MSP nanopores and their applications

Abstract

This article provides information on mutant single-stranded smegma mycobacterium porin (Msp) and its applications.

Description

[0001] This application claims the benefits of U.S. Provisional Application No. 61 / 980,415, filed April 16, 2014, and U.S. Provisional Application No. 61 / 980,393, filed April 16, 2014, which are incorporated herein by reference in their entirety.

[0002] Government Rights Statement

[0003] This invention is supported by the government and is carried out under license number R01HG005115 issued by the National Institutes of Health in the United States. The government holds certain rights to this invention.

[0004] background

[0005] The identification and characterization of analytes often involve expensive and time-consuming methodologies. For example, current technologies require costly custom reagents and / or detection methods to determine nucleic acid sequences. Similarly, methods for determining protein sequences, such as peptide fingerprinting via mass spectrometry, can be laborious and costly. Therefore, efficient methods for the detection and analysis of nucleic acids, proteins, and other analytes are essential.

[0006] Overview

[0007] This document provides a nucleic acid sequence encoding a mutant single-stranded Mycobacterium smegmatis porin (Msp). This nucleic acid sequence optionally comprises first and second nucleotide sequences, wherein the first nucleotide sequence encodes a first Msp monomer sequence and the second nucleotide sequence encodes a second Msp monomer sequence; and a third nucleotide sequence encodes an amino acid linker sequence. In some mutant single-stranded Msps provided herein, at least one of the first and second Msp monomer sequences is a mutant Msp monomer sequence containing a mutation at position 97. In other mutant single-stranded Msps provided herein, at least one of the first and second Msp monomer sequences is a mutant Msp monomer sequence containing one or more mutations at any of the following amino acid positions: I68, S73, S116, P123, or V128.

[0008] Optionally, the nucleic acid sequence encoding the mutant single-stranded Msp comprises first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences or any subgroup thereof, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences encode the first, second, third, fourth, fifth, sixth, seventh, and eighth Msp monomer sequences, respectively. The nucleic acid sequence also comprises a ninth nucleotide sequence encoding an amino acid linker sequence optionally present at multiple sites (e.g., between Msp monomer sequences). The first Msp monomer sequence may be a mutant Msp monomer sequence containing one or more mutations at any of the following amino acid positions: I68, S73, S116, P123, or V128, or a mutant Msp monomer sequence containing a mutation at position 97.

[0009] This article also provides the nucleic acid encoding the mutant Msp monomer. The Msp monomer contains mutations at one or more of the following positions: I68, S73, P97, S116, P123, or V128.

[0010] Also provided are polypeptides comprising the mutant Msp described herein and polypeptides encoded by nucleic acids described herein. A system comprising the mutant Msp described herein is also provided, wherein the Msp has a pre-hall and a constriction region defining a channel, wherein the channel is located between a first conductive liquid medium and a second conductive liquid medium, wherein at least one conductive liquid medium contains the analyte, and wherein the system is operable to detect the analyte when subjected to an electric field sufficient to translocate the analyte from one conductive liquid medium to the other.

[0011] A method for detecting the presence of an analyte is also provided. This method involves applying an electric field sufficient to cause the analyte to translocate from a first conductive medium to a liquid-connected second conductive medium via the mutation Msp described herein, and measuring an ion current, wherein a decrease in the ion current indicates the presence of the analyte in the first medium. Attached Figure Description

[0012] Figure 1 Alignments of MspA, MspB, MspC, and MspD monomers of Mycobacterium smegmatis are shown. Each protein is numbered starting with the first amino acid of the mature portion of the sequence. The sequences of the MspA, MspB, MspC, and MspD monomers without the signal / leader sequence are provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

[0013] Figure 2 A schematic diagram of the plasmid used to construct Mycobacterium ML712 smegma is shown. pML1611–mspB is the deletion vector: mspBup, mspBdown- Upstream and downstream regions homologous to mspB chromosome genes; loxP -Recombinase recombination site hyg - Hygromycin resistance; sacB -Fructansucrase; xylE -Catechol-2,3-dioxygenase; gfp2+ -Green fluorescent protein; tsPAL5000 Thermosensitive origin of replication of mycobacteria; ColE1 -Escherichia coli ( E. coli ) Replication start point. pML2622 – used to express cpnT The N-terminal channel forms an integrative plasmid with a structural domain (d1). hyg Hygromycin resistance gene; pBR322-Ori : E. coli replication origin; for cpnT Nitrile-induced expression requires pNIT1 and pNIT 2, attP : Chromosomal attachment site of bacteriophage L5; int L5 integrase; FRT : Flp recombination site.

[0014] Figure 3 Among the strains of Mycobacterium smegmatis msp Southern blotting of loci. Chromosomal DNA was isolated from SMR5(1), ML16(2), and ML712(3) Mycobacterium smegmatis strains. DNA fragments were isolated on 1% agarose gels, blotted onto nitrocellulose membranes, and detected using digoxigenin-labeled specific probes. The size of the DNA fragments hybridizing to the probes was consistent with the predicted values. Expected fragment size: mspA Loci: 1-1500bp, 2,3-1250bp; mspB Loci: 1, 2-1140bp, 3-1850bp; mspC Loci: 1-1200bp, 2,3-2100bp; mspD Loci: 1-1730bp, 2,3-1090bp.

[0015] Figure 4 Western blot analysis of Msp porin expression in a Mycobacterium smegmatis porin gene deletion mutant. Msp porin was extracted with 0.5% octylpolyoxyethylene and loaded into each lane with 10 µl. Proteins were isolated on 8% SDS-PAGE and detected by Western blot using a polyclonal antibody against MspA. Lanes: 1, SMR5 (wild type; mspA ); 2, ML16 (tripore protein deletion mutant); mspBExpression); 3, ML712 / pMS2 ​​(tetrapore protein deletion mutant); empty carrier ); 4, ML712 / pMN016(+ mspA Expression plasmid); 5, ML712 / pML904(+M1 mspA Expression plasmids).

[0016] Figure 5 This is a schematic diagram of the mycobacterial expression plasmid pML3213 encoding single-stranded M18-MspA. The treatment includes the following: hyg Hygromycin resistance gene; ory myc The origin of replication in mycobacteria; COLE1 ORI The origin of replication in E. coli; psmyc Constitutive promoters of mycobacteria; m1-1, m1-2 Etc., the index number corresponds to the position in the single-stranded gene construct. m1 mspA Gene. Tetrad A is attached to the first... m1-mspA The starting point of PacI and in the fourth m1-mspA The end of MluI; the tetrad B side is connected to the fifth m1-mspA The starting point of EcoRV and in the eighth m1-mspA HindII at the end. Individual within the tetrad. m1-mspA Genes are lateralized to KpnI, NsiI, NdeI, PstI, ScaI, and NheI. The linker connecting (GGGGS)3 (SEQ ID NO: 5) is located between restriction sites but is not shown.

[0017] Figure 6 Western blot analysis was performed to visualize the expression of single-chain MspA in Mycobacterium smegmae ML714. Mspporin was extracted with 0.5% octyl polyethylene oxide and loaded into each lane with 10 µl. Proteins were separated on 10% SDS-PAGE, transferred to a PVDF membrane, and detected with an αMspA monoclonal antibody. Lane labels were as follows: M, protein ladder; 1, expressing wild-type MspA (SMR5); 2, M12-MspA; 3, M14-MspA; 4, M18-MspA (single-chain M1-MspA). The amount of MspA was determined by quantitative image analysis.

[0018] Figure 7The thermal stability of single-chain M18-MspA was demonstrated. Single-chain M18-MspA porous protein was extracted with 0.5% n-octyl polyethylene oxide. 15 µl of sample was incubated for 15 min in buffer containing 2% SDS at a temperature higher than the indicated temperature for each lane. Proteins were separated by 8% SDS PAGE, then transferred to a PVDF membrane and detected with an αMspA monoclonal antibody. Lane labels were as follows: M, protein ladder; RT, room temperature; 40, 50, etc., indicating incubation temperature. An equal volume of protein sample was loaded into each lane.

[0019] Figure 8A The results of lipid bilayer experiments using M18-MspA are shown. Approximately 70 ng of protein was added to the bilayer chamber. The M18-MspA content in the diphyllylphosphatidylcholine lipid bilayer was recorded at -10 mV in 1 M KCl. 1-8 The current trajectory of M18-MspA was observed. This resulted in a gradual increase in current through the lipid bilayer, indicating that the M18-MspA channel was inserted into the membrane.

[0020] Figure 8B The conductivity histogram of M18-MspA is shown. 269 pores on four different membranes were analyzed. Single-chain M... 1-8 -MspA exhibits the main conductance peak at 1.1 nS.

[0021] Figure 9 The results of the voltage-gated experiment are shown. M18-MspA was added to the same side of the diphyllylphosphatidylcholine membrane. When approximately 220 channels were reconstructed into the membrane, a positive ( ) was gradually applied to the membrane. Upper trajectory ) and negative ( Lower trajectory Voltage. Record the membrane current at each applied voltage. Determine the critical voltage at which the channel begins to close. V c) The voltage at which the conductivity of the lipid bilayer decreases after its initial maximum. Critical voltage of M18-MspA. V c is +90mV. The M18-MspA channel is stable under all applied negative voltages.

[0022] Figure 10 A illustrates an example of a positive ramp generated in a single-stranded Msp containing a first mutant Msp monomer and a seventh mutant Msp monomer, the first mutant Msp monomer containing mutations at positions D56, I68, S73, D118, D134, and E139, and the seventh mutant Msp monomer containing mutations at positions L88 and I105.

[0023] Figure 10B shows a top view of the positive ramp generated in a single-stranded Msp containing a first mutant Msp monomer and a seventh mutant Msp monomer, the first mutant Msp monomer containing mutations at positions D56, I68, S73, D118, D134 and E139 and the seventh mutant Msp monomer containing mutations at positions L88 and I105.

[0024] Figure 10 C is a schematic diagram of a single-chain Msp. The numbers #1 and #7 under the subunits indicate the positions of the positive ramps.

[0025] Figure 11 For expression of single strands in E. coli m2-mspA A schematic diagram of plasmid pML3216. bla Ampicillin resistance gene; pBR322-Ori The origin of replication in E. coli; lacI lac repressor protein; T7 P T7 starter; SD The Shine-Dalgarno sequence; m2-1-m2-8 , m2 mspA Codon.

[0026] Figure 12 show scm2-mspA It can be generated in *E. coli*. *E. coli* Omp8 cells were induced with 1.5 mM IPTG at 0.5 OD600. Cells were collected and lysed at different time points. Equal volumes of protein samples were loaded onto a 10% polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue and quantified using LabWorks software (Waltham, Massachusetts). Lanes: M The molecular weight (kDa) is indicated on the left. Un Uninduced cells; after 2, 4 and 6 hours of IPTG induction.

[0027] Figure 13 Western blots of scMspA M2 refolding are shown. Samples before and after refolding were loaded onto 8% polyacrylamide gels and then transferred overnight to PVDF membranes. The membranes were stained with MspA-specific rabbit antibodies. Lanes: M The molecular weight (kDa) is indicated on the left. Un Unfolded samples after anion exchange chromatography; F , Folded scMspA M2.

[0028] Figure 14(AB) illustrates the channel-forming properties of scMspA M2. (A) Current trajectory recording of scMspA M2 in a planar lipid bilayer. With the addition of... Figure 3 As shown, after refolding the scMspA M2 protein, a gradual increase in current was observed, indicating channel insertion. (B) Histogram of single-channel conductance distribution. A total of 392 channels from 8 membranes were analyzed. The dashed lines represent Gaussian fits to the data distribution. The main conductance of scMspA M2 is 2.3 nS.

[0029] Figure 15 The voltage-dependent channel closure of scMspA M2 is shown. Protein was added to the same side of the cuvette. After inserting approximately 200 controls, the positive or negative voltage was increased in 10 mV increments, and the current was recorded for 2 minutes. Horizontal lines indicate open wells, while diagonal lines indicate closed wells. The critical voltage of scMspA M2 was determined to be approximately +80 mV / -70 mV.

[0030] Figure 16 This demonstrates the method for expressing single strands in E. coli. scmspA PN1 ( scmspA PN1 A schematic diagram of plasmid pML3222. bla Ampicillin resistance gene; pBR322-Ori The origin of replication in E. coli; lacI lac repressor protein; T7 P T7 starter; SD The Shain-Dalgano sequence; m2-97-1 – m2-97-8 Having the P97F mutation m2 mspA Codon optimization genes.

[0031] Figure 17 show scmspA PN1 It can be generated in E. coli. At an OD of 0.5... 600 E. coli Omp8 cells were induced with 1.5 mM IPTG. After 2 hours of induction, cells were collected and lysed. Equal volumes of protein samples were loaded onto an 8% polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue and quantified using LabWorks software (Waltham, Massachusetts). Lanes: M The molecular weight (kDa) is indicated on the left. Un Non-induced cells; induced with IPTG for 2 hours.

[0032] Figure 18 The Western blot of scMspA PN1 refolding is shown. Samples were loaded onto an 8% polyacrylamide gel, stained with Coomassie Brilliant Blue, and quantified by scanning using LabWorks software. Lanes: MThe molecular weight marker (kDa) on the left indicates the mass; IB, inclusion bodies purified from E. coli Omp8; AE, sample after anion exchange chromatography; D, sample after dialysis; R, folded scMspA PN1 protein.

[0033] Figure 19 The insertion of scMspA PN1 into the lipid membrane is shown. DPhPC liposomes were loaded with 30 mM carboxyfluorescein dye. Dye release was monitored using a Varian Cary fluorometer (Palo Alto, CA) at 517 nm emission (492 nm excitation). Symbols: Open square - Triton X-100 (0.1% v / v); Closed circle - LDAO (0.1% v / v); Open circle - OPOE (0.5% v / v); Closed triangle - wtMspA (60 ng / ml); Closed square - scMspA PN1 (60 ng / ml); Open triangle - scMspA M2 (120 ng / ml). No significant dye release was observed when the liposomes were in PBS buffer alone.

[0034] Figure 20 The time distribution of pore remodeling in the artificial DPhPC membrane is shown. Cuvettes were filled with KCl of indicated concentrations. A final 100 ng / ml protein sample was added to both sides. A -10 mV potential was applied, and data were recorded using TestPoint software. Each point represents the time of the first pore insertion. The median insertion time for scMspA PN1 in 1 M KCl was 399 seconds (analyzed on 9 membranes with 89% insertion success). The median insertion time for scMspA PN1 in 0.3 M KCl was 859 seconds (analyzed on 8 membranes with 50% insertion success). The median insertion time for scMspA M2 in 1 M KCl was 695 seconds (analyzed on 10 membranes with 40% insertion success). The insertion time for scMspA M2 in 0.3 M KCl was 1270 seconds (8 membranes, 12% insertion success). The results were determined by the Mann-Whitney Rank Sum test. - P=0.028.

[0035] Figure 21(AD) Shows the single-channel conductance of scMspA PN1 and scMspA M2 in 1.0 M KCl. (A) Current trajectory recording of scMspA PN1 in a planar lipid bilayer. A gradual increase in current was observed after the addition of refolded scMspA PN1 protein, indicating channel insertion. (B) Histogram of single-channel conductance distribution. A total of 137 channels from 4 membranes were analyzed. Dashed lines represent Gaussian fits to the data distribution. The principal conductance of scMspA PN1 is 2.0 nS. (C) Current trajectory recording of scMspA M2 in a planar lipid bilayer. (D) Histogram of single-channel conductance distribution. A total of 238 channels from 6 membranes were analyzed. Dashed lines represent Gaussian fits to the data distribution. The principal conductance of scMspA M2 is 1.3 nS.

