4) the second voltage or change thereof to determine a second characteristic of the at least the portion of the second analyte; and (e) characterizing one or more properties of the sample using the first characteristic or the second characteristic determined in (d).

[0324] Characterizing one or more properties of the sample may comprise determining a feature. The feature may be a feature of a proteome. The proteome may be associated with the sample. The proteome may refer to a set of proteins expressed by the organism from which the same may be derived. Characterizing one or more properties of the sample may comprise a proteome coverage of a proteome. The proteome coverage can refer to extrapolation of the number of protein discoveries by future measurements conditioned on a sequence of already performed measurements. Characterizing one or more properties of the sample may comprise a sequence coverage of the first analyte (e.g., a first polypeptide) and / or a second analyte (e.g., a second polypeptide). Sequence coverage can refer to a number of sequencing reads that may be uniquely mapped to a reference sequence and may be applied to a known part of the genome.

[0325] In some embodiments, a first nanopore and a second nanopore may be the same nanopore. In some embodiments, a first nanopore and a second nanopore may be different nanopores. In some embodiments, a first membrane and a second membrane may be the same membrane. In some embodiments, a first membrane and a second membrane may be different membranes.WSGR Docket Number: 64828-710.601

[0326] One or more properties of a sample can comprise identification of a type of a sample (e.g., a type associated with the sample). The sample type can comprise any sample type described herein. One or more properties of a sample can comprise identification of an origin of a sample. The sample origin can comprise any sample origin described herein. In some embodiments, one or more properties may comprise one or more dynamic changes in a sample. Dynamic changes may be determined through continuous measurement of a sample (e.g., one or more analytes in a sample). Sensors of a nanopore system described herein may be exposed to the sample and detect changes as the sample changes over time in situ. Dynamic changes may be observed via continuous measurement of a sample. In some embodiments, dynamic changes may be observed via repeated measurements of a sample. An interaction may be determined. The interaction can comprise an interaction of a first analyte and / or second analyte of a sample. The interaction can comprise an interaction of a first analyte and / or second analyte with one or more molecules. A sample (e.g., a cell of a biological sample) may comprise a set of one or more interactions. A first analyte and / or a second analyte (e.g., a first non-nucleic acid based polymer analyte and a second non-nucleic acid based polymer analyte) may have one or more protein-protein interactions with one or more molecules of the sample. In some embodiments, a plurality of analytes may have one or more interactions with one or more molecules (e.g., one or more polypeptides, one or more proteins, one or more peptides, one or more nucleic acids, or one or more small molecules, or any combination thereof). For example, following translocation of an analyte through a nanopore, a characterized property may comprise an interaction between the analyte and a bound entity (e.g., a leader construct, a recognition element, a PTM, or a conjugation, or any combination thereof). The characterized property can provide information on the analyte (e.g., the bound entity) and the sample (e.g., the sample containing binding partners to at least the analyte). As another example, characterizing properties of the sample may be performed through binding partners (e.g., binding molecules) to bind to and / or isolate an analyte from a sample. The binding partners (e.g., binding molecules) may bind to an analyte via affinity binding. The binding may be measured by extracting the analyte from the sample and translocating through a nanopore described herein. PORES

[0327] In some aspects, the present disclosure provides pores for analyzing analytes.

[0328] In some embodiments, a pore can be a biological pore. In some embodiments, a biological pore comprises a biological molecule. In some embodiments, a pore comprises a protein. In some embodiments, a pore comprises an assembly of proteins. In some embodiments, a pore comprises an assembly of subunits. In some embodiments, a pore comprises an assembly of fused proteins. In some embodiments, a pore comprises DNA. In some embodiments, a pore comprises a DNA origami structure. In some embodiments, a pore comprises a hybrid of DNA and peptides. In some embodiments, a pore comprises a G-quadruplex. In someWSGR Docket Number: 64828-710.601 embodiments, a pore comprises a polymer or a covalent organic framework. In some embodiments, a pore comprises a helical self-assembling pore or a chemically synthesized pore. In some embodiments, a pore comprises silicon, carbon, metal, metallic oxide, a metal-organic-framework, or a MXene. In some embodiments, a DNA based pore carries a large surface charge that can be used to create strong ion-selective electro-osmotic gradients. In some embodiments, a pore can be comprised in a membrane. In some embodiments, a pore comprises a toroidal protein. In some embodiments, a pore comprises a non- transmembrane protein. In some embodiments, a pore comprises cyclic peptides. In some embodiments, a pore comprises an assembly of cell penetrating molecules. In some embodiments, a pore comprises cell penetrating peptides. In some embodiments, a pore comprises portions of phage portal complexes. In some embodiments, a pore comprises portions of cellular transmembrane transport complexes.

[0329] In some aspects, the present disclosure provides pores for detecting and / or characterizing an analyte (e.g., a biopolymer). A pore may be a wild-type pore and / or a pore may be an engineered pore. In some embodiments, the pore (e.g., nanopore) comprises a transmembrane region. In some embodiments, the pore comprises a hydrophilic portion. In some embodiments, the pore comprises a hydrophobic portion. In some embodiments, the pore comprises a hydrophilic and a hydrophobic portion. In some embodiments, a pore comprises an opening (e.g., an entrance). In some embodiments, a pore comprises at least one opening. In some embodiments, a pore can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) openings. An entrance to a nanopore may be defined by a widest dimension (e.g., a measure from a first edge of an entrance to a second edge of the entrance). A pore may be measured by a diameter, or a circumference, or any combination thereof.

[0330] A pore can comprise a channel through which an analyte may enter. Herein the terms “channel”, “lumen” and / or “vestibule” may be used interchangeably. The channel may be of the wild-type biological nanopore or the engineered biological nanopore. In some embodiments, an analyte may be trapped in the channel of the nanopore. In some embodiments, an analyte may translocate through the channel of the nanopore. In some embodiments, an analyte may partially translocate through the channel of the nanopore. The channel may be a same width through the entire channel or a channel may have two or more different widths through the entire channel.

[0331] The channel may comprise at least one region. For example, the channel of the pore (e.g., the biological nanopore) may comprise a first region, a second region, and / or a third region. In some embodiments, the channel of the nanopore comprises a constriction (e.g., a constriction region). The constriction region may be a region of the channel different in size (e.g., width, length, diameter, circumference, or a widest dimension, or any combination thereof) than one or more other regions of the channel. The second region of the channel may have the constriction region. The first region and / or third region of the channel and the second region of the channel (e.g., comprising the constriction region) may be adjacent (e.g., immediately adjacent) to one another. In otherWSGR Docket Number: 64828-710.601 cases, the first region and / or third region and second region of the channel may be separated by a distance of at most about 4.0 nm, at most about 3.0 nm, at most about 2.0 nm, at most about 1.5 nm, at most about 1.0 nm, at most about 0.9 nm, at most about 0.8 nm, at most about 0.7 nm, at most about 0.6 nm, at most about 0.5 nm, at most about 0.4 nm, at most about 0.3 nm, at most about 0.2 nm, at most about 0.1 nm, or less than about 0.1 nm. In some embodiments, a first region and / or third region of the channel and a second region of the channel (e.g., comprising the constriction region) are separated by a distance of at least about 0.001 nm, at least about 0.01 nm, at least about 0.05 nm, at least about 0.1 nm, at least about 0.5 nm, at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 10 nm, at least about 15 nm, or greater than about 15 nm. In some embodiments, a first region and / or third region of the channel and a second region of the channel (e.g., comprising the constriction region) are separated by a distance of at most about 15 nm, at most about 10 nm, at most about 5 nm, at most about 4 nm, at most about 3 nm, at most about 2 nm, at most about 1 nm, at most about 0.5 nm, at most about 0.1 nm, at most about 0.05 nm, at most about 0.01 nm, at most about 0.001 nm, or less than about 0.001 nm. In some embodiments, a first region and / or third region of the channel and a second region of the channel (e.g., comprising the constriction region) are separated by a distance from about 0.001 nm to about 15 nm. In some embodiments, a first region and / or third region of the channel and a second region of the channel (e.g., comprising the constriction region) are separated by a distance from at least about 0.001 nm. In some embodiments, a first region and / or third region of the channel and a second region of the channel (e.g., comprising the constriction region) are separated by a distance from about 0.001 nm to about 0.01 nm, about 0.001 nm to about 0.05 nm, about 0.001 nm to about 0.1 nm, about 0.001 nm to about 0.5 nm, about 0.001 nm to about 1 nm, about 0.001 nm to about 2 nm, about 0.001 nm to about 3 nm, about 0.001 nm to about 4 nm, about 0.001 nm to about 5 nm, about 0.001 nm to about 10 nm, about 0.001 nm to about 15 nm, about 0.01 nm to about 0.05 nm, about 0.01 nm to about 0.1 nm, about 0.01 nm to about 0.5 nm, about 0.01 nm to about 1 nm, about 0.01 nm to about 2 nm, about 0.01 nm to about 3 nm, about 0.01 nm to about 4 nm, about 0.01 nm to about 5 nm, about 0.01 nm to about 10 nm, about 0.01 nm to about 15 nm, about 0.05 nm to about 0.1 nm, about 0.05 nm to about 0.5 nm, about 0.05 nm to about 1 nm, about 0.05 nm to about 2 nm, about 0.05 nm to about 3 nm, about 0.05 nm to about 4 nm, about 0.05 nm to about 5 nm, about 0.05 nm to about 10 nm, about 0.05 nm to about 15 nm, about 0.1 nm to about 0.5 nm, about 0.1 nm to about 1 nm, about 0.1 nm to about 2 nm, about 0.1 nm to about 3 nm, about 0.1 nm to about 4 nm, about 0.1 nm to about 5 nm, about 0.1 nm to about 10 nm, about 0.1 nm to about 15 nm, about 0.5 nm to about 1 nm, about 0.5 nm to about 2 nm, about 0.5 nm to about 3 nm, about 0.5 nm to about 4 nm, about 0.5 nm to about 5 nm, about 0.5 nm to about 10 nm, about 0.5 nm to about 15 nm, about 1 nm to about 2 nm, about 1 nm to about 3 nm, about 1 nm to about 4 nm, about 1 nm to about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 15 nm, about 2 nm to about 3 nm, about 2 nm to about 4 nm, about 2 nm to about 5 nm, about 2 nm to about 10 nm,WSGR Docket Number: 64828-710.601 about 2 nm to about 15 nm, about 3 nm to about 4 nm, about 3 nm to about 5 nm, about 3 nm to about 10 nm, about 3 nm to about 15 nm, about 4 nm to about 5 nm, about 4 nm to about 10 nm, about 4 nm to about 15 nm, about 5 nm to about 10 nm, about 5 nm to about 15 nm, or about 10 nm to about 15 nm.

[0332] The constriction region of the nanopore may be a narrower region of the channel than another region of the channel. In some embodiments, the constriction region of the nanopore can contribute to the electrical resistance of the nanopore. A modulation of electrical resistance may allow the nanopore to differentiate between analytes in a complex sample. Therefore, modifying a constriction region of a nanopore to shift an electrical resistance may modulate the electro-osmotic force and / or may improve the ability of the nanopore to characterize an analyte. Characterization of an analyte may occur at the constriction region. In the constriction region, the current flow may be modulated most by the composition (e.g., local composition, e.g., amino acid composition) of the analyte within. The electro-osmotic flow (EOF) may be maximally created at a narrow region (e.g., a constriction region). The EOF may be maximally created at a constriction region due to a maximal electrostatic effect on cation or anion flux in the constrained dimensions of the constriction.

[0333] In some embodiments, the nanopore comprises a shape (e.g., a geometry). For example, a nanopore may be cylindrical. In some embodiments, the nanopore can be conical shape. In some embodiments, the nanopore can be globular shape. In some embodiments, the nanopore can be hourglass shape. In some embodiments, the nanopore can be a toroidal shape, comprising a ring and a channel. In some embodiments, a nanopore comprises a biological nanopore or a solid state nanopore. The toroidal shape may comprise a toroidal polyhedral shape comprising a ring and a channel. The ring may comprise the protein or proteins that form the nanopore. The ring may comprise a cross sectional geometry similar to the protein or proteins that form the nanopore. The ring may be wider at a first side (e.g., a cis side) than a second side (e.g., a trans side), or wider at the second side (e.g., the trans side) than the first side (e.g., the cis side). The ring can comprise a portion comprising a conical geometry, a cylindrical geometry, or an amorphous geometry, or combinations thereof. The channel can comprise the central portion of the nanopore geometry that does not comprise the proteins or peptides of the nanopore. The channel may allow molecules to translocate through the nanopore (i.e. through the channel).

[0334] A channel may restrict molecules from translocating through the nanopore. The restriction may be based on a width of the channel or a charge of the channel. The channel can comprise a channel length. The channel length can be the length of the channel as measured along a longitudinal axis of the channel. This longitudinal axis may run perpendicular to a membrane. The length may be measured perpendicular to the ring of the shape (e.g., the toroidal shape) of the geometry of the nanopore. The channel length can be measured as the distance along the longitudinal axis of the channel between the most distant points of the nanopore along the longitudinal axis of the channel. In some embodiment, a channel may have a start point on a first side (e.g.,WSGR Docket Number: 64828-710.601 a cis side) of a nanopore, and an end point on a second side (e.g., a trans side) of a nanopore, or a start point on a second side (e.g., a trans side) of a nanopore, and an end point on a first side (e.g., a cis side) of a nanopore. In some embodiments a channel length can be less than a linear length or a contour length of an analyte. In some embodiments a channel length can be greater than a linear length or a contour length of an analyte.

