Nanopores and uses thereof for analysis of analytes

Abstract

The present disclosure provides methods, systems, and compositions for characterizing an analyte using a nanopore. The system may comprise a a fluidic chamber. The system may also comprise a membrane comprising an engineered biological nanopore. The membrane may separate the fluidic chamber into a first side and a second side. The engineered biological nanopore may comprise a channel. The channel may comprise a first region and a second region. The second region may comprise a constriction region. The first region may be modified to be more net negative than a respective region of a wild-type biological nanopore. The first region may also be modified to be more net neutral or more net negative than a respective region of the wild-type biological nanopore. The first region may be adjacent to the second region. The engineered biological nanopore may be contacted with a biopolymer.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of Greek Patent Application No. 20240100311, filed April 30, 2024, which is herein incorporated by reference in its entirety. BACKGROUND

[0002] Determination of analytes is an important part of scientific studies. Improvements in the characterization of analytes can be important for further scientific studies or clinical aspects. 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, proteins, 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 comprising: (a) providing a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which the second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region of the channel has a negative charge, wherein the second region of the channel has a neutral charge; and (b) contacting the engineered biological nanopore with a biopolymer.

[0005] In some embodiments, the engineered biological nanopore generates an electro-osmotic force (EOF) greater than an EOF of a wild-type biological nanopore. In some embodiments, the negative charge of the first region of the channel and the neutral charge of the second region of the channel generates the EOF.

[0006] In some embodiments, the engineered biological nanopore has a cation-selectivity P(+) / P(-) of at least about 1.5.

[0007] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0008] In some embodiments, the first region is more negative as compared to another region adjacent to a constriction region of the wild-type biological nanopore. In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the constriction region of the wild-type biological nanopore.

[0009] In some embodiments, the second region is more neutral as compared to the constriction region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to the constriction region of a wild-type biological nanopore.

[0010] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0011] In some embodiments, a shortest distance between a first ring of charge in the first region and a second ring of charge in the second region is at most about 5 nm.

[0012] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue.

[0013] In some embodiments, the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue.

[0014] In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, the mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0015] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi-conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0016] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0017] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0018] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (PlyAB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0019] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0020] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0021] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0022] In some embodiments, the biopolymer comprises a non-nucleic acid based polymer analyte. In some embodiments, the non-nucleic acid based polymer analyte comprises a protein, a polypeptide, a peptide, a saccharide, a lipid, a polymer, an inorganic material, or any combination thereof. In some embodiments, the non-nucleic acid based polymer analyte is the peptide, the protein, or the polypeptide.

[0023] In some embodiments, the first side of the fluidic chamber comprises a first solution and the second side of the fluidic chamber comprises a second solution. In some embodiments, the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of the solute. In some embodiments, the solute comprises an ion or an osmolyte. In some embodiments, a difference between the first concentration of the solute and the second concentration of the solute is configured to generate an electroosmotic force.

[0024] In some embodiments, the method further comprises measuring a signal generated by translocating the biopolymer through the engineered biological nanopore. In some embodiments, the measuring the signal comprises measuring the signal for a state of (a) an open channel of the engineered biological nanopore; (b) capture of the biopolymer by a first opening of the engineered biological nanopore; or (c) exit of the biopolymer through a second opening of the engineered biological nanopore. In some embodiments, the measuring comprises detecting differences in the signal between states (a), (b), and (c). In some embodiments, the signal comprises an ionic current, a change in ionic current, or derivations thereof. In some embodiments, the measuring comprises detecting a presence of the biopolymer, a concentration of the biopolymer, or any combination thereof. In some embodiments, the measuring comprises detecting one or more characteristics of the biopolymer. In some embodiments, the one or more characteristics of the biopolymer comprise a shape of the biopolymer, a structure of the biopolymer, one or more mutations of the biopolymer, a surface charge of the biopolymer, one or more post-translation modifications of the biopolymer, one or more ligands coupled to the biopolymer, or any combination thereof.

[0025] In some embodiments, (b) comprises contacting the biopolymer with the first side of the fluidic chamber.

[0026] In some embodiments, (b) comprises contacting the biopolymer with the second side of the fluidic chamber.

[0027] In some embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force. In some embodiments, the applied voltage is a negative voltage on the first side of the fluidic chamber. In some embodiments, the applied voltage is a negative voltage on the second side of the fluidic chamber.

[0028] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, the engineered MspA nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position D90 or D91 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88,1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0029] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0030] In some embodiments, the engineered biological nanopore is an engineered CsgG / F nanopore. In some embodiments, the engineered CsgG / F nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0031] In another aspect, the present disclosure provides a method comprising: (a) providing a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel -4- comprises a first region and a second region, which the second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region of the channel has a negative charge, wherein a net charge of the second region of the channel is at least about 50% more neutral as compared to a respective region of a wild-type biological nanopore; and (b) contacting the engineered biological nanopore with a biopolymer.

[0032] In some embodiments, the net charge of the second region of the channel is at least about 70% more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, the net charge of the second region of the channel is at least about 85% more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, the net charge of the second region of the channel is at least about 90% more neutral as compared to the respective region of the wild-type biological nanopore.

[0033] In some embodiments, the net charge of the second region of the channel is less cationic as compared to the respective region of the wild-type biological nanopore.

[0034] In some embodiments, the net charge of the second region of the channel is less anionic as compared to the respective region of the wild-type biological nanopore.

[0035] The method of any one of claims 13-18, wherein the engineered biological nanopore generates an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, a negative charge of the first region of the channel and the net charge of the second region of the channel generates the EOF.

[0036] In some embodiments, the engineered biological nanopore has a cation-selectivity P(+) / P(-) of at least about 1.5.

[0037] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0038] In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore.

[0039] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0040] In some embodiments, a shortest distance between a first ring of charge in the first region and a second ring of charge in the second region is at most about 5 nm.

[0041] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue.

[0042] In some embodiments, the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue.

[0043] In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, the mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0044] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi-conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0045] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0046] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0047] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (PlyAB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0048] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0049] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0050] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0051] In some embodiments, the biopolymer comprises a non-nucleic acid based polymer analyte. In some embodiments, the non-nucleic acid based polymer analyte comprises a protein, a polypeptide, a peptide, a saccharide, a lipid, a polymer, an inorganic material, or any combination thereof. In some embodiments, the non-nucleic acid based polymer analyte is the peptide, the protein, or the polypeptide.

[0052] In some embodiments, the first side of the fluidic chamber comprises a first solution and the second side of the fluidic chamber comprises a second solution. In some embodiments, the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of the solute. In some embodiments, the solute comprises an ion or an osmolyte. In some embodiments, a difference between the first concentration of the solute and the second concentration of the solute is configured to generate an electroosmotic force.

[0053] In some embodiments, the method further comprises measuring a signal generated by translocating the biopolymer through the engineered biological nanopore. In some embodiments, the measuring the signal comprises measuring the signal for a state of (a) an open channel of the engineered biological nanopore; (b) capture of the biopolymer by a first opening of the engineered biological nanopore; or (c) exit of the biopolymer through a second opening of the engineered biological nanopore. In some embodiments, the measuring comprises detecting differences in the signal between states (a), (b), and (c). In some embodiments, the signal comprises an ionic current, a change in ionic current, or derivations thereof. In some embodiments, the measuring comprises detecting a presence of the biopolymer, a concentration of the biopolymer, or any combination thereof. In some embodiments, the measuring comprises detecting one or more characteristics of the biopolymer. In some embodiments, the one or more characteristics of the biopolymer comprise a shape of the biopolymer, a structure of the biopolymer, one or more mutations of the biopolymer, a surface charge of the biopolymer, one or more post-translation modifications of the biopolymer, one or more ligands coupled to the biopolymer, or any combination thereof.

[0054] In some embodiments, (b) comprises contacting the biopolymer with the first side of the fluidic chamber.

[0055] In some embodiments, (b) comprises contacting the biopolymer with the second side of the fluidic chamber.

[0056] In some embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force. In some embodiments, the applied voltage is a negative voltage on the first side of the fluidic chamber. In some embodiments, the applied voltage is a negative voltage on the second side of the fluidic chamber.

[0057] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, the engineered MspA nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position D90 or D91 of a wild-type amino acid sequence -7- as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88,1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0058] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0059] In some embodiments, the engineered biological nanopore is an engineered CsgG / F nanopore. In some embodiments, the engineered CsgG / F nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0060] In another aspect, the present disclosure provides a system comprising: (a) a fluidic chamber; and (b) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region of the channel has a negative charge, wherein the second region of the channel has a neutral charge, wherein the engineered biological nanopore is configured to contact a biopolymer.

[0061] In some embodiments, the engineered biological nanopore is configured to generate an electro-osmotic force (EOF) greater than an EOF of a wild-type biological nanopore. In some embodiments, the negative charge of the first region of the channel and the neutral charge of the second region of the channel is configured to generate the EOF. In some embodiments, the engineered biological nanopore has a cation-selectivity P(+) / P(-) of at least about 1.5.

[0062] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0063] In some embodiments, the first region is more negative as compared to another region adjacent to a constriction region of the wild-type biological nanopore. In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the constriction region of the wild-type biological nanopore.

[0064] In some embodiments, the second region is more neutral as compared to a constriction region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to the constriction region of a wild-type biological nanopore.

[0065] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0066] In some embodiments, a shortest distance between a first ring of charge in the first region and a second ring of charge in the second region is at most about 5 nm.

[0067] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue.

[0068] In some embodiments, wherein the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue.

[0069] In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, the mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0070] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi-conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0071] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0072] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0073] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (PlyAB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0074] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0075] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0076] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0077] In some embodiments, the biopolymer comprises a non-nucleic acid based polymer analyte. In some embodiments, the non-nucleic acid based polymer analyte comprises a protein, a polypeptide, a peptide, a saccharide, a lipid, a polymer, an inorganic material, or any combination thereof. In some embodiments, the non-nucleic acid based polymer analyte is the peptide, the protein, or the polypeptide.

[0078] In some embodiments, the first side of the fluidic chamber comprises a first solution and the second side of the fluidic chamber comprises a second solution. In some embodiments, the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of the solute. In some embodiments, the solute comprises an ion or an osmolyte. In some embodiments, a difference between the first concentration of the solute and the second concentration of the solute is configured to generate an electroosmotic force.

[0079] In some embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force. In some embodiments, the applied voltage is a negative voltage on the first side of the fluidic chamber. In some embodiments, the applied voltage is a negative voltage on the second side of the fluidic chamber.

[0080] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, the engineered MspA nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position D90 or D91 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88,1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0081] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, -10- the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0082] In some embodiments, the engineered biological nanopore is an engineered CsgG / F nanopore. In some embodiments, the engineered CsgG / F nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0083] In another aspect, the present disclosure provides a system comprising: (a) a fluidic chamber; and (b) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region of the channel has a negative charge, wherein a net charge of the second region of the channel is at least about 50% more neutral as compared to a respective region of a wild-type biological nanopore, wherein the engineered biological nanopore is configured to a biopolymer.

[0084] In some embodiments, the net charge of the second region of the channel is at least about 70% more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, the net charge of the second region of the channel is at least about 85% more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, the net charge of the second region of the channel is at least about 90% more neutral as compared to the respective region of the wild-type biological nanopore.

[0085] In some embodiments, the net charge of the second region of the channel is less cationic as compared to the respective region of the wild-type biological nanopore.

[0086] In some embodiments, the net charge of the second region of the channel is less anionic as compared to the respective region of the wild-type biological nanopore.

[0087] In some embodiments, the engineered biological nanopore is configured to generate an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, the negative charge of the first region of the channel and the net charge of the second region of the channel is configured to generate the EOF.

[0088] In some embodiments, the engineered biological nanopore has a cation-selectivity P(+) / P(-) of at least about 1.5.

[0089] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0090] In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore.

[0091] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0092] In some embodiments, a shortest distance between a first ring of charge in the first region and a second ring of charge in the second region is at most about 5 nm.

[0093] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue.

[0094] In some embodiments, wherein the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue.

[0095] In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, the mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0096] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi-conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0097] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0098] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0099] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (PlyAB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0100] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0101] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0102] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0103] In some embodiments, the biopolymer comprises a non-nucleic acid based polymer analyte. In some embodiments, the non-nucleic acid based polymer analyte comprises a protein, a polypeptide, a peptide, a saccharide, a lipid, a polymer, an inorganic material, or any combination thereof. In some embodiments, the non-nucleic acid based polymer analyte is the peptide, the protein, or the polypeptide.

[0104] In some embodiments, the first side of the fluidic chamber comprises a first solution and the second side of the fluidic chamber comprises a second solution. In some embodiments, the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of the solute. In some embodiments, the solute comprises an ion or an osmolyte. In some embodiments, a difference between the first concentration of the solute and the second concentration of the solute is configured to generate an electroosmotic force.

[0105] In some embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force. In some embodiments, the applied voltage is a negative voltage on the first side of the fluidic chamber. In some embodiments, the applied voltage is a negative voltage on the second side of the fluidic chamber.

[0106] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, the engineered MspA nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position D90 or D91 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88,1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0107] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0108] In some embodiments, the engineered biological nanopore is an engineered CsgG / F nanopore. In some embodiments, the engineered CsgG / F nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0109] In another aspect, the present disclosure provides an engineered biological nanopore comprising a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region of the channel has a negative charge, wherein the second region of the channel has a neutral charge.

[0110] In some embodiments, the engineered biological nanopore is configured to generate an electro-osmotic force (EOF) greater than an EOF of a wild-type biological nanopore. In some embodiments, the negative charge of the first region of the channel and the neutral charge of the second region of the channel is configured to generate the EOF.

[0111] In some embodiments, the engineered biological nanopore has a cation-selectivity P(+) / P(-) of at least about 1.5.

[0112] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0113] In some embodiments, the first region is more negative as compared to another region adjacent to a constriction region of the wild-type biological nanopore. In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the constriction region of the wild-type biological nanopore

[0114] In some embodiments, the second region is more neutral as compared to the constriction region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to a constriction region of a wild-type biological nanopore.

[0115] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0116] In some embodiments, a shortest distance between a first ring of charge in the first region and a second ring of charge in the second region is at most about 5 nm.

[0117] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue.

[0118] In some embodiments, the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue

[0119] In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, the mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0120] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi-conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0121] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0122] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0123] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (PlyAB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0124] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0125] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0126] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0127] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, the engineered MspA nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position D90 or D91 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88,1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0128] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0129] In some embodiments, the engineered biological nanopore is an engineered CsgG / F nanopore. In some embodiments, the engineered CsgG / F nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0130] In another aspect, the present disclosure provides an engineered biological nanopore comprising a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region of the channel has a negative charge, wherein a net charge of the second region of the channel is at least about 50% more neutral as compared to a respective region of a wild-type biological nanopore.

[0131] In some embodiments, the net charge of the second region of the channel is at least about 70% more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, the -16- net charge of the second region of the channel is at least about 85% more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, the net charge of the second region of the channel is at least about 90% more neutral as compared to the respective region of the wild-type biological nanopore.

[0132] In some embodiments, the net charge of the second region of the channel is less cationic as compared to the respective region of the wild-type biological nanopore.

[0133] In some embodiments, the net charge of the second region of the channel is less anionic as compared to the respective region of the wild-type biological nanopore.

[0134] In some embodiments, the engineered biological nanopore is configured to generate an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, the negative charge of the first region of the channel and the net charge of the second region of the channel is configured to generate the EOF.

[0135] In some embodiments, the engineered biological nanopore has a cation-selectivity P(+) / P(-) of at least about 1.5.

[0136] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0137] In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore.

[0138] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0139] In some embodiments, a shortest distance between a first ring of charge in the first region and a second ring of charge in the second region is at most about 5 nm.

[0140] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue.

[0141] In some embodiments, the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue.

[0142] In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, the mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0143] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi-conical geometry, a straight geometry, or a vestibule geometry.

[0144] In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0145] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0146] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0147] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (PlyAB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0148] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0149] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0150] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0151] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, the engineered MspA nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position D90 or D91 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88,1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0152] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0153] In some embodiments, the engineered biological nanopore is an engineered CsgG / F nanopore. In some embodiments, the engineered CsgG / F nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0154] In some embodiments, the CsgG nanopore further comprises an additional monomer comprising a mutation corresponding to position N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wildtype amino acid sequence as set forth in SEQ ID NO: 5.

[0155] In another aspect, the present disclosure provides a method comprising: (a) providing a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region is modified to be more net negative as compared to a respective region of a wild-type biological nanopore, wherein a first ring of charge in the first region and a second ring of charge in the second region comprises a distance of at most about 3 nm, wherein the second region comprises a width of at most about 2.5 nm; and contacting the engineered biological nanopore with a biopolymer.

[0156] In some embodiments, the first region may be more net negative than the second region. In some embodiments, one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids. In some embodiments, one or more amino acids in the first region is mutated to one or more negative amino acids. In some embodiments, when one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids, then one or more amino acids in the first region is mutated to one or more negative amino acids. In some embodiments, the first region comprises at least one amino acid that is mutated to exhibit an increased net negative charge. In some embodiments, the first region comprises at least one amino acid that is mutated to exhibit the increased net negative charge as compared to a respective region of a wild-type biological nanopore. In some embodiments, the first region comprises at least one amino acid that is mutated to a negative amino -19- acid to exhibit the increased net negative charge as compared to the respective region of the wild-type biological nanopore. In some embodiments, the mutated at least one amino acid in the first region is at most 10 nm away from a mutated at least one amino acid in the second region. In some embodiments, the first ring of charge comprising the mutated at least one amino acid in the first region is at most 10 nm away from the mutated at least one amino acid in the second region. In some embodiments, the first ring of charge comprising the mutated at least one amino acid in the first region is at most 10 nm away from the second ring of charge comprising the mutated at least one amino acid in the second region. In some embodiments, the second region comprise a C(alpha)-C(alpha) diameter of at most 10 nm.

[0157] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the engineered biological nanopore comprises a first portion corresponding to the first region and a second portion corresponding to the second region. In some embodiments, a monomer of the engineered biological nanopore comprises a net charge in the first portion that is more negative as compared to a net charge in the second portion. In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit an increased net negative charge. In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit the increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore. In some embodiments, the first portion comprises at least one amino acid that is mutated to a negative amino acid to exhibit the increased net negative charge as compared to the respective portion of the monomer of the wild-type biological nanopore. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit an increased net neutral charge or an increased net negative charge. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit the increased net neutral charge or increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore. In some embodiments, the second portion comprises at least one amino acid that is mutated to a neutral amino acid or a negative amino acid to exhibit the increased net neutral charge or increased net negative charge as compared to the respective portion of the monomer of the wild-type biological nanopore. In some embodiments, the at least one mutated amino acid in the first portion is at most 10 nm away from the at least one mutated amino acid in the second portion. In some embodiments, the at least one mutated amino acid in the first portion of each of one or more monomers (or less than all monomers) forms the first ring of charge, wherein the at least one mutated amino acid in the second portion of each of one or more monomer (or less than all monomers) forms the second ring of charge, wherein each monomer of the engineered biological nanopore comprises a first portion corresponding to the first region and a second portion corresponding to the second region. In some embodiments, each monomer of the engineered biological nanopore comprises a net charge in the first portion that is more negative as compared to a net charge in the second portion.