[0036] Figure 22 The single-channel conductance of single-chain MspA PN1 in 0.3 M KCl and 1.0 M KCl is shown, as well as the increase in channel insertion into the membrane of scMspA PN1 in 0.3 M KCl after contact with the membrane. Detailed Implementation

[0037] This article describes mutant Mycobacterium smegmatis porin (Msp). A mutant Msp can be a multimeric complex composed of two or more Msp monomers, at least one of which is a mutant Msp monomer. Msp monomers are encoded by genes in Mycobacterium smegmatis. Mycobacterium smegmatis has four identified Msp genes, denoted as MspA, MspB, MspC, and MspD. Figure 1 The image shows alignments of wild-type polypeptide sequences for MspA, MspB, MspC, and MspD monomers of *Mycobacterium smegmatis*. Each protein is numbered starting with the first amino acid of the mature portion of the sequence, as indicated by the number "1" on the first amino acid of the mature amino acid sequence. The amino acid sequences of the MspA, MspB, MspC, and MspD monomers without a signal sequence, i.e., the mature portions of the sequence, are provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. The amino acid sequences of the MspA, MspB, MspC, and MspD monomers with a signal / leader sequence are provided as SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

[0038] Furthermore, the sequences of modifiable wild-type Msp monomers are published in GenBank, and these sequences, along with other sequences, are incorporated herein by reference in their entirety, including individual subsequences or fragments. For example, the nucleotide and amino acid sequences of wild-type MspA monomers can be found under GenBank accession numbers AJ001442 and CAB56052, respectively. Similarly, the nucleotide and amino acid sequences of wild-type MspB monomers can be found under GenBank accession numbers NC_008596.1 (nucleotides 600086 to 600370) and YP 884932.1, respectively. Furthermore, the nucleotide and amino acid sequences of wild-type MspC monomers can be found under GenBank accession numbers AJ299735 and CAC82509, respectively. Finally, the nucleotide and amino acid sequences of wild-type MspD monomers can be found under GenBank accession numbers AJ300774 and CAC83628, respectively.

[0039] Mutant Msp monomers can be nucleic acids encoded by MspA, MspB, MspC, or MspD, such as mRNA or full-length monomers or functional fragments thereof encoded by genomic sequences encoding MspA, MspB, MspC, or MspD, wherein the monomer contains one or more modifications.

[0040] Optionally, the mutant Msp is a mutant single-stranded Msp or a polymer of several single-stranded Msp, wherein the polymer contains at least one mutant single-stranded Msp. The mutant Msp may also be a polymer of several Msp monomers, wherein at least one Msp monomer is a mutant Msp monomer.

[0041] Single-chain Msp can, for example, comprise a multimer formed by two or more Msp monomers (e.g., eight monomers) linked by one or more amino acid linker peptides. Partially single-chain Msp refers to single-chain multimeric complexes that form porins through dimerization, trimerization, etc. Full-length single-chain Msp porins are single-chain multimeric complexes that form porins without the need for dimerization, trimerization, etc. In other words, compared to porins that must associate with other partially single-chain Msp or monomeric Msp monomers to form porins, single-chain folding forms porins, but all components are on a single amino acid chain.

[0042] Mutant single-stranded Msp and nucleic acid encoding mutant single-stranded Msp

[0043] This document provides nucleic acid sequences encoding mutant single-stranded Msp. For example, a nucleic acid sequence encoding a mutant single-stranded Msp comprises: (a) first and second nucleotide sequences, wherein the first nucleotide sequence encodes a first Msp monomer sequence and the second nucleotide sequence encodes a second Msp monomer sequence; and (b) a third nucleotide sequence encoding an amino acid linker sequence, wherein at least one of the first and second Msp monomer sequences is a mutant Msp monomer sequence having a mutation at position P97. Optionally, the mutant Msp monomer sequence may contain a mutation at position P97, wherein the mutation is not a P97S or P97C mutation. Optionally, the mutant Msp monomer sequence may contain a P97F mutation. As shown in the examples, additional hydrophobic residues, such as phenylalanine, located in loop 6 (amino acids 91-103) of scMspA facilitate faster and more efficient insertion of the pore into the lipid bilayer. For a description of loop 6 of MspA and the residues contained therein, see Huffe et al. J. Biol. Chem. 284: 10223-10231 (2009), which is incorporated herein by reference in its entirety. Thus, this paper provides for single-chain Msp containing one or more hydrophobic substitutions in ring 6 (amino acids 91-103) of Msp.

[0044] For example, this article provides a nucleic acid sequence encoding a mutant single-stranded Mycobacterium smegma porin (Msp), wherein the nucleic acid sequence comprises: (a) first and second nucleotide sequences, wherein the first nucleotide sequence encodes a first Msp monomer sequence and the second nucleotide sequence encodes a second Msp monomer sequence; and (b) a third nucleotide sequence encoding an amino acid linker sequence, wherein at least one of the first and second Msp monomer sequences is a mutant Msp monomer sequence containing one or more mutations at any of the following amino acid positions: D91, G92, D93, I94, T95, A96, P97, P98, F99, G100, L101, N102, or S103, wherein one or more of D91, G92, D93, I94, T95, A96, P97, P98, F99, G100, L101, N102, or S103 are substituted with hydrophobic amino acids. For example, the hydrophobic amino acid may be selected from the group consisting of: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tyrosine, tryptophan, proline, and glycine. By way of example, and not limitation, the mutant Msp monomer sequence may contain (i) D90N, D91N, and D93N mutations; and (ii) one or more of the G92F, T95F, A96F, P97F, P98F, G100F, L101F, N102F, or S103F mutations. As mentioned above, the substitution at positions G92, T95, A96, P97, P98, G100, L101, N102, or S103 is not limited to phenylalanine, as one or more of these amino acids may be substituted with another hydrophobic residue, such as alanine, valine, leucine, isoleucine, proline, methionine, tyrosine, tryptophan, proline, and glycine. One or more of G92, T95, A96, P97, P98, G100, L101, N102, or S103 may be substituted with the same or different hydrophobic amino acids.

[0045] As used throughout, mutations at specific amino acid positions are indicated by a single-letter code for the amino acid at that position, followed by the amino acid position number in the Msp polypeptide sequence (e.g., the amino acid position in SEQ ID NO: 1) and a single-letter code indicating the substitution of that amino acid at that position. Therefore, it should be understood that the P97 mutation is the substitution of proline for phenylalanine at amino acid 97 of SEQ ID NO: 1. Similarly, the D90N mutation is the substitution of aspartic acid for arginine at amino acid 90 of SEQ ID NO: 1, the D91N mutation is the substitution of aspartic acid for arginine at amino acid 91 of SEQ ID NO: 1, and so on. It should also be understood that the amino acids corresponding to the positions in SEQ ID NO: 1 are also provided herein (see [link to documentation]). Figure 1For example, and not to limit, those skilled in the art will understand that the corresponding amino acids for E139 of SEQ ID NO: 1 in MspB (SEQ ID NO: 2), MspC (SEQ ID NO: 3), and MspD (SEQ ID NO: 4) are A139, A139, and K138, respectively.

[0046] Optionally, any mutant Msp monomer sequence described herein may also include a mutation at amino acid position D118, a mutation at position D134, or a mutation at position E139. Optionally, the mutation at position E139 may be an E substitution for R (arginine) or an E substitution for K (lysine). Optionally, the mutation at position D118 may be a D substitution for R or a D substitution for K. Optionally, the mutation at position D134 may be a D substitution for R or a D substitution for K. For example, any mutant Msp monomer sequence described herein may include one or more mutations selected from the group consisting of: D118R mutation, D134R mutation, and E139K mutation. Optionally, any mutant Msp monomer sequence described herein may also include at least one of: (i) a mutation at position 93 and (ii) mutations at positions D90, D91, or both D90 and D91. Optionally, the amino acid at position 90, 91, or 93 may be substituted with arginine, lysine, histidine, glutamine, methionine, threonine, phenylalanine, tyrosine, or tryptophan. Optionally, any mutant Msp monomer sequence described herein may also contain D90N, D91N, and D93N mutations.

[0047] For example, a mutant Msp monomer sequence containing a mutation at position 97 may also contain (i) mutations at amino acid positions D118, D134, and / or E139, (ii) a mutation at position D93, and / or (iii) mutations at positions D90, D91, or both D90 and D91. For example, a mutant MspA monomer sequence may contain D90N, D91N, D93N, P97F, D118R, D134R, and E139K mutations. A mutant MspA monomer sequence may also contain D90N, D91N, D93N, P97F, D118R, D134R, and E139K mutations.

[0048] This document also provides a nucleic acid sequence encoding a mutant single-stranded Msp, comprising (a) first and second nucleotide sequences, wherein the first nucleotide sequence encodes a first Msp monomer sequence and the second nucleotide sequence encodes a second Msp monomer sequence; and (b) a third nucleotide sequence encoding an amino acid linker sequence, wherein at least one of the first and second Msp monomer sequences is a mutant Msp monomer sequence containing one or more mutations at any of the following amino acid positions: I68, S73, S116, P123, or V128. This document provides a nucleic acid sequence encoding a mutant single-stranded Msp, wherein the mutant Msp monomer sequence contains one or more mutations at any of I68, S73, S116, P123, or V128; wherein said mutation is not an I68V mutation or an S73C mutation.

[0049] This document provides a mutant Msp monomer sequence containing one or more mutations at I68, S73, S116, P123, and / or V128, which further includes (i) a mutation at amino acid position D118, (ii) a mutation at position D93, and / or (iii) mutations at positions D90, D91, or both D90 and D91. For example, a mutant MspA monomer sequence may contain one or more mutations at amino acid positions I68, S73, S116, P123, or V128, a mutation at D93, a mutation at D118, a mutation at D134, and a mutation at E139, or any subgroup thereof. In another example, a mutant MspA monomer sequence may contain one or more mutations at amino acid positions I68, S73, S116, P123, or V128, a mutation at D118, a mutation at D134, a D90N mutation, and / or a D91N mutation. In another embodiment, the mutant MspA monomer sequence may contain one or more mutations at amino acid positions I68, S73, S116, P123, or V128, a mutation at D118, a mutation at D134, a mutation at E139, a D90N mutation, a D91N mutation, and a D93N mutation. This document provides a nucleic acid encoding a mutant single-stranded Msp, wherein the mutant Msp monomer sequence contains one or more mutations at any of I68, S73, S116, P123, or V128, wherein the mutation is not an I68V mutation or an S73C mutation. In any mutant single-stranded Msp provided herein, the mutant Msp monomer sequence may contain one or more mutations at any of I68, S73, S116, P123, or V128, wherein the mutation is not an I68V mutation or an S73C mutation.

[0050] Optionally, any mutant Msp monomer sequence described herein may also contain one or more mutations at any of the following amino acid positions: D13, A55, D56, E57, F58, E63, S136, G137, or D172. Optionally, one or more of D13, A55, D56, E57, F58, E63, S136, G137, or D172 in the mutant Msp monomer sequence provided herein may be substituted with lysine or arginine. Optionally, any mutant Msp monomer sequence described herein containing one or more mutations at D13, A55, D56, E57, F58, E63, S136, G137, or D172 may also contain one or more mutations at the following positions: D118, D134, or E139. Optionally, any mutated Msp monomer sequence described herein containing one or more mutations at D13, A55, D56, E57, F58, E63, S136, G137, or D172 may also contain a mutation at position 93 and / or a mutation at positions 90, 91, or both positions 90 and 91.

[0051] Therefore, this document provides a nucleic acid sequence encoding a mutant single-stranded Mycobacterium smegma porin (Msp), wherein the nucleic acid sequence comprises (a) first and second nucleotide sequences, wherein the first nucleotide sequence encodes a first Msp monomer sequence and the second nucleotide sequence encodes a second Msp monomer sequence; and (b) a third nucleotide sequence encoding an amino acid linker sequence, wherein at least one of the first and second Msp monomer sequences is a mutant Msp monomer sequence containing one or more mutations at any of the following amino acid positions: D13, A55, D56, E57, F58, E63, S136, G137, or D172. Optionally, the third nucleotide sequence encoding the linker is located between the first and second nucleotide sequences.

[0052] Also provided is a nucleic acid sequence encoding a mutant single-stranded Mycobacterium smegmatis pore protein (Msp), wherein the nucleic acid sequence comprises (a) first and second nucleotide sequences, wherein the first nucleotide sequence encodes a first Msp monomer sequence and the second nucleotide sequence encodes a second Msp monomer sequence; and (b) a third nucleotide sequence encoding an amino acid linker sequence, wherein at least one of the first and second Msp monomer sequences is a mutant Msp monomer sequence comprising (i) a mutation at position 93, and / or (ii) a mutation at position 90, position 91, or both positions 90 and 91, and (iii) one or more mutations at any of the following amino acid positions: D13, A55, D56, E57, F58, E63, S136, G137, or D172.

[0053] Also provided is a nucleic acid encoding a mutant Msp monomer, wherein the Msp monomer contains mutations at one or more of the following amino acid positions: D13, A55, D56, E57, F58, E63, S136, G137, or D172. Optionally, the mutant Msp monomer sequence containing one or more mutations at D13, A55, D56, E57, F58, E63, S136, G137, or D172 may also contain a mutation at position 93 and / or mutations at positions 90, 91, or both positions 90 and 91. Optionally, the mutant Msp monomer sequence containing one or more mutations at positions D13, A55, D56, E57, F58, E63, S136, G137, or D172 may also contain D90N, D91N, and D93N mutations.

[0054] In the mutant single-stranded mutant Msp provided herein, the first monomer sequence can be any mutant monomer sequence described herein. For example, the mutant monomer sequence can be the mutant MspA sequence. The second monomer can be selected from the group consisting of: wild-type Msp monomer, second mutant Msp monomer, wild-type Msp paralogous or homologous monomer, and mutant Msp paralogous or homologous monomer. It should be understood that the second mutant Msp monomer may be the same as or different from the first mutant Msp monomer. These include, but are not limited to, MspA / Msmeg0965, MspB / Msmeg0520, MspC / Msmeg5483, MspD / Msmeg6057, MppA, PorM1, PorM2, PorM1, Mmcs4296, Mmcs4297, Mmcs3857, Mmcs4382, Mmcs4383, Mjls3843, Mjls3857, Mjls3931, Mjls4674, Mjls4675, and Mjls 4677, Map3123c, Mav3943, Mvan1836, Mvan4117, Mvan4839, Mvan4840, Mvan5016, Mvan5017, Mvan5768, MUL_2391, Mflv1734, Mflv1735, Mflv2295, Mflv1891, MCH4691c, MCH4689c, MCH4690c, MAB1080, MAB1081, MAB2800, RHA1 ro08561, RHA1 ro04074, and RHA1 ro03127. Paralogous or homologous monomers of wild-type MspA can be wild-type MspB monomers. Paralogous and homologous monomers of wild-type MspA are well known in the art. Table 1 provides a non-restrictive list of such paralogs and homologs.

[0055] Table 1. Paralogous and homologous monomers of wild-type MspA and wild-type MspA

[0056]

[0057]

[0058] Only proteins with significant amino acid similarity across their full length were included. Data were obtained from the NIH Gene Bank database at ncbi.nlm.nih.gov / blast / Blast.cgi using the PSI-Blast algorithm (BLOSUM62 matrix).

[0059] nd: "Not determined"

[0060] Stahl et al. Mol. Microbial. 40:451 (2001)

[0061] Domer et al., Biochim. Biophys. Acta. 1667:47-55 (2004)

[0062] As used herein, a mutant single-chain Msp is a polypeptide comprising at least two Msp monomers or functional fragments thereof linked by one or more amino acid linker peptides, wherein at least one Msp monomer is a mutant Msp monomer. For example, a mutant single-chain Msp may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more Msp monomers linked by one or more amino acid linker peptides, wherein at least one Msp monomer is a mutant Msp monomer. As described above, a single-chain mutant Msp can, for example, form a porin by folding, without the need for dimerization, trimerization, etc., to form a porin. Optionally, a mutant single-chain Msp may be a partially single-chain mutant Msp comprising at least two Msp monomers or fragments thereof linked by one or more amino acid linker peptides that can dimerize, trimerize, etc., to form a porin.