[0335] In some embodiments, a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) measured from an alpha-carbon position of an amino acid backbone. The dimension of the constriction region may be measured from a first alpha-carbon position to a second alpha-carbon position. In some embodiments, a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) measured from a first alpha-carbon position to a second alpha-carbon position of at least about 0.2 nm, at least about 0.3 nm, at least about 0.4 nm, at least about 0.5 nm, at least about 0.6 nm, at least about 0.7 nm, at least about 0.8 nm, at least about 0.9 nm, at least about 1.0 nm, at least about 1.1 nm, at least about 1.2 nm, at least about 1.3 nm, at least about 1.4 nm, at least about 1.5 nm, at least about 1.6 nm, at least about 1.7 nm, at least about 1.8 nm, at least about 1.9 nm, at least about 2.0 nm, at least about 2.1 nm, at least about 2.2 nm, at least about 2.3 nm, at least about 2.4 nm, at least about 2.5 nm, at least about 2.6 nm, at least about 2.7 nm, at least about 2.8 nm, at least about 2.9 nm, at least about 3.0 nm, at least about 3.1 nm, at least about 3.2 nm, at least about 3.3 nm, at least about 3.4 nm, at least about 3.5 nm, at least about 3.6 nm, at least about 3.7 nm, at least about 3.8 nm, at least about 3.9 nm, at least about 4.0 nm, or greater than about 4.0 nm. In some embodiments, a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) measured from a first alpha-carbon position to a second alpha-carbon position of at most about 4.0 nm, at most about 3.9 nm, at most about 3.8 nm, at most about 3.7 nm, at most about 3.6 nm, at most about 3.5 nm, at most about 3.4 nm, at most about 3.3 nm, at most about 3.2 nm, at most about 3.1 nm, at most about 3.0 nm, at most about 2.9 nm, at most about 2.8 nm, at most about 2.7 nm, at most about 2.6 nm, at most about 2.5 nm, at most about 2.4 nm, at most about 2.3 nm, at most about 2.2 nm, at most about 2.1 nm, at most about 2.0 nm, at most about 1.9 nm, at most about 1.8 nm, at most about 1.7 nm, at most about 1.6 nm, at most about 1.5 nm, at most about 1.4 nm, at most about 1.3 nm, at most about 1.2 nm, at most about 1.1 nm, at most about 1.0 nm, at most about 0.9 nm, at most about 0.8 nm, at most about 0.7 nm, at most about 0.6 nm, at most about 0.5 nm, at most about 0.4 nm, at most about 0.3 nm, at most about 0.2 nm, or less than about 0.2 nm.

[0336] In some embodiments, a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) measured from a first alpha-carbon position to a second alpha-carbon position from about 0.2 nm to about 4 nm. In some embodiments, a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) measured from a first alpha-carbon position to a second alpha-carbon position from about 0.2 nmWSGR Docket Number: 64828-710.601 to about 0.3 nm, about 0.2 nm to about 0.4 nm, about 0.2 nm to about 0.5 nm, about 0.2 nm to about 1 nm, about 0.2 nm to about 1.5 nm, about 0.2 nm to about 2 nm, about 0.2 nm to about 2.5 nm, about 0.2 nm to about 3 nm, about 0.2 nm to about 3.5 nm, about 0.2 nm to about 4 nm, about 0.3 nm to about 0.4 nm, about 0.3 nm to about 0.5 nm, about 0.3 nm to about 1 nm, about 0.3 nm to about 1.5 nm, about 0.3 nm to about 2 nm, about 0.3 nm to about 2.5 nm, about 0.3 nm to about 3 nm, about 0.3 nm to about 3.5 nm, about 0.3 nm to about 4 nm, about 0.4 nm to about 0.5 nm, about 0.4 nm to about 1 nm, about 0.4 nm to about 1.5 nm, about 0.4 nm to about 2 nm, about 0.4 nm to about 2.5 nm, about 0.4 nm to about 3 nm, about 0.4 nm to about 3.5 nm, about 0.4 nm to about 4 nm, about 0.5 nm to about 1 nm, about 0.5 nm to about 1.5 nm, about 0.5 nm to about 2 nm, about 0.5 nm to about 2.5 nm, about 0.5 nm to about 3 nm, about 0.5 nm to about 3.5 nm, about 0.5 nm to about 4 nm, about 1 nm to about 1.5 nm, about 1 nm to about 2 nm, about 1 nm to about 2.5 nm, about 1 nm to about 3 nm, about 1 nm to about 3.5 nm, about 1 nm to about 4 nm, about 1.5 nm to about 2 nm, about 1.5 nm to about 2.5 nm, about 1.5 nm to about 3 nm, about 1.5 nm to about 3.5 nm, about 1.5 nm to about 4 nm, about 2 nm to about 2.5 nm, about 2 nm to about 3 nm, about 2 nm to about 3.5 nm, about 2 nm to about 4 nm, about 2.5 nm to about 3 nm, about 2.5 nm to about 3.5 nm, about 2.5 nm to about 4 nm, about 3 nm to about 3.5 nm, about 3 nm to about 4 nm, or about 3.5 nm to about 4 nm.

[0337] In some embodiments, a distance or dimension (e.g., diameter) may be measured from an atom to a nearest atom of the side chain of the amino acid residue. The side chain (e.g., atom of the side chain) may protrude into the constriction region of the channel and / or constriction-forming portion of the monomer. In some embodiments, a distance or dimension (e.g., diameter) of an atom to a nearest atom of an amino acid residue of an engineered monomer and / or engineered biological described herein may be at least about 0.0001 nm, at least about 0.0005 nm, at least about 0.001 nm, at least about 0.005 nm, at least about 0.01 nm, at least about 0.02nm, at least about 0.03 nm, at least about 0.04 nm, at least about 0.05 nm, at least about 0.06 nm, at least about 0.07 nm, at least about 0.08 nm, at least about 0.09 nm, at least about 0.1 nm, at least about 0.2 nm, at least about 0.3 nm, at least about 0.4 nm, at least about 0.5 nm, at least about 0.6 nm, at least about 0.7 nm, at least about 0.8 nm, at least about 0.9 nm, at least about 1.0 nm, at least about 1.1 nm, at least about 1.2 nm, at least about 1.3 nm, at least about 1.4 nm, at least about 1.5 nm, at least about 1.6 nm, at least about 1.7 nm, at least about 1.8 nm, at least about 1.9 nm, at least about 2.0 nm or greater than about 2.0 nm. In some embodiments, a distance or dimension (e.g., diameter) of an atom to a nearest atom of an amino acid residue of an engineered monomer and / or engineered biological described herein may be at most about 2.0 nm, at most about 1.9 nm, at most about 1.8 nm, at most about 1.7 nm, at most about 1.6 nm, at most about 1.5 nm, at most about 1.4 nm, at most about 1.3 nm, at most about 1.2 nm, at most about 1.1 nm, at most about 1.0 nm, at most about 0.9 nm, at most about 0.8 nm, at most about 0.7 nm, at most about 0.6 nm, at most about 0.5 nm, at most about 0.4 nm, at most about 0.3 nm, at most about 0.2 nm, at most about 0.1 nm, at most about 0.09 nm, at mostWSGR Docket Number: 64828-710.601 about 0.08 nm, at most about 0.07 nm, at most about 0.06 nm, at most about 0.05 nm, at most about 0.04 nm, at most about 0.03 nm, at most about 0.02 nm, at most about 0.01 nm, at most about 0.005 nm, at most about 0.001 nm, at most about 0.0005 nm, at most about 0.0001 nm, or less than about 0.0001 nm.

[0338] In some embodiments, a distance or dimension (e.g., diameter) of an atom to a nearest atom of an amino acid residue of an engineered monomer and / or engineered biological described herein may be from about 0.0001 nm to about 2 nm. In some embodiments, a distance or dimension (e.g., diameter) of an atom to a nearest atom of an amino acid residue of an engineered monomer and / or engineered biological described herein may be from about 0.0001 nm to about 0.001 nm, about 0.0001 nm to about 0.005 nm, about 0.0001 nm to about 0.01 nm, about 0.0001 nm to about 0.05 nm, about 0.0001 nm to about 0.1 nm, about 0.0001 nm to about 0.2 nm, about 0.0001 nm to about 0.3 nm, about 0.0001 nm to about 0.4 nm, about 0.0001 nm to about 0.5 nm, about 0.0001 nm to about 1 nm, about 0.0001 nm to about 2 nm, about 0.001 nm to about 0.005 nm, about 0.001 nm to about 0.01 nm, about 0.001 nm to about 0.05 nm, about 0.001 nm to about 0.1 nm, about 0.001 nm to about 0.2 nm, about 0.001 nm to about 0.3 nm, about 0.001 nm to about 0.4 nm, about 0.001 nm to about 0.5 nm, about 0.001 nm to about 1 nm, about 0.001 nm to about 2 nm, about 0.005 nm to about 0.01 nm, about 0.005 nm to about 0.05 nm, about 0.005 nm to about 0.1 nm, about 0.005 nm to about 0.2 nm, about 0.005 nm to about 0.3 nm, about 0.005 nm to about 0.4 nm, about 0.005 nm to about 0.5 nm, about 0.005 nm to about 1 nm, about 0.005 nm to about 2 nm, about 0.01 nm to about 0.05 nm, about 0.01 nm to about 0.1 nm, about 0.01 nm to about 0.2 nm, about 0.01 nm to about 0.3 nm, about 0.01 nm to about 0.4 nm, about 0.01 nm to about 0.5 nm, about 0.01 nm to about 1 nm, about 0.01 nm to about 2 nm, about 0.05 nm to about 0.1 nm, about 0.05 nm to about 0.2 nm, about 0.05 nm to about 0.3 nm, about 0.05 nm to about 0.4 nm, about 0.05 nm to about 0.5 nm, about 0.05 nm to about 1 nm, about 0.05 nm to about 2 nm, about 0.1 nm to about 0.2 nm, about 0.1 nm to about 0.3 nm, about 0.1 nm to about 0.4 nm, about 0.1 nm to about 0.5 nm, about 0.1 nm to about 1 nm, about 0.1 nm to about 2 nm, about 0.2 nm to about 0.3 nm, about 0.2 nm to about 0.4 nm, about 0.2 nm to about 0.5 nm, about 0.2 nm to about 1 nm, about 0.2 nm to about 2 nm, about 0.3 nm to about 0.4 nm, about 0.3 nm to about 0.5 nm, about 0.3 nm to about 1 nm, about 0.3 nm to about 2 nm, about 0.4 nm to about 0.5 nm, about 0.4 nm to about 1 nm, about 0.4 nm to about 2 nm, about 0.5 nm to about 1 nm, about 0.5 nm to about 2 nm, or about 1 nm to about 2 nm.

[0339] In some embodiments, a pore can be a non-biological pore. In some embodiments, a pore can be a solid state pore.

[0340] In some embodiments, a pore can be a nanopore. In some embodiments, a pore comprises a width of at least about 0.5 nanometers (nm), 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, a pore comprises a width of at most about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm,WSGR Docket Number: 64828-710.601 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, a pore comprises a diameter of at least about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, a pore comprises a diameter of at most about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, a pore comprises an outer diameter of at least about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, a pore comprises an outer diameter of at most about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nm. In some embodiments, a pore comprises an inner diameter of at least about 0.1 nm, 0.2, nm, 0.3 nm, 0.4 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, a pore comprises an inner diameter of at most about 0.1 nm, 0.2, nm, 0.3 nm, 0.4 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, an inner diameter can be a diameter of one or more constrictions (e.g., constriction regions) of a pore.

[0341] In some embodiments, a pore can function at a temperature of at least about 4, 10, 20, 30, 40, 50, 60, 70, 80, or about 90 degrees Celsius. In some embodiments, a pore can function at a temperature of at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 degrees Celsius.

[0342] In some embodiments, a pore can be disposed in a membrane. In some embodiments, a pore comprises transmembrane region. In some embodiments, a transmembrane region can be formed upon assembly of multiple transmembrane region sequences present in a plurality of subunits that together form a pore. In some embodiments the transmembrane region can be partially or fully composed of beta-strands. The beta strands can partially or fully comprise amphipathic surfaces that can interface with an amphipathic membrane. In some embodiments the transmembrane region can be partially or fully composed of alpha-helicases. The alpha- helicases can partially or fully comprise amphipathic surfaces that can interface with the amphipathic membranes. In some embodiments, a transmembrane region sequence comprises an alternation of hydrophobic residues and hydrophilic residues. In some embodiments, a pore comprises a hydrophobic portion and a hydrophilic portion. In some embodiments, a transmembrane region of the pore comprises a hydrophobic portion. In some embodiments, a ring portion of a pore comprises a hydrophilic portion. In some embodiments, a pore comprises a protein that controls the translocation of an analyte (e.g. polypeptide or polynucleotide) across the pore. In some embodiments, a molecular motor can cause the translocation of an analyte across the pore. In some embodiments, the translocation of an analyte across the pore can be NTP-driven or ATP-driven. In some embodiments, the translocation of an analyte across the pore does not depend on NTP or ATP. In some embodiments, the translocation of an analyte across the pore does not depend on a molecular motor.WSGR Docket Number: 64828-710.601

[0343] In some embodiments the pore can be a monomer. In some embodiments the pore can be formed from multiple monomeric units. In some embodiments, the monomers that comprise the oligomeric pore may be identical. In some embodiments the monomers that comprise the oligomeric pore may be different.

[0344] In some embodiments, the nanopore may be an artificial biological nanopore. For example, the monomers of the nanopore may comprise proteins designed de novo (e.g., designed using machine learning algorithms). In some embodiments, portions of the nanopore (e.g., portions of the one or more monomers of the nanopore) may comprise proteins designed de novo (e.g., designed using machine learning algorithms).

[0345] In some embodiments, the nanopore may comprise an assembly of monomers. The nanopore may comprise a number of monomers. Monomers may be arranged vertically, horizontally, and / or layered as rings to form a nanopore described herein. In some embodiments, a nanopore (e.g., biological nanopore) comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, or greater than 50 monomers. In some embodiments, a nanopore (e.g., biological nanopore) comprises at most about 50, 40, 30, 25, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 2 monomers. In some embodiments, a nanopore may comprise 1 monomer. In some embodiments, a nanopore (e.g., biological nanopore) comprises from about 3 monomers to about 40 monomers. In some embodiments, a nanopore (e.g., biological nanopore) comprises from about 3 monomers to about 4 monomers, about 3 monomers to about 5 monomers, about 3 monomers to about 6 monomers, about 3 monomers to about 7 monomers, about 3 monomers to about 8 monomers, about 3 monomers to about 9 monomers, about 3 monomers to about 10 monomers, about 3 monomers to about 15 monomers, about 3 monomers to about 20 monomers, about 3 monomers to about 30 monomers, about 3 monomers to about 40 monomers, about 4 monomers to about 5 monomers, about 4 monomers to about 6 monomers, about 4 monomers to about 7 monomers, about 4 monomers to about 8 monomers, about 4 monomers to about 9 monomers, about 4 monomers to about 10 monomers, about 4 monomers to about 15 monomers, about 4 monomers to about 20 monomers, about 4 monomers to about 30 monomers, about 4 monomers to about 40 monomers, about 5 monomers to about 6 monomers, about 5 monomers to about 7 monomers, about 5 monomers to about 8 monomers, about 5 monomers to about 9 monomers, about 5 monomers to about 10 monomers, about 5 monomers to about 15 monomers, about 5 monomers to about 20 monomers, about 5 monomers to about 30 monomers, about 5 monomers to about 40 monomers, about 6 monomers to about 7 monomers, about 6 monomers to about 8 monomers, about 6 monomers to about 9 monomers, about 6 monomers to about 10 monomers, about 6 monomers to about 15 monomers, about 6 monomers to about 20 monomers, about 6 monomers to about 30 monomers, about 6 monomers to about 40 monomers, about 7 monomers to about 8 monomers, about 7 monomers to about 9 monomers, about 7 monomers to about 10 monomers, about 7 monomers to about 15 monomers, about 7 monomers to about 20 monomers, about 7 monomers to about 30 monomers, about 7 monomers to about 40 monomers, about 8 monomers to about 9WSGR Docket Number: 64828-710.601 monomers, about 8 monomers to about 10 monomers, about 8 monomers to about 15 monomers, about 8 monomers to about 20 monomers, about 8 monomers to about 30 monomers, about 8 monomers to about 40 monomers, about 9 monomers to about 10 monomers, about 9 monomers to about 15 monomers, about 9 monomers to about 20 monomers, about 9 monomers to about 30 monomers, about 9 monomers to about 40 monomers, about 10 monomers to about 15 monomers, about 10 monomers to about 20 monomers, about 10 monomers to about 30 monomers, about 10 monomers to about 40 monomers, about 15 monomers to about 20 monomers, about 15 monomers to about 30 monomers, about 15 monomers to about 40 monomers, about 20 monomers to about 30 monomers, about 20 monomers to about 40 monomers, or about 30 monomers to about 40 monomers.