[0158] In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit an increased net negative charge. In some embodiments, the first portion comprises at least one amino acid that -20- is mutated to exhibit the increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore. In some embodiments, the first portion comprises at least one amino acid that is mutated to a negative amino acid to exhibit the increased net negative charge as compared to the respective portion of the monomer of the wild-type biological nanopore. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit an increased net neutral charge or an increased net negative charge. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit the increased net neutral charge or increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore. In some embodiments, the second portion comprises at least one amino acid that is mutated to a neutral amino acid or a negative amino acid to exhibit the increased net neutral charge or the increased net negative charge, respectively, as compared to the respective portion of the monomer of the wild-type biological nanopore. In some embodiments, the at least one mutated amino acid in the first portion of each monomer forms the first ring of charge. In some embodiments, the at least one mutated amino acid in the second portion of each monomer forms the second ring of charge. In some embodiments, the at least one mutated amino acid in the first portion is at most 10 nm away from the at least one mutated amino acid in the second portion. In some embodiments, one or more amino acids in the first portion is mutated to one or more negative amino acids. In some embodiments, one or more amino acids in the second portion is modified to one or more neutral amino acids or one or more negative amino acids. In some embodiments, when one or more amino acids in the second portion is modified to one or more neutral amino acids or one or more negative amino acids, then one or more amino acids in the first portion is mutated to one or more negative amino acids. In some embodiments, the engineered biological nanopore generates an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, the first region of the channel and the second region of the channel generates the EOF. In some embodiments, the EOF acts in an opposite direction to an electrophoretic force in the nanopore system. In some embodiments, the engineered biological nanopore has a cation-selectivity P(+) / P(-) of at least about 1.5.

[0159] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, an increased net negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the increased net negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the increased net negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel. In some embodiments, the first region is more negative as compared to the respective region of the wild-type biological nanopore. In some embodiments, a net charge of the first region is at least about 50% more negative as compared to the respective region of the wild-type biological nanopore. In some embodiments, the second region is more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, a net neutral charge of the constriction region is increased. In some embodiments, a net negative charge of the constriction region is increased. In some embodiments, the channel comprises a length of at least about 2 nanometers. In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges. In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue. In some embodiments, the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue. In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0160] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi-conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0161] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0162] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0163] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (PlyAB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0164] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0165] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0166] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0167] In some embodiments, a negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0168] In some embodiments, the biopolymer comprises a non-nucleic acid based polymer analyte. In some embodiments, the non-nucleic acid based polymer analyte comprises a protein, a polypeptide, a peptide, a saccharide, a lipid, a polymer, an inorganic material, or any combination thereof. In some embodiments, the non-nucleic acid based polymer analyte is the peptide, the protein, or the polypeptide.

[0169] In some embodiments, the first side of the fluidic chamber comprises a first solution and the second side of the fluidic chamber comprises a second solution. In some embodiments, the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of the solute. In some embodiments, the solute comprises an ion or an osmolyte. In some embodiments, a difference between the first concentration of the solute and the second concentration of the solute is configured to generate an electroosmotic force.

[0170] In some embodiments, the method further comprises measuring a signal generated by translocating the biopolymer through the engineered biological nanopore. In some embodiments, the measuring the signal comprises measuring a signal for a state of (a) an open channel of the engineered biological nanopore; (b) capture of the biopolymer by a first opening of the engineered biological nanopore; or (c) exit of the biopolymer through a second opening of the engineered biological nanopore. In some embodiments, the measuring comprises detecting differences in the signal between states (a), (b), and (c). In some embodiments, the signal comprises an ionic current, a change in ionic current, or derivations thereof. In some embodiments, the measuring comprises detecting a presence of the biopolymer, a concentration of the biopolymer, or any combination thereof. In some embodiments, the measuring comprises detecting one or more characteristics of the biopolymer. In some embodiments, the one or more characteristics of the biopolymer comprise a shape of the biopolymer, a structure of the biopolymer, one or more mutations of the biopolymer, a surface charge of the biopolymer, one or more post-translation modifications of the biopolymer, one or more ligands coupled to the biopolymer, or any combination thereof.

[0171] In some embodiments, (b) comprises contacting the biopolymer with the first side of the fluidic chamber.

[0172] In some embodiments, (b) comprises contacting the biopolymer with the second side of the fluidic chamber.

[0173] In some embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force. In some embodiments, the applied voltage is a negative voltage on the first side of the fluidic chamber. In some embodiments, the applied voltage is a negative voltage on the second side of the fluidic chamber.

[0174] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, the engineered MspA nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position D90 or D91 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88,1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0175] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0176] In some embodiments, the engineered biological nanopore is an engineered CsgG / F nanopore. In some embodiments, the engineered CsgG / F nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0177] In another aspect, the present disclosure provides a system comprising: (a) a fluidic chamber; and (b) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region is modified to be more net negative as compared to a respective region of a wild-type biological nanopore, wherein a first ring of charge in the first region and a second ring of charge in the second region comprises a distance of at most about 3 nm, wherein the second region comprises a width of at most about 2.5 nm, wherein the engineered biological nanopore is configured to contact a biopolymer.

[0178] In some embodiments, the engineered biological nanopore is configured to generate an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, the first region of the channel and the second region of the channel is configured to generate the EOF. In some embodiments, EOF acts in an opposite direction to an electrophoretic force in the system.

[0179] In some embodiments, the engineered biological nanopore has a cation-selectivity P(+) / P(-) of at least about 1.5.

[0180] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, an increased net negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the increased net negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the increased net negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0181] In some embodiments, the first region is more net negative as compared to the respective region of the wild-type biological nanopore. In some embodiments, a net charge of the first region is at least about 50% more negative as compared to the respective region of the wild-type biological nanopore.

[0182] In some embodiments, the second region is more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to the respective region of a wild-type biological nanopore.

[0183] In some embodiments, a net neutral charge of the constriction region is increased.

[0184] In some embodiments, a net negative charge of the constriction region is increased.

[0185] In some embodiments, the channel comprises a length of at least about 2 nanometers.

[0186] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0187] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue. In some embodiments, the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue. In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0188] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi-conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0189] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0190] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0191] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (PlyAB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0192] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0193] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0194] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0195] In some embodiments, a negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0196] In some embodiments, the biopolymer comprises a non-nucleic acid based polymer analyte. In some embodiments, the non-nucleic acid based polymer analyte comprises a protein, a polypeptide, a peptide, a saccharide, a lipid, a polymer, an inorganic material, or any combination thereof. In some embodiments, the non-nucleic acid based polymer analyte is the peptide, the protein, or the polypeptide.

[0197] In some embodiments, the first side of the fluidic chamber comprises a first solution and the second side of the fluidic chamber comprises a second solution. In some embodiments, the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of the solute. In some embodiments, the solute comprises an ion or an osmolyte. In some embodiments, a difference between the first concentration of the solute and the second concentration of the solute is configured to generate an electroosmotic force.

[0198] In some embodiments, the system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force. In some embodiments, the applied voltage is a negative voltage on the first side of the fluidic chamber. In some embodiments, the applied voltage is a negative voltage on the second side of the fluidic chamber.

[0199] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, the engineered MspA nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position D90 or D91 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88,1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0200] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0201] In some embodiments, the engineered biological nanopore is an engineered CsgG / F nanopore. In some embodiments, the engineered CsgG / F nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0202] In another aspect, the present disclosure provides an engineered biological nanopore comprising a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region is modified to be more net negative as compared to a respective region of a wild-type biological nanopore, wherein a first ring of charge in the first region and a second ring of charge in the second region comprises a distance of at most about 3 nm, wherein the second region comprises a width of at most about 2.5 nm.

[0203] In some embodiments, the engineered biological nanopore is configured to generate an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, the first region of the channel and the second region of the channel are configured to generate the EOF. In some embodiments, the EOF acts in an opposite direction to an electrophoretic force in the engineered biological nanopore.

[0204] In some embodiments, the engineered biological nanopore has a cation-selectivity P(+) / P(-) of at least about 1.5.

[0205] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, a negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0206] In some embodiments, the first region is more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore. In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore.

[0207] In some embodiments, the second region is more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to the respective region of the wild-type biological nanopore.

[0208] In some embodiments, a net neutral charge of the constriction region is increased.

[0209] In some embodiments, a net negative charge of the constriction region is increased.

[0210] In some embodiments, the channel comprises a length of at least about 2 nanometers.

[0211] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0212] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue. In some embodiments, the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue. In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0213] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi-conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0214] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0215] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0216] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (PlyAB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0217] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0218] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0219] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0220] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, the engineered MspA nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position D90 or D91 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88,1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0221] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises -29- a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0222] In some embodiments, the engineered biological nanopore is an engineered CsgG / F nanopore. In some embodiments, the engineered CsgG / F nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0223] 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.

[0224] 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.

[0225] In some aspects, the present disclosure provides a method comprising: (a) providing a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which the second region has a constriction region, wherein (i) the first region is modified to be more net negative than a respective region of a wild-type biological nanopore and / or (ii) the second region is modified to be more net neutral or more net negative than a respective region of the wild-type biological nanopore, wherein the first region of the channel is adjacent to the second region of the channel; and (b) contacting the engineered biological nanopore with a biopolymer.

[0226] In some aspects, the present disclosure provides an engineered biological nanopore comprising a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein (i) the first region is modified to be more net negative than a respective region of a wild-type biological nanopore and / or (ii) the second region is modified to be more net neutral or more net negative than a respective region of the wild-type biological nanopore, and wherein the first region of the channel is adjacent to the second region of the channel.

[0227] In some embodiments, the first region is more net negative than the second region. In some embodiments, one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids. In some embodiments, one or more amino acids in the first region is mutated to one or more negative amino acids. In some embodiments, when one or more amino acids in the -30- second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids, then one or more amino acids in the adjacent region is mutated to one or more negative amino acids. In some embodiments, the first region comprises at least one amino acid that is mutated to exhibit an increased net negative charge. In some embodiments, the first region comprises at least one amino acid that is mutated to exhibit the increased net negative charge as compared to a respective region of a wild-type biological nanopore. In some embodiments, the first region comprises at least one amino acid that is mutated to a negative amino acid to exhibit the increased net negative charge as compared to the respective region of the wild-type biological nanopore. In some embodiments, the mutated at least one amino acid in the first region is at most 10 nm away from a mutated at least one amino acid in the second region. In some embodiments, a first ring of charge comprising the mutated at least one amino acid in the first region is at most 10 nm away from the mutated at least one amino acid in the second region. In some embodiments, the first ring of charge comprising the mutated at least one amino acid in the first region is at most 10 nm away from a second ring of charge comprising the mutated at least one amino acid in the second region. In some embodiments, the second region comprise a C(alpha)-C(alpha) diameter of at most 5 nm.

[0228] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the engineered biological nanopore comprises a first portion corresponding to the first region and a second portion corresponding to the second region. In some embodiments, a monomer of the engineered biological nanopore comprises a net charge in the first portion that is more negative as compared to a net charge in the second portion. In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit an increased net negative charge. In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit the increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore. In some embodiments, the first portion comprises at least one amino acid that is mutated to a negative amino acid to exhibit the increased net negative charge as compared to the respective portion of the monomer of the wild-type biological nanopore. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit an increased net neutral charge or an increased net negative charge. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit the increased net neutral charge or increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore. In some embodiments, the second portion comprises at least one amino acid that is mutated to a neutral amino acid or a negative amino acid to exhibit the increased net neutral charge or increased net negative charge as compared to the respective portion of the monomer of the wild-type biological nanopore.

[0229] In some embodiments, the at least one mutated amino acid in the first portion is at most 10 nm away from the at least one mutated amino acid in the second portion. In some embodiments, one or more amino acids in the first portion is mutated to one or more negative amino acids. In some embodiments, one or more amino acids in the second portion is modified to one or more neutral amino acids or one or more negative amino acids. In some embodiments, when one or more amino acids in the second portion is modified to one or more neutral amino acids or one or more negative amino acids, then one or more amino acids in the first portion is mutated to one or more negative amino acids.

[0230] In some embodiments, the at least one mutated amino acid in a first portion of each of one or more monomers (or less than all monomers) forms a first ring of charge, wherein the at least one mutated amino acid in a second portion of each of one or more monomer (or less than all monomers) forms a second ring of charge, wherein each monomer of the engineered biological nanopore comprises a first portion corresponding to the first region and a second portion corresponding to the second region. In some embodiments, each monomer of the engineered biological nanopore comprises a net charge in the first portion that is more negative as compared to a net charge in the second portion. In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit an increased net negative charge. In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit the increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore. In some embodiments, the first portion comprises at least one amino acid that is mutated to a negative amino acid to exhibit the increased net negative charge as compared to the respective portion of the monomer of the wild-type biological nanopore. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit an increased net neutral charge or an increased net negative charge. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit the increased net neutral charge or increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore.

[0231] In some embodiments, the second portion comprises at least one amino acid that is mutated to a neutral amino acid or a negative amino acid to exhibit the increased net neutral charge or the increased net negative charge, respectively, as compared to the respective portion of the monomer of the wild-type biological nanopore. In some embodiments, the at least one mutated amino acid in the first portion of each monomer forms the first ring of charge. In some embodiments, the at least one mutated amino acid in the second portion of each monomer forms the second ring of charge. In some embodiments, the at least one mutated amino acid in the first portion is at most 10 nm away from the at least one mutated amino acid in the second portion. In some embodiments, one or more amino acids in the first portion is mutated to one or more negative amino acids. In some embodiments, one or more amino acids in the second portion is modified to one or more neutral amino acids or one or more negative amino acids. In some embodiments, when one or more amino acids in the second portion is modified to one or more neutral amino acids or one or more negative amino acids, then one or more amino acids in the first portion is mutated to one or more negative amino acids. In some embodiments, one or more amino acids in the first portion is mutated to one or more negative amino acids. In some embodiments, one or more amino acids in the second portion is modified to one or more neutral amino acids or one or more negative amino acids. In some embodiments, when one or more amino acids in the second portion is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids, then one or more amino acids in the first portion is mutated to one or more negative amino acids.

[0232] In some embodiments, the engineered biological nanopore generates an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, the first region modified to be more net negative and the second region modified to be more net neutral or more net negative generate the EOF. In some embodiments, the engineered biological nanopore has a cation-selectivity P(+) / P(-) of at least about 1.5. In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, a negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel.

[0233] In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel. In some embodiments, the first region is more negative as compared to another region adjacent to a constriction region of the wild-type biological nanopore. In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore. In some embodiments, the second region is more net neutral as compared to a respective region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to the respective region of the wild-type biological nanopore.

[0234] In some aspects, the present disclosure provides a system comprising: (a) a fluidic chamber; and (b) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein (i) the first region is modified to be more net negative than a respective region of a wild-type biological nanopore and / or (ii) the second region is modified to be more net neutral or more net negative than a respective region of the wild-type biological nanopore, wherein the first region of the channel is adjacent to the second region of the channel, wherein the engineered biological nanopore is configured to contact a biopolymer.

[0235] In some embodiments, the engineered biological nanopore is configured to generate an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, the first region modified to be more net negative and the second region modified to be more net neutral or more net negative is configured to generate the EOF. In some embodiments, the engineered biological nanopore has a cationselectivity P(+) / P(-) of at least about 1.5. In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, a negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge -33- of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0236] In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore. In some embodiments, the second region is more neutral as compared to a respective region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to the respective region of a wild-type biological nanopore.

[0237] In some aspects, the present disclosure provides a method comprising (a) providing a mixture containing or suspected of containing an analyte comprising polypeptide or protein, and (b) using an engineered biological nanopore to (1) determine a sequence or (2) generate a measure of a concentration or relative amount of said analyte in said mixture at an accuracy of at least 80%.

[0238] In some embodiments, said mixture contains or is suspected of containing an additional analyte comprising an additional polypeptide or protein. In some embodiments, the method further comprises using said engineered biological nanopore to generate a measure of a concentration or relative amount of said additional analyte in said mixture at an accuracy of greater than 80%. In some embodiments, (1) said sequence is determined or (2) said measure of said concentration or relative amount of said analyte is generated at an accuracy of at least 90%. In some embodiments, (1) said sequence is determined or (2) said measure of said concentration or relative amount of said analyte is generated at an accuracy of at least 95%.

[0239] 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 without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCORPORATION BY REFERENCE

[0240] 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

[0241] 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:

[0242] FIGs. 1A-1B shows schematic representations of nanopore geometries. FIG. 1A depicts a nanopore with a first opening (101) and a second opening (102). The nanopore has a channel and constriction (103) with a channel region on each side of the constriction (103). FIG. IB depicts a nanopore with example areas of charge. The figure also shows a dimension of a first entrance (107), a dimension of a second entrance (108), and a dimension of a constriction region (109).

[0243] FIGs. 2A-2B show a schematic representation of a nanopore with a constriction region and channel. FIG. 2A shows the structure of the MspA nanopore with an octameric configuration comprising 8 monomers. FIG. 2B shows the locations of amino acid residues T83, L88, N108,1105, D90, and D91. Residues D90 and D91 are in the constriction region. Residues T83, L88, N108, and 1105 are in the channel regions.

[0244] FIGs. 3A-3G show electrical current (picoAmps) versus time (seconds) recordings of protein translocations through Msp nanopores. FIG. 3A shows a current recording from a wild-type MspA pore. FIG. 3B shows a current recording from a MspA pore with D90N mutation. FIG. 3C shows a current recording from a MspA pore with D90N, D91N, and I105E mutations. FIG. 3D shows a current recording from a MspA pore with D90N, D91N, and N108E mutations. FIG. 3E shows a current recording from a MspA pore with D90N, D91N, I105E, and N108E mutations. FIG. 3F shows a current recording from a MspA pore with D90N, D91N, and L88E mutations. FIG. 3G shows a current recording from a MspA pore with D90N, D91N, L88E, and T83E. FIG. 3H shows a current reading from a MspA pore with D90N.

[0245] FIG. 4 shows a schematic representation of a CsgG nanopore. The nanopore has a wide channel region and a narrower constriction. The constriction is near the center of the channel.

[0246] FIG. 5 shows a schematic representation of a CsgG-CsgF (CsgG / F) nanopore. The nanopore has a wide channel region (501) with a narrower constriction region (502). The nanopore has an internal CsgF peptide adapter (503) in the trans entrance of the CsgG channel that creates another channel (504).

[0247] FIG. 6 shows a depiction of a computer system that is programmed or otherwise configured to implement the methods provided herein.

[0248] FIG. 7 shows a schematic representation of a nanopore comprising one or more regions and diameter. DETAILED DESCRIPTION

[0249] While various cases of the invention have been shown and described herein, it will be obvious to those skilled in the art that such cases 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 cases of the invention described herein can be employed.

[0250] 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.

[0251] 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.”

[0252] It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0253] The term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is 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.

[0254] 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” is 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” is 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.

[0255] The term “variant” can refer to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence. In some cases, the variant is a functional variant.

[0256] The term “functional variant” can refer to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence, and is capable of having one or more activities of the reference amino acid sequence.

[0257] 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 cases, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 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 is 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” is 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, functional homolog, functional ortholog, or Junctional paralog thereof. “Homologs” can refer to proteins, peptides, oligopeptides, polypeptides having amino acid substitutions, deletions, insertions, or any combination thereof relative to an unmodified (e.g., wild-type) protein and having similar biological and / or Junctional 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 is a product of gene duplication of a common ancestral gene.

[0258] 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 cases, the percent identity between two amino acid sequences is 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 yet another preferred embodiment, the percent identity between two nucleotide sequences is 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 particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0259] 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. 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.

[0260] 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.

[0261] A “conservative amino acid substitution” can be one in which the amino acid residue is 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), aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), or any combination thereof.

[0262] 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 cases, 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 -38- inversions, non-homologous chromosomal crossover, and interstitial deletions), and loss of heterozygosity. In some cases, 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. The term “negative mutation” and “negatively-charged mutation” may be used interchangeably herein. The terms “positive mutation” and “positively-charged mutation” may be used interchangeably herein. The terms “neutral mutation” and “neutrally charged mutation” may be used interchangeably herein.