[0063] Optionally, Msp porins containing mutant single-stranded Msp may, for example, comprise two or more single-stranded Msp porin dimers, two or more single-stranded Msp porin trimers, two or more single-stranded Msp porin tetramers, two or more single-stranded Msp porin pentamers, one or more single-stranded Msp porin hexamers, one or more single-stranded Msp porin heptamers, one or more single-stranded Msp porin octamers, or combinations thereof. For example, an Msp porin may comprise one single-stranded Msp porin dimer and two single-stranded Msp porin trimers. As another example, an Msp porin may comprise one single-stranded Msp porin tetramer and two single-stranded Msp porin dimers.

[0064] This document describes amino acid linker sequences. In any single-stranded Msp described herein, the linker sequence may, for example, contain 10 to 20 amino acids. For example, an amino acid linker sequence contains 15 amino acids. Optionally, the amino acid linker sequence contains a (GGGGS)3 (SEQ ID NO: 5) peptide sequence. The same or different nucleic acids encoding the linker sequence may be provided between nucleic acid sequences encoding two or more Msp monomers. Optionally, the linker sequence may be provided between all or some nucleic acid sequences encoding the Msp monomers in the single-stranded Msp provided herein.

[0065] Also provided is a nucleic acid sequence encoding a mutant single-stranded Msp, wherein the nucleic acid sequence comprises (a) a first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequence or any subgroup thereof, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences encode the first, second, third, fourth, fifth, sixth, seventh, and eighth Msp monomer sequences, respectively; and (b) a ninth nucleotide sequence encoding an amino acid linker sequence, wherein the first Msp monomer sequence is a mutant Msp monomer sequence containing a mutation at position P97.

[0066] The mutant Msp monomer sequence may contain a mutation at P97, wherein the mutation is not a P97S or P97C mutation. The mutant Msp monomer sequence may contain a P97F mutation. As described above, any mutant Msp monomer sequence described herein may also contain a mutation at amino acid position D118, a mutation at position D134, or a mutation at position E139. For example, any mutant Msp monomer sequence described herein may contain a D118R mutation, a D134R mutation, and / or an E139K mutation. Any mutant Msp monomer sequence described herein may also contain (i) a mutation at position 93, and / or (ii) mutations at positions D90, D91, or both D90 and D91. Optionally, the amino acid at positions 90, 91, or 93 may be substituted with arginine, lysine, histidine, glutamine, methionine, threonine, phenylalanine, tyrosine, or tryptophan. Any mutant Msp monomer sequence described herein may also contain D90N, D91N, and D93N mutations. For example, this document provides a nucleic acid sequence encoding a mutant single-stranded Msp, wherein the nucleic acid sequence comprises (a) a first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequence or any subgroup thereof, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences encode the first, second, third, fourth, fifth, sixth, seventh, and eighth Msp monomer sequences, respectively; and (b) a ninth nucleotide sequence encoding an amino acid linker sequence, wherein the first Msp monomer sequence is a mutant Msp monomer sequence containing a mutation at position P97, and may further contain (i) a mutation at amino acid positions D118, D134, or E139, (ii) a mutation at position D93, and / or (iii) a mutation at positions D90, D91, or both D90 and D91. For example, the first Msp monomer sequence may be a mutant Msp monomer sequence containing the D90N mutation, D91N mutation, D93N mutation, P97F mutation, D118R mutation, D134R mutation, and E139K mutation.

[0067] Also provided is a nucleic acid sequence encoding a mutant single-stranded Msp, wherein the nucleic acid sequence comprises (a) a first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequence or any subgroup thereof, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences encode the first, second, third, fourth, fifth, sixth, seventh, and eighth Msp monomer sequences, respectively; and (b) a ninth nucleotide sequence encoding an amino acid linker sequence, wherein the first Msp monomer sequence is a mutant Msp monomer sequence containing one or more mutations at any of the following amino acid positions: I68, S73, S116, P123, or V128.

[0068] The first Msp monomer sequence may also be a mutant Msp monomer sequence containing one or more mutations at any of the following amino acid positions: I68, S73, S116, P123, or V128, and also containing a mutation at amino acid position D118, optionally having (i) a mutation at position 93, and / or (ii) mutations at positions D90, D91, or both D90 and D91. In any mutant Msp monomer sequence described herein, the amino acid at position 91 or at position 90 may be substituted with arginine, lysine, histidine, glutamine, methionine, threonine, phenylalanine, tyrosine, or tryptophan. The mutant Msp monomer sequence may also contain D90N, D91N, and D93N mutations. The mutant Msp monomer containing one or more mutations at amino acids I68, S73, S116, P123, or V128 may also contain mutations at one or more amino acids at positions D13, A55, D56, E57, F58, E63, S136, D134, G137, E139, or D172. In the mutant Msp monomer sequence described herein, D13, A55, D56, E57, F58, E63, S136, D134, G137, E139, or D172 may be substituted with lysine or arginine. Therefore, a mutant Msp monomer containing one or more mutations at amino acid I68, S73, S116, P123, or V128 may, for example, also contain (i) a mutation at amino acid position D118, (ii) a mutation at position D93, (iii) a mutation at position D90, position D91, or both positions D90 and D91, (iv) mutations at D90N, D91N, and D93N, and / or (v) a mutation in one or more amino acids at positions D13, A55, D56, E57, F58, E63, S136, D134, G137, E139, or D172.

[0069] For example, and not limited thereto, the first mutant Msp monomer may be a mutant Msp monomer containing mutations at positions D56, I68, S73, D118, D134, and E139. Optionally, the mutant Msp monomer may also contain D90N, D91N, and D93N mutations. Optionally, one or more amino acids selected from the group consisting of D56, I68, S73, D118, D134, and E139 may be substituted with lysine or arginine.

[0070] Also provided is a nucleic acid sequence encoding a mutant single-stranded Msp, wherein the nucleic acid sequence comprises (a) a first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequence or any subgroup thereof, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences encode the first, second, third, fourth, fifth, sixth, seventh, and eighth Msp monomer sequences, respectively; and (b) a ninth nucleotide sequence encoding an amino acid linker sequence, wherein the first Msp monomer sequence is a mutant Msp monomer sequence containing one or more mutations at any of the following amino acid positions: I68, S73, S116, P123, or V128, and wherein one or more of the first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences encode a mutant Msp monomer sequence containing mutations at one or more of the following positions: T83, N86, L88, I105, D90, D91, G92, D93, or A96. For example, and not limited to, the seventh nucleotide can encode a mutant Msp monomer sequence containing a mutation at one or more of the following positions: T83, N86, L88, I105, D90, D91, G92, D93, or A96.

[0071] It should be understood that the first and seventh nucleotide sequences can be arranged, but not necessarily in the order of the first and seventh nucleotide sequences in a nucleic acid sequence containing the first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences. In this case, the first nucleotide sequence is the nucleotide sequence encoding the first or initiating monomer of the single-stranded Msp, and can be the first, second, third, fourth, fifth, sixth, seventh, or eighth nucleotide sequence of the single-stranded Msp. The initiating nucleotide sequence is referred to as the first nucleotide sequence regardless of where it appears in the single-stranded Msp. For example, if the initiating subunit of the single-stranded Msp is the first Msp monomer (first nucleotide sequence), then the seventh Msp monomer (seventh nucleotide sequence) contains mutations at one or more of the following positions: T83, N86, L88, I105, D90, D91, G92, D93, or A96. In another example, if the initiating subunit of the single-stranded Msp is the second Msp monomer (first nucleotide sequence), then the eighth Msp monomer (seventh nucleotide sequence) contains a mutation at one or more of the following positions: T83, N86, L88, I105, D90, D91, G92, D93, or A96. In another example, if the initiating subunit of the single-stranded Msp is the third Msp monomer (first nucleotide sequence), then the first Msp monomer (seventh nucleotide sequence) contains a mutation at one or more of the following positions: T83, N86, L88, I105, D90, D91, G92, D93, or A96. In another example, if the initiating subunit of the single-stranded Msp is the fourth Msp monomer (first nucleotide sequence), then the second Msp monomer (seventh nucleotide sequence) contains a mutation at one or more of the following positions: T83, N86, L88, I105, D90, D91, G92, D93, or A96. In another example, if the initiating subunit of a single-stranded Msp is the fifth Msp monomer (first nucleotide sequence), then the third Msp monomer (seventh nucleotide sequence) contains a mutation at one or more of the following positions: T83, N86, L88, I105, D90, D91, G92, D93, or A96. In another example, if the initiating subunit of a single-stranded Msp is the sixth Msp monomer (first nucleotide sequence), then the fourth Msp monomer (seventh nucleotide sequence) contains a mutation at one or more of the following positions: T83, N86, L88, I105, D90, D91, G92, D93, or A96. In another example, if the initiating subunit of a single-stranded Msp is the seventh Msp monomer (first nucleotide sequence), then the fifth Msp monomer (seventh nucleotide sequence) contains a mutation at one or more of the following positions: T83, N86, L88, I105, D90, D91, G92, D93, or A96.In another instance, if the starting subunit of a single-stranded Msp is the eighth Msp monomer (first nucleotide sequence), then the sixth Msp monomer (seventh nucleotide sequence) contains a mutation at one or more of the following positions: T83, N86, L88, I105, D90, D91, G92, D93, or A96.

[0072] For example, and not limited thereto, the first Msp monomer may be a mutant Msp monomer containing mutations at positions D56, I68, S73, D118, D134, and E139, and the seventh monomer may be a mutant Msp monomer containing mutations at positions L88 and I105. Optionally, each amino acid at positions D56, I68, S73, D118, D134, and E139 of the first mutant Msp monomer may be substituted with lysine or arginine. Optionally, each amino acid at positions D56, I68, S73, D118, D134, and E139 of the first mutant Msp monomer may be substituted with phenylalanine, tryptophan, histidine, or tyrosine. Optionally, each amino acid position at positions L88 and I105 of the seventh mutant Msp monomer may be substituted with lysine or arginine. Optionally, each amino acid position at positions L88 and I105 of the seventh mutant Msp monomer may be substituted with phenylalanine, tryptophan, histidine, or tyrosine. Substitution of D56, I68, S73, D118, D134, E139, L88, and / or I105 with aromatic amino acids such as phenylalanine, tryptophan, histidine, or tyrosine can promote p-stacking interactions with analytes, such as nucleotides, to reduce translocation rates. Optionally, the first, second, third, fourth, fifth, sixth, seventh, and eighth Msp monomer sequences, or subgroups thereof, may contain D90N, D91N, and D93N mutations.

[0073] Figure 10 A and 10B illustrate non-limiting examples of positive ramps generated in single-stranded Msp monomers containing a first mutant Msp monomer and a seventh mutant Msp monomer, the first mutant Msp monomer containing mutations at positions D56, I68, S73, D118, D134, and E139, and the seventh mutant Msp monomer containing mutations at positions L88 and I105. This positively charged ramp within the precordial chamber of MspA guides single-stranded nucleic acids, such as DNA, through the Msp. The electrostatic interaction between the nucleic acid and the ramp allows for controlled translocation of DNA through the pores. This reduces the Brownian motion and translocation rate of the nucleic acid. It also increases precision and the interaction between nucleic acid bases and amino acids within the constricted regions. Figure 10 C is a schematic diagram of a single-chain Msp. The numbers #1 and #7 under the subunits indicate the positions of the positive ramps.

[0074] In any mutant single-stranded Msp described herein, the constricted region may be modified to enhance the nucleobase, protein, or analyte recognition properties of MspA. Modification of the constricted region can produce reads that enhance, for example, base-specific interactions. Reads can be generated by introducing amino acids with longer side chains extending into the DNA or another analyte pathway. For example, and not limited to, to generate one or more reads, the amino acid at positions 90 and / or 91 in any mutant Msp monomer of the single-stranded Msp described herein may be substituted with arginine, lysine, histidine, glutamine, methionine, threonine, phenylalanine, tyrosine, tryptophan, or a non-natural amino acid. Positioning heads may also be generated to increase the efficiency of one or more reads. For example, an amino acid with a longer side chain, preferably hydrophobic or negatively charged, may be introduced opposite the read to reduce the escape movement of DNA or another analyte within the constricted region. Suitable amino acids include, but are not limited to, aspartic acid, glutamine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, and non-natural amino acids. To further reduce the translocation rate, stacking sides can be generated by mutating one or more amino acids at positions 83, 86, 88, and 105. For example, and not limited thereto, one or more amino acids at positions 83, 86, 88, and 105 can be substituted with tryptophan, tyrosine, or phenylalanine. Optionally, the stacking sides are positioned near the positive slope.

[0075] One or more of the first, second, third, fourth, fifth, sixth, seventh, and eighth Msp monomer sequences, or any subgroup thereof, may be independently selected from the group consisting of: wild-type MspA monomers, mutant MspA monomers, wild-type MspA paralogous or homologous monomers, and mutant MspA paralogous or homologous monomers. It should be understood that when the second, third, fourth, fifth, sixth, seventh, and / or eighth Msp monomer sequences are mutant MspA monomer sequences, the mutant MspA monomer sequence may be identical or different from the first mutant MspA monomer sequence. Optionally, the second, third, fourth, fifth, sixth, seventh, and eighth Msp monomer sequences, or any subgroup thereof, are wild-type MspA paralogous or homologous monomers. These include, but are not limited to, MspA / Msmeg0965, MspB / Msmeg0520, MspC / Msmeg5483, MspD / Msmeg6057, MppA, PorM1, PorM2, PorM1, Mmcs4296, Mmcs4297, Mmcs3857, Mmcs4382, Mmcs4383, Mjls3843, Mjls3857, and Mjls3931. Mjls4674, Mjls4675, Mjls4677, Map3123c, Mav3943, Mvan1836, Mvan4117, Mvan4839, Mvan4840, Mvan5016, Mvan5017, Mvan5768, MUL_2391, Mflv1734, Mflv1735, Mflv2295, Mflv1891, MCH4691c, MCH4689c, MCH4690c, MAB1080, MAB1081, MAB2800, RHA1 ro08561, RHA1 ro04074, and RHA1 ro03127. Paralogous or homologous monomers of wild-type MspA can be wild-type MspB monomers.

[0076] Mutant Msp monomer and nucleic acid encoding mutant Msp monomer

[0077] Also provided is a nucleic acid encoding a mutant Msp monomer, wherein the Msp monomer contains a mutation at position 97. Optionally, the mutant Msp monomer may contain a mutation at P97, wherein the mutation is not a P97S or P97C mutation. Optionally, the mutant Msp monomer may contain a P97F mutation. Optionally, the mutant Msp monomer may also contain a mutation at amino acid position D118, a mutation at position D134, or a mutation at position E139. For example, a mutant Msp monomer containing a mutation at position 97 may also contain a D118R mutation, a D134R mutation, and / or an E139K mutation. Optionally, a mutant Msp monomer containing a mutation at position 97 may also contain (i) a mutation at position 93, and / or (ii) mutations at positions D90, D91, or both D90 and D91. Optionally, the amino acid at position 90 or 91 is substituted with arginine, lysine, histidine, glutamine, methionine, threonine, phenylalanine, tyrosine, or tryptophan. Optionally, the mutant Msp monomer may also contain D90N, D91N, and D93N mutations. For example, and not limited thereto, the mutant MspA monomer sequence may contain D90N, D91N, D93N, P97F, D118R, D134R, and E139K mutations.