[0346] As the monomers of the nanopore arranged vertically, horizontally, and / or layered, the amino acid residues (e.g., positively-charged amino acid residues, negatively-charged amino acid residues, or neutral amino acid residues, or any combination thereof) may form one or more rings of charges. In some embodiments, a pore may be engineered to contain regions of separate rings of charges along the longitudinal length of the channel. For example, a nanopore may be engineered to contain regions of at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, or greater than about 20 separate rings of charges along the longitudinal length of the channel. A nanopore may be engineered to contain regions of at most about 20, at most about 19, at most about 18, at most about 17, at most about 16, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or less than about 2 separate rings of charges along the longitudinal length of the channel. A nanopore may be engineered to contain regions from about 2 to about 20 separate rings of charges along the longitudinal length of the channel. A nanopore may be engineered to contain regions from about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 6, about 2 to about 7, about 2 to about 8, about 2 to about 9, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 3 to about 4, about 3 to about 5, about 3 to about 6, about 3 to about 7, about 3 to about 8, about 3 to about 9, about 3 to about 10, about 3 to about 15, about 3 to about 20, about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 6 to about 15, about 6 to about 20, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 7 to about 15, about 7 to about 20, about 8 to about 9, about 8 to about 10, about 8 to about 15, about 8 to about 20, about 9 to about 10, about 9 to about 15, about 9 to aboutWSGR Docket Number: 64828-710.601 20, about 10 to about 15, about 10 to about 20, or about 15 to about 20 separate rings of charges along the longitudinal length of the channel.

[0347] In some embodiments, a polypeptide can translocate through a pore at a rate of about 10 amino acids per second (aa / sec), 20 aa / sec, 30 aa / sec, 40 aa / sec, 50 aa / sec, 60 aa / sec, 70 aa / sec, 80 aa / sec, 90 aa / sec, 100 aa / sec, 110 aa / sec, 120 aa / sec, 130 aa / sec, 140 aa / sec, 150 aa / sec, 200 aa / sec, 250 aa / sec, 300 aa / sec, 350 aa / sec, 400 aa / sec, 450 aa / sec, or about 500 aa / sec. In some embodiments, a polypeptide can translocate through a pore at a rate greater than about 10 aa / sec, 20 aa / sec, 30 aa / sec, 40 aa / sec, 50 aa / sec, 60 aa / sec, 70 aa / sec, 80 aa / sec, 90 aa / sec, 100 aa / sec, 110 aa / sec, 120 aa / sec, 130 aa / sec, 140 aa / sec, 150 aa / sec, 200 aa / sec, 250 aa / sec, 300 aa / sec, 350 aa / sec, 400 aa / sec, 450 aa / sec, or about 500 aa / sec. In some embodiments, a polypeptide can translocate through a pore at a rate less than about 10 aa / sec, 20 aa / sec, 30 aa / sec, 40 aa / sec, 50 aa / sec, 60 aa / sec, 70 aa / sec, 80 aa / sec, 90 aa / sec, 100 aa / sec, 110 aa / sec, 120 aa / sec, 130 aa / sec, 140 aa / sec, 150 aa / sec, 200 aa / sec, 250 aa / sec, 300 aa / sec, 350 aa / sec, 400 aa / sec, 450 aa / sec, or about 500 aa / sec.

[0348] In some embodiments, a pore can be configured to provide a condition for dominant electro-osmotic capture of an analyte. For example, for a nanopore system (e.g., an Aerolysin nanopore system or another nanopore system described herein), low pH conditions may increase the net positive charge inside the pore channel, resulting in (i) increased anion selectivity, (ii) a strong net anion-selective pore, or (iii) an increased electrostatic repulsion of mostly positively charged analytes, or (iv) any combination thereof. Low pH can comprise a pH of at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, at most about 1, or less than about 1. The resulting strong electro-osmotic flux through the pore can be exploited to capture analytes against the direction of the electrophoretic forces (EPF) acting upon them (e.g. with a positive applied potential at the trans electrode for a system with mostly positively charged peptides in the cis solution). It can be advantageous to exploit electro-osmotic forces to capture analytes since it can be less sensitive to charge composition. It can be advantageous for capturing and / or detecting a diverse composition of unlabeled peptides (e.g. neutral, net positive, net negative).

[0349] In some embodiments, an electro-osmotic force (EOF) may be in a first side (e.g., cis side) to second side (e.g., trans side) direction. In some embodiments, an electro-osmotic force may be in a second side (e.g., trans side) to first side (e.g., cis side) direction. In some embodiments, an electrophoretic force may be in a first side (e.g., cis side) to second side (e.g., trans side) direction. In some embodiments, an electrophoretic force may be in a second side (e.g., trans side) to first side (e.g., cis side) direction. In some embodiments, an EOF may be in a first side (e.g., cis side) to second side (e.g., trans side) direction and an EPF may be in a first side (e.g., cis side) to second side (e.g., trans side) direction. In some embodiments, an EOF may be in a first side (e.g., cis side) to second side (e.g., trans side) direction and an EPF may be in a second side (e.g., trans side) to first side (e.g., cis side) direction. In some embodiments, an EOF may be in a second side (e.g., trans side) toWSGR Docket Number: 64828-710.601 first side (e.g., cis side) direction and an EPF may be in a second side (e.g., trans side) to first side (e.g., cis side) direction. In some embodiments, an EOF may be in a second side (e.g., trans side) to first side (e.g., cis side) direction and an EPF may be in a first side (e.g., cis side) to second side (e.g., trans side) direction.

[0350] The strength of electro-osmotic force (EOF) acting on the analytes can be tuned (e.g. by mutagenesis). A pore (e.g., nanopore) described herein can comprise one or more mutations. The mutation may be a substitution, an insertion, a deletion, or a chemical modification, or any combination thereof. The mutation may comprise a positively-charged amino acid residue, or a negatively-charged amino acid reside, or any combination thereof. For example, the electro-osmotic force can be reduced to increase the duration for which the analytes may be retained in the pore. For example, the anion ion-selectivity bias and resulting net anionic electro-osmotic flux that results from low pH conditions can be reduced by introducing acidic residues (e.g., by substitution adjacent to the aromatic mutations). Acidic mutation substitutions that reduce net positive charge can reduce electrostatic repulsion of mostly positively charge analytes. Net positive charge can also be reduced by replacing basic residues with neutral or acidic residues. Without wishing to be bound by theory, one or more mutations of negatively charged amino acid residues may increase a negative charge of a pore described herein. The increased negative charge may increase a duration that a positively-charged analyte resides in the pore. As another example, one or more mutations of positively charged amino acid residues may increase a positively charge of a pore described herein. The increased positive charge may increase a duration that a negatively- charged analyte resides in the pore. In some embodiments, one or more aromatic mutations (e.g., insertion of one or more aromatic amino acid residues and / or substitution of one or more aromatic amino acid residues) may affect a diameter of a nanopore described herein. The one or more aromatic mutations may decrease a diameter of a nanopore described herein due to the bulky side chain configuration of aromatic amino acid residues. Mutations of one or more amino acid residues in a constriction region and / or lumen-facing region of a nanopore described herein may provide for greater accuracy of determining one or more characteristics of an analyte. In some embodiments, one or more aromatic mutations (e.g., insertion of one or more aromatic amino acid residues and / or substitution of one or more aromatic amino acid residues) may affect one or more charges of a nanopore described herein.

[0351] In some embodiments, a membrane can provide a partition for providing a voltage difference between a first side (e.g., cis side) and a second side (e.g., trans side) of a pore. In some embodiments, an EOF can result from a net ionic current flow cis to trans. In some embodiments, a cis to trans EOF results from a net ionic current flow cis to trans over a total ionic current flow, also referred to as a relative net current flow cis to trans, of greater than about 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, or about 0.99. In some embodiments, a cis to trans EOF results from a net ionic current flow trans to cis over a total ionic current flow, also referredWSGR Docket Number: 64828-710.601 to as a relative net current flow cis to trans, of less than about 0.0, -0.1, -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, - 0.9, -0.95, or about -0.99.

[0352] In some embodiments, the electro-osmotic force comprises a net ionic current flow from the first side (e.g., cis side) to the second side (e.g., trans side). In some embodiments, the electro-osmotic force can be modulated by a pH, a type of a salt, a concentration of a salt, an osmotic pressure across the membrane of the system, or a modification of the nanopore, or any combinations thereof. In some embodiments, the electro- osmotic force can be modulated by a modification of a charge of the nanopore. In some embodiments, the electro-osmotic force can be modulated by an asymmetric salt distribution between the first side (e.g., cis side) and second side (e.g., trans side) of the membrane. In some embodiments, the electro-osmotic force can be modulated by modification of a charge of the nanopore.

[0353] In some embodiments, a pore can be configured to provide a dominant EOF in the direction cis to trans across the membrane of a pore system. In some embodiments, a pore can be configured to provide a dominant EOF in the direction trans to cis across the membrane of a pore system. In some embodiments, a pore can be configured to provide an EOF that acts against an electrophoretic force (EPF) across the membrane of a pore system. As shown in FIGs. 1A-1C, nanopores can have a strong net Electro-Osmotic Force (EOF) in the direction cis-to-trans across a membrane as indicated by the arrow. The Electrophoretic Forces (EPF) acting on the analyte can depend on the composition of charges on the analyte in the sections in and near the nanopore channel, and therefore can sometimes act in the net direction cis-to-trans or trans-to-cis. A strong and dominant cis-to-trans EOF can cause the capture, stretching, and efficient translocation of long polymer analytes from the cis compartment to the trans compartment regardless of the net direction of the EPF. The net flow can arise from a large cis-to-trans ion flow dominating over any trans-to-cis ion flows (e.g., a lower flow or counter- charged ions under an applied potential). In some embodiments, a positive charge may be applied to a compartment of the nanopore system (e.g., a trans compartment). With a positive voltage applied to a compartment (e.g., a trans compartment) across the membrane, nanopores with net positive internal charge may be used to limit the flow of cations from trans to cis. In some embodiments, a negative charge may be applied to a compartment of the nanopore system (e.g., a trans compartment). With a negative voltage applied to a compartment of the nanopore system (e.g., a trans compartment) across the membrane, nanopores with net negative internal charge may be used to limit the flow of anions from trans to cis.

[0354] In some embodiments, a pore can be configured to provide an EOF that acts with an EPF across the membrane of a pore system. In some embodiments, a pore can comprise a relative ion selectivity P(+) / P(-) of greater than about 5.0 or less than about 0.1. In some embodiments, a pore can comprise a relative ion selectivity P(+) / P(-) of greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3, 3.2, 3.4, 3.6, 3.8, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or about 5.0. In some embodiments, a pore can comprise aWSGR Docket Number: 64828-710.601 relative ion selectivity P(+) / P(-) of less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3, 3.2, 3.4, 3.6, 3.8, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or about 5.0.

[0355] In some embodiments, a pore can comprise a relative ion selectivity P(+) / P(-)of at least about 0.1, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, or greater than about 5 under an applied voltage difference across the membrane. In some embodiments, a pore can comprise a relative ion selectivity P(+) / P(-)of at most about 5, at most about 4, at most about 3, at most about 2, at most about 1, at most about 0.9, at most about 0.8, at most about 0.7, at most about 0.6, at most about 0.5, at most about 0.4, at most about 0.3, at most about 0.2, at most about 0.1, or less than about 0.1 under an applied voltage difference across the membrane.

[0356] In some embodiments, a pore can comprise a relative ion selectivity P(+) / P(-) from about 0.1 to about 5 under an applied voltage difference across the membrane. In some embodiments, a pore can comprise a relative ion selectivity P(+) / P(-) from about 0.1 to about 0.2, about 0.1 to about 0.3, about 0.1 to about 0.4, about 0.1 to about 0.5, about 0.1 to about 1, about 0.1 to about 1.5, about 0.1 to about 2, about 0.1 to about 2.5, about 0.1 to about 3, about 0.1 to about 4, about 0.1 to about 5, about 0.2 to about 0.3, about 0.2 to about 0.4, about 0.2 to about 0.5, about 0.2 to about 1, about 0.2 to about 1.5, about 0.2 to about 2, about 0.2 to about 2.5, about 0.2 to about 3, about 0.2 to about 4, about 0.2 to about 5, about 0.3 to about 0.4, about 0.3 to about 0.5, about 0.3 to about 1, about 0.3 to about 1.5, about 0.3 to about 2, about 0.3 to about 2.5, about 0.3 to about 3, about 0.3 to about 4, about 0.3 to about 5, about 0.4 to about 0.5, about 0.4 to about 1, about 0.4 to about 1.5, about 0.4 to about 2, about 0.4 to about 2.5, about 0.4 to about 3, about 0.4 to about 4, about 0.4 to about 5, about 0.5 to about 1, about 0.5 to about 1.5, about 0.5 to about 2, about 0.5 to about 2.5, about 0.5 to about 3, about 0.5 to about 4, about 0.5 to about 5, about 1 to about 1.5, about 1 to about 2, about 1 to about 2.5, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1.5 to about 2, about 1.5 to about 2.5, about 1.5 to about 3, about 1.5 to about 4, about 1.5 to about 5, about 2 to about 2.5, about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2.5 to about 3, about 2.5 to about 4, about 2.5 to about 5, about 3 to about 4, about 3 to about 5, or about 4 to about 5 under an applied voltage difference across the membrane.

[0357] In some embodiments, a pore can comprise a relative ion selectivity P(+) / P(-)of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, or about 5 under an applied voltage difference across the membrane.