[0263] Nanopores selective for ions (e.g., cation-selective nanopores for positively-charged ions) may generate strong EOFs that can facilitate the capture and / or translocation of heterogeneously charged biological molecules. The constriction region can also represent a sensing region of a nanopore described herein, wherein the sensing region may be where signal is generated (e.g., signal generated from capture and / or translocation of an analyte). A constriction region engineered with charged amino acid residues (e.g., negatively-charged amino acid residues) may generate a strong EOF and be selective for cation and / or anion species. However, without wishing to be bound by theory, an engineered biological nanopore with a charged constriction may also result in a small pool of available modifications (e.g., modifications to the nanopore) to tune a current signal.

[0264] It can be advantageous to engineer a nanopore (e.g., a biological nanopore) and / or a monomer of a nanopore to generate strong EOF with modifications outside of the constriction region. For example, the present disclosure provides engineered nanopores (e.g., biological nanopores) with modifications to the constriction region to contain one or more neutral charges and / or negative charges. These engineered biological nanopores may then be modified to introduce one or more negatively-charged amino acid residues to a region other than the constriction region (e.g., a region adjacent to the constriction region). This combination of charges can result in a strong EOF, and methods of generating a strong EOF that may not involve engineering a charged constriction region.

[0265] In some aspects, the present disclosure provides nanopores, systems, methods, or any combination thereof for analysis of an analyte (e.g., a biopolymer or a non-nucleic acid-based polymer analyte). The nanopores, systems, methods, or any combination thereof described herein may be used to determine one or more characteristics of an analyte. A characteristic of an analyte can comprise a length of the analyte (e.g., a contour length, in the case of polymeric analyte), a volume of the analyte, a mass of the analyte, a shape of the analyte, a secondary structure of the analyte, a tertiary structure of the analyte, a charge distribution of the analyte, an identity of the analyte, a sequence of the analyte, any chemical modifications of the analyte, or any combination thereof. The nanopores, systems, methods, or any combination thereof described herein may be used for single molecule analysis. The single molecule analysis may be analysis of a nucleic acid analyte or a non-nucleic acid analyte (e.g., a peptide, polypeptide, protein, or any combination thereof). The methods, systems, nanopores, or any combination thereof described herein may be used for characterizing at least one feature of a target analyte (e.g., protein, polypeptide, nucleic acid conjugated to a protein / polypeptide / peptide, or combination thereof). The detection and / or analysis may be of one or more analytes. As an example, the detection and / or analysis of the one or more analytes may be at a single molecule level. Nanopores

[0266] 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 cases, the pore (e.g., nanopore) comprises a transmembrane region. In some cases, the pore comprises a hydrophilic portion. In some cases, the pore comprises a hydrophobic portion. In some cases, the pore comprises a hydrophilic and a hydrophobic portion. In some cases, a pore comprises an opening (e.g., an entrance). In some cases, a pore comprises at least one opening. In some cases, 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, a circumference, or any combination thereof.

[0267] 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 cases, an analyte may be trapped in the channel of the nanopore. In some cases, an analyte may translocate through the channel of the nanopore. In some cases, 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.

[0268] 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 cases, 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, 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 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. The 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. A first region of the channel may be adjacent to a first end of a second region (e.g., constriction region) and a third region may be adjacent to a second end of a second region (e.g., constriction region). As an example, a first region of the channel may be adjacent to a first entrance of a second region (e.g., constriction region) and a third region may be adjacent to a second entrance of a second region (e.g., constriction region). In other cases, the first region and / or third region may be separated from the second region of the channel by a distance of at most about 4.0 run, at most about 3.0 nm, at most about 2.0 rim, at most about 1.5 run, at most about 1.0 run, 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 cases, a first region and / or third region of the channel may be separated from a second region of the channel (e.g., comprising the constriction region) 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 cases, a first region and / or third region of the channel may be separated from a second region of the channel (e.g., comprising the constriction region) 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 cases, a first region and / or third region of the channel may be separated from a second region of the channel (e.g., comprising the constriction region) by a distance from about 0.001 nm to about 15 nm. In some cases, a first region and / or third region of the channel may be separated from a second region of the channel (e.g., comprising the constriction region) by a distance from at least about 0.001 nm. In some cases, a first region and / or third region of the channel may be separated from a second region of the channel (e.g., comprising the constriction region) 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, 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 run, about 5 run to about 10 run, about 5 run to about 15 nm, or about 10 nm to about 15 nm.

[0269] Unless specifically indicated otherwise, when referring to “diameter” herein, a diameter may be determined by measuring center-to-center distances or atomic surface-to-surface distances. A diameter may be measured along a plane from a first alpha carbon of a first amino acid to a second alpha carbon of a second amino acid (e.g., between a first alpha carbon to a second alpha carbon that is opposite to the first alpha carbon). The second region (e.g., comprising a constriction region) of the nanopore may be a narrower region of the channel than another region of the channel (e.g., a first region and / or third region). In some cases, 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. Without wishing to be bound by theory, modifying a constriction region of a nanopore to shift an electrical resistance may modulate the electro-osmotic force (EOF) and / or may improve the ability of the nanopore to characterize an analyte. Characterization of an analyte may occur at the second region (e.g., constriction region). In the second region (e.g., constriction region), a current flow may be modulated by a composition of the analyte within. For example, a current flow may be modulated by local composition of an analyte and / or an amino acid composition of the analyte. The electro-osmotic flow (EOF) may be maximally created at a second region (e.g., a constriction region or narrowest region). For example, the EOF may be maximally created at a second region (e.g., a constriction region or narrowest region) due to a maximal electrostatic effect on cation or anion flux in the constrained dimensions of the second region.

[0270] The constriction region of the nanopore may be a narrower region of the channel than another region of the channel. In some cases, the constriction region of the nanopore can contribute to the electrical resistance of the nanopore. For example, the presence of charged amino acid residues may contribute to an electrical resistance. A constriction region comprising a net negative charge from one or more negatively-charged amino acid residues may affect a flow of electric current of the nanopore system and / or translocation of an analyte (e.g., a charged or uncharged analyte). Alternatively, a constriction region comprising a net negative charge from one or more negatively-charged amino acid residues may affect a flow of electric current of the nanopore system and / or translocation of an analyte (e.g., a charged or uncharged analyte). 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.

[0271] In some cases, the nanopore comprises a shape (e.g., a geometry). For example, a nanopore may be cylindrical. In some cases, the nanopore can be conical shape. In some cases, the nanopore can be globular shape. In some cases, the nanopore can be hourglass shape. In some cases, the nanopore can be a toroidal shape, comprising a ring and a channel. In some cases, 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 toroidal shape can comprise a ring or a donut shape. 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, 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).

[0272] In some cases in which the nanopore comprises an hourglass shape, the nanopore may have two or more flanking regions. The regions may be the first region and the third region described herein. For example, the first region and third region may flank a second region of the nanopore. The second region may be a constriction region of the nanopore. The first and third regions (e.g., funnel regions) can have the same diameter or different (e.g., distinct) diameters. The first and third regions can each have a diameter of at least about 2.2, at least about 2.4, at least about 2.6, at least about 2.8, at least about 3.0, at least about 3.2, at least about 3.4, at least about 3.6, at least about 3.8, at least about 4.0, at least about 4.2, at least about 4.4, at least about 4.6, at least about 4.8, at least about 5.0, at least about 5.2, at least about 5.4, at least about 5.6, at least about 5.8, at least about 6.0, at least about 6.2, at least about 6.4, at least about 6.6, at least about 6.8, at least about 7.0, at least about 7.5, at least about 8.0, at least about 8.5, at least about 9.0, at least about 9.5, at least about 10, or greater than about 10 times the diameter of the second region (e.g., constriction region).

[0273] 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 (e.g., run substantially 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., 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 cases a channel length is less than a linear length or a contour length of an analyte. In some cases a channel length is greater than a linear length or a contour length of an analyte.

[0274] In some cases, a channel comprises a channel length from about 0.5 nm to about 40 nm. In some cases, the channel can comprise a channel length of at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 5 nm, at least about 10, at least about 15 nm, at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, or more than 40 nm. In some cases, the channel can comprise a channel length of at most about 40 nm, at most about 35 nm, at most about 30 nm, at most about 25 nm, at most about 20 nm, at most about 15 nm, at most about 10 nm, at most about 5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5nm, or less than 0.5 nm. In some cases, the channel can comprise a channel length of about 0.5 nm, about 1 nm, about 1.5 nm, about 2 nm, about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, or about 40 nm.

[0275] In some cases, a first region and / or third region of the channel of the engineered biological nanopore described herein may comprise a dimension (e.g., width, length, diameter, circumference, or widest dimension) measured along a plane from a first alpha carbon of a first amino acid to a second alpha carbon of a second amino acid (e.g., between a first alpha carbon to a second alpha carbon that is opposite to the first alpha carbon). The dimension may be a diameter of the nanopore (e.g., the engineered biological nanopore). The diameter may be expressed as a Ca-Ca distance (e.g., the alpha-carbon to alpha-carbon distance). The diameter can be of a widest region of the first region and / or third region. In some cases, the diameter may be of a narrowest region of the first region and / or third region. In some cases, the dimension (e.g., width, length, diameter, circumference, or widest dimension) may be at least about 1 nanometer (nm), at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 5.5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm, wherein the dimension (e.g., diameter) is expressed as the Ca-Ca distance (e.g., the alpha-carbon to alpha-carbon distance). In some cases, the first region and / or third region of the channel of the engineered biological nanopore described herein may comprise a diameter of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5.5 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1 nm, or less than about 1 nm, wherein the diameter may be expressed as the Ca-Ca distance (e.g., the alpha-carbon to alpha-carbon distance). In some cases, the first region and / or third region of the channel of the engineered biological nanopore described herein may comprise a diameter of about 1 nm, 2 nm, 2.5 nm, 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or 10 nm. A first region and / or third region of the channel (e.g., a region adjacent to a nanopore) may be a wider than a second region of the nanopore (e.g., comprising the constriction region).In some cases, the first region and / or third region may have one or more mutations (e.g., a plurality of mutations). The one or more mutations may be at an area of the first region and / or third region with a diameter (e.g., narrowest diameter) wider than a second region (e.g., a narrowest region). A dimension (e.g., width, length, diameter, circumference, or widest dimension) may be determined by measuring from a first alpha carbon of a first amino acid (e.g., a first mutated amino acid) to a second alpha carbon of a second amino acid (e.g., a second mutated amino acid). As an example, one or more mutations of a first region and / or third region may be positioned in an area of the first region and / or third region comprising a diameter (e.g., narrowest diameter) of at least about 1 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 5.5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm, wherein the diameter is expressed as the Ca-Ca distance (e.g., the alpha-carbon to alpha-carbon distance). As another example, one or more mutations of a first region and / or third region may be positioned in an area of the first region and / or third region comprising a diameter (e.g., narrowest diameter) of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5.5 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1 nm, or less than about 1 nm, wherein the diameter may be expressed as the Ca-Ca distance (e.g., the alpha-carbon to alpha-carbon distance). In some cases, the one or more mutations of a first region and / or third region may be positioned in an area of the first region and / or third region comprising a diameter (e.g., narrowest diameter) of about 1 nm, 2 nm, 2.5 nm, 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or 10 nm. For example, as shown in FIG. 7, a nanopore 700 may comprise a first region 710 and a second region (e.g., constriction region) 720. The first region can be adjacent to the second region. The first region can have a diameter 730. This diameter 730 can be expressed as a dimension from a first alpha carbon of a first amino acid to a second alpha carbon of a second amino acid (e.g., between a first alpha carbon to a second alpha carbon that is opposite to the first alpha carbon). A mutation 740 can be introduced at the area of the first region 710 where there is a diameter (e.g., narrowest diameter) 730.

[0276] A nanopore can comprise at least one opening (e.g., entrance). The opening can be a first opening. The nanopore may comprise two or more opening (e.g., entrances). For example, a nanopore described herein may comprise a first opening and a second opening. An opening of a nanopore can face a side (e.g., compartment) of a nanopore system described herein. For example, a first opening of a nanopore may face a first side (e.g., cis side) of a nanopore system. A second opening of a nanopore may face a second side (e.g., trans side) of a nanopore system. As shown in FIG. 1A, a nanopore described herein may comprise a first opening (101). The first opening (101) can face a first side (e.g., a cis side) of the nanopore system. The engineered biological nanopore may comprise a second opening (102). The second opening (102) can face a second side (e.g., a trans side) of the nanopore system.

[0277] In some cases, a first region and / or third region of a nanopore may comprise a larger dimension (e.g., width, length, diameter, circumference, or widest dimension) compared to a second region (e.g., comprising a constriction region) of the channel of the nanopore. For example, the first region and / or third region of the channel of the nanopore may comprise a dimension (e.g., width, length, diameter, circumference, or widest dimension) that may be at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than about 90% larger than that of a second region (e.g., comprising a constriction region) of the channel of the nanopore. For example, the first region and / or third region of the channel of the nanopore may comprise a dimension (e.g., width, length, diameter, circumference, or widest dimension) that may be at most about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less than about 5% larger than that of a second region (e.g., comprising a constriction region) of the channel of the nanopore. In some cases, an engineered biological nanopore described herein may comprise a first region and / or third region of a channel (e.g., adjacent to a constriction region) that can be at least about 1.5x, 2x, 3x, 4x, 5x, lOx, 50x, or greater than about 50x wider than a second region of a channel (e.g., comprising a constriction region). In some cases, an engineered biological nanopore described herein may comprise a first region and / or third region of a channel (e.g., adjacent to a constriction region) that can be at most about 50x, lOx, 5x, 4x, 3x, 2x, 1.5x or less than about 1.5x wider than a second region of a channel (e.g., comprising a constriction region). In some cases, an engineered biological nanopore described herein may comprise a first region and / or third region of a channel (e.g., adjacent to a constriction region) that can be from about 2x to about 50x wider than a second region of a channel (e.g., comprising a constriction region). In some cases, an engineered biological nanopore described herein may comprise a first region and / or third region of a channel (e.g., adjacent to a constriction region) that can be from about 2x to about 3x, about 2x to about 4x, about 2x to about 5x, about 2x to about 6x, about 2x to about 7x, about 2x to about 8x, about 2x to about 9x, about 2x to about lOx, about 2x to about 20x, about 2x to about 25x, about 2x to about 5 Ox, about 3x to about 4x, about 3x to about 5x, about 3x to about 6x, about 3x to about 7x, about 3x to about 8x, about 3x to about 9x, about 3x to about lOx, about 3x to about 20x, about 3x to about 25x, about 3x to about 5Ox, about 4x to about 5x, about 4x to about 6x, about 4x to about 7x, about 4x to about 8x, about 4x to about 9x, about 4x to about lOx, about 4x to about 20x, about 4x to about 25x, about 4x to about 50x, about 5x to about 6x, about 5x to about 7x, about 5x to about 8x, about 5x to about 9x, about 5x to about lOx, about 5x to about 20x, about 5x to about 25x, about 5x to about 5 Ox, about 6x to about 7x, about 6x to about 8x, about 6x to about 9x, about 6x to about lOx, about 6x to about 20x, about 6x to about 25x, about 6x to about 5Ox, about 7x to about 8x, about 7x to about 9x, about 7x to about lOx, about 7x to about 20x, about 7x to about 25x, about 7x to about 50x, about 8x to about 9x, about 8x to about lOx, about 8x to about 20x, about 8x to about 25x, about 8x to about 5Ox, about 9x to about lOx, about 9x to about 20x, about 9x to about 25x, about 9x to about 50x, about lOx to about 20x, about lOx to about 25x, about lOx to about 50x, about 20x to about 25x, about 20x to about 50x, or about 25x to about 50x wider than a second region of a channel (e.g., comprising a constriction region).

[0278] In some cases, a first opening of a nanopore may be the same dimension (e.g., diameter, circumference, and / or widest dimension) as a second opening. In some cases, a first opening of a nanopore may be a different dimension (e.g., diameter, circumference, and / or widest dimension) as a second opening. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) of a first opening of 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 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, at least about 16 nm, at least about 17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm, at least about 25 nm, at least about 30 nm, or greater than about 30 nm. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) of a first opening of at most about 30 nm, at most about 25 nm, at most about 20 nm, at most about 19 nm, at most about 18 nm, at most about 17 nm, at most about 16 nm, at most about 15 nm, at most about 14 nm, at most about 13 nm, at most about 12 nm, at most about 11 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 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, or less than about 0.1 nm. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) of a first opening (e.g., cis opening) from about 1 nm to about 8 nm. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) of a first opening (e.g., cis opening) from 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 4.5 nm, about 1 nm to about 5 nm, about 1 nm to about 6 nm, about 1 nm to about 7 nm, about 1 nm to about 8 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 4.5 nm, about 1.5 nm to about 5 nm, about 1.5 nm to about 6 nm, about 1.5 nm to about 7 nm, about 1.5 nm to about 8 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 4.5 nm, about 2 nm to about 5 nm, about 2 nm to about 6 nm, about 2 nm to about 7 nm, about 2 nm to about 8 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 4.5 nm, about 2.5 nm to about 5 nm, about 2.5 nm to about 6 nm, about 2.5 nm to about 7 nm, about 2.5 nm to about 8 nm, about 3 nm to about 3.5 nm, about 3 nm to about 4 nm, about 3 nm to about 4.5 nm, about 3 nm to about 5 nm, about 3 nm to about 6 nm, about 3 nm to about 7 nm, about 3 nm to about 8 nm, about 3.5 nm to about 4 nm, about 3.5 nm to about 4.5 nm, about 3.5 nm to about 5 nm, about 3.5 nm to about 6 nm, about 3.5 nm to about 7 nm, about 3.5 nm to about 8 nm, about 4 nm to about 4.5 nm, about 4 nm to about 5 nm, about 4 nm to about 6 nm, about 4 nm to about 7 nm, about 4 nm to about 8 nm, about 4.5 nm to about 5 nm, about 4.5 nm to about 6 nm, about 4.5 nm to about 7 nm, about 4.5 nm to about 8 nm, about 5 nm to about 6 nm, about 5 nm to about 7 run, about 5 run to about 8 run, about 6 run to about 7 run, about 6 run to about 8 run, or about 7 nm to about 8 nm. In some cases, a nanopore provided herein may comprise a dimension of a first opening (e.g., cis opening) (e.g., diameter, circumference, and / or widest dimension) from about 8 nm to about 30 nm. In some cases, a nanopore provided herein may comprise a dimension of a first opening (e.g., cis opening) (e.g., diameter, circumference, and / or widest dimension) from at most about 30 nm. In some cases, a nanopore provided herein may comprise a dimension of a first opening (e.g., cis opening) (e.g., diameter, circumference, and / or widest dimension) from about 8 nm to about 9 nm, about 8 nm to about 10 nm, about 8 nm to about 11 nm, about 8 nm to about 12 nm, about 8 nm to about 13 nm, about 8 nm to about 14 nm, about 8 nm to about 15 nm, about 8 nm to about 20 nm, about 8 nm to about 25 nm, about 8 nm to about 30 nm, about 9 nm to about 10 nm, about 9 nm to about 11 nm, about 9 nm to about 12 nm, about 9 nm to about 13 nm, about 9 nm to about 14 nm, about 9 nm to about 15 nm, about 9 nm to about 20 nm, about 9 nm to about 25 nm, about 9 nm to about 30 nm, about 10 nm to about 11 nm, about 10 nm to about 12 nm, about 10 nm to about 13 nm, about 10 nm to about 14 nm, about 10 nm to about 15 nm, about 10 nm to about 20 nm, about 10 nm to about 25 nm, about 10 nm to about 30 nm, about 11 nm to about 12 nm, about 11 nm to about 13 nm, about 11 nm to about 14 nm, about 11 nm to about 15 nm, about 11 nm to about 20 nm, about 11 nm to about 25 nm, about 11 nm to about 30 nm, about 12 nm to about 13 nm, about 12 nm to about 14 nm, about 12 nm to about 15 nm, about 12 nm to about 20 nm, about 12 nm to about 25 nm, about 12 nm to about 30 nm, about 13 nm to about 14 nm, about 13 nm to about 15 nm, about 13 nm to about 20 nm, about 13 nm to about 25 nm, about 13 nm to about 30 nm, about 14 nm to about 15 nm, about 14 nm to about 20 nm, about 14 nm to about 25 nm, about 14 nm to about 30 nm, about 15 nm to about 20 nm, about 15 nm to about 25 nm, about 15 nm to about 30 nm, about 20 nm to about 25 nm, about 20 nm to about 30 nm, or about 25 nm to about 30 nm.