[0078] Also provided is a nucleic acid encoding a mutant Msp monomer, wherein the Msp monomer contains mutations at one or more of the following positions: I68, S73, S116, P123, or V128. Optionally, the mutant monomer also contains a mutation at amino acid position D118. Optionally, the Msp monomer also contains mutations at positions D90, D91, or both D90 and D91. Optionally, the amino acid at position 91 or at position 90 may be substituted with arginine, lysine, histidine, glutamine, methionine, threonine, phenylalanine, tyrosine, or tryptophan. Optionally, the mutant Msp monomer sequence may also contain mutations at D90N, D91N, and D93N. Optionally, the mutant Msp monomer sequence may also contain mutations at one or more amino acids at positions D13, A55, D56, E57, F58, E63, S136, D134, G137, E139, or D172. Optionally, one or more of D13, A55, D56, E57, F58, E63, S136, D134, G137, E139, or D172 may be substituted with lysine or arginine.

[0079] Also provided is a nucleic acid encoding a mutant Msp monomer, wherein the Msp monomer contains mutations at one or more of the following positions: T83, N86, G92, or A96. Optionally, the mutant Msp monomer sequence also contains a mutation at position L88 or I105. Optionally, the mutant monomer also contains a mutation at amino acid position D118. Optionally, the Msp monomer also contains mutations at positions D90, D91, or both D90 and D91. Optionally, the amino acid at position 91 or at position 90 may be substituted with arginine, lysine, histidine, glutamine, methionine, threonine, phenylalanine, tyrosine, or tryptophan. Optionally, the mutant Msp monomer sequence may also contain D90N, D91N, and D93N mutations.

[0080] As used herein, a mutant Msp monomer is an Msp monomer that has at least or at most 70, 75, 80, 85, 90, 95, 98, or 99% or higher identity with a wild-type Msp monomer, or any range thereof, but less than 100% identity, and retains channel-forming ability when associated with one or more other Msp monomers (wild-type or mutant). Therefore, any mutant Msp provided herein may contain additional modifications such as substitution, insertion, deletion, and / or addition, in addition to the mutations described herein, provided that the mutant Msp monomer has at least or at most 70, 75, 80, 85, 90, 95, 98, or 99% or higher identity with a wild-type Msp monomer, or any range thereof, but less than 100%, and retains channel-forming ability when associated with one or more other Msp monomers.

[0081] Any mutant Msp described herein may contain 2-15 identical or different Msp monomers, wherein at least one Msp monomer is a mutant Msp monomer. Optionally, the mutant Msp may contain 7-9 identical or different Msp monomers. Optionally, at least the second monomer is selected from the group consisting of: wild-type Msp monomer, second mutant Msp monomer, wild-type Msp paralogous or homologous monomer, and mutant Msp paralogous or homologous monomer, wherein the second mutant Msp monomer may be identical or different from the first mutant Msp monomer. For example, any mutant Msp described herein may contain 2-15 Msp monomers, wherein at least one Msp monomer is a mutant MspA monomer. Optionally, at least the second monomer is selected from the group consisting of: wild-type MspA monomer, second mutant MspA monomer, wild-type MspA paralogous or homologous monomer, and mutant MspA paralogous or homologous monomer, wherein the second mutant MspA monomer may be identical or different from the first mutant MspA monomer. Optionally, the second monomer is a wild-type MspA paralogous or homologous monomer.

[0082] For example, a mutant Msp may comprise one or more Msp monomers containing a mutation at position 97. In another instance, a mutant Msp may comprise one or more Msp monomers containing mutations at one or more of I68, S73, S116, P123, or V128, and one or more Msp monomers containing mutations at one or more of the following positions: T83, N86, L88, I105, D90, D91, G92, D93, or A96. In yet another instance, a mutant Msp may comprise one or more Msp monomers having mutations at positions D56, I68, S73, D118, D134, and E139, and one or more Msp monomers having mutations at positions L88 and I105.

[0083] Modifications to amino acid sequences can occur as allelic variations (e.g., due to genetic polymorphism) or due to environmental influences (e.g., due to exposure to ultraviolet radiation) or other human interventions (e.g., through mutagenesis of cloning DNA sequences), such as induction site, deletion, insertion, and substitution mutations. These modifications can lead to changes in the amino acid sequence, provide silent mutations, modify restriction sites, or provide other specific mutations. Amino acid sequence modifications generally fall into one or more of three categories: substitution, insertion, or deletion. Insertions include amino and / or terminal fusions and the insertion of one or more amino acid residues into the sequence. Insertions are generally smaller than those with amino or carboxyl terminal fusions, for example, on the order of 1 to 4 residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, deletions at any site within the protein molecule do not exceed about 2 to about 6 residues. Amino acid substitutions are usually single residues, but can occur at many different positions at once; insertions are typically on the order of about 1 to about 10 amino acid residues; and deletions will range from about 1 to about 30 residues. Deletions or insertions are preferably generated in adjacent pairs, i.e., the deletion of 2 residues or the insertion of 2 residues. Substitution, deletion, insertion, or any combination thereof can be combined to obtain the final construct. Mutations may or may not place the sequence outside the reading frame and may or may not produce complementary regions that can generate mRNA secondary structures. Substitution modification is a modification in which at least one residue has been removed and a different residue has been inserted at its position.

[0084] Modifications, including the specific amino acid substitutions disclosed herein, can be produced by known methods. For example, modifications can be produced by site-directed mutagenesis of nucleotides in DNA encoding a protein, resulting in DNA encoding that modification, which is then expressed in recombinant cell culture to produce Msp monomers or single-stranded multimers. Techniques for producing substitution mutations at predetermined sites in DNA with known sequences are well known, such as M13 primer mutagenesis and PCR mutagenesis.

[0085] The amino acids in the Msp protein described herein may be any of the following 20 naturally occurring amino acids, D-stereomers of naturally occurring amino acids, non-natural amino acids, and chemically modified amino acids. Non-natural amino acids (i.e., those not naturally occurring in proteins) are also known in the art, as described, for example, in Williams et al., Mol. Cell. Biol. 9:2574 (1989); Evans et al., J. Amer. Chem. Soc. 112:4011-4030 (1990); Pu et al., J. Amer. Chem. Soc. 56:1280-1283 (1991); Williams et al., J. Amer. Chem. Soc. 113:9276-9286 (1991); and all references cited therein. β and γ amino acids are known in the art and are also considered as non-natural amino acids herein.

[0086] As used herein, chemically modified amino acids refer to amino acids whose side chains have been chemically modified. For example, the side chain may be modified to include a signal moiety, such as a fluorophore or radiolabel. The side chain may also be modified to include a new functional group, such as a thiol, carboxylic acid, or amino group. Post-translational modified amino acids are also included in the definition of chemically modified amino acids.

[0087] Conservative amino acid substitutions are also considered. For example, conserved amino acid substitutions can occur in one or more amino acid residues of any Msp monomer provided herein. Those skilled in the art will understand that a conserved substitution is the replacement of one amino acid residue with another amino acid that is biologically and / or chemically similar. The following eight groups each contain amino acids that are conservedly substituted for each other:

[0088] 1) Alanine (A), glycine (G);

[0089] 2) Aspartic acid (D), glutamine (E);

[0090] 3) Asparagine (N), glutamine (Q);

[0091] 4) Arginine (R), Lysine (K);

[0092] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0093] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0094] 7) Serine (S), threonine (T); and

[0095] 8) Cysteine ​​(C), Methionine (M)

[0096] Non-conservative substitutions, such as glycine replacing proline, are also taken into consideration.

[0097] Those skilled in the art will readily understand how to determine the identity of two peptides or nucleic acids. For example, identity can be calculated after aligning two sequences, ensuring that the identity is at its highest level. Another way to calculate identity is through publicly available algorithms. Smith and Waterman algorithms can be used. Adv. Appl. Math The algorithm of . 2: 482 (1981); by Needleman and Wunsch, J. Mol. Biol The alignment algorithm of . 48: 443 (1970); by Pearson and Lipman, Proc. Natl. Acad. Sci. USA Similarity search methods of . 85: 2444 (1988); implementation of these algorithms by computer (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package, GeneticsComputer Group, 575 Science Dr., Madison, WI; BLAST algorithm of Tatusova and Madden FEMS Microbiol. Lett. 174: 247-250 (1999) available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / bl2.html); or by observation of the best alignment of sequences for comparison.

[0098] For nucleic acids, for example, Zuker, whose work is incorporated into this paper by citing at least the material on nucleic acid alignment, Science 244:48-52, 1989; Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989; Jaeger et al. Methods Enzymol The algorithms disclosed in 183:281-306, 1989 can obtain the same type of identity. It should be understood that any method can generally be used and, in some cases, the results of these different methods can differ, but those skilled in the art will understand that if identity is found using at least one of these methods, the sequence will be said to have the prescribed identity.

[0099] For example, as used herein, a sequence described as having a specific percentage of identity with another sequence refers to a sequence that, when calculated using one or more of the methods described above, has the described identity. For instance, if the Zuker method calculates that the first sequence and the second sequence have 80% identity, then even if any other method calculates that the first sequence and the second sequence do not have 80% identity, as defined herein, the first sequence and the second sequence still have 80% identity. As another example, if each method calculates that the first sequence and the second sequence have 80% identity (although in practice, different methods often produce different calculated percentages of identity), then, as defined herein, the first sequence and the second sequence have 80% identity.

[0100] Furthermore, any Msp or Msp monomer can also be chemically or biologically modified. For example, as those skilled in the art know, Msp or Msp monomer can be chemically modified to produce disulfide bridges.

[0101] Msp may contain nucleotide binding sites. As used herein, a nucleotide binding site is a site in the Msp where a nucleotide contacts or remains on an amino acid for a relatively long period of time due to diffusion, such as greater than 1 picosecond or 1 nanosecond. These temporary rest times can be assessed using molecular dynamics calculations.

[0102] Also provided are peptides encoded by nucleic acids described herein. Therefore, peptides comprising mutant Msp monomers or functional fragments thereof are provided. Non-limiting examples of mutant Msp monomers include, but are not limited to, peptides comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 containing any mutation described herein. Also provided are Msp monomers comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity or any percentage between these percentages with the peptides comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, wherein said peptides contain any mutation described herein. Also provided are peptides comprising mutant single-stranded Msp or functional fragments thereof. Also provided are peptides comprising mutant single-stranded Msp or fragments thereof, wherein the mutant single-stranded Msp comprises any mutant Msp monomer described herein.

[0103] Table 2 provides non-limiting examples of mutant Msp monomers containing the mutations described herein. Each exemplary mutant Msp monomer contains all the mutations listed for each monomer. For example, D90N / D91N / D93N / P97F means that all four mutations are present. This document also provides single-stranded Msp containing any of the exemplary mutant Msp monomer sequences provided in Table 2. It should be understood that the amino acids listed in parentheses are listed as substitutes for that position. For example, P97(A / V / L / IF / M / Y / W or G) means that P97 can be substituted with A, V, L, I, F, M, YW, or G.

[0104] Table 2

[0105]

[0106] Channel forming protein

[0107] Methods for determining whether a protein is a channel-forming protein are well known in the art. Whether an Msp forms a channel can be determined by determining whether the protein is inserted into the bilayer, as described, for example, in Example 2 of U.S. Patent Publication No. 20120055792, which is incorporated herein by reference in its entirety. All methods for preparing and using porins described in U.S. Patent Publication No. 20120055792 can be used to prepare and use the Msp porins described herein. If the protein is inserted into the bilayer, the porin is a channel-forming protein. Typically, channel formation is detected by observing discrete changes in conductivity. See U.S. Patent Publication No. 20120055792, and Niederweis et al. Mol. Microbiol. 33:933 (1999), both are incorporated into this paper by reference.

[0108] This document describes bilayers. Msp proteins are typically capable of being intercalated into lipid bilayers or other films known in the art. An example of intercalating a mutant MspA into a lipid bilayer is provided in U.S. Patent Publication No. 20120055792; this technique can also be applied to other Msp proteins. Additionally, various lipid bilayers and films, including inorganic materials that can be used with respect to the Msp discussed herein, are described by reference to U.S. Patent No. 6,746,594, which is incorporated herein by reference. The methods, apparatus, and techniques described by reference to U.S. Patent No. 6,267,872, which is incorporated herein by reference, can also be used with respect to the Msp discussed herein. Moreover, more than one Msp protein can be contained in a lipid bilayer. For example, 2, 3, 4, 5, 10, 20, 200, 2000, or more Msp proteins can be contained in a lipid bilayer. Optionally, from 2 to 10 Msp proteins can be used in the methods described herein. 10Any number of Msp. Such a variety of Msp can form Msp clusters. Clusters can be randomly assembled or can adopt a pattern. As used in this paper, a cluster refers to molecules that are clustered together and move as a unit, but are not covalently bonded to each other.

[0109] Optionally, the Msp is not spontaneously gating. As used herein, gating refers to a spontaneous change in the conductance through a protein channel that is typically temporary (e.g., lasting as little as 1–10 milliseconds or up to 1 second). Long-lasting gating events can often be reversed by changing polarity. In most cases, the probability of gating increases with the application of higher voltages. The degree of gating and the change in conductance through the channel is highly variable between Msps, depending on, for example, the composition of the precordial and constricted regions and the nature of the liquid medium immersing the protein. Typically, the protein becomes less conductive during gating, and the conductance can therefore stop permanently (i.e., the channel can be permanently closed), making the process irreversible. Optionally, gating refers to the spontaneous change in the conductance through the protein channel to below 75% of its open-state current.

[0110] Various conditions, such as light and liquid media, including their pH, buffer composition, detergent composition, and temperature, can affect the behavior of Msp, particularly in terms of its conductivity through the channel and the temporary or permanent movement of the analyte relative to the channel.

[0111] As used throughout, a channel refers to the central, empty portion of an Msp (Medium Spindle) defined by a vestibule and constriction, through which gases, liquids, ions, or analytes can pass. As used herein, "cis" refers to the passage of an analyte through its entry channel or the movement of an analyte across its surface along the side of the Msp channel. As used herein, "trans" refers to the passage of an analyte (or a fragment thereof) through its exit channel or the unmoved movement of an analyte across its surface along the side of the Msp channel.

[0112] Any mutant Msp described herein, such as mutant MspA, may include a precordial and constricted regions defining a channel. Further, the diameter of the mutant Msp or its paralog or homolog may be smaller than the diameter of the constricted region of the corresponding wild-type Msp or its paralog or homolog. The mutant Msp or its paralog or homolog may have a mutation within the precordial or constricted region that allows the analyte to translocate through the channel of the mutant Msp or its paralog or homolog by electrophoresis or otherwise, at a translocation velocity or mean translocation velocity lower than the translocation velocity or mean translocation velocity of the analyte through the channel of the wild-type Msp or its paralog or homolog. Similarly, any mutant Msp described herein may comprise a precordial having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and a constriction 5 having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm, wherein the precordial and the constriction together define a channel. It should be understood that one or more mutations may be generated in the precordial or constriction of any Msp described herein to increase or decrease the conductivity through the Msp channel. For example, any mutant Msp described herein may also include amino acid deletions, substitutions, or insertions within the precordial and / or constriction to alter conductivity.

[0113] As used throughout, the vestibule refers to the conical section within the Msp, whose diameter typically decreases from one end to the other along the central axis, with the narrowest part of the vestibule connecting to the constriction. The vestibule can also be called the goblet region. Together, the vestibule and the constriction define the passageway of the Msp. When referring to the diameter of the vestibule, it should be understood that because the vestibule is conical in shape, the diameter varies along the path of the central axis, with the diameter being larger at one end than at the other. The diameter ranges from approximately 2 nm to approximately 6 nm. Optionally, the diameter is about, at least about, or at most about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 nm, or any range thereof. The length of the central axis can range from about 2 nm to about 6 nm. Optionally, the length is about, at least about, or at most about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 nm, or any range thereof. When the diameter is mentioned herein, it can be determined by measuring the center-to-center distance or the atomic surface-to-surface distance.