[0358] In some embodiments, a pore comprises a lumen. In some embodiments, a nanopore lumen comprises a net charge of at least about 2 coulombs, at least about 3 coulombs, at least about 4 coulombs, at least about 5 coulombs, at least about 10 coulombs, at least about 15 coulombs, at least about 20 coulombs, at least about 25 coulombs, at least about 30 coulombs, at least about 35 coulombs, at least about 40 coulombs, at least about 45WSGR Docket Number: 64828-710.601 coulombs, at least about 50 coulombs, at least about 55 coulombs, at least about 60 coulombs, at least about 70 coulombs, at least about 80 coulombs, at least about 90 coulombs, at least about 100 coulombs, at least about 150 coulombs, at least about 200 coulombs, or greater than about 200 coulombs. In some embodiments coulombs, a nanopore lumen comprises a net charge of at most about 200 coulombs, at most about 150 coulombs, at most about 100 coulombs, at most about 90 coulombs, at most about 80 coulombs, at most about 70 coulombs, at most about 60 coulombs, at most about 55 coulombs, at most about 50 coulombs, at most about 45 coulombs, at most about 40 coulombs, at most about 35 coulombs, at most about 30 coulombs, at most about 25 coulombs, at most about 20 coulombs, at most about 15 coulombs, at most about 10 coulombs, at most about 5 coulombs, at most about 4 coulombs, at most about 3 coulombs, at most about 2 coulombs, or less than about 2 coulombs. In some embodiments, a nanopore lumen comprises a net charge from about 2 to about 200 coulombs. In some embodiments, a nanopore lumen comprises a net charge from at most about 200. In some embodiments, a nanopore lumen comprises a net charge from about 2 to about 5, about 2 to about 10, about 2 to about 20, about 2 to about 30, about 2 to about 40, about 2 to about 50, about 2 to about 75, about 2 to about 100, about 2 to about 125, about 2 to about 150, about 2 to about 200, about 5 to about 10, about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 5 to about 75, about 5 to about 100, about 5 to about 125, about 5 to about 150, about 5 to about 200, about 10 to about 20, about 10 to about 30, about 10 to about 40, about 10 to about 50, about 10 to about 75, about 10 to about 100, about 10 to about 125, about 10 to about 150, about 10 to about 200, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 20 to about 75, about 20 to about 100, about 20 to about 125, about 20 to about 150, about 20 to about 200, about 30 to about 40, about 30 to about 50, about 30 to about 75, about 30 to about 100, about 30 to about 125, about 30 to about 150, about 30 to about 200, about 40 to about 50, about 40 to about 75, about 40 to about 100, about 40 to about 125, about 40 to about 150, about 40 to about 200, about 50 to about 75, about 50 to about 100, about 50 to about 125, about 50 to about 150, about 50 to about 200, about 75 to about 100, about 75 to about 125, about 75 to about 150, about 75 to about 200, about 100 to about 125, about 100 to about 150, about 100 to about 200, about 125 to about 150, about 125 to about 200, or about 150 to about 200 coulombs. In some embodiments, a pore lumen comprises a net positive charge. In some embodiments, a pore lumen comprises a net negative charge.

[0359] In some embodiments, a pore comprises a recognition region. A recognition region can be identified structurally by the dimensions of the central channel, e.g., X-ray diffraction structures, electron-microscopy structures, and / or computer modeling (e.g., molecular modeling, or homology modeling, or any combination thereof). A recognition region may be a region where electric field lines concentrate. A recognition region may be where a presence of an analyte disrupts the most the ionic current flowing through a pore, e.g., under an applied potential. A recognition region may comprise one or more narrow cross-sections of a pore channel,WSGR Docket Number: 64828-710.601 which can be, e.g., a diameter of less than 2 nanometers or 1 nanometer. In some embodiments, a pore can be engineered to have one or more narrow sections of an internal diameters (constrictions) within the recognition region, which can provide higher sensitivity / ionic current deflection to analytes. In some embodiments, a pore can be engineered to provide longer or shorter residence time of analytes.

[0360] In some embodiments, a lumen-facing recognition region of a pore can be engineered (by one or more natural or non-natural amino acid substitutions, deletions, or modifications) to manipulate the internal dimensions, hydrophobicity, or aromaticity, or combinations thereof, of the pore. In some embodiments, engineering the lumen-facing recognition region of the pore increases the dwell time and / or resolution for peptides traversing the pore. In some embodiments, a lumen-facing recognition region of a pore can be engineered to decrease a translocation speed of an analyte through a pore. In some embodiments, a lumen- facing region of the pore can be engineered to reduce the analyte interacting or binding to the pore lumen. In some embodiments, a lumen-facing region of the pore can be engineered to increase the analyte interacting or binding to the pore lumen. In some embodiments, a lumen-facing recognition region of a pore can be engineered by modifying outwards facing residues to perturb the nearby lumen-facing residues.

[0361] In some embodiments, a charge in a channel of a pore can adapted to alter the selectivity of the pore. In some embodiments, a pore may be modified by one or more mutations. In some embodiments, a mutation comprises one or more point mutations. In some embodiments, a point mutation can be at a non-conserved position. In some embodiments, a point mutation can be a lumen-facing mutation. In some embodiments, a point mutation can be a membrane-facing mutation. In some embodiments, a point mutation can alter a characteristic of a pore. In some embodiments, a point mutation can alter a pore channel charge, conductance at a set pH, ion selectivity, electro-osmotic flux, conductivity, shape, or structure, or combinations thereof. In some embodiments, a point mutation can allow for a conductance or analyte translation at a pH of less than about 1, 2, 3, 3.8, 4, 4.5, 6, 7, 8, 9, 1011, 12, 13, or about 14. In some embodiments, a point mutation can allow for a conductance or analyte translation at a pH of greater than about 1, 2, 3, 3.8, 4, 4.5, 6, 7, 8, 9, 1011, 12, 13, or about 14. In some embodiments, one or more point mutations may affect a diameter of a pore described herein. The one or more point mutations may modulate (e.g., widen or narrow) a diameter of a constriction region of a pore described herein. The constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) measured from an alpha-carbon position of an amino acid backbone. The dimension of the constriction region may be measured from a first alpha-carbon position to a second alpha-carbon position. In some embodiments, one or more point mutations may modulate a constriction region of a nanopore to comprise a diameter of at least about 0.2 nm, 0.3 nm, 0.4 nm, 0.5 nm, 1.0 nm, 1.5 nm, 2.0 nm, 2.5 nm, 3.0 nm, 3.5 nm, 4.0 nm, 4.5 nm, 5.0 nm, or greater than about 5.0 nm. In some embodiments, one or more point mutations may modulate a constriction region of a nanopore to comprise aWSGR Docket Number: 64828-710.601 diameter of at most about 5.0 nm, at most about 4.5 nm, at most about 4.0 nm, at most about 3.5 nm, at most about 3.0 nm, at most about 2.5 nm, at most about 2.0 nm, at most about 1.5 nm, at most about 1.0 nm, at most about 0.5 nm, at most about 0.4 nm, at most about 0.3 nm, at most about 0.2 nm, or less than about 0.2 nm. In some embodiments, one or more point mutations may modulate a constriction region of a nanopore to comprise a diameter between about 0.2 nm to about 5 nm. In some embodiments, one or more point mutations may modulate a constriction region of a nanopore to comprise a diameter between about 0.2 nm to about 0.3 nm, about 0.2 nm to about 0.4 nm, about 0.2 nm to about 0.5 nm, about 0.2 nm to about 1 nm, about 0.2 nm to about 1.5 nm, about 0.2 nm to about 2 nm, about 0.2 nm to about 2.5 nm, about 0.2 nm to about 3 nm, about 0.2 nm to about 3.5 nm, about 0.2 nm to about 4 nm, about 0.2 nm to about 5 nm, about 0.3 nm to about 0.4 nm, about 0.3 nm to about 0.5 nm, about 0.3 nm to about 1 nm, about 0.3 nm to about 1.5 nm, about 0.3 nm to about 2 nm, about 0.3 nm to about 2.5 nm, about 0.3 nm to about 3 nm, about 0.3 nm to about 3.5 nm, about 0.3 nm to about 4 nm, about 0.3 nm to about 5 nm, about 0.4 nm to about 0.5 nm, about 0.4 nm to about 1 nm, about 0.4 nm to about 1.5 nm, about 0.4 nm to about 2 nm, about 0.4 nm to about 2.5 nm, about 0.4 nm to about 3 nm, about 0.4 nm to about 3.5 nm, about 0.4 nm to about 4 nm, about 0.4 nm to about 5 nm, about 0.5 nm to about 1 nm, about 0.5 nm to about 1.5 nm, about 0.5 nm to about 2 nm, about 0.5 nm to about 2.5 nm, about 0.5 nm to about 3 nm, about 0.5 nm to about 3.5 nm, about 0.5 nm to about 4 nm, about 0.5 nm to about 5 nm, about 1 nm to about 1.5 nm, about 1 nm to about 2 nm, about 1 nm to about 2.5 nm, about 1 nm to about 3 nm, about 1 nm to about 3.5 nm, about 1 nm to about 4 nm, about 1 nm to about 5 nm, about 1.5 nm to about 2 nm, about 1.5 nm to about 2.5 nm, about 1.5 nm to about 3 nm, about 1.5 nm to about 3.5 nm, about 1.5 nm to about 4 nm, about 1.5 nm to about 5 nm, about 2 nm to about 2.5 nm, about 2 nm to about 3 nm, about 2 nm to about 3.5 nm, about 2 nm to about 4 nm, about 2 nm to about 5 nm, about 2.5 nm to about 3 nm, about 2.5 nm to about 3.5 nm, about 2.5 nm to about 4 nm, about 2.5 nm to about 5 nm, about 3 nm to about 3.5 nm, about 3 nm to about 4 nm, about 3 nm to about 5 nm, about 3.5 nm to about 4 nm, about 3.5 nm to about 5 nm, or about 4 nm to about 5 nm.

[0362] In some embodiments, a charge in a channel of a pore can be adapted to alter an EOF. In some embodiments, a charge in a channel of a pore can be altered by changing the type of charged residue, the location of a charge, or the dimensions of the pore, or combinations thereof. In some embodiments, a pore can be modified to create a high overlap between Debye layers (alternatively termed Stern layers, the Gouy-Chapman diffuse layer or the electric double layer) or double layers to create energy barriers to limit the flow of a specific ion. In some embodiments, increased positive charge in the pore channel can increase transport of anionic species and / or decrease the transport of cationic species. In some embodiments, increased negative charge in the pore channel can increase transport of cationic species and / or decreases the transport of anionic species. This can, in turn, alter the net electro-osmotic flux of hydrated ions flowing through the pore under an appliedWSGR Docket Number: 64828-710.601 potential. In some cases, electro-osmotic forces may act against an electrophoretic force during analyte capture. In some embodiments, electro-osmotic forces may dominate an electrophoretic force during analyte capture. In some embodiments which use electrophoretic mechanisms to sense polymers, it can be advantageous to reduce the net ion-selectivity and / or electro-osmotic flux to a level where electrophoretic forces dominate analyte capture. For example, the anion ion-selectivity bias and resulting net anionic electro-osmotic flux can be reduced by introducing acidic residues by substitution adjacent to the aromatic mutations. In another example, net positive charge can be also reduced by replacing basic residues with neutral or acidic residue(s), optionally by substitution with aromatic residue(s) that also separately and additively improve peptide capture and / or discrimination (e.g. CytK-K128F and Aer-K238F). In some embodiments, a pore may be engineered to contain regions of 1, 2, 3, 4, 5, 6, or more separate rings of charges along the longitudinal length of the channel. In some embodiments, the rings may be spaced 0.5 nm, 1.0 nm, 1.5 nm, 2.0 nm, 3.0 nm or further from each other.

[0363] In some embodiments, a pore comprising a point mutation described herein can have an open pore current of at least about 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or about 400 pA. In some embodiments, a pore comprising a point mutation described herein can have an open pore current of at least about 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or about 400 pA at a pH of less than about 2, 3, 3.8, 4, 4.5, 7, 7.5, or about 8. In some embodiments, a pore comprising a point mutation described herein can have an open pore current of at most about 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or about 400 pA. In some embodiments, a pore comprising a point mutation described herein can have an open pore current of at most about 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or about 400 pA at a pH of less than about 2, 3, 3.8, 4, 4.5, 7, 7.5, or 8.

[0364] In some embodiments, a pore comprises an aromatic amino acid within a lumen of the pore. In some embodiments, a pore comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 aromatic amino acids within a lumen of the pore. In some embodiments, a pore comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 aromatic amino acids within a lumen of the pore. In some embodiments, an aromatic amino acid within a lumen of a pore increases an analyte interaction with a lumen of a pore. In some embodiments, an aromatic amino acid within a lumen of a pore increases an analyte residence time withing a pore. In some embodiments, an aromatic amino acid within a lumen of a pore increases signal differences between different analytes.WSGR Docket Number: 64828-710.601

[0365] In some embodiments, a pore comprises a monomer. In some embodiments, a pore comprises a dimer, a trimer, a tetramer, pentamer, a hexamer, a heptamer, an octamer, a nonamer, a decamer, an undecamer, or a dodecamer . In some embodiments, a pore comprises an oligomer. In some embodiments, a pore comprise a homo-oligomer or a hetero-oligomer. In some embodiments, a pore can comprise a plurality of subunits. In some embodiments, a pore comprises several repeating subunits, such as at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, a pore comprises several repeating subunits, such as at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, a plurality of subunits of a pore may be axially aligned. In some embodiments, a plurality of subunits of a pore comprise an approximately central axis. In some embodiments, a pore comprises a channel through which an ion can flow. In some embodiments, a pore comprises a plurality of subunits. In some embodiments, a pore comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 subunits. In some embodiments, a pore comprises at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 subunits. In some embodiments, a pore comprises a ring of multiple identical mutations. In some embodiments, a pore comprises a ring of multiple identical mutations in a recognition region that can be co-planar with a membrane and orthogonal to the direction of analyte passage. In some embodiments, a pore comprises different mutations for its subunits to comprise a hetero-oligomeric assembly. In some embodiments, a pore comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or more mutant monomers. The number of mutated units can be adapted to modulate the extent / magnitude of the change to the recognition region. In some embodiments, a pore comprises mutations on one or more beta-strands. In some embodiments, beta-strands can comprise mutations either in the down strand or in the up strand of a beta strand, or in both. In some embodiments the pore comprises mutations to one or more alpha-helices. In some embodiments a mutation can be inward or lumen facing. In some embodiments a mutation can be outward or membrane facing.