[0279] In some cases, a nanopore provided herein may comprise a dimension of a first opening of about 1 nanometer (nm), about 2 nm, about 3 nm, about 4 nm, or about 5 nm. In some cases, a nanopore provided herein may comprise a dimension of a second opening of about 1 nm, about 2 nm, about 3 nm, about 4 nm, or about 5 nm.

[0280] In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) of a second opening (e.g., trans opening) of at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 5.5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, or greater than about 15 nm. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) of a second opening (e.g., trans opening) of at most about 15 nm, at most about 14 nm, at most about 13 nm, at most about 12 nm, at most about 11 nm, at most about 10 nm, at most about 9 nm, at most -48- about 8 nm, at most about 7 run, at most about 6 run, at most about 5.5 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, or less than about 1 nm.

[0281] In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) of a second opening (e.g., trans opening) from about 0.5 nm to about 6 nm. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) of a second opening (e.g., trans opening) from 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 4.5 nm, about 0.5 nm to about 5 nm, about 0.5 nm to about 5.5 nm, about 0.5 nm to about 6 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 4.5 nm, about 1 nm to about 5 nm, about 1 nm to about 5.5 nm, about 1 nm to about 6 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 4.5 nm, about 1.5 nm to about 5 nm, about 1.5 nm to about 5.5 nm, about 1.5 nm to about 6 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 4.5 nm, about 2 nm to about 5 nm, about 2 nm to about 5.5 nm, about 2 nm to about 6 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 4.5 nm, about 2.5 nm to about 5 nm, about 2.5 nm to about 5.5 nm, about 2.5 nm to about 6 nm, about 3 nm to about 3.5 nm, about 3 nm to about 4 nm, about 3 nm to about 4.5 nm, about 3 nm to about 5 nm, about 3 nm to about 5.5 nm, about 3 nm to about 6 nm, about 3.5 nm to about 4 nm, about 3.5 nm to about 4.5 nm, about 3.5 nm to about 5 nm, about 3.5 nm to about 5.5 nm, about 3.5 nm to about 6 nm, about 4 nm to about 4.5 nm, about 4 nm to about 5 nm, about 4 nm to about 5.5 nm, about 4 nm to about 6 nm, about 4.5 nm to about 5 nm, about 4.5 nm to about 5.5 nm, about 4.5 nm to about 6 nm, about 5 nm to about 5.5 nm, about 5 nm to about 6 nm, or about 5.5 nm to about 6 nm. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) of a second opening (e.g., trans opening) from about 6 nm to about 15 nm. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and / or widest dimension) of a second opening (e.g., trans opening) from about 6 nm to about 7 nm, about 6 nm to about 8 nm, about 6 nm to about 9 nm, about 6 nm to about 10 nm, about 6 nm to about 11 nm, about 6 nm to about 12 nm, about 6 nm to about 13 nm, about 6 nm to about 14 nm, about 6 nm to about 15 nm, about 7 nm to about 8 nm, about 7 nm to about 9 nm, about 7 nm to about 10 nm, about 7 nm to about 11 nm, about 7 nm to about 12 nm, about 7 nm to about 13 nm, about 7 nm to about 14 nm, about 7 nm to about 15 nm, about 8 nm to about 9 nm, about 8 nm to about 10 nm, about 8 nm to about 11 nm, about 8 nm to about 12 nm, about 8 nm to about 13 nm, about 8 nm to about 14 nm, about 8 nm to about 15 nm, about 9 nm to about 10 nm, about 9 nm to about 11 nm, about 9 nm to about 12 nm, about 9 nm to about 13 nm, about 9 nm to about 14 run, about 9 run to about 15 run, about 10 nm to about 11 nm, about 10 run to about 12 nm, about 10 nm to about 13 nm, about 10 nm to about 14 nm, about 10 nm to about 15 nm, about 11 nm to about 12 nm, about 11 nm to about 13 nm, about 11 nm to about 14 nm, about 11 nm to about 15 nm, about 12 nm to about 13 nm, about 12 nm to about 14 nm, about 12 nm to about 15 nm, about 13 nm to about 14 nm, about 13 nm to about 15 nm, or about 14 nm to about 15 nm.

[0282] The constriction region of the nanopore may also have a dimension (e.g., diameter, circumference, and / or widest dimension). The constriction region may comprise a length, where the length can be a length along a longitudinal axis of a channel region of a nanopore. In some cases, the length of the constriction region of the nanopore (e.g., biological nanopore) can be 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 2.0 nm, at least about 3.0 nm, at least about 4.0 nm, at least about 5.0 nm, or greater than about 5.0 nm. In some cases, the length of the constriction region of the nanopore (e.g., biological nanopore) can be at most about 5.0 nm, at most about 4.0 nm, at most about 3.0 nm, at most about 2.0 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 cases, the length of the constriction region of the nanopore (e.g., biological nanopore) can be from about 0.1 nm to about 1.2 nm. In some cases, the length of the constriction region of the nanopore (e.g., biological nanopore) can be from 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 0.6 nm, about 0.1 nm to about 0.7 nm, about 0.1 nm to about 0.8 nm, about 0.1 nm to about 0.9 nm, about 0.1 nm to about 1 nm, about 0.1 nm to about 1.1 nm, about 0.1 nm to about 1.2 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 0.6 nm, about 0.2 nm to about 0.7 nm, about 0.2 nm to about 0.8 nm, about 0.2 nm to about 0.9 nm, about 0.2 nm to about 1 nm, about 0.2 nm to about 1.1 nm, about 0.2 nm to about 1.2 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 0.6 nm, about 0.3 nm to about 0.7 nm, about 0.3 nm to about 0.8 nm, about 0.3 nm to about 0.9 nm, about 0.3 nm to about 1 nm, about 0.3 nm to about 1.1 nm, about 0.3 nm to about 1.2 nm, about 0.4 nm to about 0.5 nm, about 0.4 nm to about 0.6 nm, about 0.4 nm to about 0.7 nm, about 0.4 nm to about 0.8 nm, about 0.4 nm to about 0.9 nm, about 0.4 nm to about 1 nm, about 0.4 nm to about 1.1 nm, about 0.4 nm to about 1.2 nm, about 0.5 nm to about 0.6 nm, about 0.5 nm to about 0.7 nm, about 0.5 nm to about 0.8 nm, about 0.5 nm to about 0.9 nm, about 0.5 nm to about 1 nm, about 0.5 nm to about 1.1 nm, about 0.5 nm to about 1.2 nm, about 0.6 nm to about 0.7 nm, about 0.6 nm to about 0.8 nm, about 0.6 nm to about 0.9 nm, about 0.6 nm to about 1 nm, about 0.6 nm to about 1.1 nm, about 0.6 nm to about 1.2 nm, about 0.7 nm to about 0.8 nm, about 0.7 nm to about 0.9 nm, about 0.7 nm to about 1 nm, about 0.7 nm to about 1.1 nm, about 0.7 nm to about 1.2 nm, about 0.8 nm to about 0.9 nm, about 0.8 nm to about 1 nm, about 0.8 nm to about 1.1 nm, about 0.8 nm to about 1.2 run, about 0.9 nm to about 1 run, about 0.9 run to about 1.1 run, about 0.9 nm to about 1.2 nm, about 1 nm to about 1.1 nm, about 1 nm to about 1.2 nm, or about 1.1 nm to about 1.2 nm.

[0283] In some cases, a second region of the channel of the engineered biological nanopore described herein may comprise a dimension (e.g., width, length, diameter, circumference, or widest dimension) measured along a plane from a first alpha carbon of a first amino acid to a second alpha carbon of a second amino acid. The dimension may be a diameter of the nanopore (e.g., the engineered biological nanopore) expressed as a Ca-Ca distance (e.g., the alpha-carbon to alpha-carbon distance). The diameter can be of any region of the second region, for example a narrowest region. In some cases, the dimension (e.g., width, length, diameter, circumference, or widest dimension) of the second region may be at least about 0.1 nm, at least about 0.5 nm, at least about 1.0 nm, at least about 1.5 nm, at least about 2.0 nm, at least about 2.5 nm, at least about 3.0 nm, at least about 3.5 nm, at least about 4.0 nm, at least about 4.5 nm, at least about 5.0 nm, or greater than about 5.0 nm. In some cases, the dimension (e.g., width, length, diameter, circumference, or widest dimension) of the second region may be 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 0.1 nm, or less than about 0.1 nm. In some cases, the dimension (e.g., width, length, diameter, circumference, or widest dimension) of the second region may be about 0.1 nm, 0.5 nm, 1.0 nm, 2.0 nm, 2.5 nm, 3.0 nm, 3.5 nm, 4.0 nm, 4.5 nm, or 5.0 nm. In some cases, the dimension (e.g., width, length, diameter, circumference, or widest dimension) of the second region may be from about 0.5 nm to 2.0 nm.

[0284] In some cases, a first region and / or third region can comprise a length. The length of the first region and / or third region can be a length as measured along a longitudinal axis of the channel. This longitudinal axis may run perpendicular to a membrane (e.g., run substantially perpendicular to a membrane). The length of the first region and / or third region can be measured as the distance along the longitudinal axis of the first region and / or third region between the most distant points of the first region and / or third region along the longitudinal axis of the channel. In some cases, a length of a first region and / or third region of the nanopore may be 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, at least about 20 nm, or greater than about 20 nm. In some cases, a length of a first region and / or third region of the nanopore may be at most about 20 nm, 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, or less than about 0.5 nm. In some cases, a length of a first region and / or third region of the nanopore may be about 0.5 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 10 nm, about 15 nm, or about 20 nm. In some cases, a length of the first region and / or third region may be from about 3 nm to 10 nm.

[0285] In some cases, a second region (e.g., constriction region) can comprise a length. The length of the second region (e.g., constriction region) can be a length as measured along a longitudinal axis of the channel. This longitudinal axis may run perpendicular to a membrane (e.g., run substantially perpendicular to a membrane). The length of the second region (e.g., constriction region) can be measured as the distance along the longitudinal axis of the second region (e.g., constriction region) between the most distant points of the second region (e.g., constriction region) along the longitudinal axis of the channel. In some cases, a length of a second region (e.g., constriction region) of the nanopore may be at least about 0.1 run, at least about 0.5 run, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 run, at least about 3 run, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 10 nm, or greater than about 10 nm. In some cases, a length of a second region (e.g., constriction region) of the nanopore may be at most about 10 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, at most about 0.1 nm, or less than about 0.1 nm. In some cases, a length of a second region (e.g., constriction region) of the nanopore may be about 0.5 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 10 nm, about 15 nm, or about 20 nm. In some cases, a length of the second region (e.g., constriction region) may be from about 0.1 nm to 5 nm.

[0286] In some cases, one or more amino acid mutations may be present in a region of the nanopore comprising a constriction region (e.g., a narrowest region). The one or more amino acid mutations may be present in a region (e.g., constriction region) comprising 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. For example, one or more amino acid mutations may be present in a region (e.g., constriction region) comprising 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 1.0 nm, at least about 1.5 nm, at least about 2.0 nm, at least about 2.5 nm, at least about 3.0 nm, at least about 3.5 nm, at least about 4.0 nm, at least about 4.5 nm, at least about 5.0 nm, or greater than about 5.0 nm. As another example, one or more amino acid mutations may be present in a region (e.g., constriction region) comprising 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 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 0.1 nm, or less than about 0.1 nm.

[0287] As another example, the one or more amino acid mutations may be present in a region (e.g., constriction region) comprising 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 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 run to about 1 nm, about 0.3 run to about 1.5 run, about 0.3 nm to about 2 run, 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.

[0288] In some cases, 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 cases, 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 nanopore 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 cases, 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 nanopore 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, atmost 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 most 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.

[0289] In some cases, 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 nanopore described herein may be from about -53- 0.0001 nm to about 2 nm. In some cases, 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 nanopore 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.

[0290] A constriction region may be located at any region of a nanopore (e.g., a region of the channel of the biological nanopore). In some cases, the constriction region can be located at a distance from a first entrance of the nanopore (e.g., the biological nanopore). For example, the constriction region can be adjacent to a first entrance of the nanopore. A constriction region may be located at least about 0.0001 nm (nanometers), 0.001 nm, 0.01 nm, 0.05 nm, 0.1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, or greater than about 20 nm from a first entrance of a nanopore. A constriction region may be located at most about 20 nm, 15 nm, 10 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm, 0.1 nm, 0.05 nm, 0.01 nm, 0.001 nm, 0.0001 nm, or less than about 0.0001 nm from a first entrance of a nanopore. In some cases, a constriction region may be located from about 0.0001 nm to about 10 nm from a first entrance of a nanopore. In some cases, a constriction region may be located from about 0.0001 nm to about 0.0005 nm, 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 1 nm, about 0.0001 nm to about 2 nm, about 0.0001 nm to about 3 nm, about 0.0001 nm to about 4 nm, about 0.0001 nm to about 5 nm, about 0.0001 nm to about 10 nm, about 0.0005 nm to about 0.001 nm, about 0.0005 run to about 0.005 run, about 0.0005 nm to about 0.01 run, about 0.0005 nm to about 0.05 nm, about 0.0005 nm to about 1 nm, about 0.0005 nm to about 2 nm, about 0.0005 nm to about 3 nm, about 0.0005 nm to about 4 nm, about 0.0005 nm to about 5 nm, about 0.0005 nm to about 10 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 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 0.005 nm to about 0.01 nm, about 0.005 nm to about 0.05 nm, about 0.005 nm to about 1 nm, about 0.005 nm to about 2 nm, about 0.005 nm to about 3 nm, about 0.005 nm to about 4 nm, about 0.005 nm to about 5 nm, about 0.005 nm to about 10 nm, about 0.01 nm to about 0.05 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 0.05 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 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 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, about 3 nm to about 4 nm, about 3 nm to about 5 nm, about 3 nm to about 10 nm, about 4 nm to about 5 nm, about 4 nm to about 10 nm, or about 5 nm to about 10 nm from a first entrance of a nanopore.

[0291] In some cases, the constriction region can be located at a distance from a second entrance of the nanopore (e.g., the biological nanopore). For example, the constriction region can be located adjacent to the second entrance of the nanopore. A constriction region may be located at least about 0.0001 nm, 0.001 nm, 0.01 nm, 0.05 nm, 0.1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, or greater than about 20 nm from a second entrance of a nanopore. A constriction region may be located at most about 20 nm, 15 nm, 10 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm, 0.1 nm, 0.05 nm, 0.01 nm, 0.001 nm, 0.0001 nm, or less than about 0.0001 nm from a second entrance of a nanopore. In some cases, a constriction region may be located from about 0.0001 nm to about 10 nm from a second entrance of a nanopore. In some cases, a constriction region may be located from about 0.0001 nm to about 0.0005 nm, 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 1 nm, about 0.0001 nm to about 2 nm, about 0.0001 nm to about 3 nm, about 0.0001 nm to about 4 nm, about 0.0001 nm to about 5 nm, about 0.0001 nm to about 10 nm, about 0.0005 nm to about 0.001 nm, about 0.0005 nm to about 0.005 nm, about 0.0005 nm to about 0.01 nm, about 0.0005 nm to about 0.05 nm, about 0.0005 nm to about 1 nm, about 0.0005 nm to about 2 nm, about 0.0005 nm to about 3 nm, about 0.0005 nm to about 4 nm, about 0.0005 nm to about 5 nm, about 0.0005 nm to about 10 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 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 0.005 nm to about 0.01 nm, about 0.005 run to about 0.05 run, about 0.005 run to about 1 run, about 0.005 run to about 2 run, about 0.005 nm to about 3 nm, about 0.005 nm to about 4 nm, about 0.005 nm to about 5 nm, about 0.005 nm to about 10 nm, about 0.01 nm to about 0.05 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 0.05 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 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 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, about 3 nm to about 4 nm, about 3 nm to about 5 nm, about 3 nm to about 10 nm, about 4 nm to about 5 nm, about 4 nm to about 10 nm, or about 5 nm to about 10 nm from a second entrance of a nanopore.

[0292] A constriction region can be located between the first and second entrance of a nanopore (e.g., the biological nanopore). For example, a constriction region may reside in a channel of a nanopore described herein at any distance between a first entrance and a second entrance of the nanopore. A nanopore may comprise a constriction region with an adjacent channel region. An adjacent channel region can comprise a region of the nanopore channel that may be at least about 0.0001 nm, 0.001 nm, 0.01 nm, 0.05 nm, 0.1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, or greater than about 10 nm from a constriction region. An adjacent channel region can comprise a region of the nanopore channel that may be at most about 10 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm, 0.1 nm, 0.05 nm, 0.01 nm, 0.001 nm, 0.0001 nm, or less than about 0.0001 nm from a constriction region.

[0293] The methods, systems, nanopores, or any combination thereof described herein can comprise increasing the net negative charge at a constriction area and / or in a funnel region just outside of the at least one constriction area. The distance can be a length between a first region (e.g., a funnel region) and a second region (e.g., a constriction region). The distance (e.g., length) of a funnel region to the constriction area (measured along the central pore axis) can be from about 0.2 nm, about 0.3 nm, about 0.4 nm, about 0.5 nm, about 0.6 nm, about 0.7 nm, about 0.8 nm, about 0.9 nm, about 1.0 nm, about 1.2 nm, about 1.5 nm, about 2.0 and an upper limit independently selected from about 3 nm, about 2.9 nm, about 2.8 nm, about 2.7 nm, about 2.6 nm, about 2.5 nm, about 2.4 nm, about 2.3 nm, about 2.2 nm, or about 2.1 nm.

[0294] In some cases, the nanopore can be a transmembrane protein pore derived from beta-barrel pores or alpha-helix bundle pores, beta-barrel pores comprising a barrel or channel that is formed from beta-strands. Examples of beta-barrel pores can include, but are not limited to, beta-toxins, such as alpha-hemolysin, anthrax toxin and leukocidins, and outer membrane proteins / porins of bacteria, such as Mycobacterium smegmatis porin (Msp), for example MspA, MspB, MspC or MspD, CsgG from the E. colt curb secretion system, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, outer membrane protein FhuA, outer membrane protein A (OmpA) and Neisseria autofransporter lipoprotein (NalP) -56- and other pores, such as lysenin, bacterial nucleoside transporter Tsx. The nanopore (e.g., engineered biological nanopore) can comprise one or more monomeric units. The one or more monomeric units may be from a betabarrel pore or alpha-helix pore. The terms “monomer” and “monomeric unit” may be used interchangeably herein. The monomeric unit can be the individual subunit protein of a pore described herein. One or more monomeric units can assemble to form a functional porin channel. As an example, a nanopore (e.g., engineered biological nanopore) described herein may comprise one or more monomeric units of a MspA pore, CsgG pore, CsgF pore, OmpF pore, OmpG pore, outer membrane phospholipase A pore, outer membrane protein FhuA pore, OmpA pore, NalP pore, aHL pore, FraC pore, lysenin pore, bacterial nucleoside transporter Tsx pore, or any combinations thereof, or any functional homologs thereof, or any functional paralogs thereof, or any functional orthologs thereof. In some cases, an engineered biological nanopore described herein may comprise one or more monomers (e.g., mutant monomers) of MspA / MsmegO965, MspB / Msmeg0520, MspC / Msmeg5483, MspD / Msmeg6057, MppA, PorMl, PorM2, PorMl, Mmcs4296, Mmcs4297, Mmcs3857, Mmcs4382, Mmcs4383, Mjls3843, Mjls3857, Mjls3931 Mjls4674, Mjls4675, Mjls4677, Map3123c, Mav3943, Mvanl836, Mvan4117, Mvan4839, Mvan4840, Mvan5016, Mvan5017, Mvan5768, MUL —2391, Mflvl734, Mflvl735, Mflv2295, Mflvl891, MCH4691c, MCH4689c, MCH4690c, MAB1080, MAB1081, MAB2800, RHA1 ro08561, RHA1 ro04074, RHA1 ro03127, or any combinations thereof.