[0114] As used throughout, the constriction refers to the narrowest part of the Msp channel in terms of diameter, which connects to the vestibule. The length of the constriction can range from about 0.3 nm to about 2 nm. Optionally, the length is about, at most about, or at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or 3 nm, or any range from which it can be inferred. The diameter of the constriction can range from about 0.3 nm to about 2 nm. Optionally, the diameter is about, at most about or at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or 3 nm, or any range thereof.

[0115] Any Msp discussed herein may be contained within a lipid bilayer. Optionally, the analyte translocates from the same side through the channel to the opposite side by electrophoresis or otherwise. Optionally, the analyte translocates from the opposite side through the channel to the same side by electrophoresis or otherwise. Optionally, the analyte is driven into the channel from the same side or the opposite side by electrophoresis or otherwise and remains in the channel or subsequently regresses to the same side or the opposite side, respectively. It should be understood that the analyte may translocate through the channel in the presence or absence of an electric field.

[0116] Single-chain Msp functions at various electrolyte concentrations, such as approximately 0.3–1 M KCl (see [link to relevant documentation]). Figure 22 To optimize channel activity, lipid association can occur before the Msp inserts into the membrane or lipid bilayer. In a non-limiting example, Figure 22 No channel activity was observed in a buffer solution containing only 0.3 M KCl at pH 8.0. However, rupturing the membrane and subsequently recoating it resulted in enhanced channel activity of scMspA PN1 in an electrolyte containing 0.3 M KCl at pH 8.0. Therefore, in any of the methods described herein, Msp can be contacted with one or more lipids or pre-incubated to optimize channel activity.

[0117] carriers and cells

[0118] Vectors containing nucleic acids encoding the polypeptides described herein are also provided. The vectors may also contain promoter sequences, such as constitutive or inducible promoters. Examples of constitutive promoters include, but are not limited to, p... smyc Promoters and Phsp60. Examples of inducible promoters include, but are not limited to, acetamide-inducible promoters and tetracycline-inducible promoters.

[0119] Cultured cells, or their progeny, transfected with any of the vectors described herein, are also provided, wherein the cells are capable of expressing Msp (as a single-chain Msp, an Msp containing Msp monomers, or an Msp monomer). Mycobacterium smegmatis strains containing any of the vectors described herein are also provided. Mycobacterium smegmatis strains lacking endogenous porins are also considered and may also contain any of the vectors described herein. "Lacks" means that endogenous porins are undetectable in an immunoblot when used with appropriate Msp-specific antiserum, or contain less than 1% endogenous porins.

[0120] Any Msp monomer or single-stranded Msp disclosed herein can be generated by transforming a mutant bacterial strain containing the deletions of wild-type MspA, wild-type MspB, wild-type MspC, or wild-type MspD into a vector containing an inducible promoter operatively linked to a nucleic acid sequence encoding the Msp monomer or single-stranded Msp porin; and purifying the Msp monomer or single-stranded Msp porin (see, for example, U.S. Patent No. 6,746,594, incorporated herein by reference). Optionally, the mutant bacterial strain contains recA Gene deletion. Optionally, the vector contains any nucleic acid encoding the Msp monomer or single-stranded Msp described herein. The bacterial strain may also contain Mycobacterium smegmatis strains ML16, ML714, or ML712.

[0121] System and usage

[0122] A system is also provided comprising a mutant Msp having a defined channel and a constriction region as described herein, wherein the channel is located between a first liquid medium and a second liquid medium, wherein at least one liquid medium contains an analyte, and wherein the system is operable to detect the properties of the analyte. The system is operable to detect the properties of any analyte by subjecting the Msp to an electric field, causing the analyte to interact with the Msp. The system is operable to detect the properties of an analyte by subjecting the Msp to an electric field, causing the analyte to translocate through the channel of the Msp by electrophoresis. A system is also provided comprising an Msp having a defined channel and a constriction region, wherein the channel is located in a lipid bilayer between a first liquid medium and a second liquid medium, and a unique liquid connection point between the first and second liquid media occurs in the channel. Furthermore, any system described herein may comprise any mutant Msp described herein.

[0123] The first and second liquid media may be the same or different, and either or both may contain one or more salts, detergents, or buffers. In fact, any liquid media described herein may contain one or more of salts, detergents, or buffers. Optionally, at least one liquid media is conductive. Optionally, at least one liquid media is non-conductive. Any liquid media described herein may contain a viscosity-changing substance or a velocity-changing substance. The liquid media may contain any analyte described herein.

[0124] The properties analyzed can be electrochemical, chemical, or physical. The Msp can be contained in a lipid bilayer of the system or any other embodiment described herein. The system can contain multiple Msps. The system can contain any Msp described herein, such as a single-stranded mutant MspA or a mutant Msp containing at least 2-15 monomers, wherein at least one monomer is a mutant MspA monomer. The mutant Msp contained in the system may contain an anterior chamber with a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and a constricted region with a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm, wherein the anterior chamber and the constricted region together define a channel.

[0125] Any MSP described herein, including those included in a system, may also include a molecular engine. A molecular engine in a system enables analytes to translocate into or through channels at a lower translocation rate or average translocation rate than the translocation rate or average translocation rate of analytes into or through channels without a molecular engine. A molecular engine can be, for example, an enzyme such as a polymerase, exonuclease, or helicase (e.g., DnaB) or a phage nucleic acid packaging engine (see, for example, Serwer). Viruses 3(7): 1249-80 (2011)). Any system described herein may also include a patch-clamp amplifier or data acquisition device. The system may also include one or more temperature control devices in communication with a first liquid medium, a second liquid medium, or both. Any system described herein is operable to translocate analytes through Msp channels by electrophoresis or otherwise.

[0126] The mutant MspA may have a mutation in the precordial or constricted region, allowing the analyte to translocate across the channel, for example by electrophoresis, at an average translocation rate of less than 0.5 nm / µs or less than 0.05 nm / µs. The analyte can be selected from the group consisting of nucleotides, nucleic acids, amino acids, peptides, proteins, polymers, drugs, ions, contaminants, nanoscale objects, or combinations or clusters thereof. Optionally, the analyte may be further defined as a nucleic acid. Nucleic acids may translocate across the channel by electrophoresis or otherwise, at an average translocation rate of less than 1 nucleotide / µs or less than 0.1 nucleotide / µs. Nucleic acids may be further defined as ssDNA, dsDNA, RNA, or combinations thereof.

[0127] As used herein, electrophoretic translocation of analytes refers to applying an electric field to an Msp porin in contact with one or more solutions (e.g., immersion in a solution), causing a current to flow through the Msp channel. The electric field causes the analyte to move and interact with the channel. As used herein, “interact” means the analyte moves into and, optionally, across the channel, where “across the Msp channel” (or “translocation”) means entering one side of the channel and moving toward and out of the other side. In particular, it is considered that in any embodiment discussed herein, any analyte discussed herein may translocate across the Msp channel by electrophoresis or otherwise. In this regard, it is particularly considered that, unless specifically indicated, any embodiment including translocation herein may refer to electrophoretic translocation or non-electrophoretic translocation. Optionally, methods without electrophoretic translocation are considered.

[0128] As used throughout, liquid media include aqueous media, organic aqueous media, and organic liquid-only media. Organic media include, for example, methanol, ethanol, dimethyl sulfoxide, and mixtures thereof. Liquids that can be used in the methods described herein are well known in the art. Such media, including descriptions and examples of conductive liquid media, are provided, for example, in U.S. Patent No. 7,189,503, which is incorporated herein by reference in its entirety. Salts, detergents, or buffers may be added to such media. Such agents can be used to change the pH or ionic strength of the liquid media. Viscosity-modifying substances, such as glycerol, or various polymers (e.g., polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, cellulose polymers), and mixtures thereof, may be included in the liquid media. Methods for measuring viscosity are well known in the art.

[0129] Any reagent that can be added to the liquid medium can also alter the rate of the analyte being studied. Thus, rate modifiers can be salts, detergents, buffers, viscosity modifiers, or any other reagent added to the liquid medium to increase or decrease the rate of the analyte. Typically, the analytes used herein are soluble or partially soluble in at least one liquid medium in contact with the Msp described herein.

[0130] As used herein, nucleic acids refer to deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, encompass known analogs of natural nucleic acids that hybridize with nucleic acids in a manner similar to naturally occurring nucleotides, such as peptide nucleic acids (PNAs) and phosphate-thioester DNA. Unless otherwise indicated, a particular nucleic acid sequence includes its complementary sequence. Nucleotides include, but are not limited to, ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP, 5-methyl-CTP, 5-methyl-dCTP, ITP, diTP, 2-amino-adenosine-TP, 2-amino-deoxyadenosine-TP, 2-thiothymidine triphosphate, pyrrole-pyrimidine triphosphate, and 2-thiocytidine, as well as all of the above α-thiotriphosphates, and 2'-O-methyl-ribonucleoside triphosphates of all the above bases. The modified bases include, but are not limited to, 5-Br-UTP, 5-BrdUTP, 5-F-UTP, 5-F-dUTP, 5-propynyl-dCTP, and 5-propynyl-dUTP.

[0131] As used herein, a drug is any substance that can alter the biological processes of a subject. Drugs may be designed or used for the diagnosis, treatment, or prevention of a subject's disease, condition, syndrome, or other health problem. A drug may be of a therapeutic nature, meaning it is used solely to alter biological processes and not for the diagnosis, treatment, or prevention of a subject's disease, condition, syndrome, or other health problem. Biologics, referring to substances produced through biological mechanisms involving recombinant DNA technology, are also covered by the term drug. Drugs include, for example, antibacterial agents, anti-inflammatory drugs, anticoagulants, antiviral agents, antihypertensive drugs, antidepressants, antimicrobial agents, analgesics, anesthetics, beta-blockers, bisphosphonates, chemotherapeutic agents, contrast agents, fertility drugs, hormones, sedatives, statins, steroids, and vasodilators. Merck Index: an Encyclopedia of Chemicals, Drugs, and Biologicals Non-limiting examples of drugs can also be found in the 15th edition of New Jersey: Merck, 2013. Antibacterial drugs used to treat tuberculosis include, for example, isoniazid, rifampin, pyrazinamide, and ethambutol. Methods using drugs as analytes can also include drug screening. For example, Msp can be used to study drug uptake into cells or organisms by observing ion current blocking. Specific Msp pore protein constrictions and / or precordial chambers with different sizes, electrostatic properties, and chemical properties can be constructed to precisely mimic the pathways required for drugs to proceed or leave cells or organisms. These methods can greatly accelerate drug screening and drug design (see, for example, Pagel et al.). J. Bacteriology 189:8593 (2007)).

[0132] As used herein, a pollutant is a substance that pollutes the air, water, or soil. Non-limiting examples of pollutants include fertilizers, pesticides, insecticides, detergents, petroleum hydrocarbons, smoke, and substances containing heavy metals, such as those containing zinc, copper, or mercury (e.g., methylmercury).

[0133] This document may use any analyte, including, for example, nucleotides, nucleic acids, amino acids, peptides, proteins, polymers, drugs, ions, contaminants, nanoscale objects, or any other molecule containing one or a combination of these analytes. Analytes may be subgroups (e.g., 2-10 nucleotides or amino acids), as a cluster as a whole is considered an analyte. Typically, the size of the analyte will not be so large that it cannot enter the Msp channel. In other words, a typical analyte will be smaller than the Msp channel opening. However, analytes larger than the channel opening can be used, and it can be determined that the analyte is too large to enter the channel. Optionally, the molecular weight of the analyte is less than 1,000,000 Da. Optionally, the molecular weight of the analyte is about, at most about, or at least about 1,000,000, 950,000, 900,000, 850,000, 800,000, 750,000, 700,000, 650,000, 600,000, 550,000, 500,000, 450,000, 400,000, 350,000, or 30 0,000, 250,000, 200,000, 150,000, 100,000, 75,000, 50,000, 25,000, 20,000, 15,000, 10,000, 7,500, 5,000, 2,500, 2,000, 1,500, 1,000, or 500 Da or less, or any range thereof.

[0134] Analytes can also be nanoscale objects, i.e., objects smaller than 100 nm in either of their two dimensions. As used herein, analytes may also contain magnetic beads. Magnetic beads can be further defined as streptavidin-coated magnetic beads. Analytes may also contain optical beads. Any analyte described herein may be ionic or neutral. Analytes may contain biotin.

[0135] The beads that can be used include magnetic beads and optical beads. For example, streptavidin-coated magnetic beads can be used to apply a counterforce to the electrostatic force pulling DNA through the Msp channel. In this latter technique, the magnetic beads are attached to the biotinylated DNA, and a strong magnetic field gradient is used to apply a force equivalent to the electrostatic driving force (-10 pN). See Gosse and Croquette. Biophys. J.82:3314 (2002). In this way, the blocking current readout will not be affected, but the forces on the DNA can be controlled independently. Then, dozens or hundreds of complete independent readouts for each DNA molecule can be correlated and aggregated to reconstruct an accurate DNA sequence.

[0136] Optical beads manipulated with “optical tweezers” are also known in the art, and such methods are applicable to the Msp described herein. Optical tweezers are common tools for applying forces to nanoscale objects. An analyte is attached to one end of the bead, while the other end can be inserted into a channel of a porin. The position and force of the bead are controlled and measured using optical tweezers. Such methods control the flow of the analyte into the channel and allow for greater control over readings of the analyte, such as readings of polymer units. For a description of such methods in the case of artificial nanopores, see, for example, Trepagnier et al., Nano Lett. 7:2824 (2007). The use of optical tweezers is also discussed by reference to U.S. Patent No. 5,795,782, which is incorporated herein by reference.

[0137] Fluorescence resonance energy transfer (FRET), a well-known technique, can be used in the analytical methods described herein. For example, a fluorescent FRET acceptor or FRET donor molecule can be incorporated into the Msp. The analyte is then labeled with a matched FRET donor or FRET acceptor. Energy transfer is likely to occur when the matched FRET donor is within the Forster distance of the FRET acceptor. The resulting signal can be used for analytical purposes, replacing or supplementing methods using ion currents as described herein. Therefore, detection, identification, or sequencing methods can include FRET technology. Other optical methods that can be employed include introducing optically active molecules into the interior of the Msp (e.g., the precordial or constricted region). External light is applied to affect the interior of the protein. Such methods can be used to influence the translocation rate of the analyte or to allow the analyte to enter or leave the channel, providing a controlled channel for the analyte. Alternatively, a light pulse focused on the pore can be used to heat the pore to influence how it interacts with the analyte. Such control can be very fast because the heat from the small focal point will dissipate rapidly. Therefore, methods for controlling the translocation rate of the analyte can employ such optically active molecules or light pulses. Manipulation of the transposition velocity can also be achieved by attaching an object to one end of the analyte, and then having the other end of the analyte interact with the Msp. The object can be a bead (e.g., polystyrene beads), a cell, a macromolecule (e.g., streptavidin, neutral avidin, DNA, etc.), or a nanoscale object. The object can then be subjected to fluid flow or passive viscous resistance.

[0138] Molecular engines are well known in the art and refer to molecules (e.g., enzymes) that physically interact with analytes, such as polymers (e.g., 15 polynucleotides), and are capable of physically moving the analyte relative to a fixed location, such as the precordial, constricted region, or channel of an Msp. While not wishing to be theoretically constrained, molecular engines utilize chemical energy to generate mechanical forces. Molecular engines can interact sequentially with each unit (or “mer”) of a polymer. Non-limiting examples of molecular engines include DNA polymerases, RNA polymerases, helicases, ribosomes, and exonucleases. Non-enzymatic engines are also known, such as viral engines that encapsulate DNA. See Smith et al., Nature 413:748 (2001). Various molecular engines and the desired properties of such engines are described in U.S. Patent No. 7,238,485, which is incorporated herein by reference in its entirety.