[0366] In some embodiments, a pore comprises a plurality of protomers. In some embodiments, a plurality of protomers can be comprised in an assembly that forms at least a portion of the pore, wherein the protomers form a channel region of the pore. In some embodiments, a plurality of protomers can comprise identical sequences or different sequences. In some embodiments, a pore comprises a plurality of mixed protomers. In some embodiments, a plurality of protomers may be separated from a pore. In some embodiments, a plurality of protomers may be fused to a pore. In some embodiments, a pore lumen comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 charged amino acids pointing towards the lumen of the pore per protomer. In some embodiments, a pore lumen comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 charged amino acids pointing towards the lumen of the pore per protomer. In some embodiments, a pore lumen comprises a plurality of negatively charged amino acids. In some embodiments, a pore lumen comprises a plurality of positively charged amino acids. In someWSGR Docket Number: 64828-710.601 embodiments, a pore can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, or about 100 individual proteins. In some embodiments, a pore can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, or about 100 individual proteins. In some embodiments, a pore can comprise charged residues either in rings around the pore in plane with the membrane, and / or vertically up the pore channel perpendicular to plane of the membrane. In some embodiments, a pore comprises charged residues at a pore entry. In some embodiments, a pore comprises charged residues at a pore exit. In some embodiments, a pore comprises charged residues at a pore constriction. In some embodiments, charged residues can comprise Asp, Glu, Arg, Lys, His, or non-natural amino acids.

[0367] In some embodiments, a pore can be configured to detect analytes larger than 40 kDa. The size of a pore opening, channel, or constriction region, or any combination thereof, may be large enough to accommodate a large analyte (e.g., an analyte larger than 40 kDa). In some embodiments, a pore can be configured to detect analytes larger than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or about 10000 kDa. In some embodiments, a pore can be configured to detect analytes smaller than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or about 10000 kDa. In some embodiments, a pore comprises a cylindrically-shaped region. In some embodiments, a pore comprises a cone-shaped region. In some embodiments, a pore comprises a cylindrical chamber on a second side (e.g., trans side). In some embodiments, a pore comprises a cone chamber on a first side (e.g., cis side). In some embodiments, a pore comprises a cylindrical chamber on a second side (e.g., trans side) and a cone chamber on a first side (e.g., cis side) separated by an inner constriction. In some embodiments a pore comprises an hourglass shape. In some embodiments a pore comprises a cone shape on a first side (e.g., cis side) and a cone shape on a second side (e.g., trans side), separated by an inner constriction.

[0368] In some embodiments, a pore comprises an affinity tag, e.g., a His-tag or Strep-tag. In some embodiments, an affinity tag can be appended to a N- or C-terminus of a pore or a subunit thereof. In some embodiments, an affinity tag can be appended to a pore or a subunit thereof via a linker, e.g., a GSA linker. An affinity tag may comprise a Glutathione-S-transferase (GST) tag, a Maltose-Binding Protein (MBP) tag, a FLAG tag, a c-myc tag, a hemagglutinin (HA) tag, a T7 tag, a calmodulin-binding peptide (CBP) tag, a biotinylation tag, or any combination thereof. In some embodiments, a linker can comprise (GGGGS)n and / or (SG)n, where n may comprise any integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10). In some embodiments, a linker described herein may comprise (GGGGS)3, (Gly)8, (Gly)6, (EAAAK)3, (EAAAK)n, VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSNSY, GGIEGRGS, TRHRQPRGWE,WSGR Docket Number: 64828-710.601 AGNRVRRSVG, RRRRRRRRR, GFLG, A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro)n, disulfide bond, or cysteine linkages, or any combination thereof.

[0369] Gated nanopores can comprise nanopores described herein that may act as rapid, closable channels to gate transport of one or more analytes across a membrane. In some embodiments, a nanopore may exhibit spontaneous gating, in which the channel may not be open for an analyte to enter, translocate through, or any combination thereof. In some embodiments, a pore may exhibit no spontaneous gating for a period of at least about 10, 20, 30, or 60 s at an applied potential of less than about -50 mV or greater than about 50mV. In some embodiments, a pore may exhibit no spontaneous gating for a period of at most about 10, 20, 30, or 60 s at an applied potential of -50 mV. In some embodiments, a pore may exhibit no spontaneous gating for a period of at least about 5, 10, 15, or 20s at an applied potential of -150 mV. In some embodiments, a pore may exhibit no spontaneous gating for a period of at most about 5, 10, 15, or 20s at an applied potential of -150 mV.

[0370] In some embodiments, a pore comprises residues configured to anchor the pore to a membrane. In some embodiments, a pore comprises a mutation that increases the solubility of the pore or a subunit thereof in a lipid bilayer. In some embodiments, a pore comprises a mutation that increases the solubility of the pore or a subunit thereof in a lipid bilayer in an external water environment. In some embodiments, a pore comprises residues configured to improve insertion efficiency into a membrane. In some embodiments, a pore comprises residues configured to reduce spontaneous gating. In some embodiments, a pore comprises residues configured to reduce signal noise. The signal noise can be noise in a signal comprising ionic current, impedance, current rectification, potential, tunnelling, conductivity, light, or mechanical deformation.

[0371] In some embodiments, a pore comprises a protease. In some embodiments, a protease can be configured to degrade a polypeptide into a fragment. In some embodiments, one or more proteases degrade a polypeptide into one or more fragments. In some embodiments, a fragment enters a channel of a pore. In some embodiments, a polypeptide fragment comprises a portion comprising a non-natural amino acid, polyethylene glycol, PNA, DNA, or RNA, or combinations thereof. In some embodiments, a protease can be active. In some embodiments, a protease can be inactive. In some embodiments, a polypeptide can be degraded into a fragment before translocation through a pore.

[0372] In some embodiments, a pore comprises an unfoldase. In some embodiments, an unfoldase linearizes a polymer which enters the pore. In some embodiments, a polymer threads through a pore while it can be recognized by ionic currents. In some embodiments, an unfoldase comprises a prokaryotic AAA+ unfoldase, ClpX, PAN unfoldase, or Valosin-containing protein-like ATPase. In some embodiments, an unfoldase may be modulated by an adaptor protein or other accessory proteins or chaperones. In some embodiments, the AAA+ enzyme (e.g., AAA+ unfoldase) is selected from the group consisting of ClpX, ClpA, Pan, LON, VAT, AMA,WSGR Docket Number: 64828-710.601 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, and functional homologs, orthologs, paralogs thereof.

[0373] In some embodiments, a pore comprises a ring-forming protein. In some embodiments, a ring-forming protein can be configured to transport a polymer, e.g. a polypeptide, across a transmembrane region of a pore. In some embodiments, a ring-forming protein comprises a toroidal or donut-shaped multi-subunit protein. In some embodiments, a ring-forming protein comprises a ring-forming multimeric protein, such as an octameric, heptameric or hexameric protein. In some embodiments, a ring-forming protein comprises a heptameric protein. In some embodiments, a heptameric protein include those submitted to the Protein Data Bank (PDB) under one of the following unique accession or identification code codes: lg31, 1h64, 1hx5, 1i4k, 1i5l, 1i8f, 1i81, 1iok, 1j2p, 1jri, 1lep, 1lnx, 1loj, 1mgq, 1n9s, 1ny6, 1p3h, 1tzo, 1wnr, 1xck, 2cb4, 2cby, 2yf2, 3bpd, 3cf0, 3j83, 3ktj, 3m0e, 3st9, 4b0f, 4emg, 4gm2, 4hnk, 4hw9, 4jcq, 4ki8, 4owk, 4qhs, 4xq3, 5jzh, 5msj, 5msk, 5mx5 and 5uw8e.

[0374] In some embodiments, a transmembrane portion of a pore comprises a linker. The linker may be a helical linker, a non-helical linker, a flexible linker, or a cleavable linker, or any combination thereof. In some embodiments, a linker can by hydrophilic or mostly hydrophilic. In some embodiments, a transmembrane portion of a pore comprises a flexible hydrophilic linker. A flexible linker can comprise a linker rich in small and / or polar amino acids (e.g., glycine, serine, or threonine, or any combination thereof), which can provide good flexibility and solubility. For example, a transmembrane portion can comprise on the N-and / or C-terminal side a flexible hydrophilic linker of at least about 3, 4, 5, 10, 15, or 20 amino acids. A transmembrane portion can comprise on the N-and / or C-terminal side a flexible hydrophilic linker of at most about 3, 4, 5, 10, 15, or 20 amino acids. In some embodiments, a hydrophilic linker can interact with the charged head groups of membrane (phospho)lipids. For example, hydrophilic residues include serine, threonine, asparagine, glutamine, aspartate, glutamate, lysine and arginine. In some embodiments, a hydrophilic linker comprises at least a portion comprising non-hydrophilic or non-natural amino acids to tune the linker’s properties.

[0375] In some embodiments, a pore comprises a protease. In some embodiments, a pore comprises a protease- unfoldase pair. In some embodiments, a protease-unfoldase pair can be attached on a first side (e.g., cis side) of a FraC pore. Then, cleaved peptides can be sequentially recognized and translocated across the pore. In some embodiments, a protease-unfoldase pair can comprise a barrel-shaped ATP-dependent ClpXP protease. In some embodiments, a protease can encase digested peptides, preventing their release in solution.

[0376] In some embodiments, a pore can be configured to proteolytically cleave analytes. In some embodiments, a pore can be configured to not to proteolytically cleave analytes. The conditions of a pore and / or nanopore system may comprise ATP concentration, or buffer types, or any combination thereof. The conditions of the pore and / or nanopore system can be configured to cleave or not to cleave analytes. For example, VATWSGR Docket Number: 64828-710.601 can be capable of feeding the polypeptide through the pore at a speed that can be tuned by the concentration of ATP. A transmembrane proteasome can simultaneously process and identify different analytes. In some embodiments, translocated peptides may be proteolytically degraded. In some embodiments, the pore can be employed with an inactivated protease / proteasome which recognizes proteins as they are linearized and transported across the pore at a controlled speed. In some embodiments, the activity of the protease / proteasome can be monitored at the single molecule level. In some embodiments, translocated peptides may not be proteolytically degraded. In some embodiments, a proteasome and / or a portion of a proteasome may comprise a protease domain, or a translocase domain, or any combination thereof.

[0377] In some embodiments, a pore comprises natural or non-natural aromatic amino acid residues. In some embodiments, a non-natural aromatic amino acid can be selected from the group consisting of 3,4-dihydroxy- L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine, L-thyroxine, phenylglycine (Phg) or nor-tyrosine (norTyr). In some embodiments, a non-natural aromatic amino acid can be a D-amino acid, a Homo-amino acid (methylene), a Beta-homo-amino acid, a N-methyl amino acid, or an Alpha-methyl amino acid. In some embodiments, a non-natural aromatic amino acid can be a derivatized Phe / Tyr / Trp amino acid, e.g., a ring- substituted Phe / Tyr / Trp amino acids. In some embodiments, a non-natural aromatic amino acid can be a derivative of Phe, Tyr or Trp, substituted by, e.g., a halogen, -CH3, OH, -CH2NH3, -C(O)H, -CH2CH3,-CN, - CH2CH2CH3, -SH, or another group. Non-natural aromatic amino acids include, but are not limited to, O- methyl-L-tyrosine; 3-methyl- phenylalanine; a p-acetyl-L-phenylalanine; O-4-allyl-L-tyrosine; 4-propyl-L- tyrosine; fluorinated phenylalanine; isopropyl-L-phenylalanine; ap-azido-L- phenylalanine; a p-acyl-L- phenylalanine; a p-benzoyl-L-phenylalanine; a phosphonotyrosine; a p-iodo-phenylalanine; p- bromophenylalanine; p- amino-L-phenylalanine; an isopropyl-L-phenylalanine; an amino-, isopropyl-, or O- allyl-containing phenylalanine analogue; a p-(propargyloxy) phenylalanine; 3-nitro-tyrosine; 5-fluoro- tryptophan, 5-hydroxy-tryptophan, 5-methoxy- tryptophan, 5-methyl-tryptophan, trifluoromethyl-tryptamine ethyl ester.

[0378] In some embodiments, a non-naturally-occurring amino acids may be introduced by including synthetic aminoacyl-tRNAs in the IVTT system used to express a mutant monomer. In some embodiments, a non- naturally-occurring amino acids may be introduced by expressing a mutant monomer in E. coli that may be auxotrophic for specific amino acids in the presence of synthetic (i.e. non-naturally-occurring) analogues of those specific amino acids. In some embodiments, a non-naturally-occurring amino acids may be introduced using synthetic peptide chemistry methods. Monomeric units of the pores may be formed entirely from synthetic peptides constructed using conjugation methods, e.g., chemical ligation, or cysteine coupling. In some embodiments, monomers of the pore may comprise partially synthetic units coupled to naturally expressed peptide units using coupling methods.WSGR Docket Number: 64828-710.601

[0379] In some embodiments, a pore comprises an alpha-helical or beta-barrel oligomeric pore forming toxin or porin. A pore can comprise a beta-barrel pore forming protein and / or peptide and / or an alpha helical pore forming protein and / or peptide. In some embodiments, a pore can be selected from the group consisting of Aerolysin (Aer), Cytolysin K (CytK), Mycobacterium smegmatis porin A (MspA), alpha-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, phage derived portal proteins (Phi29, or G20c, or any combination thereof) or a mutant thereof. In some embodiments, a pore comprises a phage portal complex, or a cellular transmembrane transport complex. In some embodiments, a pore comprises an alpha-helix bundle or channel. In some embodiments, a pore comprises a transmembrane protein pore derived from beta-barrel pores or alpha- helix bundle pores. In some embodiments, a beta-barrel pore or beta-barrel pore forming protein and / or peptide comprises a barrel or channel structure comprising beta-strands. In some embodiments, a pore comprises charged residues on both the “up” strands and the “down” of a beta-strand. In some embodiments, charged residues can be located at sequence positions that co-locate them approximately co-planar in a beta-barrel.

[0380] In some embodiments, the nanopore comprises a pore-forming toxin. The nanopore can comprise an α- pore-forming toxin, or a β-pore-forming toxin, or any combination thereof. The nanopore can comprise a pore- forming toxin derived from a bacterium. The bacterium can be of a genus of bacteria including, but not limited to, Xenorhabdus, Yersinia, Providencia, Pseudomonas, Proteus, Morganella, or Photorhabdus. In some embodiments, the nanopore comprises a pore-forming toxin derived from a bacterial species selected from the group consisting of Escherichia coli, Mycobacterium smegmatis, Staphylococcus aureus, Salmonella typhi, P. aeruginosa, A. baumanii, Klebsiella oxytoca, Bacillus cereus, A. hydrophila, S. marcescens, V. cholerae, P. entomophila, C. perfringens, and Y. enterocolitica. In some embodiments, a nanopore described herein may comprise one or more monomers of a T7 pore, a PN pore, a stable protein 1 (SP1) pore, a Phi29 pore, a PlyAB pore, an alpha-hemolysin (α-HL) pore, a SPP1 pore, a FraC pore, a MspA pore, a CsgG pore, an OmpG pore, an aerolysin pore, a cytolysin A (ClyA) pore, a FhuA pore, a PFO pore, a TMH4C4 pore, any combination thereof, or homolog, paralog, ortholog, or any combination thereof.