[0295] In some cases, alpha-helix bundle pores comprise a barrel or channel that is formed from alpha-helices. Examples of alpha-helix bundle pores can include, but are not limited to, inner membrane proteins and outer membrane proteins, such as WZA polysaccharide transporter and FraC. In a specific embodiment, the nanopore is selected from the group consisting of Aerolysin (Aer), Cytolysin K (CytK), Mycobacterium smegmatis porin A (MspA), alpha-hemolysin (aHL), E. colt curli secretion system component CsgG, Fragaceatoxin C (FraC) or an engineered mutant thereof. In one embodiment the nanopore is 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).

[0296] In some cases, 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. The nanopore may comprise a T7 nanopore, a SPP1 nanopore, a Phi29 nanopore, a Mycobacterium smegmatis porin A (MspA) nanopore, a fragaceatoxin C (FraC) nanopore, a cytolysin A (ClyA) nanopore, a TMH4C4 nanopore, or any combination thereof. In some cases, 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. The nanopore may comprise a stable protein 1 (SP1) nanopore, a pleurotolysin toxin (PlyAB) nanopore, an outer membrane protein G (OmpG) nanopore, an aerolysin nanopore, a ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof. In some cases, the nanopore (e.g., the biological nanopore) can comprise -57- a vestibule geometry (e.g., a globular geometry or goblet geometry). The nanopore may comprise an alphahemolysin nanopore, a curli specific gene G (CsgG) nanopore, or any combination thereof.

[0297] In some cases, the nanopore comprises a pore-forming toxin. The nanopore can comprise an a-helical pore-forming toxin, a P-barrel 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 cases, 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.

[0298] In some cases, the nanopore can be a T7 pore, a PN pore, a SP1 pore, a Phi29 pore, a PlyAB pore, an alpha-hemolysin (a-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. In some cases, the nanopore described herein may comprise one or more monomers from T7, PN, SP1, Phi29, PlyAB, a-HL, SPP1, FraC, MspA, CsgG, OmpG, aerolysin, ClyA, FhuA, PFO, TMH4C4, or any combination thereof. In some cases, 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 (a-HL) pore, a SPP 1 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. In some cases, the nanopore described herein may comprise one or more monomers from T7, PN, SP1, Phi29, PlyAB, a-HL, SPP1, FraC, MspA, CsgG, OmpG, aerolysin, ClyA, FhuA, PFO, TMH4C4, or any combination thereof. In some cases, the nanopore may comprise a one or more monomers from T7, PN, SP1, Phi29, PlyAB, a-HL, SPP1, FraC, MspA, CsgG, OmpG, aerolysin, ClyA, FhuA, PFO, or TMH4C4, or a mutant thereof, or a functional homolog thereof, or a Junctional ortholog thereof, or a functional paralog thereof. The monomer described herein can be a pore-forming protein. Pore-forming proteins can represent a group of molecules that create channels or pores in membranes. The proteins can assemble into pores and allow the passage of analytes (e.g., molecules, ions, nucleic acids, peptides, polypeptides, proteins, or combinations thereof) through a membrane (e.g., lipid bilayer).

[0299] In some cases, the nanopore may comprise an assembly of monomers. The nanopore may comprise a number of monomers in an arrangement. Monomers may be arranged vertically, horizontally, layered as rings, or any combination thereof, to form a nanopore described herein. In some cases, 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 cases, 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 cases, a nanopore may comprise 1 monomer. For example, the nanopore may be an OmpG nanopore. In some cases, a nanopore (e.g., biological nanopore) comprises from about 3 monomers to about 40 monomers. In some cases, 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 9 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.

[0300] A monomer can comprise one or more portions. For example, a monomer may comprise a first portion, a second portion, a third portion, or any combination thereof. In some cases, a monomer may comprise more than three portions. A first portion of a monomer can correspond to a first region of a nanopore described herein (e.g., an engineered biological nanopore). A second portion of a monomer can correspond to a second region of a nanopore described herein (e.g., an engineered biological nanopore). The second portion of the monomer may be a constriction region-forming portion. A third portion of a monomer can correspond to a third region of a nanopore described herein (e.g., an engineered biological nanopore).

[0301] In some cases, a nanopore described herein may comprise a homogeneous monomeric composition. A homogeneous monomeric composition can comprise, any of the monomers disclosed herein, with the same amino acid composition. For example, a nanopore may be comprised of 8 monomers. Each monomer may comprise at least one portion. For example, each monomer may comprise a first portion, a second portion, a third portion, or any combination thereof. In some cases, the first portions of monomers of the nanopore may be the same amino acid composition. The same amino acid composition can refer to two or more portions of monomers (e.g., first portions of the monomers) with the same amino acid sequence. In some cases, the second portions of monomers of the nanopore may be the same amino acid composition. In some cases, the third portions of monomers of the nanopore may be the same amino acid composition. In some cases, the first, second, and third portions between each monomer of the nanopore may be the same amino acid composition (e.g., comprise the same charge). In some cases, a nanopore described herein may comprise a heterogeneous monomeric composition. A heterogeneous monomeric composition can comprise monomers with different amino acid compositions. A heterogeneous monomeric composition can comprise one or more of any of the monomers disclosed herein. A heterogeneous monomeric composition can comprise, any of the monomers disclosed here, wherein a monomer the nanopore has different amino acid composition than another monomer of the nanopore. Monomers comprising different amino acid compositions may have different charges. As an example, a different amino acid composition between two monomers (e.g., one or more portions of two monomers) may be monomers (e.g., one or more portions of two monomers) with different amino acid sequences. As another example, a different amino acid composition between two monomers (e.g., one or more portions of two monomers) may be monomers (e.g., one or more portions of two monomers) with different net charges. In some cases, a first portion of at least one monomer of the nanopore may have a different amino acid composition than a first portion of another monomer of the nanopore. In some cases, a second portion of at least one monomer of the nanopore may have a different amino acid composition than a second portion of another monomer of the nanopore. In some cases, a third portion of at least one monomer of the nanopore may have a different amino acid composition than a third portion of another monomer of the nanopore. In some cases, a first portion and a second portion of at least one monomer of the nanopore may have a different amino acid composition than a first portion and a second portion of another monomer of the nanopore. In some cases, a first portion and a third portion of at least one monomer of the nanopore may have a different amino acid composition than a first portion and a third portion of another monomer of the nanopore. In some cases, a second portion and a third portion of at least one monomer of the nanopore may have a different amino acid composition than a second portion and a third portion of another monomer of the nanopore. In some cases, a first portion, a second portion, and a third portion of at least one monomer of the nanopore may have a different amino acid composition than a first portion, a second portion, and a third portion of another monomer of the nanopore.

[0302] In some cases, if the nanopore comprises a single monomer (e.g., from one proteinaceous chain), there may be a ring of charge if there are multiple mutations around the single monomer (e.g., proteinaceous chain). For example, a monomer described herein (e.g., a single monomer) may comprise two or more mutations forming a ring of charge around the single monomer (e.g., proteinaceous chain). The monomer may comprise a plurality of mutations forming the ring of charge. In some cases, the monomer may comprise at least about 2 mutations, at least about 3 mutations, at least about 4 mutations, at least about 5 mutations, at least about 10 mutations, or greater than about 10 mutations. In some cases, the monomer may comprise at most about 10 mutations, at most about 5 mutations, at most about 4 mutations, at most about 3 mutations, at most about 2 mutations, or less than about 2 mutations.

[0303] One or more of the monomers of the nanopore may be arranged vertically, horizontally, layered, or any combination thereof, the amino acid residues (e.g., positively-charged amino acid residues, negatively-charged amino acid residues, neutral amino acid residues, or any combination thereof) to form one or more rings of charge. In some cases, a pore may be engineered to contain regions of separate rings of charge 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 charge 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 charge along the longitudinal length of the channel. A nanopore may be engineered to contain regions of charges (e.g., rings of charge). For example, a nanopore may have about 2 to about 20 separate rings of charge in regions 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 about 20, about 10 to about 15, about 10 to about 20, or about 15 to about 20 separate rings of charge along the longitudinal length of the channel.

[0304] A region of a channel of a nanopore described herein may comprise one or more rings of charge. A first region of the nanopore may have a number of rings of charge. A third region of a nanopore may have a number of rings of charge. In some cases, a total number of rings of charge of a nanopore may be rings of charge in both a first region and a third region of the nanopore. In some cases, a first region and / or third region of the channel may comprise at least about 1, 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 15, at least about 20, or greater than about 20 rings of charge. In some cases, a first region and / or third region of the channel may comprise at most about 20, at most about 15, 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, at most about 1, or less than about 1 ring of charge. In some cases, a first region and / or third region of the channel may comprise from about 1 ring of charge to about 20 rings of charge. In some cases, a first region and / or third region of the channel may comprise from about 1 ring of charge to about 2 rings of charge, about 1 ring of charge to about 3 rings of charge, about 1 ring of charge to about 4 rings of charge, about 1 ring of charge to about 5 rings of charge, about 1 ring of charge to about 6 rings of charge, about 1 ring of charge to about 7 rings of charge, about 1 ring of charge to about 8 rings of charge, about 1 ring of charge to about 9 rings of charge, about 1 ring of charge to about 10 rings of charge, about 1 ring of charge to about 15 rings of charge, about 1 ring of charge to about 20 rings of charge, about 2 rings of charge to about 3 rings of charge, about 2 rings of charge to about 4 rings of charge, about 2 rings of charge to about 5 rings of charge, about 2 rings of charge to about 6 rings of charge, about 2 rings of charge to about 7 rings of charge, about 2 rings of charge to about 8 rings of charge, about 2 rings of charge to about 9 rings of charge, about 2 rings of charge to about 10 rings of charge, about 2 rings of charge to about 15 rings of charge, about 2 rings of charge to about 20 rings of charge, about 3 rings of charge to about 4 rings of charge, about 3 rings of charge to about 5 rings of charge, about 3 rings of charge to about 6 rings of charge, about 3 rings of charge to about 7 rings of charge, about 3 rings of charge to about 8 rings of charge, about 3 rings of charge to about 9 rings of charge, about 3 rings of charge to about 10 rings of charge, about 3 rings of charge to about 15 rings of charge, about 3 rings of charge to about 20 rings of charge, about 4 rings of charge to about 5 rings of charge, about 4 rings of charge to about 6 rings of charge, about 4 rings of charge to about 7 rings of charge, about 4 rings of charge to about 8 rings of charge, about 4 rings of charge to about 9 rings of charge, about 4 rings of charge to about 10 rings of charge, about 4 rings of charge to about 15 rings of charge, about 4 rings of charge to about 20 rings of charge, about 5 rings of charge to about 6 rings of charge, about 5 rings of charge to about 7 rings of charge, about 5 rings of charge to about 8 rings of charge, about 5 rings of charge to about 9 rings of charge, about 5 rings of charge to about 10 rings of charge, about 5 rings of charge to about 15 rings of charge, about 5 rings of charge to about 20 rings of charge, about 6 rings of charge to about 7 rings of charge, about 6 rings of charge to about 8 rings of charge, about 6 rings of charge to about 9 rings of charge, about 6 rings of charge to about 10 rings of charge, about 6 rings of charge to about 15 rings of charge, about 6 rings of charge to about 20 rings of charge, about 7 rings of charge to about 8 rings of charge, about 7 rings of charge to about 9 rings of charge, about 7 rings of charge to about 10 rings of charge, about 7 rings of charge to about 15 rings of charge, about 7 rings of charge to about 20 rings of charge, about 8 rings of charge to about 9 rings of charge, about 8 rings of charge to about 10 rings of charge, about 8 rings of charge to about 15 rings of charge, about 8 rings of charge to about 20 rings of charge, about 9 rings of charge to about 10 rings of charge, about 9 rings of charge to about 15 rings of charge, about 9 rings of charge to about 20 rings of charge, about 10 rings of charge to about 15 rings of charge, about 10 rings of charge to about 20 rings of charge, or about 15 rings of charge to about 20 rings of charge.

[0305] In some cases, a second region of the channel may comprise at least about 1, 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 15, at least about 20, or greater than about 20 rings of charge. In some cases, a second region of the channel may comprise at most about 20, at most about 15, 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, at most about 1, or less than about 1 ring of charge. In some cases, a second region of the channel may comprise from about 1 ring of charge to about 20 rings of charge. In some cases, a second region of the channel may comprise from about 1 ring of charge to about 2 rings of charge, about 1 ring of charge to about 3 rings of charge, about 1 ring of charge to about 4 rings of charge, about 1 ring of charge to about 5 rings of charge, about 1 ring of charge to about 6 rings of charge, about 1 ring of charge to about 7 rings of charge, about 1 ring of charge to about 8 rings of charge, about 1 ring of charge to about 9 rings of charge, about 1 ring of charge to about 10 rings of charge, about 1 ring of charge to about 15 rings of charge, about 1 ring of charge to about 20 rings of charge, about 2 rings of charge to about 3 rings of charge, about 2 rings of charge to about 4 rings of charge, about 2 rings of charge to about 5 rings of charge, about 2 rings of charge to about 6 rings of charge, about 2 rings of charge to about 7 rings of charge, about 2 rings of charge to about 8 rings of charge, about 2 rings of charge to about 9 rings of charge, about 2 rings of charge to about 10 rings of charge, about 2 rings of charge to about 15 rings of charge, about 2 rings of charge to about 20 rings of charge, about 3 rings of charge to about 4 rings of charge, about 3 rings of charge to about 5 rings of charge, about 3 rings of charge to about 6 rings of charge, about 3 rings of charge to about 7 rings of charge, about 3 rings of charge to about 8 rings of charge, about 3 rings of charge to about 9 rings of charge, about 3 rings of charge to about 10 rings of charge, about 3 rings of charge to about 15 rings of charge, about 3 rings of charge to about 20 rings of charge, about 4 rings of charge to about 5 rings of charge, about 4 rings of charge to about 6 rings of charge, about 4 rings of charge to about 7 rings of charge, about 4 rings of charge to about 8 rings of charge, about 4 rings of charge to about 9 rings of charge, about 4 rings of charge to about 10 rings of charge, about 4 rings of charge to about 15 rings of charge, about 4 rings of charge to about 20 rings of charge, about 5 rings of charge to about 6 rings of charge, about 5 rings of charge to about 7 rings of charge, about 5 rings of charge to about 8 rings of charge, about 5 rings of charge to about 9 rings of charge, about 5 rings of charge to about 10 rings of charge, about 5 rings of charge to about 15 rings of charge, about 5 rings of charge to about 20 rings of charge, about 6 rings of charge to about 7 rings of charge, about 6 rings of charge to about 8 rings of charge, about 6 rings of charge to about 9 rings of charge, about 6 rings of charge to about 10 rings of charge, about 6 rings of charge to about 15 rings of charge, about 6 rings of charge to about 20 rings of charge, about 7 rings of charge to about 8 rings of charge, about 7 rings of charge to about 9 rings of charge, about 7 rings of charge to about 10 rings of charge, about 7 rings of charge to about 15 rings of charge, about 7 rings of charge to about 20 rings of charge, about 8 rings of charge to about 9 rings of charge, about 8 rings of charge to about 10 rings of charge, about 8 rings of charge to about 15 rings of charge, about 8 rings of charge to about 20 rings of charge, about 9 rings of charge to about 10 rings of charge, about 9 rings of charge to about 15 rings of charge, about 9 rings of charge to about 20 rings of charge, about 10 rings of charge to about 15 rings of charge, about 10 rings of charge to about 20 rings of charge, or about 15 rings of charge to about 20 rings of charge.

[0306] In some cases, the rings of charge of the first region and / or third region may all be of the same charge (e.g., all the rings may be negatively-charged or neutral charged). One or more rings of charge of a region (e.g., a first region, second region, third region, or any combination thereof) may comprise a negative charge. For example, one or more rings of charge may comprise one or more negatively-charged amino acids. To achieve a negative charge, one or more amino acids may be modified to be one or more negatively -charged amino acids. One or more amino acids (e.g., one or more positively-charged amino acids and / or neutral amino acids) may be substituted for one or more negatively-charged amino acids. One or more negatively-charged amino acids may be added. One or more rings of charge of a region (e.g., a first region, second region, third region, or any combination thereof) may be more net negative as compared to one or more rings of charge of a respective region of a wild-type nanopore. One or more rings of charge of a region (e.g., a first region, second region, third region, or any combination thereof) may comprise more negatively-charged amino acids as compared to one or more rings of charge of a respective region of a wild-type nanopore. As an example, one or more amino acids (e.g., one or more positively-charged amino acids and / or neutral amino acids) may be substituted for one or more negatively-charged amino acids. As another example, one or more negatively-charged amino acids may be added to the region (e.g., a first region, second region, third region, or any combination thereof). As another example, one or more amino acids (e.g., one or more positively-charged amino acids and / or neutral amino acids) may be deleted from the region (e.g., a first region, second region, third region, or any combination thereof). As another example, one or more positively-charged amino acids may be substituted for one or more neutral amino acids in the region (e.g., a first region, second region, third region, or any combination thereof). One or more rings of charge of a first region and / or third region may be net negative. A net negative charge may be achieved when there may be a greater number of negatively-charged amino acids compared to a number of positively-charged amino acids and / or a number of neutral charged amino acids. A net negative charge of one or more rings of charge of the region (e.g., a first region, second region, third region, or any combination thereof) may be achieved by (i) substituting one or more amino acids (e.g., one or more positively-charged amino acids and / or neutral amino acids) for one or more negatively-charged amino acids; (ii) deleting one or more positively-charged amino acids and / or neutral amino acids; (iii) adding one or more negatively-charged amino acids; or (iv) any combinations thereof.