[0139] The molecular engine can be positioned on the same side or opposite side of the Msp porin and can optionally be fixed, as described in the '485 patent. The method of incorporating the molecular engine into the Msp can be performed using the method described in the '485 patent. The systems and apparatus described in the '485 patent can also be employed with respect to the Msp described herein. In fact, as described herein, any embodiment discussed in the '485 patent can be employed with the Msp. Molecular engines are also discussed, for example, in Cockroft et al., J. Amer. Chem. Soc. 130:818 (2008); Benner et al., Nature Nanotech. 2:718 (2007); and Gyarfas et al., ACS Nano 3:1457 (2009).

[0140] Molecular engines are typically used to modulate the rate of interaction or translocation velocity of an analyte with an Msp. Any Msp described herein may contain a molecular engine. Optionally, the molecular engine is used to reduce the rate of analyte entry into the Msp porin channel or to reduce the translocation velocity of the analyte across the Msp channel. Optionally, the translocation velocity or average translocation velocity is less than 0.5 nm / µs. Optionally, the translocation velocity or average translocation velocity is less than 0.05 nm / µs. Optionally, the translocation velocity or average translocation velocity is less than 1 nucleotide / µs. Optionally, the translocation velocity or average translocation velocity is less than 0.1 nucleotide / µs.

[0141] Optionally, the migration rate of the analyte ranges from greater than 0 Hz to 2000 Hz. Here, rate refers to the number of subunits (or “mers”) that a regular polymer advances in 1 second (Hz). Optionally, the range is between about 50-1500 Hz, 100-1500 Hz, or 350-1500 Hz. Optionally, the migration rate is about, at most about, or at least about 25, 75, 100, 150, 200, 250, 300, 15, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, or 2000 Hz, or any range thereof. For at least a portion of the characterization period, the rate is controlled by using a molecular engine that moves the analyte at a substantially constant rate. Additionally, the range of migration rates may depend on the molecular engine. For example, the range is 350-1500 Hz for RNA polymerase; 75-1500 Hz for DNA polymerase; and 50-1500 Hz for ribosomes, helicases, and exonucleases.

[0142] Recording and detection techniques can be employed in the methods described herein. Additionally, recording methods and instruments that can be used with respect to Msp, as well as methods for optimizing conductivity readings, are described in their entirety by reference to U.S. Patent Nos. 5,795,782 and 7,189,503. U.S. Patent No. 6,746,594, also incorporated herein by reference in its entirety, describes supports for thin films containing nanopores and methods for using such supports with respect to the Msp described herein.

[0143] Methods for preparing single-chain Msp

[0144] Msp pores, such as MspA, are currently the best available channel proteins for nucleic acid nanopore sequencing. However, the composition of its eight subunits makes it impossible to introduce asymmetric changes into the pores that optimize the properties of Msp for nucleic acid sequencing. To overcome this difficulty, this paper presents methods for preparing single-stranded Msp. These methods can be used to generate complete or partial single-stranded Msp. Typically, the method involves transforming mutant bacterial strains. Mutant strains contain deletions of wild-type MspA, wild-type MspB, wild-type MspC, wild-type MspD, and optionally... recAGene deletion. The mutant strain was transformed with a vector containing nucleic acid encoding a single-stranded Msp porin. The single-stranded Msp porin was then purified from the bacteria. Optionally, the single-stranded Msp porin comprised a single-stranded MspA porin. Optionally, the vector comprised any nucleic acid described herein.

[0145] As described in the examples, to combine the excellent sequencing capabilities of MspA with the enhanced ability to adapt the pre-hall and constriction properties to DNA sequencing, a single-stranded MspA porin octamer was constructed, which allows for optimal properties of the pre-hall and constriction regions for DNA sequencing. In the MspA porin protein, the MspA strands are clustered together and linked by short peptide linkers. A (GGGGS)3 (SEQ ID NO: 3) peptide, for example, is used to link the carboxyl terminus of a previous MspA monomer (or multimer) to the amino terminus of the next MspA monomer (or multimer) lacking a signal peptide. To generate vectors containing MspA porin sequences, each MspA monomer sequence is side-mounted with a unique restriction site, which allows for the mutation of any individual monomer. To generate MspA porin sequences, each MspA sequence can be stepwise assembled using the unique restriction sites to form dimers, tetramers, and octamers of single-stranded MspA. To avoid recombination problems in the generation of single-stranded MspA multimers, seven synthetic MspA genes are used with different codons, i.e., these genes encode identical amino acid sequences, but the DNA sequence has been altered from the native MspA nucleotide sequence (SEQ ID NO: 10). To generate the MspA porin sequence, the nucleotide sequence encoding the first Msp monomer may optionally contain a nucleic acid sequence encoding a leader sequence (e.g., amino acids 1-27 of SEQ ID NO: 6). Each of the seven Msp monomers following the first Msp monomer sequence may contain SEQ ID NO: 1 or SEQ ID NO: 1 having one or more mutations described herein. The vector containing the MspA porin sequence is transformed into a tetraporin mutant bacterial strain, as described in the examples. Optionally, the single-stranded Msp can be purified and subjected to a refolding process. For example, anion exchange chromatography in the presence of 8M urea can be used to obtain a purified component of the single-stranded Msp, which is then dialyzed with a buffer to remove urea. Following dialysis, a refolding buffer containing a refolding agent (e.g., L-arginine) and a detergent is added to the sample, and the sample is purified to obtain refolded single-chain Msp. Refolding agents are known to those skilled in the art. These include, but are not limited to, arginine hydrochloride, arginineamide, glycineamide, proline, glycerol, and cyclodextrin (see, for example, Yamaguchi et al.). Biomolecules4: 235-251 (2014); and the expression level and oligomeric state of MspA pore protein can be examined by Western blotting or other immunohistochemical techniques known to those skilled in the art. The channel activity of MspA pore protein can be determined by lipid bilayer assays, as described in the examples and as known to those skilled in the art.

[0146] Single-stranded M18-MspA pore insertion into lipid bilayers occurs far more frequently than similar amounts of octamer M1-MspA. Octamer MspA insertion is a lengthy process. Therefore, single-stranded Msp, such as those described herein, is advantageous for the detection and identification of analytes using Msp, for example, in the setup of systems and methods for nucleic acid sequencing.

[0147] Methods to increase Msp insertion in lipid bilayers

[0148] This document provides a method for increasing the number of Msp insertions in a lipid bilayer, comprising contacting any Msp described herein with lipids to form a lipid-associated Msp and inserting the lipid-associated Msp of this step into the lipid bilayer. Optionally, the contacting step includes inserting the Msp into the lipid bilayer and disrupting the lipid bilayer to form the lipid-associated Msp. For example, an Msp may be inserted into a lipid bilayer, followed by disruption of the lipid bilayer. The disrupted lipid bilayer contains the Msp. Thus, the Msp is associated with the lipid. The lipid-associated Msp can then be contacted with other lipids to form another lipid bilayer containing the lipid-associated Msp. As used herein, a lipid bilayer is a thin film containing lipid molecules, such as phospholipids, that can be used for insertion of any Msp provided herein. Therefore, in the method provided herein, an Msp may be contacted with phospholipids that are part of or not part of the lipid bilayer to form the lipid-associated Msp.

[0149] As described above, those skilled in the art can determine whether Msp is inserted into a bilayer using techniques such as those described in Example 2 of U.S. Patent No. 20120055792, which is incorporated herein by reference in its entirety. All methods of preparing and using porins described in U.S. Patent No. 20120055792 can be used to prepare and use the Msp porins described herein. If the protein is inserted into the bilayer, the porin is a channel-forming protein. Typically, channel formation is detected by observing discrete changes in conductivity. See U.S. Patent Publication No. 20120055792, and Niederweis et al. Mol. Microbiol. 33:933 (1999), both of which are incorporated herein by reference. The increase in Msp insertion may be approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or more.

[0150] Detection methods

[0151] A method for detecting the presence of an analyte is also provided, comprising: (a) applying an electric field sufficient to cause the analyte to translocate from a first conductive medium to a liquid-connected second conductive medium via any mutant Msp described herein; and (b) measuring an ion current, wherein a decrease in the ion current indicates the presence of the analyte in the first medium. Optionally, the first and second liquid conductive media are identical. Optionally, the first and second liquid conductive media are different. The mutant Msp porin can be any mutant Msp porin discussed herein. For example, an Msp porin can be a mutant single-stranded Msp, a mutant Msp comprising 2-15 monomers, or a combination thereof. As mentioned above, various Msps can also be used in the method described herein.

[0152] In the methods disclosed herein, the Msp may also include a molecular engine. The molecular engine enables the analyte to move into or through the channel at a translocation velocity or average translocation velocity lower than the translocation velocity or average translocation velocity of the analyte by electrophoresis into or through the channel without the molecular engine. Therefore, in any embodiment including the application of an electric field herein, the electric field may be sufficient to cause the analyte to translocate through the channel by electrophoresis. Any liquid medium discussed herein, such as a conductive liquid medium, may contain the analyte. In methods including the measurement of ionic current, the analyte interacts with the Msp porin channel to provide a current pattern, wherein the appearance of blockage in the current pattern indicates the presence of the analyte.

[0153] The methods disclosed herein may also include identifying analytes. For example, such methods may include comparing a current pattern obtained with respect to an unknown analyte with a known current pattern obtained using a known analyte under the same conditions. In another instance, and not limitingly, identifying an analyte may include (a) measuring an ion current to provide a current pattern, wherein a decrease in the current defines a blockage in the current pattern, and (b) comparing one or more blockages in the current pattern with (i) one or more blockages in the current pattern, or (ii) one or more blockages in a known current pattern obtained using a known analyte.

[0154] The analyte can be any analyte described herein. For example, the analyte can be a nucleotide, nucleic acid, amino acid, peptide, protein, polymer, drug, ion, contaminant, or nanoscale object. Optionally, in the methods provided herein, at least one of the first or second conductive liquid media contains a variety of different analytes.

[0155] In methods where the analyte is a polymer, such as a protein, peptide, or nucleic acid, the method may further include identifying one or more units of the polymer. For example, identifying one or more units of a polymer may include measuring an ionic current to provide a current pattern containing blockages for each polymer unit, and comparing one or more blockages in that current pattern with (i) one or more other blockages in that current pattern or (ii) one or more blockages in a current pattern obtained using a polymer having known units. These methods may include identifying sequential units of the polymer, such as, but not limited to, sequences or consecutive nucleotides in nucleic acids. In another example, the methods described herein may be used to identify sequences or consecutive amino acids in peptides.

[0156] The method provided herein may include distinguishing at least a first unit within a polymer from at least a second unit within the polymer. Distinguishing may include measuring the ionic currents generated when the first and second units individually translocate through a channel to generate first and second current modes, respectively, wherein the first and second current modes are distinct from each other.

[0157] The methods described herein may also include sequencing polymers. Sequencing may include measuring the ion current or optical signal as each unit of the polymer translocates individually through the channel to provide a current pattern associated with each unit, and comparing each current pattern with the current pattern of a known unit obtained under the same conditions, in order to sequence the polymer.

[0158] A method for sequencing nucleic acids or peptides using any mutated Msp provided herein is also provided. The method includes generating a lipid bilayer comprising first and second sides, adding purified Msp to the first side of the lipid bilayer, applying a positive voltage to the second side of the lipid bilayer to translocate the experimental nucleic acid or peptide sequence through the Msp pore protein, comparing the experimental blocking current with a blocking current standard, and determining the experimental sequence.

[0159] Any detection methods provided herein may also include determining the concentration, size, molecular weight, shape, or orientation of an analyte, or any combination thereof.

[0160] As used herein, a polymer is a molecule comprising two or more linear units (also called “mers”), where each unit may be identical or different. Non-limiting examples of polymers include nucleic acids, peptides, and proteins, as well as a variety of hydrocarbon polymers (e.g., polyethylene, polystyrene) and functionalized hydrocarbon polymers, wherein the polymer backbone comprises carbon chains (e.g., polyvinyl chloride, polymethacrylate). Polymers include copolymers, block copolymers, and branched polymers such as star polymers and dendritic polymers.

[0161] This document describes a method for sequencing polymers using an Msp. Alternatively, the sequencing method can be performed in a manner similar to those described in U.S. Patent No. 7,189,503, which is incorporated herein by reference in its entirety. See also U.S. Patent No. 6,015,714, which is incorporated herein by reference in its entirety. In such sequencing methods, more than one reading can be performed to improve accuracy. Methods for analyzing polymer characteristics (e.g., size, length, concentration, identity) and identifying discrete units (or “mers”) of polymers are also discussed in the '503 patent and can be employed for this Msp. In fact, an Msp can be employed for any of the methods discussed in the '503 patent.

[0162] Currently, several types of observable signals can be used as readout mechanisms in nanopore sequencing and analyte detection. Exemplary readout methods rely on ion blocking currents or common-pass currents, which uniquely pass through nucleotides or other analytes occupying the narrowest contracted portion of the pore for identity determination. This method is known as blocking current nanopore sequencing, or BCNS. Blocking current detection and characterization of nucleic acids have been demonstrated in both protein pore α-hemolysin (aHL) and solid nanopores.

[0163] Blocking current detection and characterization have been shown to provide a wealth of information about DNA structures that pass through or remain in nanopores under various conditions. Generally, blocking is demonstrated by changes in ion current, clearly identifiable by noise fluctuations and typically correlated with the presence of analyte molecules at the pore's central opening. The strength of the blocking will depend on the type of analyte present. More specifically, blocking is defined as an interval in which the ion current decreases to approximately 5-100% of the unblocked current level, remains below a threshold for at least 1.0 µs, and spontaneously recovers to the unblocked level. For example, the ion current can decrease to approximately, at least approximately, or at most approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or any range thereof below a threshold that can be inferred from it. Blocking is rejected if the average current of the unblocked signal deviates from the typical unblocked level by more than twice the square root noise (rms noise) of the unblocked signal, either before or after it. Deep blockage is defined as an interval where the ion current decreases to less than 50% of the unblocked level. Intervals where the current is maintained between 80% and 50% of the unblocked level are defined as partial blockage.

[0164] Disclosed are materials, compositions, and components that can be used in conjunction with the disclosed methods and compositions, and can be used in their preparation or as products. These and other materials are disclosed herein, and it should be understood that when combinations, subgroups, interactions, groupings, etc., of these materials are disclosed, although a specific reference to every different individual and collective combination and arrangement may not be explicitly disclosed, each is specifically considered and described herein. For example, if a method is disclosed and discussed, and many modifications that can be made to the many compositions included in the method are discussed, then every combination and arrangement of the method and possible modifications are specifically considered unless explicitly stated otherwise. Similarly, any subgroups or combinations of these are also specifically considered and disclosed. This concept applies to all aspects of this disclosure, including but not limited to steps in the method. Therefore, if there are multiple additional steps that can be performed, it should be understood that each of these additional steps can be performed using any particular method step or combination of method steps of the disclosed method, and each such combination or subgroup of combinations is specifically considered and should be considered disclosed. It is also considered that any implementation discussed in this specification can be realized for any method, compound, protein, porin, peptide, polypeptide, polymer, monomer, nucleic acid, vector, strain, cultured cell, system or combination thereof described herein, and vice versa.

[0165] The publications cited in this article and the materials cited therein are hereby explicitly incorporated in their entirety by reference.

[0166] Many embodiments have been described. However, it should be understood that various modifications can be made. Therefore, other embodiments are within the scope of the following claims.