[0381] In some embodiments, the nanopore (e.g., the biological nanopore) can comprise a conical geometry or a semi-conical geometry. A conical geometry can comprise a shape in which a nanopore tapers over a longitudinal axis, wherein a first entrance of a nanopore is larger (e.g., comprises a wider dimension) than a second entrance. In some embodiments, the nanopore (e.g., the biological nanopore) can comprise a straight geometry (e.g., a cylindrical geometry). A straight geometry may comprise a shape in which a channel of a nanopore can be the same width (e.g., diameter) over its longitudinal axis. In some embodiments, the nanopore can be a T7 pore, a PN pore, a stable protein 1 (SP1) pore, a Phi29 pore, a PlyAB pore, an alpha-hemolysin (α- HL) pore, a SPP1 pore, a FraC pore, a MspA pore, a CsgG pore, an OmpG pore, an aerolysin pore, a cytolysin A (ClyA) pore, a FhuA pore, a PFO pore, or a TMH4C4 pore, or any combination thereof. In someWSGR Docket Number: 64828-710.601 embodiments, the nanopore described herein may comprise one or more monomers from T7, PN, SP1, Phi29, PlyAB, α-HL, SPP1, FraC, MspA, CsgG, OmpG, aerolysin, ClyA, FhuA, PFO, or TMH4C4, or any combination thereof. In some embodiments, an engineered biological nanopore described herein may comprise one or monomers from a T7 pore, a PN pore, a SP1 pore, a Phi29 pore, a PlyAB pore, an alpha-hemolysin (α- HL) pore, a SPP1 pore, a FraC pore, a MspA pore, a CsgG pore, an OmpG pore, an aerolysin pore, a ClyA pore, a FhuA pore, a PFO pore, or a TMH4C4 pore, or any combination thereof. In some embodiments, the nanopore described herein may comprise one or more monomers from T7, PN, SP1, Phi29, PlyAB, α-HL, SPP1, FraC, MspA, CsgG, OmpG, aerolysin, ClyA, FhuA, PFO, or TMH4C4, or any combination thereof. In some embodiments, the nanopore (e.g., the biological nanopore) can comprise a vestibule geometry (e.g., a globular geometry or goblet geometry). The nanopore may comprise an alpha-hemolysin nanopore, or a curli specific gene G (CsgG) nanopore, or any combination thereof.

[0382] In some embodiments, a pore comprises comprise beta-toxins, such as alpha-hemolysin, anthrax toxin and leukocidins. In some embodiments, a pore comprises outer membrane proteins / porins of bacteria, such as Mycobacterium smegmatis porin (Msp), for example MspA, MspB, MspC or MspD, CsgG. In some embodiments, a pore comprises outer membrane porin F (OmpF). In some embodiments, a pore comprises outer membrane porin G (OmpG). In some embodiments, a pore comprises outer membrane phospholipase A. In some embodiments, a pore comprises outer membrane protein FhuA. In some embodiments, a pore comprises outer membrane protein A (OmpA). In some embodiments, a pore comprises Neisseria autotransporter lipoprotein (NalP). In some embodiments, a pore comprises lysenin. In some embodiments, a pore comprises bacterial nucleoside transporter Tsx. In some embodiments, a pore comprises inner membrane proteins and outer membrane proteins, such as WZA and FraC. In some embodiments, a pore comprises Aer, CytK, MspA, aHL, CsgG, or FraC or an engineered mutant thereof. In some embodiments, a pore comprises a transmembrane pore derived from or based on Msp, e.g. MspA, a-hemolysin (a-HL), lysenin, CsgG, ClyA, Spl or haemolytic protein fragaceatoxin C (FraC). In some embodiments, a pore comprises an oligomeric accessory protein coupled to a transmembrane domain of the pore. In some embodiments, a pore comprises alpha-helical or beta- barrel transmembrane regions. In some embodiments, a beta-barrel pore or beta-barrel pore forming protein and / or peptide can include, but may not be limited to, beta-toxins, such as alpha-hemolysins, aerolysins, lysenin, cytolysins, cytolysin K, anthrax toxin and leukocidins, and outer membrane proteins / porins of bacteria, such as Mycobacterium smegmatis porin (Msp), for example MspA, MspB, MspC or MspD, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A (OMPLA), ferric hydroxamate uptake component A (FhuA), Curli production transport component CsgG, and Neisseria autotransporter lipoprotein (NalP). In some embodiments, an alpha-helix pore or alpha-helical forming protein and / or peptide can include, but may not be limited to, inner membrane proteins and outer membrane proteins, such asWSGR Docket Number: 64828-710.601 Actinoporins, the outer membrane core complex (OMCC) of H. pylori Cag T4SS particles, and the transmembrane domain of the E. coli polysaccharide transporter Wza. In some embodiments, a pore comprises CytK or a genetically engineered mutant thereof. In some embodiments, a pore can comprise a porin. In some embodiments, a pore can comprise OmpF, OmpG, or FhuA. In some embodiments, a pore comprises a pore forming protein (PFP). In some embodiments, a PFP comprises an α-PFP. In some embodiments, a PFP comprises a β-PFPs. In some embodiments, a PFP comprises a bundle of α-helices. In some embodiments, a PFP comprises a transmembrane β-barrel.

[0383] In some embodiments, the nanopore comprises CytK or a genetically engineered mutant thereof. In some embodiments, the mutant CytK comprises one or more of the amino acid substitutions selected from the group consisting of K128D, K128F, K155D, S120D, Q122D, G122D and S151D. FIGs. 3A-3D depict representations of a CytK nanopore and one or more mutations. FIG. 3A shows a surface representation of a pore (e.g., a CytK pore) and FIG. 3B depicts a cartoon representation of a β-barrel region, with N-terminal strands depicted as dark gray and C-terminal strands depicted as light gray. Residues include E112, T116, S120, Q122, S126, K128, E139, T143, Q145, T147, S151, and K155 (charged residues in bold). FIG. 3C shows residues in each beta strand of the transmembrane beta-barrel region of wild-type CytK, marking water-facing residues of the down- and up- strands most suitable for mutagenesis. In some embodiments, as shown in FIG. 3D, a mutant pore (e.g., a mutant CytK pore) can be a high ion selectivity mutant (e.g., CytK-2E-4D). The mutant pore can be in 1 M KCl, pH 7.5 {(p(K) / p(Cl) of 4.04 ± 0.07, and p(K) / p(Cl) of 1.3 at pH 3.8)}. Substitutions of the CytK nanopore can comprise one or more amino acid substitutions comprising K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, S151D, K155Q, T116D, S126D, T143D, Q145D, T147D, or any combination thereof.

[0384] In some embodiments, a nanopore described herein can comprise one or more CytK monomers. A CytK monomer may comprise a wildtype CytK monomer. A CytK monomer may comprise a mutant CytK monomer. A CytK monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NOs.: 8 or 107. In some embodiments, a CytK monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NOs.: 8 or 107. In some embodiments, a CytK monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution) in an amino acid sequence as set forth in SEQ ID NOs.: 8 or 107.WSGR Docket Number: 64828-710.601

[0385] In some embodiments, a CytK monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs.: 9-26 or 108-112. In some embodiments, a CytK monomer of a nanopore described here may comprise an amino acid sequence as set forth in any one of SEQ ID NOs.: 9-26 or 108-112. In some embodiments, a CytK monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in any one of SEQ ID NOs.: 9-26 or 108-112.

[0386] In some embodiments, a pore comprises engineered pores. In some embodiments, a pore comprises an oligomeric accessory protein coupled to a transmembrane domain. In some embodiments, a pore can be derived from naturally existing nanopores. In some embodiments, a pore comprises a protein pore templated by a DNA structure. In some embodiments, a pore comprises a de novo pore based on de novo alpha-helical or beta-barrel transmembrane regions. In some embodiments, a pore can comprise a de novo pore based on de novo beta- barrel pore forming protein and / or peptide and / or alpha-helical pore forming protein and / or peptide.

[0387] In some embodiments, a pore can be a portion of an existing pore or mutations thereof. In some embodiments, a pore comprises a combination of elements of distinct pores or mutations thereof. In some embodiments, a pore comprises an assembly of genetically engineered pleurotolysin (Ply) A and B subunits. In some embodiments, a pore comprises a protein of the Membrane Attack Complex PerForin / Cholesterol Dependent Cytolysin (MACPF / CDC) protein superfamily. In some embodiments, a pore comprises perforin, complement C9, pneumolysin, or lysteriolysin. In some embodiments, a pore comprises a recognition or a binding portion configured to recognize or bind to an analyte. In some embodiments, a pore comprises a transmembrane portion comprising a channel for an analyte to pass through. In some embodiments the pore comprises a molecular or proteinaceous adapter. In some embodiments the molecular or proteinaceous adapter acts as a recognition or binding site for analytes. In some embodiments, the molecular or proteinaceous adapter can be a cyclic adapter. The adapter can be bound covalently or non-covalently.

[0388] In some embodiments, a pore comprises a proteasome. In some embodiments, the proteasome can be a S20 proteasome, a 26S proteasome, a 11S proteasome, a ClpXP proteasome, open reading frame number 854 in the M. mazei genome, or combinations thereof. In some embodiments, the proteasome comprises a subunit or components of a proteasome. In some embodiments, a proteasome can be a fused proteasome. In some embodiments, a C-terminus of a subunit of a ring-forming (multimeric) protein comprising a flanked transmembrane sequence can be genetically fused to a N-terminus of a proteasome subunit. In someWSGR Docket Number: 64828-710.601 embodiments, a ring-forming protein subunit can be fused to an N-terminally truncated proteasome subunit such that the proteasome gate can be left open towards. In some embodiments, a pore comprises a proteasome fused to the pore. In some embodiments, a pore comprises a proteasome fused to the pore such that the proteasome can be located on the first side (e.g., cis side) of the pore when the pore can be disposed in a membrane. In some embodiments, a proteasome can be coupled to a pore. In some embodiments, a proteasome can be coupled non-covalently to a pore. A proteasome may be fused and / or coupled to a pore by a linker described herein. The linker may be a helical linker, a non-helical linker, a flexible linker, a cleavable linker, or any combination thereof. In some embodiments, a proteasome can be coupled to a pore on a first side (e.g., cis side) of a membrane. In some embodiments, a proteasome can be coupled to a pore on a second side (e.g., trans side) of a membrane. In some embodiments, a pore comprises one or more proteasome subunits. In some embodiments, a pore comprises a proteasome α-subunit. In some embodiments, a pore comprises a proteasome β-subunit. In some embodiments, a pore comprises a ring of proteasome α-subunits. In some embodiments, a pore comprises a ring of proteasome β-subunits. In some embodiments, a pore comprises a catalytically active subunit. In some embodiments, a pore comprises a protease. In some embodiments, a pore comprises a protease having a trypsin-type or chymotrypsin-type of activity.

[0389] In some embodiments, a pore comprises prokaryotic AAA+ unfoldase ClpX. ClpX can unfold analytes by NTP-driven translocation of the polypeptide chain through the central pore of its hexameric assembly. In complex with the ClpP peptidase, ClpX can carry out protein degradation by translocating unfolded analytes directly into the ClpP proteolytic chamber. In some embodiments, a pore comprises a multi-protein pore sensor complex comprising an artificial ClpP pore, e.g. by fusion to PA, which sensor complex further comprises ClpX or a homologous protein unfoldase.

[0390] In some embodiments, a pore comprises an oligomeric Fragaceatoxin C (FraC) pore. In some embodiments, FraC can be a type II pore. In some embodiments, a type II FraC pore comprises an apparent heptameric stoichiometry, and / or a conductance of about 1.22-1.26 nS when assayed at pH 7.5 in a 1M NaCl solution or about 0.99-1.08 nS when assayed at pH 4.5 in a 1 M KC solution. Conductance values can be determined by collecting single channels under -50 mV applied potential using 1 M NaCl, 15 mM Tris pH 7.5, or 1 M KCl, 0.1 M citric acid, 180 mM Tris base pH 4.5. In some embodiments, a type II FraC pore can comprise a pore size (at the narrowest constriction) of about 1.1 nm, which can be determined from homology modeling.

[0391] In some embodiments, FraC can be a type III pore. In some embodiments, a type III FraC pore comprises an apparent hexameric stoichiometry, and / or a conductance of about 0.37-0.43 nS when assayed at pH 4.5 in a 1M KC solution. In some embodiments, a type III FraC pore comprises a pore size (at the narrowest constriction) of about 0.8 nm, which can be determined from homology modeling.WSGR Docket Number: 64828-710.601

[0392] In some embodiments, a nanopore described herein can comprise one or more FraC monomers. A FraC monomer may comprise a wildtype FraC monomer. A FraC monomer may comprise a mutant FraC monomer. A FraC monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NOs.: 51 or 52. In some embodiments, a FraC monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NOs.: 51 or 52. In some embodiments, a FraC monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NOs.: 51 or 52.

[0393] In some embodiments, a nanopore described herein can comprise one or more alpha-hemolysin (α- hemolysin) monomers. An alpha-hemolysin (α-hemolysin) monomer may comprise a wildtype alpha- hemolysin (α-hemolysin) monomer. An alpha-hemolysin (α-hemolysin) monomer may comprise a mutant alpha-hemolysin (α-hemolysin) monomer. An alpha-hemolysin (α-hemolysin) monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO.: 106. In some embodiments, an alpha-hemolysin (α-hemolysin) monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NO.: 106. In some embodiments, an alpha-hemolysin (α- hemolysin) monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NO.: 106.

[0394] In some embodiments, a nanopore described herein can comprise one or more CsgG monomers. A CsgG monomer may comprise a wildtype CsgG monomer. A CsgG monomer may comprise a mutant CsgG monomer. A CsgG monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO.: 128. In some embodiments, a CsgG monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NO.: 128. In some embodiments,WSGR Docket Number: 64828-710.601 a CsgG monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NO.: 128.