[0307] One or more rings of charge of a region (e.g., a first region, second region, third region, or any combination thereof) may comprise a neutral charge. For example, one or more rings of charge may comprise one or more neutral-charged amino acids. To achieve a neutral charge, one or more amino acids may be modified to be one or more neutral-charged amino acids. One or more amino acids (e.g., one or more positively-charged amino acids and / or negatively-charged amino acids) may be substituted for one or more neutral-charged amino acids. One or more neutral-charged amino acids may be added to the region (e.g., a first region, second region, third region, or any combination thereof). One or more rings of charge of a region (e.g., a first region, second region, third region, or any combination thereof) may be more net neutral as compared to one or more rings of charge of a respective region of a wild-type nanopore. One or more rings of charge of a region (e.g., a first region, second region, third region, or any combination thereof) may comprise more neutral-charged amino acids as compared to one or more rings of charge of a respective region of a wild-type nanopore. As an example, one or more amino acids (e.g., one or more positively-charged amino acids and / or negatively-charged amino acids) may be substituted for one or more neutral-charged amino acids. As another example, one or more neutral-charged amino acids may be added to the region (e.g., a first region, second region, third region, or any combination thereof). A number of one or more positively-charged amino acids and / or negatively-charged amino acids may be added to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and achieve a neutral charge). As another example, one or more amino acids (e.g., one or more positively-charged amino acids and / or negatively-charged amino acids) may be deleted from the region (e.g., a first region, second region, third region, or any combination thereof). A number of one or more positively-charged amino acids and / or negatively-charged amino acids may be deleted to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and achieve a neutral charge). One or more rings of charge of a first region and / or third region may be net neutral. A net neutral charge may be achieved when there may be a same number of positively-charged amino acids and negatively-charged amino acids. A net neutral charge may be achieved when there may be a same number of positively-charged amino acids and negatively-charged amino acids when the ring of charge comprises all neutral charged amino acids. A net neutral charge of one or more rings of charge of the region (e.g., a first region, second region, third region, or any combination thereof) may be achieved by (i) substituting one or more amino acids (e.g., one or more positively-charged amino acids and / or negatively-charged amino acids) for one or more neutral-charged amino acids; (ii) deleting one or more positively-charged amino acids and / or negatively-charged amino acids; (iii) adding one or more neutral-charged amino acids; or (iv) any combinations thereof. A net neutral charge of one or more rings of charge of the region (e.g., a first region, second region, third region, or any combination thereof) may also be achieved by (i) adding one or more positively-charged amino acids and / or negatively-charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and thus comprise an overall neutral charge) and / or (ii) deleting one or more positively-charged amino acids and / or negatively-charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and thus comprise an overall neutral charge).

[0308] In some cases, the rings of charge of the second region may all be of the same charge (e.g., all the rings may be negatively-charged, neutral charged, or any combination thereof). In some cases, a ring of charge of the one or more rings of charge of the first region may comprise at least one negative charge, at least one positive charge, at least one neutral charge, or any combination thereof. In some cases, a ring of charge of the one or more rings of charge of the third region may comprise at least one negative charge, at least one positive charge, at least one neutral charge, or any combination thereof. In some cases, a ring of charge of the one or more rings of charge of the second region may comprise at least one negative charge, at least one positive charge, at least one neutral charge, or any combination thereof. In some cases, one or more rings of charge of the rings of the charges in the channel of the engineered biological nanopore may be above a second region of the channel (e.g., comprising the constriction region). In some cases, one or more rings of charge of the rings of the charges in the channel of the engineered biological nanopore may be below a second region of the channel (e.g., comprising the constriction region). In some cases, one or more rings of charge of the rings of the charges in the channel of the engineered biological nanopore may be immediately above (e.g., adjacent to) a second region of the channel (e.g., comprising the constriction region). For example, a first ring of charge of the first region and / or third region may comprise a net negative charge (e.g., greater amount of negatively-charged amino acid residues than an amount of positively-charged amino acid residues and / or neutrally-charged amino acid residues). A first ring of charge of the first region and / or third region may comprise a greater negative charge distribution as compared to a charge distribution of a second ring of charge of the first region and / or third region. In some cases, one or more rings of charge of the rings of the charges in the channel of the engineered biological nanopore may be immediately below (e.g., adjacent to) a second region of the channel (e.g., comprising the constriction region). For example, a first ring of charge of the second region may comprise a net neutral charge (e.g., equal (e.g., balanced) amount of negatively-charged amino acid residues and positively-charged amino acid residues or 100% neutral amino acid residue composition). A first ring of charge of the second region may comprise a more neutral charge distribution as compared to a charge distribution of a second ring of charge of the first region and / or third region. A neutral charge distribution of the rings of charge of the second region of the channel may be 50% more than a respective region of a wild-type nanopore. For example, a first ring of charge may comprise a neutral charge distribution and a second ring of charge may comprise a positive charge distribution or a negative charge distribution. A neutral charge distribution of the rings of charge of the second region of the channel may be 100% more than a respective region of a wild-type nanopore. For example, a first ring of charge may comprise a neutral charge distribution and a second ring of charge may comprise a neutral charge distribution.

[0309] A separation of the rings may modulate an EOF of the nanopore. The number of rings, spacing of rings, or any combination thereof may affect a net charge of a channel of a nanopore. In some cases, the rings of charge (e.g., between a ring of charge in the first region and / or third region and a second ring of charge in the second region) may each be spaced 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 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at last about 9 nm, or greater than about 10 nm apart from each other along the longitudinal length of the channel. In some cases, the rings of charge (e.g., between a ring of charge in the first region and / or third region and a second ring of charge in the second region) may each be spaced at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 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 0.9 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 apart from each other along the longitudinal length of the channel.

[0310] In some cases, the rings of charge (e.g., between a ring of charge in the first region and / or third region and a second ring of charge in the second region) may each be spaced from about 0.1 nm to about 5 nm apart from each other along the longitudinal length of the channel. In some cases, the rings of charge (e.g., between a ring of charge in the first region and / or third region and a second ring of charge in the second region) may each be spaced from 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 1.5 nm, about 0.1 nm to about 2 nm, about 0.1 nm to about 2.5 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 0.2 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 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 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 run, about 0.4 run to about 4 run, about 0.4 run to about 5 run, about 0.5 run 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 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 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 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 4 nm, about 2 nm to about 5 nm, about 2.5 nm to about 3 nm, about 2.5 nm to about 4 nm, about 2.5 nm to about 5 nm, about 3 nm to about 4 nm, about 3 nm to about 5 nm, about 4 nm to about 5 nm, about 5 nm to about 6 nm, about 6 nm to about 7 nm, about 7 nm to about 8 nm, about 8 nm to about 9 nm, or about 9 nm to about 10 nm apart from each other along the longitudinal length of the channel.

[0311] In some cases, a ring of charge may be spaced 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 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at last about 9 nm, or greater than about 10 nm apart from another ring of charge along the longitudinal length of the channel. In some cases, a ring of charge may be spaced at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 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 0.9 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 apart from another ring of charge along the longitudinal length of the channel.

[0312] In some cases, a ring of charge may be spaced from about 0.1 nm to about 5 nm apart from another ring of charge along the longitudinal length of the channel. In some cases, a ring of charge may be spaced from 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 1.5 nm, about 0.1 nm to about 2 nm, about 0.1 nm to about 2.5 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 0.2 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 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 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 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 run to about 2 run, about 0.5 run to about 2.5 run, about 0.5 run to about 3 run, 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 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 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 4 nm, about 2 nm to about 5 nm, about 2.5 nm to about 3 nm, about 2.5 nm to about 4 nm, about 2.5 nm to about 5 nm, about 3 nm to about 4 nm, about 3 nm to about 5 nm, about 4 nm to about 5 nm, about 5 nm to about 6 nm, about 6 nm to about 7 nm, about 7 nm to about 8 nm, about 8 nm to about 9 nm, or about 9 nm to about 10 nm apart from another ring of charge along the longitudinal length of the channel.

[0313] In some cases, a first ring of charge may comprise one or more mutations described herein. The first ring of charge may be separated from a second ring of charge comprising one or more mutations described herein. The first ring of charge and the second ring of charge may be in the same region (e.g., a first region, a second region, or a third region described herein). The first ring of charge (e.g., first ring of charge comprising one or more mutations described herein) and the second ring of charge (e.g., second ring of charge comprising one or more mutations described herein) may be separated by 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 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 15 nm, or greater than about 15 nm apart from each other along the longitudinal length of the channel. The first ring of charge (e.g., first ring of charge comprising one or more mutations described herein) and the second ring of charge (e.g., second ring of charge comprising one or more mutations described herein) may be separated by at most about 15 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 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 0.9 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 apart from each other along the longitudinal length of the channel.

[0314] As another example, the first ring of charge (e.g., first ring of charge comprising one or more mutations described herein) and the second ring of charge (e.g., second ring of charge comprising one or more mutations described herein) may be in different regions. For example, a first ring of charge may be in a first region and / or third region, and a second ring of charge may be in a second region. The first ring of charge (e.g., first ring of charge comprising one or more mutations described herein) and the second ring of charge (e.g., second ring of charge comprising one or more mutations described herein) may be separated by 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 nm, -69- at least about 2 nm, at least about 3 run, at least about 4 run, at least about 5 run, at least about 6 run, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 15 nm, or greater than about 15 nm apart from each other along the longitudinal length of the channel. The first ring of charge (e.g., first ring of charge comprising one or more mutations described herein) and the second ring of charge (e.g., second ring of charge comprising one or more mutations described herein) may be separated by at most about 15 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 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 0.9 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 apart from each other along the longitudinal length of the channel.

[0315] An engineered biological nanopore described herein may comprise two or more monomers (e.g., 8 monomers or an octameric pore). Each monomer of the nanopore may comprise at least a first portion, a second portion, and a third portion. A portion of a monomer may comprise one or more mutations of one or more residues to one or more negative charges. The first portion of a monomer can contribute to a first region of a channel (e.g., a region of the channel adjacent to the constriction region). The second portion of a monomer (e.g., the constriction-forming portion) can contribute to a second region of the channel (e.g., the region comprising the constriction region). The third portion of a monomer can contribute to a third region of the channel (e.g., another region of the channel adjacent to the constriction region). In the engineered biological nanopore, the second portion of the monomer may comprise one or more neutral charges. In the engineered biological nanopore, the second portion of the monomer may comprise one or more negative charges. In the engineered biological nanopore, the second portion of the monomer may comprise one or more neutral charges and negative charges. In some cases, a first portion of a first monomer of the engineered biological nanopore may comprise a mutation of one or more residues to negative charges to increase a negative charge distribution in the first portion (e.g., a portion forming a first region of the channel). A third portion of a first monomer of the engineered biological nanopore may comprise a mutation of one or more residues to a negative charge to increase a negative charge distribution in the third portion (e.g., a portion forming a third region of the channel). A first portion of a second monomer of the engineered biological nanopore may comprise the same mutation(s) as compared to the first portion of the first monomer. In some cases, a first portion of a second monomer of the engineered biological nanopore may comprise different mutations as compared to the first portion of the first monomer. For example, a first portion of a second monomer of the engineered biological nanopore may comprise one or more mutations of one or more residues to a negative charge to increase a negative charge distribution. The second monomer may comprise one or more mutations of one or more residues in a third portion to increase a negative charge distribution in the third portion (e.g., a portion forming a third region of the channel). As an example, the one or more monomers comprising an engineered biological nanopore (e.g., oligomeric nanopore) described herein may comprise one or more mutations of one or more second portions (e.g., constriction region-forming portions) to contribute to a neutral constriction region and / or negative constriction region of the nanopore, one or more mutations of first portions to contribute to a greater negative charge of a first region of the channel (e.g., a region of the channel adjacent to the constriction region), one or more mutations of third portions to contribute to a greater negative charge of a third region of the channel (e.g., another region of the channel adjacent to the constriction region), or any combination thereof. The neutral second region (e.g., constriction region) may comprise a neutral charge. The neutral second region (e.g., constriction region) may be modified to be more neutral as compared to a respective region of a wild-type nanopore. The neutral second region (e.g., constriction region) may have a net neutral charge. As another example, the monomers comprising an engineered biological nanopore (e.g., oligomeric nanopore) described herein may comprise one or more mutations of one or more second portions (e.g., constriction region-forming portions) to contribute to a negative second region (e.g., constriction region) of the nanopore, one or more mutations of first portions to contribute to a greater negative charge of a first region of the channel (e.g., a region of the channel adjacent to the constriction region), one or more mutations of third portions to contribute to a greater negative charge of a third region of the channel (e.g., another region of the channel adjacent to the constriction region), or any combination thereof. The second region (e.g., negatively-charged second region, e.g., constriction region) may comprise a negative charge. The second region (e.g., negatively-charged second region, e.g., constriction region) may be modified to be more negatively-charged as compared to a respective region of a wild-type nanopore. The second region (e.g., negatively-charged second region, e.g., constriction region) may have a net negative charge.

[0316] In some cases, the second region of the channel (e.g., comprising the constriction region) may comprise a charge (e.g., a neutral charge). The second region of the channel may have a net neutral charge. The net neutral charge may result from an equal number of positively charged amino acid residues and negatively charged amino acid residues. A balance of positively charged amino acid residues and negatively charged amino acid residues may provide for a net neutral charge of a constriction region of a nanopore described herein. A net neutral charge may result from an increase in neutral amino acid residues in the second region of the channel. The charges may be distributed in rings (e.g., rings of charge). The rings can be co-planar with a membrane. An engineered biological nanopore and / or monomers described herein may comprise a number of unitary charges. The unitary charges may be the amino acid residues (e.g., the charged amino acid residues) that comprise the rings of charge of the nanopore.

[0317] A unit of charge carried by a single proton and / or a single electron may be referred to as an “elementary charge”. An elementary charge may be a smallest charge that can exist freely. Elementary charge may be a value representing a charge of an electron or proton. Unitary charge may be a quantization of charge (e.g., a quantization of charge into one or more rings of charge).

[0318] In some cases, the engineered biological nanopore comprises a number of unitary charges. In some cases, the channel of the engineered biological nanopore described herein comprises a number of unitary charges. In some cases, the first region of the channel and / or the third region of the channel of the engineered biological nanopore described herein comprise a number of unitary charges. The channel of the engineered biological nanopore may comprise at least about 2 unitary charges, at least about 3 unitary charges, at least about 4 unitary charges, at least about 5 unitary charges, at least about 6 unitary charges, at least about 7 unitary charges, at least about 8 unitary charges, at least about 9 unitary charges, at least about 10 unitary charges, at least about 12 unitary charges, at least about 15 unitary charges, at least about 18 unitary charges, at least about 20 unitary charges, at least about 25 unitary charges, at least about 30 unitary charges, at least about 40 unitary charges, at least about 50 unitary charges, at least about 75 unitary charges, at least about 100 unitary charges, at least about 200 unitary charges, or greater than about 200 unitary charges. The channel of the engineered biological nanopore may comprise at most about 200 unitary charges, at most about 100 unitary charges, at most about 75 unitary charges, at most about 50 unitary charges, at most about 40 unitary charges, at most about 30 unitary charges, at most about 25 unitary charges, at most about 20 unitary charges, at most about 18 unitary charges, at most about 15 unitary charges, at most about 12 unitary charges, at most about 10 unitary charges, at most about 9 unitary charges, at most about 8 unitary charges, at most about 7 unitary charges, at most about 6 unitary charges, at most about 5 unitary charges, at most about 4 unitary charges, at most about 3 unitary charges, at most about 2 unitary charges, or less than about 2 unitary charges.

[0319] The channel of the engineered biological nanopore may comprise from about 5 unitary charges to about 250 unitary charges, he channel of the engineered biological nanopore may comprise from about 5 unitary charges to about 10 unitary charges, about 5 unitary charges to about 20 unitary charges, about 5 unitary charges to about 30 unitary charges, about 5 unitary charges to about 40 unitary charges, about 5 unitary charges to about 50 unitary charges, about 5 unitary charges to about 75 unitary charges, about 5 unitary charges to about 100 unitary charges, about 5 unitary charges to about 125 unitary charges, about 5 unitary charges to about 150 unitary charges, about 5 unitary charges to about 200 unitary charges, about 5 unitary charges to about 250 unitary charges, about 10 unitary charges to about 20 unitary charges, about 10 unitary charges to about 30 unitary charges, about 10 unitary charges to about 40 unitary charges, about 10 unitary charges to about 50 unitary charges, about 10 unitary charges to about 75 unitary charges, about 10 unitary charges to about 100 unitary charges, about 10 unitary charges to about 125 unitary charges, about 10 unitary charges to about 150 unitary charges, about 10 unitary charges to about 200 unitary charges, about 10 unitary charges to about 250 unitary charges, about 20 unitary charges to about 30 unitary charges, about 20 unitary charges to about 40 unitary charges, about 20 unitary charges to about 50 unitary charges, about 20 unitary charges to about 75 unitary charges, about 20 unitary charges to about 100 unitary charges, about 20 unitary charges to about 125 unitary charges, about 20 unitary charges to about 150 unitary charges, about 20 unitary charges to about 200 unitary charges, about 20 unitary charges to about 250 unitary charges, about 30 unitary charges to about 40 unitary charges, about 30 unitary charges to about 50 unitary charges, about 30 unitary charges to about 75 unitary charges, about 30 unitary charges to about 100 unitary charges, about 30 unitary charges to about 125 unitary charges, about 30 unitary charges to about 150 unitary charges, about 30 unitary charges to about 200 unitary charges, about 30 unitary charges to about 250 unitary charges, about 40 unitary charges to about 50 unitary charges, about 40 unitary charges to about 75 unitary charges, about 40 unitary charges to about 100 unitary charges, about 40 unitary charges to about 125 unitary charges, about 40 unitary charges to about 150 unitary charges, about 40 unitary charges to about 200 unitary charges, about 40 unitary charges to about 250 unitary charges, about 50 unitary charges to about 75 unitary charges, about 50 unitary charges to about 100 unitary charges, about 50 unitary charges to about 125 unitary charges, about 50 unitary charges to about 150 unitary charges, about 50 unitary charges to about 200 unitary charges, about 50 unitary charges to about 250 unitary charges, about 75 unitary charges to about 100 unitary charges, about 75 unitary charges to about 125 unitary charges, about 75 unitary charges to about 150 unitary charges, about 75 unitary charges to about 200 unitary charges, about 75 unitary charges to about 250 unitary charges, about 100 unitary charges to about 125 unitary charges, about 100 unitary charges to about 150 unitary charges, about 100 unitary charges to about 200 unitary charges, about 100 unitary charges to about 250 unitary charges, about 125 unitary charges to about 150 unitary charges, about 125 unitary charges to about 200 unitary charges, about 125 unitary charges to about 250 unitary charges, about 150 unitary charges to about 200 unitary charges, about 150 unitary charges to about 250 unitary charges, or about 200 unitary charges to about 250 unitary charges.

[0320] In some cases, the first region, second region, and / or third region of the channel of the wild-type biological nanopore or engineered biological nanopore may comprise a charge (e.g., a negative charge). In some cases, a first region and / or a third region may be more net negative than a second region of a nanopore (e.g., an engineered biological nanopore). The net negative charge of the first region, second region, and / or third region of the channel may result from a greater number of negatively charged amino acid residues compared to a number of positively charged amino acid residues, neutral amino acid residues, or combination thereof. A net negative charge or increasing a net negative charge may result from substitution of positively charged amino acid residues to neutral amino acid residues. A net negative charge or increasing a net negative charge may result from substitution of positively charged amino acid residues to negatively-charged amino acid residues. For example, a net negative charge or increasing a net negative charge may result from a region (e.g., a first region, second region, and / or third region) comprising five negatively-charged amino acid residues and five positively-charged amino acid residues and substituting one or more positively-charged residues for one or more neutral amino acid residues. For example, a net negative charge or increasing a net negative charge may result from a region comprising five negatively-charged amino acid residues and five positively-charged amino acid residues and substituting one or more positively-charged residues for one or more negatively-charged amino acid residues and / or one or more neutral charged amino acids. The first region and / or third region of the channel of the engineered biological nanopore (e.g., the region adjacent to the constriction region) may be modified to be more net negative as compared to another region adjacent to a constriction region of a wild-type biological nanopore. In some cases, the first region may be more net negative as compared to a respective region of a wild-type biological nanopore. In some cases, a net charge of a first region may be (e.g., or may be modified to be) at least about 50% more negative as compared to a respective region of a wild-type biological nanopore. The second region of the channel of the engineered biological nanopore (e.g., the region having a constriction region) may be modified to be more net negative as compared to another region having of a nanopore described herein may comprise a constriction region of a wild-type biological nanopore.