[0167] Example

[0168] A single-stranded MspA was constructed, consisting of eight covalently linked monomers. As shown in this paper, the expression plasmid encoding single-stranded M1-MspA is unstable in wild-type Mycobacterium smegma, but can be used in the absence of homologous recombination. recA The gene can proliferate in Mycobacterium smegmatis. recA The gene was deleted in *Mycobacterium smegmatis* ML712, a tetraporin mutant lacking four known Msp porins. This strain enabled the expression of single-stranded M1-MspA. The channel produced by single-stranded M1-MspA has similar conductivity to the octamer M1-MspA channel, but with significantly enhanced tolerance to voltage gating. This unexpected advantage of single-stranded MspA is crucial for nanopore sequencing of nucleic acids, such as DNA.

[0169] Construction of Msp four deletion mutant of Mycobacterium smegmatis

[0170] To isolate the mutant MspA pore protein, the tripore protein-deficient mutant Mycobacterium smegmatis ML16 strain (Δ) was used. mspA::FRT Δ mspC :: FRT Δ mspD :: FRT (See Stephan et al., Mol. Microbiol. 58: 714-730 (2005)). However, low levels of MspB were still detected in this strain in immunoblotting using MspA-specific rabbit antiserum. The presence of MspB can contribute to the heterogeneity observed in single-channel experiments and complicate data analysis. To overcome this problem and improve MspA preparation, a method lacking all four MspA molecules was constructed. msp Genetically modified Mycobacterium smegmatis strain.

[0171] Because of 4 Mycobacterium smegma msp Complete gene deletion is lethal, so the first step was to integrate the expression cassette of the N-terminal channel-forming domain of CpnT (1) into the Mycobacterium smegmatis porin triple mutant ML16. For this purpose, plasmid pML2622 was constructed, carrying the N-terminal domain of CpnT labeled with His6 and HA under the control of a nitrile-inducible promoter. Figure 2 The N-terminal domain of CpnT forms a channel in a planar bilayer experiment and supplements the porin-mutant Mycobacterium smegmatis ML16 strain in a glycerol uptake experiment. The rescue plasmid pML2622 was integrated into the mycophage L5-site on the chromosome of the porin-trimutant Mycobacterium smegmatis ML16. Integration was confirmed by PCR using a primer set complementary to the L5 genomic region. This strain was named Mycobacterium smegmatis ML709. After pML2622 integration, it was analyzed using methods such as those described by Stephan et al. (…). Gene The Flp recombinase described in 343: 181-190 (2004) removes the plasmid backbone from the chromosome to eliminate the genes encoding hygromycin phosphotransferase and L5 integrase. This strain was named Mycobacterium smegmatis ML709-234.

[0172] To ensure the remaining smegma mycobacterium ML711 mspB Gene deletion, construction mspB The deletion vector pML1611 contains two reporter genes that serve as markers of integration and isoposition. gfp and xylE ( Figure 2 ). mspB Deletion vector pML1611 carries mspB The 863bp and 946bp upstream and downstream regions are used to transfer Mycobacterium smegmatis ML709-234 via allelic exchange. mspBGene deletion. This Mspporin tetra mutant was named Mycobacterium smegmatis ML711. Lateral access was performed by excising the chromosome using the plasmid pCreSacB containing Cre recombinase. loxP site gfp and hyg Genes. Four genes were confirmed by PCR using specific primers with chromosomal DNA and by Southern blotting using specific probes with chromosomal DNA. msp All genes are missing ( Figure 3 This unlabeled Msp porin tetra mutant was named Mycobacterium smegmatis ML712 (related genotype: ΔmspA::FRT , ΔmspB::loxP , ΔmspC::FRT , ΔmspD::FRT , attB L5:: FRT -pNIT-cpnT d1 -FRT). Growth of strain ML712 on Middlebrook 7H10 agar plates was impaired compared to wild-type and ML16 strains. Next, as described by Heinz et al. (Anal. Biochem. 285: 113-120 (2000)), the performance of strain ML712 was evaluated by extracting Mycobacterium smegmatis cells using the detergent octylpolyoxyethylene. msp Gene expression. Msp tetraporin-deficient mutant Mycobacterium smegmatis ML712, grown in Middlebrook 7H9 medium, unlike the triporin mutant ML16, did not produce any Msp protein. Figure 4 This demonstrates that complete [implantation / achievement] is possible in Mycobacterium smegmatis ML712. msp Gene deletion. When wild-type genes were expressed in ML712 using plasmids pMN016 and pML904, respectively. mspA or M1mspA At that time, the expression level of MspA was similar to that of wild-type Mycobacterium smegmatis.

[0173] Construction of single-chain M1-MspA

[0174] Previously, M12-MspA subunit dimers were constructed (Pavlenok et al.) PLoS One 7(6): e38726). As a next step for single-chain MspA, make 4 mspA-M1 Gene fusion was performed to encode the M14-MspA subunit tetramer. The resulting plasmid pML2647 was transformed into a tetraporin-deleted strain of Mycobacterium smegmatis ML712 to generate and purify the protein. However, the tetramer... mspA The plasmid is unstable. To avoid recombination, recAThe gene was deleted in the tetraporin mutant ML712 and the strain Mycobacterium smegmatis ML714 was produced.

[0175] Then, the gene encoding a single-stranded MspA (M18-MspA) containing eight M1-MspA subunits was cloned into *E. coli*. Each subunit had D90N, D91N, and D93N mutations. This was achieved by fusing two genes encoding the tetramer M1 MspA (M14-MspA) together using pML2647 as a template. The individual MspA subunits were separated by a (GGGGS)3 linker. In the resulting plasmid pML3213, the two tetramers... M1-mspA Constructs are laterally attached to unique restriction sites (tetrad A: PacI, MluI; tetrad B: EcoRV, HindIII) Figure 5 Except for the first and last genes in the tetramer, the genes in the tetramer are flanked by the same restriction sites.

[0176] income m1 8 -mspA Genes in constitutive p smyc Under the control of the promoter ( Figure 5 Plasmid pML3213 was transformed into Mycobacterium smegma ML714. recA (A mutant lacking the tetraporin) was used to generate and purify the protein. Western blot experiments showed that the expression level of single-stranded M18-MspA in Mycobacterium smegmatis ML714 was lower than that of M12-MspA and reduced to approximately 7% of the wild-type MspA level. Figure 6 ).

[0177] Stability of single-chain M1-MspA

[0178] MspA pores exhibit extremely high tolerance to thermal and chemical degradation (Heinz et al.). J. Biol. Chem. 278: 8678-8685 (2003)). To test the thermostability of single-chain MspA, the M18-MspA protein was subjected to increasingly higher temperatures for 15 minutes in the presence of 2% SDS. Figure 7 Even after heating the protein to 100°C, significant amounts of M18-MspA remained stable. Figure 7 This result shows that M18-MspA is at least as stable as wild-type MspA protein in the face of heat denaturation.

[0179] Channel properties of single-stranded M1-MspA

[0180] To examine whether M18-MspA forms a functional channel, an in vitro lipid bilayer experiment was performed. No insertion was recorded when only n-octyl-POE buffer was added to the lipid bilayer. Adding approximately 70 ng of M18-MspA protein resulted in a gradual increase in the current passing through the lipid bilayer, indicating that the surface M18-MspA channel had inserted into the membrane. Figure 8A Analysis of the current record of M18-MspA showed a main peak at 1.1 nS. Figure 8B The conductivity of this channel is similar to that of those pores produced by M1-MspA dimer (1.3 nS) and M1 MspA monomer (1.4 nS) (see Pavlenok et al.).

[0181] Voltage gating

[0182] Voltage gating is defined as the spontaneous closure of a channel at a certain voltage threshold and is an inherent property of bacterial β-barrel channel proteins (Bainbridge et al.). FEBS Lett 431(3): 305-308 (1998)). Because voltages up to +180 mV are used to translocate ssDNA through MspA pores, tolerance to voltage gating is extremely important for nanopore sequencing experiments (Manrao et al.). Nat. Biotechnol. 30(4): 349-353 (2012); Derrington et al. Proc. Natl. Acad. Sci. USA 107(37): 16060-16065 (2010); Butler et al. Proc. Natl. Acad. Sci. USA 105(52): 20647-20652 (2008)). Therefore, the voltage gating of M18-MspA in lipid bilayer experiments was analyzed. After inserting approximately 220 M18-MspA pores, the voltage through the lipid bilayer was increased sequentially in increments of 10 mV, and the ionic current through the pores was measured for 3 minutes. The critical voltage Vc was defined as the voltage at which the pores begin to close, and was measured in these experiments as the ionic current decreased.

[0183] The M18-MspA channel begins to close at +90mV and is fully stable under all applied negative voltages. Figure 9 In a second experiment using gel-purified M18-MspA protein, no voltage gating up to ±100 mV was observed. Therefore, the critical voltage V for M18-MspA is... c It is more than M1-MspA or M12-MspA (for both proteins, V) critThe voltage gating (+40mV, -50mV) was twice as high. These results show that linking all eight subunits to a single polypeptide significantly increased the voltage gating tolerance of single-stranded MspA. This unexpected advantage of single-stranded MspA is crucial for nanopore sequencing of nucleic acids, such as DNA.

[0184] Construction of mutant single-stranded MspA (scMspA M2)

[0185] As described in this paper, mutations in MspA are useful for improving its interaction with DNA, its base recognition properties, and its interaction with membrane and accessory proteins, such as Phi29 DNA polymerase. Using the methods described above for single-stranded M1 MspA, a mutant single-stranded MspA (MspA M2) was constructed, in which eight mutant MspA monomers are linked together. The expression of both single-stranded M1 MspA and single-stranded MspA M2 constructs in Mycobacterium smegmatis ML712 was confirmed by Western blotting using an MspA antibody, demonstrating the feasibility of scMspA generation in Mycobacterium smegmatis. As shown in this paper, single-stranded MspA can be expressed in Escherichia coli. Single-stranded M2 MspA (scMspA M2) protein was produced in mg amounts, but it was unfolded. A folding scheme that allows the isolation of active scMspA M2 has been developed.

[0186] A single chain m2-mspA (scm2-mspA) 8 of them m2-mspA Genes (such as Butler, etc.) PNAS 105:20647-20652 (2008) describes a DNA fragment containing the mutants D90N / D91N / D93N / D118R / D134R / E139K linked via a (GGGGS)3 polypeptide linker. Additionally, each gene is flanked with a unique restriction site to enable specific modification of each MspA subunit. Genes in this sequence are named starting from the ATG start codon. m2-1 to m2-8 ( Figure 11 (See Table 3). For the protein generation and purification of single-chain MspA M2 protein in E. coli cells, the MspA signal peptide was removed. scm2-mspA The sequence was codon-optimized for optimal expression in *E. coli* and synthesized using GenScript. scm2- mspA Genes were side-ligated to EcoRI and HindIII and obtained from the pUC57 plasmid derived from GenScript. Next, the entire gene was excised. scm2-mspA It was then cloned into the pET-21(a)+ vector. scm2-mspA The gene is controlled by the T7 promoter in the resulting plasmid pML3216. Figure 11 ).

[0187] For the generation and purification of scMspA M2 protein, plasmid pML3216 was transformed into E. coli strain BL21(DE3)Omp8, which lacks the three major porins (see Prilipov et al.). FEMS Microbiol. Lett 163: 65-72 (1998)). The BL21(DE3) Omp8 strain was selected to prevent scMspA M2 from being contaminated by endogenous porins from *E. coli*. Induction was performed with 1.5 mM IPTG. scm2-mspA Following expression, cells were grown at 37°C in LB medium supplemented with ampicillin. Maximum expression of the target protein was observed 2 hours after induction, accounting for approximately 4% of the total protein in the cell lysis products. Figure 12 The protein band corresponding to scMspA M2 has an apparent mass of 170 kDa, consistent with its predicted molecular mass of 165.6 kDa. Figure 12 Next, scMspA M2 from inclusion bodies was isolated and purified, as exemplified by Sambrook et al. ( CSH Protocols As described in 2006. Inclusion bodies mainly containing scMspA M2 protein were dissolved in 8M urea. The sample was then subjected to anion exchange chromatography in the presence of 8M urea using a HiTrap QFF column (GE HealthCare, United Kingdom). The elution profile of scMspA M2 protein was very similar to that of previously published wild-type MspA (Heinz et al., 2003). This protein is likely unfolded and lacks channel activity.

[0188] Then, scMspA M2 was purified and subjected to a refolding procedure. Following anion exchange chromatography, the purified scMspA M2 at a concentration of 50 µg / mL was diluted 10-fold in a buffer solution containing 10 mM NaCl, 25 mM HEPES, 0.6 M L-arginine, and 0.1% (v / v) LDAO, pH 8.0, to produce a final volume of 1 mL. The mixture was incubated overnight at room temperature (approximately 21 °C) on a rotary mixer. The sample was then transferred to a dialysis tube with a 3.5 kDa MWCO and dialyzed overnight at room temperature with 2 L of buffer solution containing 10 mM NaCl, 25 mM HEPES (pH 8.0), and 0.023% (v / v) LDAO. The dialyzed protein was transferred to a microtube and incubated for another day at room temperature. Next, the refolding efficiency was assessed by Western blot analysis using MspA-specific rabbit antiserum. Following the refolding procedure, the band reacting with the MspA polyclonal antibody migrated from 170 kDa to approximately 130 kDa, indicating that scMspA M2 had folded into a more compact form with increased electrophoretic mobility. Figure 13 Such changes in electrophoretic mobility after folding have been previously observed in outer membrane proteins of E. coli. However, it is unclear whether MspA will show a similar phenomenon.

[0189] To examine whether scMspA M2 forms a functional channel in vitro after the refolding procedure, a lipid bilayer experiment was performed. No channel activity was observed when only 0.023% LDAO-buffer was added to the planar bilayer. Conversely, the addition of scMspA M2 protein after the refolding step resulted in a gradual increase in current, indicating channel insertion into the lipid bilayer. Figure 14 Analysis of the current trajectory shows an average conductance of 2.3 nS. Figure 14 Interestingly, analysis of MspA M2 generated from the monomer showed two peaks at 1.2 nS and 2.4 nS, indicating two distinct protein conformations. Furthermore, multichannel experiments with scMspA M2 demonstrated improved voltage-gated tolerance, with a critical voltage of +80 mV / -70 mV. Figure 15 Enhanced voltage tolerance, for example, is advantageous for ssDNA experiments performed at relatively high voltages.

[0190]

[0191] Construction of mutant single-stranded MspA (MspA PN1)

[0192] A single chain mspA pn1 (scmspA PN1) Genes, including 8 mspA Genes (such as Butler, etc.) PNAS 105: 20647-20652 (2008) describes a DNA fragment containing the P97F mutation and mutations D90N / D91N / D93N / D118R / D134R / E139K linked by a (GGGGS)3 polypeptide linker. Additionally, each gene is flanked with a unique restriction site to enable specific modification of each MspA subunit. Genes in this sequence are named starting from the ATG start codon. m2- 97-1 to m2-97-8 ( Figure 16 (See Table 3). For the protein generation and purification of single-chain MspA PN1 protein in E. coli cells, the MspA signal peptide was removed. scmspA PN1 The sequence was codon-optimized for optimal expression in *E. coli* and synthesized using GenScript. scmspA PN1 Genes were side-ligated to EcoRI and HindIII and obtained from the pUC57 plasmid derived from GenScript. Next, the entire gene was excised. scmspA PN1 It was then cloned into the pET-21(a)+ vector. scmspA PN1The gene is controlled by the T7 promoter in the resulting plasmid pML3216. Figure 16 ).