[0395] In some embodiments, a MspA monomer may comprise a wildtype MspA monomer. In some embodiments, a wildtype MspA monomer may comprise an amino acid sequence as set forth in MGLDNELSLVDGQDRTLTVQQWDTFLNGVFPLDRNRLTREWFHSGRAKYIVAGPGADEFEGTLELG YQIGFPWSLGVGINFSYTTPNILIDDGDITAPPFGLNSVITPNLFPGVSISADLGNGPGIQEVATFSVDVS GAEGGVAVSNAHGTVTGAAGGVLLRPFARLIASTGDSVTTYGEPWNMNGSAGSAWSHPQFEK (SEQ ID NO.: 171). In some embodiments, a nanopore described herein can comprise one or more MspA monomers. A MspA monomer may comprise a mutant MspA monomer. A MspA monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO.: 171. In some embodiments, a MspA monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NO.: 171. In some embodiments, a MspA monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NO.: 171.

[0396] In some cases, an MspA nanopore may comprise a mutation in a lumen-facing region, or constriction region, or any combination thereof. In some embodiments, a mutation of an MspA nanopore can comprise a in a lumen-facing region and / or constriction region from one or more amino acid residues to one or more aromatic residues (e.g., tryptophan (W) residue, phenylalanine (F) residue, tyrosine (Y) residue, or histidine (H) residue, or any combination thereof), one or more negatively-charged amino acid residues, one or more positively- charged amino acid residues, one or more neutral-charged residues, one or more acidic amino acid residues, one or more amidic amino acid residues (e.g., asparagine (N) residue and / or glutamine (Q) residue), one or more sulfur-containing amino acid residues, or any combination thereof.

[0397] In some embodiments, a monomer of a MspA nanopore may comprise a mutation at position D90, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 171. In some embodiments, a monomer of a MspA nanopore may comprise a mutation at position D91, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 171. In some embodiments, the mutation at position D90 and / or D91 of the MspA monomer can comprise one or more mutations (e.g., a substitution mutations) to one or more aromatic residues (e.g., tryptophan (W) residue, phenylalanine (F) residue, tyrosine (Y) residue, or histidine (H) residue, or any combination thereof), one or more negatively-WSGR Docket Number: 64828-710.601 charged amino acid residues, one or more positively-charged amino acid residues, one or more neutral-charged residues, one or more acidic amino acid residues, one or more amidic amino acid residues (e.g., asparagine (N) residue and / or glutamine (Q) residue), or one or more sulfur-containing amino acid residues, or any combination thereof, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 171.

[0398] In some embodiments, the MspA nanopore can comprise a mutation (e.g., a substitution mutation) at position D90 to an asparagine residue, wherein the mutation comprises D90N and wherein the residue numbering corresponds to SEQ ID NO: 171. In some embodiments, the MspA nanopore can comprise a mutation (e.g., a substitution mutation) at position D91 to an asparagine residue, wherein the mutation comprises D91N and wherein the residue numbering corresponds to SEQ ID NO: 171.

[0399] In some embodiments, a monomer of a nanopore described herein (e.g., a MspA nanopore) may comprise a mutation at a position 83, 88, 103, 105, or 108, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 171. In some embodiments, a nanopore (e.g., a MspA nanopore) can comprise one or more mutations of one or more monomers. The mutations of one or more monomers may comprise one or more mutations at position S103, I105, N108, T83, or L88, or any combination thereof, to one or more aromatic residues (e.g., tryptophan (W) residue, phenylalanine (F) residue, tyrosine (Y) residue, or histidine (H) residue, or any combination thereof), one or more negatively-charged amino acid residues, one or more positively-charged amino acid residues, one or more neutral-charged residues, one or more acidic amino acid residues, one or more amidic amino acid residues (e.g., asparagine (N) residue and / or glutamine (Q) residue), or one or more sulfur-containing amino acid residues, or any combination thereof. In some embodiments, nanopore (e.g., MspA nanopore) comprises at least one mutation (e.g., a substitution mutation) in one or more monomers, wherein the mutation comprises S103E, I105E, N108E, L88E, or T83E, or any combination thereof, and wherein the residue numbering corresponds to SEQ ID NO: 171. In some embodiments, a monomer of a nanopore (e.g., MspA nanopore) described herein may comprise one or more mutations at positions D90, D91, D93, A96, T83, L88, S103, I105, or N108, or any combination thereof.

[0400] In some embodiments, a nanopore described herein may comprise any combination of monomers comprising an amino acid sequence as set forth in any one of SEQ ID NOs.: 8-26, 51, 52, 106-112, or 128. In some embodiments, a nanopore described herein may comprise one or more monomers comprising an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs.: 8-26, 51, 52, 106-112, or 128. In some embodiments, a nanopore described herein may comprise one or moreWSGR Docket Number: 64828-710.601 monomers comprising an amino acid sequence as set forth in any one of SEQ ID NOs.: 8-26, 51, 52, 106-112, or 128. In some embodiments, a nanopore described herein may comprise one or more monomers comprising at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in any one of SEQ ID NOs.: 8-26, 51, 52, 106-112, or 128.

[0401] In some embodiments, a CsgG monomer may comprise a wildtype CsgG monomer. In some embodiments, a wildtype CsgG monomer may comprise an amino acid sequence as set forth in CLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQFKPYPASNFSTAVPQSATAML VTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMVEGSIIGYESNVKSG GVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVG YTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES (SEQ ID NO.: 211). In some embodiments, a nanopore described herein can comprise one or more CsgG monomers. A CsgG monomer may comprise a mutant CsgG monomer. A CsgG monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, a CsgG monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, a CsgG monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, a nanopore described herein can comprise an engineered CsgG nanopore.

[0402] The CsgG pore may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more monomers. The CsgG pore may be a CsgG / F pore. The CsgG / F pore may comprise a CsgG nanopore with a channel region and constriction region. The pore may further comprise a CsgF protein (e.g., a CsgF peptide adapter). In some embodiments, a monomer of a CsgG nanopore may comprise a mutation at position Y51, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, a monomer of a CsgG nanopore may comprise a mutation at position N55, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, a monomer of a CsgG nanopore may comprise a mutation at position F56, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, a monomer of a CsgG nanopore may comprise a mutation at position F48, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, a monomer of a CsgG nanopore may comprise a mutation at position F58, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, theWSGR Docket Number: 64828-710.601 mutation at position Y51, N55, F56, F48, F58, or any combination thereof of the CsgG monomer can comprise one or more mutations (e.g., insertion mutations, deletion mutations, substitution mutations) to one or more aromatic residues (e.g., tryptophan (W) residue, phenylalanine (F) residue, tyrosine (Y) residue, or histidine (H) residue, or any combination thereof), one or more negatively-charged amino acid residues, one or more positively-charged amino acid residues, one or more neutral-charged residues, one or more acidic amino acid residues, one or more amidic amino acid residues (e.g., asparagine (N) residue and / or glutamine (Q) residue), or one or more sulfur-containing amino acid residues, or any combination thereof, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211.

[0403] In some embodiments, a pore can be coupled or fused to one or more accessory partner proteins that aids the binding and / or functioning o...

Claims

WSGR Docket Number: 64828-710.601 CLAIMS WHAT IS CLAIMED IS:

1. A method, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting (1) a current or change thereof; or (2) a voltage or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%.

2. The method of claim 1, wherein (c) comprises using (1) the current or change thereof, or (2) the voltage or change thereof to determine the characteristic of the at least the portion of the analyte with an accuracy of at least 80%.

3. A method for determining a characteristic of an analyte, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof, wherein (i) an average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second, or (ii) an average rate of translocation is between about 0.1 nm / s to about 10000 nm / s; (b) detecting (1) a current or change thereof; or (2) a voltage or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte.

4. The method of claim 3, wherein the average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second.

5. The method of claim 4, wherein the average rate of translocation is between about 1 amino acids per second to about 100 amino acids per second with a motor protein.

6. The method of claim 4, wherein the average rate of translocation is between about 500 amino acids per second to about 5000 amino acids per second without a motor protein. -518- WSGR Docket No.64828-710.601WSGR Docket Number: 64828-710.601 7. The method of any one of claims 3-6, wherein the average rate of translocation is between about 0.1 nm / s to about 10000 nm / s.

8. The method of claim 7, wherein the average rate of translocation is between about 0.3 nm / s to about 30 nm / s.

9. The method of any one of claims 1-8, further comprising in (b) detecting the current or change thereof, and (c) comprises using the current or change thereof.

10. The method of any one of claims 1-9, further comprising in (b) detecting the voltage or change thereof, and (c) comprises using the voltage or change thereof.

11. The method of any one of claims 1-10, further comprising, in (c), determining / assigning the one or more characteristics of the at least the portion of the analyte based on (1) an electrical signal or change thereof of (i) the current or change thereof, or (ii) the voltage or change thereof, and (2) a database.

12. The method of claim 11, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more proteoforms thereof, or one or more variants thereof, or one or more fragments thereof, or a combination thereof.

13. A method for characterizing an analyte, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting an electrical signal or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) assigning one or more characteristics to the at least the portion of the analyte based on the electrical signal and a database, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more proteoforms thereof, or one or more variants thereof, or one or more fragments thereof, or a combination thereof.

14. The method of claim 13, wherein the database does not comprise a reference signal associated with the at least the portion of the analyte.

15. The method of claim 13, wherein the database comprises a reference signal associated with the at least the portion of the analyte.

16. The method of any one of claims 13-15, wherein the electrical signal or change thereof may be a measurement of (1) the current or change thereof or (2) the voltage or change thereof. -519- WSGR Docket No.64828-710.601WSGR Docket Number: 64828-710.601 17. The method of any one of claims 13-16, wherein the electrical signal or change thereof comprises (1) one or more reads; or (2) one or more additional portions of the electrical signal or change thereof.

18. The method of claims 17, wherein the one or more additional portions of the electrical signal or change thereof comprises one or more blocks of impurities.

19. The method of any one of claims 13-18, wherein the electrical signal or change thereof comprises measurements of (1) the current or change thereof or (2) the voltage or change thereof over a period of time.

20. The method of claims 19, wherein the period of time comprises one or more portions associated with a measurement of concentration associated with a sample comprising the at least the portion of the analyte.

21. The method of any one of claims 13-20, further comprising, prior to (c), pre-processing the electrical signal or change thereof, thereby generating a pre-processed electrical signal or change thereof.

22. The method of claims 21, wherein the one or more characteristics are assigned using the pre- processed electrical signal or change thereof.

23. The method of claims 21 or 22, further comprising extracting one or more reads from (1) the electrical signal or change thereof or (2) the pre-processed electrical signal or change thereof.

24. The method of claim 23, further comprising pre-processing the one or more reads, thereby generating one or more pre-processed reads.

25. The method of claim 24, wherein the pre-processing comprises denoising, filtering, segmenting, or scaling, or a combination thereof.

26. The method of any one of claims 22-25, wherein the one or more characteristics are assigned using (1) the one or more reads or (2) the one or more pre-processed reads (e.g., one or more segments).

27. The method of claim 26, further comprising comparing (1) the one or more reads or (2) the one or more pre-processed reads to one or more reference signals in the database.

28. The method of claim 27, wherein the comparing comprises alignment.

29. The method of claim 28, wherein the alignment comprises time warping 30. The method of any one of claims 24-29, further comprising scoring (1) the one or more reads or (2) the one or more pre-processed reads to the one or more reference signals, thereby assigning the one or more characteristics to the at least the portion of the analyte.

31. The method of claim 30, wherein scoring comprises aligning at least a portion of the electrical signal of change thereof with the at least the portion of the one or more reference signals. -520- WSGR Docket No.64828-710.601WSGR Docket Number: 64828-710.601 32. The method of any one of claims 24-31, further comprising aggregating (1) the one or more reads or (2) the one or more pre-processed reads to assignment the one or more characteristics to the at least the portion of the analyte.

33. The method of any one of claims 13-32, wherein the one or more characteristics are assigned using the electrical signal or change thereof.

34. The method of any one of claims 13-32, wherein the database is generated from one or more reference sequences.

35. The method of claim 77, wherein the one or more reference sequences are derived with genomic information or transcriptomic information of the sample.

36. The method of claim 35, wherein the genomic information comprises genome sequencing information (e.g., DNA) related to polynucleic acid sequences, abundance, number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, or cellular origin information, or any combination thereof.

37. The method of claim 35 or 36, wherein the transcriptomic information comprises genome sequencing information (e.g., RNA) related to ribopolynucleic acid sequences, abundance, number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, or cellular origin information, or any combination thereof.

38. The method of any one of claims 34-37, wherein the database is generated from the one or more reference sequences using one or more machine learning algorithms.

39. The method of any one of claims 15-38, wherein the database comprises one or more reference signals for the at least the portion of the analyte, or fragments thereof, or at least one proteoform thereof, or at least one variant thereof, or combination thereof.

40. The method of any one of claims 13-39, wherein the one or more polypeptides comprise one or more expressible polypeptides.

41. The method of any one of claims 13-40, wherein the one or more variants thereof comprise one or more post-translationally modified variants thereof.

42. The method of any one of claims 1-41, wherein translocating can comprise translocating the at least the portion of the analytes in the C-to-N direction or in the N-to-C direction relative to the at least the portion of the analytes sequence.

43. The method of any one of claims 1-42, wherein the one or more characteristics comprises a plurality of natural or unnatural post-translational modifications of the at least the portion of the analyte.

44. The method of claim 43, wherein the natural or unnatural post-translation modifications is phosphorylation, glycosylation, deamidation, or acetylation, or any combination thereof. -521- WSGR Docket No.64828-710.601WSGR Docket Number: 64828-710.601 45. The method of any one of claims 1-44, wherein the one or more characteristics comprises a length of the at least the portion of the analyte.

46. The method of any one of claims 1-45, wherein the one or more characteristics comprises an average speed of translocation of the at least the portion of the analyte through the nanopore 47. The method of any one of claims 1-46, wherein the one or more characteristics comprises determining whether the at least the portion of the analyte comprises one or more molecular entities 48. The method of claim 47, wherein the one or more molecular entities is a compound (e.g., drug, small molecule), particle, nucleic acid, polynucleic acid, peptide, polynucleotide, or protein, or fragments thereof, or any combination thereof.

49. The method of any one of claims 1-48, wherein the one or more characteristics comprises a category or identity associated with the at least the portion of the analyte.

50. The method of any one of claims 1-49, wherein the one or more characteristics comprises one or more of a secondary structure, tertiary structure, or quaternary structure, or a combination thereof associated with the at least the portion of the analyte.

51. The method of any one of claims 1-50, wherein the one or more characteristics comprises a sequence associated with the at least the portion of the analyte.

52. The method of any one of claims 1-51, wherein the one or more characteristics comprises a one or more mutations associated with the at least the portion of the analyte.

53. The method of any one of claims 1-52, wherein the one or more characteristics comprises a one or more isoforms associated with the at least the portion of the analyte.