[0321] One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein may comprise a negative charge. For example, one or more regions (e.g., a first region, second region, third region, or any combination thereof) may comprise one or more negatively-charged amino acids. To achieve a negative charge, one or more amino acids may be modified to be one or more negatively-charged amino acids. One or more amino acids (e.g., one or more positively-charged amino acids and / or neutral amino acids) may be substituted for one or more negatively-charged amino acids. One or more negatively-charged amino acids may be added. In some cases, a region (e.g., a first region, second region, third region, or any combination thereof) may comprise a plurality of amino acids modified to a negative charge. A region (e.g., a first region, second region, third region, or any combination thereof) may comprise two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids modified to a negative charge. For example, the two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids may be substituted with two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) negatively-charged amino acid residues. As another example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) negatively-charged amino acid residues may be added to the region (e.g., a first region, second region, third region, or any combination thereof). The negatively-charged amino acids may be any negatively-charged amino acid described herein (e.g., negatively-charged natural amino acids and / or negatively-charged non-natural amino acids).

[0322] In some cases, two or more mutations in two or more monomers may make up a ring of charge in one or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein. One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein may comprise two or more mutations that can form a ring of charge relative to the two or more mutations. As an example, the two or more mutations may comprise one or more mutations to negatively-charged amino acids (e.g., substitution to one or more negatively-charged amino acids and / or addition of one or more negatively-charged amino acids). Introduction of a negatively-charged amino acid may thus provide at least one ring of charge comprising a negative charge or a net negative charge (e.g., a negative charge or a net negative charge associated with a mutated amino acid).

[0323] One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) may be more net negative as compared to one or more respective regions (e.g., a first -74- region, second region, third region, or any combination thereof) of a wild-type nanopore. One or more regions (e.g., a first region, second region, third region, or any combination thereof) may comprise more negatively-charged amino acids as compared to one or more respective regions (e.g., a first region, second region, third region, or any combination thereof) of a wild-type nanopore. As an example, one or more amino acids (e.g., one or more positively-charged amino acids and / or neutral amino acids) may be substituted for one or more negatively-charged amino acids. As another example, one or more negatively-charged amino acids may be added to the region (e.g., a first region, second region, third region, or any combination thereof). As another example, one or more amino acids (e.g., one or more positively-charged amino acids and / or neutral amino acids) may be deleted from the region (e.g., a first region, second region, third region, or any combination thereof). As another example, one or more positively-charged amino acids may be substituted for one or more neutral amino acids in the region (e.g., a first region, second region, third region, or any combination thereof). In some cases, a region (e.g., a first region, second region, third region, or any combination thereof) may be more net negative as compared to a respective region of a wild-type nanopore. The region may comprise a plurality of amino acids modified to a negative charge. For example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids may be substituted with two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) negatively-charged amino acid residues. As another example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) negatively-charged amino acid residues may be added to the region (e.g., a first region, second region, third region, or any combination thereof) to make the region more net negative as compared to a respective region of a wild-type nanopore. As a further example, the region (e.g., a first region, second region, third region, or any combination thereof) may comprise a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of deleted positively-charged amino acids and / or neutral-charged amino acids to achieve a more net negative charge as compared to a respective region of a wild-type nanopore. In some cases, the region of the monomer may comprise a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of positively-charged amino acids substituted for a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of neutral-charged amino acids (e.g., to thus make the region more net negative as compared to a respective region of a wild-type nanopore). A region of a nanopore disclosed herein may comprise a ring of charge comprising a plurality of mutations. The ring of charge may be net positive, and then mutated with (1) one or more negative charges and / or (2) one or more neutral charges, so that the ring of charge is more net negative than the ring of charge in the wild type nanopore. The ring of charge may be net neutral, and then mutated with (1) one or more negative charges, so that the ring of charge is more net negative than the ring of charge in the wild type nanopore.

[0324] In some cases, if there may be a total number of rings of charge in a region, and the rings of charge are each net positive, then (i) one or more positively-charged amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (ii) one or more positively-charged amino acids in one or more rings can be substituted with one or more neutral-charged amino acids, (iii) one or more positively-charged amino acids in one or more rings can be deleted, (iv) one or more neutral-charged amino acids in one or more rings can be added, (v) one or more negatively-charged amino acids in one or more rings can be added, or (vi) any combination thereof, to arrive at the region that is more net negative (e.g., comprising more net negative rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a total number rings of charge in a region, and the rings of charge are each net neutral, then (i) one or more neutral-charged amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (ii) one or more negatively-charged amino acids in one or more rings can be added, or (iii) any combination thereof, to arrive at the region that is more net negative (e.g., comprising more net negative rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a greater number or equal number of positively-charged rings of charge in one or more regions as compared to a number of neutral-charged rings of charge, then (i) one or more neutral amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (ii) one or more positively-charged amino acids in one or more rings can be substituted with one or more neutral amino acids, (iii) one or more positively-charged amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (iv) one or more positively-charged amino acids can be deleted, (v) one or more negatively-charged amino acids in one or more rings can be added, (vi) one or more neutral-charged amino acids in one or more rings can be added, or (vii) any combination thereof, to arrive at the region that is more net negative (e.g., comprising more net negative rings of charge) as compared to a respective region of a wild-type nanopore.

[0325] In some cases, if there may be a greater number or equal number of neutral-charged rings of charge in a region as compared to a number of positively-charged rings of charge, then (i) one or more neutral amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (ii) one or more positively-charged amino acids in one or more rings can be substituted with one or more neutral amino acids, (iii) one or more positively-charged amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (iv) one or more positively-charged amino acids in one or more rings can be deleted, (v) one or more negatively-charged amino acids in one or more rings can be added, (vi) one or more neutral-charged amino acids in one or more rings can be added, or (vii) any combination thereof, to arrive at the region that is more net negative (e.g., comprising more net negative rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a greater number or equal number of neutral-charged rings of charge in a region as compared to a number of negatively-charged rings of charge, then (i) one or more neutral amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (ii) one or more negatively-charged amino acids in one or more rings can be added, (iii) one or more neutral-charged amino acids in one or more rings can be deleted, or (vii) any combination thereof, to arrive at the region that is more net negative (e.g., comprising more net negative rings of charge) as compared to a respective region of a wild-type nanopore.

[0326] In some cases, if there may be a greater number or equal number of negatively-charged rings of charge in a region as compared to a number of positively-charged rings of charge, then (i) one or more negatively-charged amino acids in one or more rings can be added, (ii) one or more positively-charged amino acids in one or more rings can be deleted, (iii) one or more positively-charged amino acids in one or more rings can be substituted with one or more neutral amino acids, (iv) one or more positively-charged amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, or (v) any combination thereof, to arrive at the region that is more net negative (e.g., comprising more net negative rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a greater number or equal number of negatively-charged rings of charge in a region as compared to a number of neutral-charged rings of charge, then (i) one or more negatively-charged amino acids in one or more rings can be added, (ii) one or more neutral-charged amino acids in one or more rings can be deleted, (iii) one or more neutral-charged amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, or (v) any combination thereof, to arrive at the region that is more net negative (e.g., comprising more net negative rings of charge) as compared to a respective region of a wild-type nanopore.

[0327] One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) may be net negative. A net negative charge may be achieved when there may be a greater number of negatively-charged amino acids compared to a number of positively-charged amino acids and / or a number of neutral charged amino acids. A net negative charge of one or more regions (e.g., a first region, second region, third region, or any combination thereof) may be achieved by (i) substituting one or more amino acids (e.g., one or more positively-charged amino acids and / or neutral amino acids) for one or more negatively-charged amino acids; (ii) deleting one or more positively-charged amino acids and / or neutral amino acids; (iii) adding one or more negatively-charged amino acids; or (iv) any combinations thereof. In some cases, a region (e.g., a first region, second region, third region, or any combination thereof) may be net negative. The region (e.g., a first region, second region, third region, or any combination thereof) may comprise negatively-charged amino acids. The portion may comprise a plurality of amino acids modified to a negative charge. For example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids may be substituted with two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) negatively-charged amino acid residues. As another example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) negatively-charged amino acid residues may be added to the region (e.g., a first region, second region, third region, or any combination thereof). In some cases, the region (e.g., a first region, second region, third region, or any combination thereof) may comprise a plurality of substituted and / or added negatively-charged amino acids such that the total amino acids of the region are negatively charged (e.g., and the region is net negative). The negatively-charged amino -77- acids may be any negatively-charged amino acid described herein. As a further example, the region (e.g., a first region, second region, third region, or any combination thereof) may comprise a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of deleted positively-charged amino acids and / or neutral-charged amino acids to achieve a negative charge (e.g., a greater number of negatively-charged amino acids in the region as compared to a number of positively-charged amino acids and / or neutral charged amino acids). In some cases, two or more monomers making up a ring of charge in a region may be net negative. For example, for an octameric nanopore (e.g., a nanopore comprising eight monomeric units) two more monomers may be net negative in a region (e.g., a first region, second region, third region, or any combination thereof). As another example, each monomer making up a ring of charge in a region may be net negative. The ring of charge may be net neutral or net positive, and then mutated so that the ring of charge is net negative. For example, the ring of charge may be net positive, then mutated with one or more negative or one or more neutral charges so that the ring of charge is net negative. As another example, the ring of charge may be net neutral, then mutated with one or more negative charges so that the ring of charge is net negative.

[0328] One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein may comprise a neutral charge. For example, one or more regions may comprise one or more neutral-charged amino acids. To achieve a neutral charge, one or more amino acids may be modified to be one or more neutral-charged amino acids. One or more amino acids (e.g., one or more positively-charged amino acids and / or negatively-charged amino acids) may be substituted for one or more neutral-charged amino acids. One or more neutral-charged amino acids may be added to the region (e.g., a first region, second region, third region, or any combination thereof). In some cases, a region (e.g., a second region, e.g., a constriction region) may comprise a plurality of amino acids modified to a neutral charge. A region (e.g., a second region, e.g., a constriction region) may comprise two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids modified to a neutral charge. For example, the two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids may be substituted with two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) neutral-charged amino acid residues. As another example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) neutral-charged amino acid residues may be added to the region (e.g., a second region, e.g., a constriction region). The neutral-charged amino acids may be any neutral-charged amino acid described herein. In some cases, two or more mutations in two or more monomers may make up a ring of charge in one or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein. One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein may comprise two or more mutations that can form a ring of charge relative to the two or more mutations. As an example, the two or more mutations may comprise one or more mutations to neutral-charged amino acids (e.g., substitution to one or more neutral-charged amino acids and / or addition of one or more neutral-charged amino acids). Introduction of a neutral-charged amino acid may thus provide at least one ring of charge comprising a neutral charge or a net negative charge (e.g., a neutral charge or a net negative charge associated with a mutated amino acid).

[0329] One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein may be more net neutral as compared to one or more respective regions of a wild-type nanopore. One or more regions (e.g., a first region, second region, third region, or any combination thereof) may comprise more neutral-charged amino acids as compared to one or more respective regions of a wild-type nanopore. As an example, one or more amino acids (e.g., one or more positively-charged amino acids and / or negatively-charged amino acids) may be substituted for one or more neutral-charged amino acids. As another example, one or more neutral-charged amino acids may be added to the region (e.g., a first region, second region, third region, or any combination thereof). A number of one or more positively-charged amino acids and / or negatively-charged amino acids may be added to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and achieve a neutral charge). As another example, one or more amino acids (e.g., one or more positively-charged amino acids and / or negatively-charged amino acids) may be deleted from the region (e.g., a first region, second region, third region, or any combination thereof). A number of one or more positively-charged amino acids and / or negatively-charged amino acids may be deleted to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and achieve a neutral charge). In some cases, a region (e.g., a second region, e.g., a constriction region) may be more net neutral as compared to a respective region (e.g., a constriction region) of a wild-type nanopore. The region (e.g., a second region, e.g., a constriction region) may comprise more neutral-charged amino acids as compared to a respective region (e.g., a constriction region) of a wild-type nanopore. The region may comprise a plurality of amino acids modified to a neutral charge. For example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids may be substituted with two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) neutral-charged amino acid residues. As another example, two or more (e.g., three or more, e.g., five or more) neutral-charged amino acid residues may be added to the region (e.g., a second region, e.g., a constriction region). The neutral-charged amino acids may be any neutral-charged amino acid described herein. As a further example, the region (e.g., a second region, e.g., a constriction region) may comprise a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of deleted positively-charged amino acids and / or negatively-charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids in the region (e.g., and thus be more net neutral as compared to a respective region (e.g., a constriction region) of a wild-type nanopore). As another example, the region (e.g., a second region, e.g., a constriction region) may comprise a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of added positively-charged amino acids and / or negatively-charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids in the region (e.g., and thus be more net neutral as compared to a respective region (e.g., a constriction region) of a wild-type nanopore). A region of a nanopore disclosed herein may comprise a ring of charge comprising a plurality of mutations. The ring of charge may be net negative, and then mutated with (1) one or more positive charges and / or (2) one or more neutral charges, so that the ring of charge is more net neutral than the ring of charge in the wild type nanopore. The ring of charge may be net positive, and then mutated with (1) one or more negative charges and / or (2) one or more neutral charges, so that the ring of charge is more net neutral than the ring of charge in the wild type nanopore.

[0330] In some cases, if there may be a total number of rings of charge in a region, and the rings of charge are each net positive, then (i) one or more positively-charged amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (ii) one or more positively-charged amino acids in one or more rings can be substituted with one or more neutral-charged amino acids, (iii) one or more positively-charged amino acids in one or more rings can be deleted, (iv) one or more neutral-charged amino acids in one or more rings can be added, (v) one or more negatively-charged amino acids in one or more rings can be added, or (vi) any combination thereof, to arrive at the region that is more net neutral (e.g., comprising more net neutral rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a total number of rings of charge in a region, and the rings of charge are each net negative, then (i) one or more negatively-charged amino acids in one or more rings can be substituted with one or more positively-charged amino acids, (ii) one or more negatively-charged amino acids in one or more rings can be substituted with one or more neutral-charged amino acids, (iii) one or more negatively-charged amino acids in one or more rings can be deleted, (iv) one or more neutral-charged amino acids in one or more rings can be added, (v) one or more positively-charged amino acids in one or more rings can be added, or (vi) any combination thereof, to arrive at the region that is more net neutral (e.g., comprising more net neutral rings of charge) as compared to a respective region of a wild-type nanopore.

[0331] In some cases, if there may be a greater number or equal number of positively-charged rings of charge in a region as compared to a number of neutral-charged rings of charge, then (i) one or more neutral amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (ii) one or more positively-charged amino acids in one or more rings can be substituted with one or more neutral amino acids, (iii) one or more positively-charged amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (iv) one or more positively-charged amino acids in one or more rings can be deleted, (v) one or more negatively-charged amino acids in one or more rings can be added, (vi) one or more neutral-charged amino acids in one or more rings can be added, or (vii) any combination thereof, to arrive at the region that is more net neutral (e.g., comprising more net neutral rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a greater number or equal number of neutral-charged rings of charge in a region as compared to a number of positively-charged rings of -80- charge, then (i) one or more neutral amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (ii) one or more positively-charged amino acids in one or more rings can be substituted with one or more neutral amino acids, (iii) one or more positively-charged amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (iv) one or more positively-charged amino acids in one or more rings can be deleted, (v) one or more negatively-charged amino acids in one or more rings can be added, (vi) one or more neutral-charged amino acids in one or more rings can be added, or (vii) any combination thereof, to arrive at the region that is more net neutral (e.g., comprising more net neutral rings of charge) as compared to a respective region of a wild-type nanopore.

[0332] In some cases, if there may be a greater number or equal number of neutral-charged rings of charge in a region as compared to a number of negatively-charged rings of charge, then (i) one or more neutral amino acids in one or more rings can be substituted with one or more positively-charged amino acids, (ii) one or more negatively-charged amino acids in one or more rings can be substituted with one or more neutral amino acids, (iii) one or more negatively-charged amino acids in one or more rings can be substituted with one or more positively-charged amino acids, (iv) one or more negatively-charged amino acids in one or more rings can be deleted, (v) one or more positively-charged amino acids in one or more rings can be added, (vi) one or more neutral-charged amino acids in one or more rings can be added, or (vii) any combination thereof, to arrive at the region that is more net neutral (e.g., comprising more net neutral rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a greater number or equal number of negatively-charged rings of charge in a region as compared to a number of positively-charged rings of charge, then (i) one or more positively-charged amino acids in one or more rings can be added, (ii) one or more neutral-charged amino acids in one or more rings can be added, (iii) one or more negatively-charged amino acids in one or more rings can be deleted, (iii) one or more negatively-charged amino acids in one or more rings can be substituted with one or more neutral amino acids, (iv) one or more negatively-charged amino acids in one or more rings can be substituted with one or more positively-charged amino acids, or (v) any combination thereof, to arrive at the region that is more net neutral (e.g., comprising more net neutral rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a greater number or equal number of negatively-charged rings of charge in a region as compared to a number of neutral-charged rings of charge, then (i) one or more positively-charged amino acids in one or more rings can be added, (ii) one or more neutral-charged amino acids in one or more rings can be added, (iii) one or more negatively-charged amino acids in one or more rings can be substituted with one or more positively-charged amino acids, (iv) one or more negatively-charged amino acids in one or more rings can be substituted with one or more neutral-charged amino acids, (v) one or more negatively charged amino acids in one or more rings can be deleted, or (v) any combination thereof, to arrive at the regions that is more net neutral (e.g., comprising more net neutral rings of charge) as compared to a respective region of a wild-type nanopore.

[0333] One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein may be net neutral. A net neutral charge may be achieved when there may be a same number of positively-charged amino acids and negatively-charged amino acids. A net neutral charge may be achieved when the one or more regions (e.g., a first region, second region, third region, or any combination thereof) comprises all neutral charged amino acids. A net neutral charge of one or more regions (e.g., a first region, second region, third region, or any combination thereof) may be achieved by (i) substituting one or more amino acids (e.g., one or more positively-charged amino acids and / or negatively-charged amino acids) for one or more neutral-charged amino acids; (ii) deleting one or more positively-charged amino acids and / or negatively-charged amino acids; (iii) adding one or more neutral-charged amino acids; or (iv) any combinations thereof. A net neutral charge of one or more regions (e.g., a first region, second region, third region, or any combination thereof) may also be achieved by (i) adding one or more positively-charged amino acids and / or negatively-charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and thus comprise an overall neutral charge) and / or (ii) deleting one or more positively-charged amino acids and / or negatively-charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and thus comprise an overall neutral charge). In some cases, a region (e.g., a second region, e.g., a constriction region) may be net neutral. The region (e.g., a second region, e.g., a constriction region) may comprise neutral-charged amino acids. The region may comprise a plurality of amino acids modified to a neutral charge. For example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids may be substituted with two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) neutral-charged amino acid residues. As another example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) neutral-charged amino acid residues may be added to the region (e.g., a second region, e.g., a constriction region). In some cases, the region (e.g., a second region, e.g., a constriction region) may comprise a plurality of substituted and / or added neutral-charged amino acids such that the total amino acids of the region are neutral charged (e.g., and the region is net neutral). The neutral-charged amino acids may be any neutral-charged amino acid described herein. As a further example, the region (e.g., a second region, e.g., a constriction region) may comprise a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of deleted positively-charged amino acids and / or negatively-charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids in the region (e.g., and thus be net neutral). As another example, the region (e.g., a second region, e.g., a constriction region) may comprise a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of added positively-charged amino acids and / or negatively-charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids in the region (e.g., and thus be net neutral). In some cases, two or more monomers making up a ring of charge in a region may be net neutral. For example, for an octameric nanopore (e.g., a nanopore comprising eight monomeric units) two more monomers (e.g., about 2, 3, 4, 5, 6, 7, or 8 monomers) may be net neutral in a region (e.g., a first region, second region, third region, or any combination thereof). As another example, each monomer making up a ring of charge in a region may be net neutral. The ring of charge may be net negative or net positive, and then mutated so that the ring of charge is net neutral. For example, the ring of charge may be net negative, then mutated with one or more positive or one or more neutral charges so that the ring of charge is net neutral. As another example, the ring of charge may be net positive, then mutated with one or more negative or one or more neutral charges so that the ring of charge is net neutral.