54. The method of any one of claims 1-53, further comprising, prior to (a), providing: (i) a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and; (2) a membrane comprising the nanopore, wherein the membrane separates the fluidic chamber into a first side and a second side.

55. The method claim 54, wherein the first side comprises a first solution and the second side comprises a second solution, wherein the first solution and the second solution are configured to translocate the at least the portion of the analyte across the nanopore.

56. The method of claim 55, wherein the first solution and the second solution are configured to generate an electro-osmotic force across the membrane.

57. The method of claim 56, wherein electro-osmotic force translocates the at least the portion of the analyte from the first side through the nanopore to the second side against an electrophoretic force acting in a direction opposite the electro-osmotic force. -522- WSGR Docket No.64828-710.601WSGR Docket Number: 64828-710.601 58. The method of any one of claims 54-57, further comprising, prior to (a) contacting a complex comprising the at least the portion of the analyte and a translocase with the first side of the nanopore.

59. The method of any one of claims 1-58, wherein the nanopore is a biological nanopore.

60. The method of claim 59, wherein the biological nanopore comprises at least a portion of an alpha helical pore forming protein or peptide.

61. The method of claim 59, wherein the biological nanopore comprises at least a portion of a beta barrel pore forming protein or peptide.

62. The method of any one of claims 59-61, wherein the biological nanopore comprises Aerolysin (Aer), Cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, OmpF, OmpG, FhuA, or phage derived portal proteins, or fragments thereof, or modified variants thereof, or ion-selective mutants thereof.

63. The method of any one of claims 1-62, wherein the nanopore comprises a engineered CytK nanopore.

64. The method of any one of claims 1-62, wherein the nanopore comprises an engineered MspA nanopore or an engineered CsgG nanopore.

65. The method of any one of claims 1-64, further comprising, prior to (a), unfolding the analyte with one or more unfoldases, translocases, unfoldase domains, or translocase domains, or fragments thereof, or any combination thereof.

66. The method of any one of claims 1-65, wherein the inner diameter of the nanopore is from about 0.5 nm to about 2 nm.

67. The method of any one of claims 1-66, wherein the at least the portion of the analyte comprises a linear length greater than a channel length of the nanopore.

68. The method of any one of claims 1-67, wherein the at least the portion of the analyte comprises one or more leader constructs at a N-terminus or a C-terminus.

69. The method of any one of claims 1-68, wherein the nanopore comprises an adaptor.

70. The method of any one of claims 1-69, wherein the nanopore is coupled to one or more recognition elements 71. The method of any one of claims 1-70, wherein the at least the portion of the analyte is at least 100 amino acids 72. A method for sample analysis, comprising: (a) providing a sample comprising a plurality of analytes, wherein the plurality of analytes comprises a first analyte and a second analyte; -523- WSGR Docket No.64828-710.601WSGR Docket Number: 64828-710.601 (b) translocating at least a portion of the first analyte through a first nanopore disposed within a first membrane and at least a portion of the second analyte through a second nanopore disposed within a second membrane, wherein the at least a portion of the first analyte comprises at least a portion of a first protein, at least a portion of a first polypeptide, or at least a portion of a first peptide, or first fragments thereof, or a combination thereof, wherein the at least a portion of the second analyte comprises at least a portion of a second protein, at least a portion of a second polypeptide, or at least a portion of a second peptide, or second fragments thereof, or a combination thereof; (c) detecting (i) (1) a first current or change thereof or (2) a first voltage or change thereof while the at least the portion of the first analyte is translocating through the first nanopore, and (ii) (3) a second current or change thereof or (4) a second voltage or change thereof while the at least the portion of the second analyte is translocating through the second nanopore; (d) using (i) (1) the first current or change thereof or (2) the first voltage or change thereof to determine a first characteristic of the at least the portion of the first analyte and (ii) (3) the second current or change thereof or (4) the second voltage or change thereof to determine a second characteristic of the at least the portion of the second analyte; and (e) characterizing one or more properties of the sample using the first characteristic or the second characteristic determined in (d).

73. The method of claim 72, wherein the at least the portion of the first analyte or the at least the portion of the second analyte is at least 100 amino acids.

74. The method of claim 72 or 73, further comprising, in (d), determining (A) the first characteristic based on (1) a first electrical signal or change thereof of the first current or change thereof or the first voltage or change thereof and (2) a database, or (B) the second characteristic based on (1) a second electrical signal or change thereof of the second current or change thereof or the second voltage or change thereof and (2) the database.

75. The method of claim 74, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more proteoforms thereof, or one or more variants thereof, or one or more fragments thereof, or a combination thereof.

76. The method of claim 74 or 75, wherein the first electrical signal or change thereof or the second electrical signal or change thereof comprises (1) one or more reads; or (2) one or more additional portions of the signal or change thereof.

77. The method of any one of claims 74-76, wherein (1) the first electrical signal or change thereof comprises measurements of the first current or change thereof or the first voltage or change thereof -524- WSGR Docket No.64828-710.601WSGR Docket Number: 64828-710.601 over a first period of time; or (2) the second electrical signal or change thereof comprises measurements of the second current or change thereof or the second voltage or change thereof over a second period of time.

78. The method of any one of claims 74-77, further comprising, in (d), pre-processing the first electrical signal or change thereof or the second electrical signal or change thereof, thereby generating (1) a pre-processed first electrical signal or change thereof or (2) a pre-processed second electrical signal or change thereof.

79. The method of claim 78, further comprising extracting (A) one or more first reads from (1) the first electrical signal or change thereof or (2) the pre-processed first electrical signal or change thereof; or (B) one or more second reads from (1) the second electrical signal or change thereof or (2) the pre- processed second electrical signal or change thereof.

80. The method of claim 78 or 79, wherein the first characteristic or the second characteristic is determined using (1) the one or more first reads or one or more pre-processed first reads; or (2) the one or more second reads or one or more pre-processed second reads.

81. The method of claim 80, further comprising comparing (1) the one or more first reads, the one or more pre-processed reads, the one or more second reads, the one or more pre-processed second reads to (2) one or more reference signals in the database 82. The method of any one of claims 72-81, wherein the database is generated from one or more reference sequences.

83. The method of claim 82, wherein the one or more reference sequences are derived with genomic information or transcriptomic information of the sample.

84. The method of claim 82 or 83, wherein the database is generated from the one or more reference sequences using one or more machine learning algorithms.

85. The method of any one of claims 72-84, wherein the database comprises one or more reference signals for the at least the portion of the analyte, or at least one fragment thereof, or at least one proteoform thereof, or at least one variant thereof, or combination thereof.

86. The method of any one of claims 72-85, wherein the characterizing the one or more properties comprises determining at least one feature of a proteome associated with the sample.

87. The method of claim 86, wherein (i) proteome coverage is at least 1% or (ii) sequence coverage of the at least the portion of the first polypeptide or the at least the portion of the second polypeptide is at least 1%.

88. The method of any one of claims 72-87, wherein the first nanopore and the second nanopore are different nanopores. -525- WSGR Docket No.64828-710.601WSGR Docket Number: 64828-710.601 89. The method of any one of claims 72-88, wherein the first membrane and the second membrane are different membranes 90. The method of any one of claims 72-89, wherein the sample comprises a first type of analyte and a second type of analyte.

91. The method of claim 90, wherein the first type of analyte and the second type of analyte are different types.

92. The method of claim 90 or 91, wherein the first type of analyte comprises the at least the portion of the first analyte and the second type of analyte comprises the at least the portion of the second analyte.

93. The method of any one of claims 72-92, wherein the characterizing the one or more properties comprises determining the number of analytes in the first type of analyte and determining the number of analytes in the second type of analyte.

94. The method of any one of claims 72-93, wherein the sample comprises one type of analyte.

95. The method of claim 94, wherein the at least the portion of the first analyte and the at least the portion of the second analyte are from the same analyte type.

96. The method of claim 94 or 95, further comprising determining one or more of a number of analytes in the sample, relative abundance of analytes in the sample, an absolute abundance of analytes in the sample, identification of origins of the analytes in the sample, analytes with secondary structures, analytes with tertiary structures, analytes with quaternary structures, or one or more impurities in the sample, or a combination thereof.

97. The method of any one of claims 72-96, further comprising characterizing one or more properties of the sample using a plurality of characteristics associated with the plurality of analytes.

98. The method of any one of claims 72-97, wherein the one or more properties comprises an absolute or relative abundance, absolute concentration, relative concentration, or origin of one or more analyte types in the sample.

99. The method of any one of claims 72-98, wherein the one or more properties comprises an absolute or relative abundance, absolute concentration, relative concentration, or origin of one or more analytes in the sample.

100. The method of any one of claims 72-99, wherein the one or more properties comprises differences between at least a subset of analytes of the plurality of analytes.

101. The method of any one of claims 72-100, wherein the one or more properties comprises differences in sequence of at most 10 units between at least a subset of analytes of the plurality of analytes. -526- WSGR Docket No.64828-710.601WSGR Docket Number: 64828-710.601 102. The method of any one of claims 72-101, wherein the one or more properties comprises a percentage of modified or different analytes in the plurality of analytes.

103. The method of any one of claims 72-102, wherein the one or more properties comprises a quantification of one or more proteins, polypeptides or peptides in the sample.

104. The method of any one of claims 72-103, wherein the one or more properties comprises a quantification of one or more protein, polypeptide or peptide types in the sample.

105. The method of any one of claims 72-104, wherein the one or more properties comprises identification of a type associated with the sample or with an origin of the sample 106. The method of any one of claims 72-105, wherein the first characteristic or second characteristic comprises a plurality of natural or unnatural post-translational modifications of the at least the portion of the analyte.

107. The method of claim 106, wherein the natural or unnatural post translational modification is phosphorylation, glycosylation, deamidation, or acetylation, or any combination thereof.

108. The method of any one of claims 72-107, wherein the first characteristic or second characteristic comprises a length of the at least the portion of the analyte 109. The method of any one of claims 72-108, wherein the first characteristic or second characteristic comprises an average speed of translocation of the at least the portion of the analyte through the nanopore 110. The method of any one of claims 72-109, wherein the first characteristic or second characteristic comprises determining whether the at least the portion of the analyte comprises one or more molecular entities.

111. The method of claim 110, wherein the at least the portion of the first analyte or the at least the portion of the second analyte comprises one or more molecular entities.

112. The method of claim 110 or 111, wherein the one or more molecular entities is a compound (e.g., drug, small molecule), particle, nucleic acid, polynucleic acid, peptide, polynucleotide, or protein, or fragments thereof, or any combination thereof.

113. The method of any one of claims 72-112, wherein the first characteristic or second characteristic comprises a category or identity associated with the at least the portion of the first analyte or the at least the portion of the second analyte.

114. The method of any one of claims 72-113, further comprising, prior to (b), providing: (i) a first nanopore system, wherein the first nanopore system comprises (1) a first fluidic chamber and (2) the first membrane comprising the first nanopore, wherein the first membrane separates the first fluidic chamber into a first side and a second side, and (ii) a second nanopore system, wherein the second -527- WSGR Docket No.64828-710.601WSGR Docket Number: 64828-710.601 nanopore system comprises (1) a second fluidic chamber and; (2) the second membrane comprising the second nanopore, wherein the second membrane separates the second fluidic chamber into a side and an additional side.

115. The method of claim 114, further comprising, providing (i) a first electrophoretic force (EPF) acting in an opposite direction to a first side to second side electro-osmotic force, or (ii) a second electrophoretic force (EPF) acting in an opposite direction to a side to additional side electro-osmotic force.

116. The method of claim 114 or 115, wherein (i) the first side comprises a first solution and the second side comprises a second solution, or (ii) the side comprises a solution and the additional side comprises an additional solution.

117. The method of claim 116, wherein (i) the first solution and the second solution are configured to translocate the at least the portion of the first analyte across the first nanopore, or (ii) the solution and the additional solution are configured to translocate the at least the portion of the second analyte across the second nanopore.

118. The method of claim 116 or 117, wherein (i) the first solution and the second solution are configured to generate a first electro-osmotic force across the first membrane; (i) the solution and the additional solution are configured to generate a second electro-osmotic force across the second membrane.

119. The method of claim 118, wherein (i) the first electro-osmotic force translocates the at least the portion of the first analyte from the first side through the first nanopore to the second side against a first electrophoretic force acting in a direction opposite the first electro-osmotic force; or (ii) a second electro-osmotic force translocates the at least the portion of the second analyte from the side through the second nanopore to the additional side against a second electrophoretic force acting in a direction opposite the second electro-osmotic force.

120. The method of any one of claims 114-119, further comprising, prior to (a) (i) contacting a first complex comprising the at least the portion of the first analyte and a first translocase with the first side of the first nanopore; or (ii) contacting a second complex comprising the at least the portion of the second analyte and a second translocase with the side of the second nanopore.

121. The method of any one of claims 72-120, wherein the first nanopore or the second nanopore is a biological nanopore.

122. The method of claim 121, wherein the biological nanopore comprises at least a portion of an alpha helical pore forming protein or peptide. -528- WSGR Docket No.64828-710.601WSGR Docket Number: 64828-710.601 123. The method of claim 121, wherein the biological nanopore comprises at least a portion of a beta barrel pore forming protein or peptide.

124. The method of any one of claims 72-123, further comprising, prior to (a), unfolding the first analyte or second analyte with one or more unfoldases, translocases, unfoldase domains, or translocase domains, or fragments thereof, or any combination thereof.

125. The method of claim 124, wherein the one or more unfoldases, translocases, unfoldase domains, or translocase domains, or fragments thereof, or any combination thereof are configured to position proximal to the nanopore upon a binding event with the polypeptide.

126. The method of any one of claims 72-125, wherein the at least the portion of the first analyte or the at least the portion of the second analyte comprises one or more post-translational modifications.

127. The method of any one of claims 72-126, wherein the at least the portion of the first analyte or the at least the portion of the second analyte comprises one or more leader constructs at a N-terminus or a C-terminus.

128. The method of any one of claims 72-127, wherein the first nanopore or the second nanopore comprises an adaptor.

129. The method of any one of claims 72-128, wherein the first nanopore or the second nanopore is coupled to one or more recognition elements.

130. The method of any one of claims 72-129, wherein the first nanopore or the second nanopore comprises an inner pore constriction from about 0.5 nanometers (nm) to about 2 nm.

131. The method of any one of claims 72-130, wherein the first nanopore or the second nanopore has an ion-selectivity P(+) / P(-) of greater than 2.

0.

132. The method of any one of claims 72-130, wherein the first nanopore or the second nanopore has an ion-selectivity P(+) / P(-) of less than 0.

50.

133. The method of claim 72-132, wherein the sample is a complex sample. -529- WSGR Docket No.64828-710.601

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