[0334] There may be a distance between an amino acid (e.g., a modified amino acid) in one region of the nanopore described herein (e.g., an engineered biological nanopore) and another amino acid (e.g., another modified amino acid) in another region of the nanopore. In some cases, a mutated amino acid in a first region may be separated by a distance from a mutated amino acid in a second region (e.g., a different region). As an example, a mutated amino acid in a region (e.g., a first region and / or a third region) may be separated by a distance from another mutated amino acid in another region (e.g., the second region). A mutated amino acid in a region (e.g., a first region and / or a third region) may be separated from another mutated amino acid in another region (e.g., the second region) by a distance of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, or less than about 0.5 nm. A mutated amino acid in a region (e.g., a first region and / or a third region) may be separated from another mutated amino acid in a region (e.g., the second region) by a distance of at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm.

[0335] Two amino acids (e.g., two mutated amino acids) may be separated from one another, wherein the two amino acids may be in the same region of a nanopore. In some cases, an amino acid (e.g., a modified amino acid) in one region of a nanopore described herein (e.g., an engineered biological nanopore) may be separated by a distance from another amino acid (e.g., another modified amino acid) in the same region. A mutated amino acid in a region (e.g., a first region, a second region, a third region, a second protein unit, or any combination thereof) may be separated from another mutated amino acid in the same region (e.g., a first region, a second region, a third region, a second protein unit, or any combination thereof) by a distance of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, or less than about 0.5 nm. A mutated amino acid in a region (e.g., a first region, a second region, a third region, a second protein unit, or -83- any combination thereof) may be separated from another mutated amino acid in the same region (e.g., a first region, a second region, a third region, a second protein unit, or any combination thereof) by a distance of at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm.

[0336] A mutated amino acid in a first region or third region may be at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, or less than about 0.5 nm away from a mutated amino acid in a second region. A mutated amino acid in a first region or third region may be at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm away from a mutated amino acid in a second region.

[0337] In some cases, a first portion, second portion, and / or third portion of a monomer described herein may comprise a charge (e.g., a negative, a neutral charge, or any combination thereof). A first portion, second portion, and / or third portion of a monomer may comprise a negative charge. A first portion, second portion, and / or third portion of a monomer may be modified to be more net negative as compared to a respective portion of a wild-type monomer. A first portion, second portion, and / or third portion of a monomer may comprise a net negative charge. The net negative charge may result from a greater number of negatively charged amino acid residues compared to a number of positively charged amino acid residues, neutral amino acid residues, or combination thereof. A net negative charge or increasing a net negative charge of a first portion of a monomer described herein may result from substitution of positively charged amino acid residues to neutral amino acid residues. A net negative charge or increasing a net negative charge of a first portion of a monomer described herein may result from substitution of positively charged amino acid residues to negatively-charged amino acid residues. For example, a net negative charge or increasing a net negative charge of a first portion, second portion, and / or third portion of a monomer described herein may result from a portion comprising five negatively-charged amino acid residues and five positively-charged amino acid residues, and substituting one or more positively-charged residues for one or more neutral amino acid residues. For example, a net negative charge or increasing a net negative charge of a first portion of a monomer described herein may result from a portion comprising five negatively-charged amino acid residues and five positively-charged amino acid residues, and substituting one or more positively-charged residues for one or more negatively-charged amino acid residues. A third portion of a monomer may comprise a net negative charge. The net negative charge may result from a greater number of negatively charged amino acid residues compared to a number of positively charged amino acid residues, neutral amino acid residues, or combination thereof. A net negative charge or increasing a net negative charge of a third portion of a monomer described herein may result from substitution of positively charged amino acid residues to neutral amino acid residues. A net negative charge or increasing a net negative charge of a third portion of a monomer described herein may result from substitution of positively charged amino acid residues to negatively-charged amino acid residues. For example, a net negative charge or increasing a net negative charge of a third portion of a monomer described herein may result from a portion comprising five negatively-charged amino acid residues and five positively-charged amino acid residues, and substituting one or more positively-charged residues for one or more neutral amino acid residues. For example, a net negative charge or increasing a net negative charge of a third portion of a monomer described herein may result from a portion comprising five negatively-charged amino acid residues and five positively-charged amino acid residues, and substituting one or more positively-charged residues for one or more negatively-charged amino acid residues. The first portion and / or third portion of an engineered monomer (e.g., the portion(s) adjacent to the constriction-forming portion of the monomer) may be more negative as compared to another portion adjacent to a constriction-forming portion of a wild-type monomer. The second portion of the engineered monomer (e.g., the constriction-forming portion) may be more negative as compared to another constriction-forming portion of a wild-type biological nanopore.

[0338] In some cases, a first portion and / or a third portion may comprise a negative charge (e.g., net negative charge). The negative charge may be from one or more negatively-charged amino acids (e.g., one or more negatively-charged natural amino acids and / or one or more negatively-charged non-natural amino acids). In some cases, a first portion or a third portion may comprise at least one amino acid that is mutated to exhibit an increased net negative charge. For example, one or more positively-charged amino acids and / or one or more neutral amino acids may be modified (e.g., mutated) to one or more negatively-charged amino acids. In some cases, a first portion and / or a third portion may comprise a plurality of amino acids that are mutated to exhibit an increased net negative charge. For example, a first portion may comprise two amino acids that are mutated to exhibit an increased net negative charge and a third portion may comprise two amino acids that are mutated to exhibit an increased net negative charge. In some cases, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, or greater than about 30 amino acids in a first portion and / or third portion may be modified to one or more amino acids that increase a net negative charge (e.g., one or more negatively-charged amino acids). In some cases, at most about 30, at most about 20, at most about 10, 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 amino acid in a first portion and / or third portion may be modified to one or more amino acids that increase a net negative charge (e.g., one or more negatively-charged amino acids). Modifications of one or more amino acids of a portion of a monomer described herein may comprise (i) substitution of one or more amino acids (e.g., natural and / or non-natural amino acids); (ii) addition of one or more amino acids (e.g., natural and / or non-natural amino acids); (iii) deletion of one or more amino acids (e.g., natural and / or non-natural amino acids); (iv) or any combinations thereof. Modifications of amino acids may also comprise post-translational modifications. For example, modifications of one or more amino acids of a portion of a monomer described herein may comprise phosphorylation, methylation, acetylation, glycosylation, addition of disulfide bonds, ubiquitination, hydroxylation, carboxylation, lipidation, amidation, or any combinations thereof. Any post-translational modification may be combined with one or more of addition of at least one amino acid, deletion of at least one amino acid, or substitution of at least one amino acid.

[0339] A first portion and / or a third portion may comprise at least one amino acid that is mutated to exhibit an increased net negative charge as compared to a respective portion of a wild-type biological nanopore. For example, a first portion of an engineered biological nanopore described herein may have an amino acid in a second portion (e.g., constriction portion). A wild-type biological nanopore may have an amino acid at an identical position in a second portion. The amino acid in the engineered biological nanopore may be mutated to increase a net negative charge. Thus, the first portion of the engineered biological nanopore comprises at least one amino acid that is mutated to exhibit an increased net negative charge as compared to a respective portion of a wild-type biological nanopore. As another example, a first portion and / or a third portion may comprise at least one amino acid that is mutated to a negatively-charged amino acid to exhibit an increased net negative charge as compared to a respective portion of a wild-type biological nanopore.

[0340] In some cases, at least one mutated amino acid in a first portion of each of one or more monomers may form a first ring of charge. For example, in an octameric nanopore, at least one mutated amino acid in a first portion and / or third portion of each of the 8 monomers may form a first ring of charge. In other cases, at least one mutated amino acid in a first portion and / or third portion of less than all monomers may form a first ring of charge. For example, in an octameric nanopore, at least one mutated amino acid in a first portion of 6 monomers may form a first ring of charge. In some cases, at least one mutated amino acid in a second portion of each of one or more monomers may form a second ring of charge. For example, in an octameric nanopore, at least one mutated amino acid in a second portion of each of the 8 monomers may form a second ring of charge. In other cases, at least one mutated amino acid in a second portion of less than all monomers may form a second ring of charge. For example, in an octameric nanopore, at least one mutated amino acid in a second portion of 6 monomers may form a second ring of charge.

[0341] One or more portions (e.g., a first portion, second portion, third portion, second protein unit, or any combination thereof) of a monomer described herein may comprise a neutral charge. For example, one or more portions may comprise one or more neutral-charged amino acids. To achieve a neutral charge, one or more amino acids may be modified to be one or more neutral-charged amino acids. One or more amino acids (e.g., one or more positively-charged amino acids and / or negatively-charged amino acids) may be substituted for one or more neutral-charged amino acids. One or more neutral-charged amino acids may be added to the portion (e.g., a first portion, second portion, third portion, or any combination thereof). In some cases, a portion (e.g., a second portion, e.g., a constriction forming portion) may comprise a plurality of amino acids modified -86- to a neutral charge. A p...

Claims

1. A method comprising:(a) providing a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, wherein the second region has a constriction region, wherein (i) the first region is modified to be more net negative than a respective region of a wild-type biological nanopore and / or (ii) the second region is modified to be more net neutral or more net negative than a respective region of the wild-type biological nanopore, wherein the first region of the channel is adjacent to the second region of the channel; and(b) contacting the engineered biological nanopore with a biopolymer.

2. The method of claim 1, wherein the first region is more net negative than the second region.

3. The method of claim 1 or 2, wherein one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids.

4. The method of any one of claims 1-3, wherein one or more amino acids in the first region is mutated to one or more negative amino acids.

5. The method of any one of claims 1-4, wherein when one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids, then one or more amino acids in the first region is mutated to one or more negative amino acids.

6. The method of claim 5, wherein the first region comprises at least one amino acid that is mutated to exhibit an increased net negative charge.

7. The method of claim 6, wherein the mutated at least one amino acid in the first region is at most 10 nm away from a mutated at least one amino acid in the second region.

8. The method of claim 6 or 7, wherein a first ring of charge comprising the mutated at least one amino acid in the first region is at most 10 nm away from a second ring of charge comprising the mutated at least one amino acid in the second region.

9. The method of any one of claims 1-8, wherein the engineered biological nanopore comprises one or more monomers.

10. The method of any one of claims 1-9, wherein a monomer of the engineered biological nanopore comprises a first portion corresponding to the first region and a second portion corresponding to the second region.

11. The method of claim 10, wherein a monomer of the engineered biological nanopore comprises a net charge in the first portion that is more negative as compared to a net charge in the second portion.

12. The method of claim 10 or 11, wherein the first portion comprises at least one amino acid that is mutated to exhibit an increased net negative charge.

13. The method of any one of claims 10-12, wherein the second portion comprises at least one amino acid that is mutated to exhibit an increased net neutral charge or an increased net negative charge.

14. The method of claim 13, wherein the at least one mutated amino acid in the first portion is at most 10 nm away from the at least one mutated amino acid in the second portion.

15. The method of any one of claims claim 1-14, wherein the engineered biological nanopore generates an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore.

16. The method of claim 15, wherein the first region modified to be more net negative and the second region modified to be more net neutral or more net negative generate the EOF.

17. The method of any one of claims 1-16, wherein the engineered biological nanopore has a cationselectivity P(+) / P(-) of at least about 1.5.

18. The method of any one of claims 1-17, wherein the second region of the channel comprises a first entrance and a second entrance.

19. The method of claim 18, wherein the first region of the channel is adjacent to the first entrance of the second region of the channel.

20. The method of claim 18, wherein the first region of the channel is adjacent to the second entrance of the second region of the channel.

21. The method of any one of claims 1-20, wherein one or more amino acid mutations in the second region of the engineered biological nanopore increase a cation selectivity p(K+ / Cl-) relative to the wild-type biological nanopore.

22. The method of any one of claims 1-21, wherein one or more amino acid mutations in the first region of the engineered biological nanopore increase a cation selectivity p(K+ / Cl-) relative to the wild-type biological nanopore.

23. The method of any one of claims 1-22, wherein the first region comprises one or more amino acid mutations that increase a negative charge in the residues that contribute to a region of the engineered biological nanopore comprising a diameter of at most 5 nm.

24. The method of any one of claims 1-23, wherein the first region comprises one or more amino acid mutations that increase a negative charge in the residues that contribute to a region of the engineered biological nanopore comprising a diameter of at most 2 nm.

25. The method of any one of claims 1-24, wherein a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore.

26. The method of any one of claims 1-25, wherein the second region is more net neutral as compared to the respective region of the wild-type biological nanopore.T1. The method of any one of claims 1-26, wherein a net charge of the second region of the channel is at least about 50% more neutral as compared to the respective region of the wild-type biological nanopore.

28. The method of any one of claims 1-27, wherein the constriction region has a neutral charge.

29. The method of any one of claims 1-28, wherein the second region comprises one or more amino acid mutations that increase a neutral charge in the residues that contribute to a narrowest region of the engineered biological nanopore comprising a diameter of at most 1.0 run.

30. The method of any one of claims 1-29, wherein the engineered biological nanopore comprises a cation selectivity P(+) / P(-) of at least about 1.8.

31. A method comprising:(a) providing a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, wherein the second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region is modified to be more net negative as compared to a respective region of a wild-type biological nanopore, wherein a first ring of charge in the first region and a second ring of charge in the second region comprises a distance of at most about 3 nm, wherein the second region comprises a width of at most about 2.5 run; and(b) contacting the engineered biological nanopore with a biopolymer.

32. The method of claim 31, wherein the first region is more net negative than the second region.

33. The method of claim 31 or 32, wherein one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids34. The method of any one of claims 31-33, wherein one or more amino acids in the first region is mutated to one or more negative amino acids.

35. The method of claim 34, wherein when one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids, then one or more amino acids in the adjacent region is mutated to one or more negative amino acids36. The method of claim 35, wherein the first region comprises at least one amino acid that is mutated to exhibit an increased net negative charge.

37. The method of claim 36, wherein the mutated at least one amino acid in the first region is at most 10 nm away from a mutated at least one amino acid in the second region.

38. The method of claim 36 or 37, wherein the first ring of charge comprising the mutated at least one amino acid in the first region is at most 10 nm away from the second ring of charge comprising the mutated at least one amino acid in the second region.

39. The method of any one of claims 31-38, wherein the engineered biological nanopore comprises one or more monomers.

40. The method of any one of claims 31-39, wherein a monomer of the engineered biological nanopore comprises a first portion corresponding to the first region and a second portion corresponding to the second region.

41. The method of claim 40, wherein a monomer of the engineered biological nanopore comprises a net charge in the first portion that is more negative as compared to a net charge in the second portion.

42. The method of claim 40 or 41, wherein the first portion comprises at least one amino acid that is mutated to exhibit an increased net negative charge.

43. The method of any one of claims 40-42, wherein the second portion comprises at least one amino acid that is mutated to exhibit an increased net neutral charge or an increased net negative charge.

44. The method of claim 43, wherein the at least one mutated amino acid in the first portion is at most 10 nm away from the at least one mutated amino acid in the second portion.

45. The method of any one of claims 31-44, wherein the engineered biological nanopore generates an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore.

46. The method of claim 45, wherein the first region of the channel and the second region of the channel generates the EOF.

47. The method of claim 45 or 46, wherein the EOF acts in an opposite direction to an electrophoretic force in the nanopore system.

48. The method of any one of claims 31-47, wherein the engineered biological nanopore has a cationselectivity P(+) / P(-) of at least about 1.5.

49. The method of any one of claims 31-48, wherein the second region of the channel comprises a first entrance and a second entrance.

50. The method of claim 49, wherein the first region of the channel is adjacent to the first entrance of the second region of the channel.

51. The method of claim 49, wherein the first region of the channel is adj acent to the second entrance of the second region of the channel.

52. The method of any one of claims 31-51, wherein one or more amino acid mutations in the second region of the engineered biological nanopore increase a cation selectivity p(K+ / Cl-) relative to the wild-type biological nanopore.

53. The method of any one of claims 31-52, wherein one or more amino acid mutations in the first region of the engineered biological nanopore increase a cation selectivity p(K+ / Cl-) relative to the wild-type biological nanopore.

54. The method of any one of claims 31-53, wherein the first region comprises one or more amino acid mutations that increase a negative charge in the residues that contribute to a region of the engineered biological nanopore comprising a diameter of at most 5 run.

55. The method of any one of claims 31-54, wherein the first region comprises one or more amino acid mutations that increase a negative charge in the residues that contribute to a region of the engineered biological nanopore comprising a diameter of at most 2 run.

56. The method of any one of claims 31-55, wherein a net charge of the first region is at least about 50% more negative as compared to the respective region of the wild-type biological nanopore.

57. The method of any one of claims 31-56, wherein the second region is more neutral as compared to the respective region of the wild-type biological nanopore.

58. The method of any one of claims 31-57, wherein a net charge of the second region of the channel is at least about 50% more neutral as compared to the respective region of the wild-type biological nanopore.

59. The method of any one of claims 31-58, wherein the constriction region has a neutral charge.

60. The method of any one of claims 31-59, wherein the second region comprises one or more amino acid mutations that increase a neutral charge in the residues that contribute to a narrowest region of the engineered biological nanopore comprising a diameter of at most 1.0 run.

61. The method of any one of claims 31-60, wherein the engineered biological nanopore comprises a cation selectivity P(+) / P(-) of at least about 1.8.

62. A system comprising:(a) a fluidic chamber; and(b) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, wherein the second region has a constriction region, wherein (i) the first region is modified to be more net negative than a respective region of a wild-type biological nanopore and / or (ii) the second region is modified to be more net neutral or more net negative than a respective region of the wild-type biological nanopore, wherein the first region of the channel is adjacent to the second region of the channel, wherein the engineered biological nanopore is configured to contact a biopolymer.

63. A system comprising:(a) a fluidic chamber; and(b) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, wherein the second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region of the channel has a negative charge, wherein a net charge of the secondregion of the channel is at least about 50% more neutral as compared to a respective region of a wild-type biological nanopore, wherein the engineered biological nanopore is configured to a biopolymer.

64. A system comprising:(a) a fluidic chamber; and(b) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, wherein the second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region is modified to be more net negative as compared to a respective region of a wild-type biological nanopore, wherein a first ring of charge in the first region and a second ring of charge in the second region comprises a distance of at most about 3 nm, wherein the second region comprises a width of at most about 2.5 nm, wherein the engineered biological nanopore is configured to contact a biopolymer.

65. A method comprising (a) providing a mixture containing or suspected of containing an analyte comprising polypeptide or protein, and (b) using an engineered biological nanopore to (1) determine a sequence or (2) generate a measure of a concentration or relative amount of said analyte in said mixture at an accuracy of at least 80%.

66. The method of claim 65, wherein said mixture contains or is suspected of containing an additional analyte comprising an additional polypeptide or protein.

67. The method of claim 65 or 66, further comprising using said engineered biological nanopore to generate a measure of a concentration or relative amount of said additional analyte in said mixture at an accuracy of greater than 80%.

68. The method of any one of claims 65-67, wherein (1) said sequence is determined or (2) said measure of said concentration or relative amount of said analyte is generated at an accuracy of at least 90%.

69. The method of any one of claims 65-68, wherein (1) said sequence is determined or (2) said measure of said concentration or relative amount of said analyte is generated at an accuracy of at least 95%.

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