[0193] For the generation and purification of scMspA PN1 protein, plasmid pML3216 was transformed into E. coli strain BL21(DE3)Omp8, which lacks the three major porins (see Prilipov et al.). FEMS Microbiol. Lett 163: 65-72 (1998)). BL21(DE3) Omp8 strain was selected to prevent scMspA PN1 from being contaminated by endogenous porins from *E. coli*. Induction was performed with 1.5 mM IPTG. scmspA PN1 Following expression, cells were grown at 37°C in LB medium supplemented with ampicillin. Maximum expression of the target protein was observed 2 hours after induction, accounting for approximately 5% of the total protein in the cell lysis products. Figure 17 The protein band corresponding to scMspA PN1 has an apparent mass of 170 kDa, consistent with its predicted molecular mass of 165.6 kDa. Figure 17 Next, scMspA PN1 from inclusion bodies was isolated and purified, as in the case of Sambrook et al. ( CSH Protocols As described in 2006. Inclusion bodies mainly containing scMspA PN1 protein were dissolved in 8M urea. The sample was then subjected to anion exchange chromatography in the presence of 8M urea using a HiTrap QFF column (GE HealthCare, United Kingdom). The elution profile of scMspA PN1 protein was very similar to that of previously published wild-type MspA (Heinz et al., 2003). This protein is likely unfolded and lacks channel activity.

[0194] Then, scMspAPN1 was purified and subjected to a refolding procedure. Following anion exchange chromatography, the purified scMspAPN1 fraction was dialyzed against 2 L of buffer containing 140 mM NaCl, 10 mM K2HPO4 / KH2PO4, and 2 mM KCl (pH 7.5) to remove urea. The mixture was incubated overnight at room temperature (approximately 21°C). After dialyzing, L-arginine and LDAO were added to the sample to produce final volumes of 0.6 M and 0.1% (v / v), respectively. The protein sample was incubated overnight on a fixed-track shaker in refolding buffer (140 mM NaCl, 10 mM K2HPO4 / KH2PO4, 2 mM KCl, 0.6 M L-arginine, 0.1% (v / v) LDAO, pH 7.5). Figure 18The concentration of the purified sample was calculated to be 1.37 mg / ml by absorbance measurement at 280 nm. The protein yield was 0.45 mg per 1 L of bacterial culture.

[0195] To test the effect of phenylalanine at position 97 on the incorporation of single-chain MspA into artificial lipid membranes, the insertion activity of different MspA constructs was measured by monitoring the release of fluorescent carboxyfluorescein dye from the liposomes, as described (see Schwarz et al., Biophys. J. 58(3):577-83 (1990); Schwarz et al., Biochim. Biophys. Acta1239(1): 51-7 (1995)). Briefly, DPhPC liposomes were prepared by extrusion in the presence of 30 mM carboxyfluorescein. Carboxyfluorescein self-quenched upon encapsulation within the lipid vesicles. Following dye-loaded liposomes with MspA pore insertion, diffusion-mediated dye efflux resulted in increased fluorescence in the reaction mixture. Figure 19 The results of these carboxyfluorescein release experiments are shown. Adding buffer containing LDAO (0.1% v / v) or OPOE (0.5% v / v) resulted only in minimal release of the dye from the liposomes, as opposed to the Triton X-100 (1% v / v) buffer used as a positive control. Importantly, adding scMspAPN1 (60 ng / ml, final) resulted in faster and greater release of carboxyfluorescein than adding scMspA M2 (120 ng / ml, final). Interestingly, wild-type MspA (60 ng / ml, final) resulted in slower dye diffusion from the liposomes than scMspA PN1. Figure 19 These data suggest that the additional phenylalanine in ring 6 of scMspA promotes faster and more efficient insertion of the lipid bilayer into the pores.

[0196] Next, the insertion time of the first pore into the DPhPC membrane was measured in a bilayer apparatus. It was assumed that the pore with enhanced insertion capacity would require less time to insert into the lipid membrane. To examine the effect of phenylalanine in ring 6 on the membrane insertion time of scMspA, scMspA PN1 was compared with scMspA M2. Simply put, electrolyte was added to the bilayer cuvettes, a potential of -10 mV was applied, and data were acquired and recorded using TestPoint software. The same cuvettes were used throughout these experiments. Protein was added to both sides at a final concentration of 100 ng / ml. Importantly, successful insertion events were observed in 89% of the experiments for scMspA PN1, but only in 40% of the experiments for scMspA M2. This is consistent with the results of the carboxyfluorescein release assay. Although the median insertion time of scMspA PN1 was 399 seconds, significantly different from the 695 seconds of scMspA M2, this difference was not statistically significant. Surprisingly, the insertion rate decreased when scMspA PN1 was analyzed in 0.3 M KCl solution (median time: 859 s, 50%). However, successful insertion was achieved in half of the experiments with scMspA PN1, while only one successful insertion was observed with scMspA M2 in 0.3 M KCl, at a time of 1270 s (12% successful insertion rate across 8 membranes analyzed). Figure 20 This result demonstrates the beneficial effect of phenylalanine in ring 6 on the insertion of single-chain MspA into the membrane.

[0197] To examine whether scMspA PN1 forms a functional channel in vitro after the refolding procedure, a lipid bilayer experiment was performed. No channel activity was observed when only 0.1% LDAO-buffer was added to the planar bilayer. Conversely, the addition of scMspA PN1 protein after the refolding step resulted in a gradual increase in current, indicating channel insertion into the lipid bilayer. Figure 21 Analysis of the current trajectory shows an average conductance of 2.0 nS (). Figure 21 This translates to a larger residual current per nucleotide and a better signal in DNA sequencing experiments.

[0198] Effects of lipids on the activity of single-chain MspA PN1 channels

[0199] scMspA PN1 was stored at room temperature at concentrations of 1 µg / mg and 0.2 µg / ml for more than one month. scMspA PN1 was diluted in 0.1% LDAO, 140 mM NaCl, 10 mM K₂HPO₄ / KH₂PO₄ (pH 7.5), and 2 mM KCl. Methods for preparing horizontal bilayers for channel experiments are known in the art. See, for example, Butler et al. (2008) and Akeson et al., Biophysical Journal; 77: 3227-3233 (1999), both incorporated herein by reference in their entirety. For channel experiments, 2% diphyranylphosphatidylcholine (DiphPC) in chloroform was used to form a membrane bilayer for MspA insertion, substantially as described by Butler et al. and Akeson et al. After MspA insertion into the bilayer, the membrane was ruptured and recoated with 1% DiphPC in n-decane. The electrolytes used in these experiments were 0.3 or 1M KCl and 10mM Hepes, at pH 8.0 or pH 7.0, respectively.

[0200] like Figure 22 As shown, single-chain MspA functions at various electrolyte concentrations, such as approximately 0.3–1 M KCl. To optimize channel activity, lipid association can be performed before MspA insertion into the membrane or lipid bilayer. Therefore, in any of the methods described herein, MspA can be contacted or pre-incubated with one or more lipids to optimize channel activity. In a non-limiting example, Figure 22 No channel activity was observed in a buffer solution containing only 0.3 M KCl at pH 8.0. However, rupturing the membrane and subsequently recoating it resulted in enhanced channel activity of scMspA PN1 in an electrolyte solution containing 0.3 M KCl at pH 8.0.

Claims

1. A nucleic acid encoding a mutant single-stranded Msp, wherein the sequence of said nucleic acid comprises: (a) The first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences encode the first, second, third, fourth, fifth, sixth, seventh, and eighth Msp monomer sequences, respectively; and (b) A ninth nucleotide sequence encoding an amino acid linker sequence consisting of SEQ ID NO: 5, wherein the ninth nucleotide sequence is located between every two consecutive nucleotide sequences of the first, second, third, fourth, fifth, sixth, seventh and eighth nucleotide sequences; Each Msp monomer sequence is a mutant Msp monomer sequence, wherein the mutant Msp monomer sequence has only the following mutations compared to SEQ ID NO:1: D90N mutation, D91N mutation, D93N mutation, P97F mutation, D118R mutation, D134R mutation and E139K mutation.

2. A mutant Msp encoded by the nucleic acid according to claim 1.

3. A vector comprising the nucleic acid according to claim 1.

4. A system comprising a single-stranded Msp encoded by a nucleic acid according to claim 1, wherein the Msp has a precordial and a constriction region defining a channel, wherein the channel is located between a first conductive liquid medium and a second conductive liquid medium, wherein at least one conductive liquid medium contains an analyte, and wherein the system is operable to detect the analyte when subjected to an electric field sufficient to translocate the analyte from one conductive liquid medium to the other.

5. The system according to claim 4 is further defined as comprising a plurality of Msp.

6. The system of claim 4, wherein the Msp is further defined as comprising a vestibule having a length of 2 to 6 nm and a diameter of 2 to 6 nm, and a constriction having a length of 0.3 to 3 nm and a diameter of 0.3 to 3 nm, wherein the vestibule and the constriction together define a channel.

7. The system of claim 5, wherein the Msp is further defined as comprising a vestibule having a length of 2 to 6 nm and a diameter of 2 to 6 nm, and a constriction having a length of 0.3 to 3 nm and a diameter of 0.3 to 3 nm, wherein the vestibule and the constriction together define a channel.

8. The system of claim 4, wherein the Msp further comprises a molecular engine, wherein the molecular engine enables the analyte to move into or through the channel at an average transposition rate lower than the average transposition rate of the analyte into or through the channel without the molecular engine.

9. The system of claim 5, wherein the Msp further comprises a molecular engine, wherein the molecular engine enables the analyte to move into or through the channel at an average transposition rate lower than the average transposition rate of the analyte into or through the channel without the molecular engine.

10. The system of claim 6, wherein the Msp further comprises a molecular engine, wherein the molecular engine enables the analyte to move into or through the channel at an average transposition rate lower than the average transposition rate of the analyte into or through the channel without the molecular engine.

11. The system of claim 7, wherein the Msp further comprises a molecular engine, wherein the molecular engine enables the analyte to move into or through the channel at an average transposition rate lower than the average transposition rate of the analyte into or through the channel without the molecular engine.

12. The system of claim 4, further comprising a patch clamp amplifier.

13. The system of claim 5, further comprising a patch clamp amplifier.

14. The system of claim 6, further comprising a patch clamp amplifier.

15. The system of claim 8, further comprising a patch clamp amplifier.

16. The system according to any one of claims 7 and 9-11, further comprising a patch clamp amplifier.

17. The system according to claim 4, further comprising a data acquisition device.

18. The system according to claim 5, further comprising a data acquisition device.

19. The system according to claim 6, further comprising a data acquisition device.

20. The system according to claim 8, further comprising a data acquisition device.

21. The system according to claim 12, further comprising a data acquisition device.

22. The system according to any one of claims 7, 9-11 and 13-15, further comprising a data acquisition device.

23. The system according to claim 16, further comprising a data acquisition device.

24. The system of claim 4, further comprising one or more temperature regulating devices in communication with the first conductive liquid medium, the second conductive liquid medium, or both.

25. The system of claim 5, further comprising one or more temperature regulating devices in communication with the first conductive liquid medium, the second conductive liquid medium, or both.

26. The system of claim 6, further comprising one or more temperature regulating devices in communication with the first conductive liquid medium, the second conductive liquid medium, or both.

27. The system of claim 8, further comprising one or more temperature regulating devices in communication with the first conductive liquid medium, the second conductive liquid medium, or both.

28. The system of claim 12, further comprising one or more temperature regulating devices in communication with the first conductive liquid medium, the second conductive liquid medium, or both.

29. The system of claim 16, further comprising one or more temperature regulating devices in communication with the first conductive liquid medium, the second conductive liquid medium, or both.

30. The system of claim 17, further comprising one or more temperature regulating devices in communication with the first conductive liquid medium, the second conductive liquid medium, or both.

31. The system of claim 22, further comprising one or more temperature regulating devices in communication with the first conductive liquid medium, the second conductive liquid medium, or both.

32. The system according to any one of claims 7, 9-11, 13-15, 18-21 and 23, further comprising one or more temperature regulating devices in communication with the first conductive liquid medium, the second conductive liquid medium or both.

33. The system of claim 4, wherein the Msp is located in a lipid bilayer between a first conductive liquid medium and a second conductive liquid medium.

34. The system of claim 5, wherein the Msp is located in a lipid bilayer between a first conductive liquid medium and a second conductive liquid medium.

35. The system of claim 6, wherein the Msp is located in a lipid bilayer between a first conductive liquid medium and a second conductive liquid medium.

36. The system of claim 8, wherein the Msp is located in a lipid bilayer between a first conductive liquid medium and a second conductive liquid medium.

37. The system of claim 12, wherein the Msp is located in a lipid bilayer between a first conductive liquid medium and a second conductive liquid medium.

38. The system of claim 16, wherein the Msp is located in a lipid bilayer between a first conductive liquid medium and a second conductive liquid medium.

39. The system of claim 17, wherein the Msp is located in a lipid bilayer between a first conductive liquid medium and a second conductive liquid medium.

40. The system of claim 22, wherein the Msp is located in a lipid bilayer between a first conductive liquid medium and a second conductive liquid medium.

41. The system of claim 24, wherein the Msp is located in a lipid bilayer between a first conductive liquid medium and a second conductive liquid medium.

42. The system of claim 32, wherein the Msp is located in a lipid bilayer between a first conductive liquid medium and a second conductive liquid medium.

43. The system according to any one of claims 7, 9-11, 13-15, 18-21, 23 and 25-31, wherein the Msp is located in a lipid bilayer between a first conductive liquid medium and a second conductive liquid medium.

44. The system of claim 43, wherein the Msp is in contact with the lipids prior to being located in the lipid bilayer.

45. The system of claim 43, wherein the Msp is refolded in a buffer containing a refolding agent and a detergent prior to being located in the lipid bilayer.

46. ​​A method for detecting the presence of an analyte, comprising: a) Apply an electric field sufficient to cause the analyte to translocate from the first conductive medium to the liquid-connected second conductive medium via the mutation Msp as described in claim 4; and b) Measure the ion current, wherein a decrease in the ion current indicates the presence of the analyte in the first medium.

47. The method of claim 46, wherein the porin further comprises a molecular engine, wherein the molecular engine enables an analyte to move into or through the channel at an average translocation rate lower than the average translocation rate of the analyte by electrophoresis into or through the channel without the molecular engine.

48. The method of claim 46, further comprising identifying the analyte.

49. The method of claim 48, wherein identifying the analyte comprises measuring the ion current to provide a current pattern, wherein a decrease in the current defines a blockage in the current pattern, and comparing one or more blockages in the current pattern with (i) one or more blockages in a current pattern or (ii) one or more blockages in a known current pattern obtained using a known analyte.

50. The method of claim 46 or 47, wherein the analyte is a nucleotide, amino acid, polymer, drug, ion, contaminant, or nanoscale object.

51. The method of claim 48, wherein the analyte is a nucleotide, amino acid, polymer, drug, ion, contaminant, or nanoscale object.

52. The method of claim 49, wherein the analyte is a nucleotide, amino acid, polymer, drug, ion, contaminant, or nanoscale object.

53. The method of claim 52, wherein the analyte is a polymer.

54. The method of claim 53, wherein the polymer is a protein, peptide, or nucleic acid.

55. The method of claim 54, wherein the polymer is a nucleic acid.

56. The method of claim 55, wherein the nucleic acid is ssDNA, dsDNA, RNA, or a combination thereof.

57. The method according to any one of claims 52-55, further comprising identifying one or more units of the polymer.

58. The method of claim 57, wherein identifying one or more units of the polymer comprises measuring the ion current to provide a current pattern containing a blockage for each polymer unit, and comparing one or more blockages in the current pattern with (i) one or more other blockages in the current pattern or (ii) one or more blockages in the current pattern obtained using a polymer having known units.

59. The method of claim 46 or 47, further comprising determining the concentration, size, molecular weight, shape, or orientation of the analyte, or any combination thereof.

60. The method of claim 48, further comprising determining the concentration, size, molecular weight, shape or orientation of the analyte, or any combination thereof.

61. The method of claim 50, further comprising determining the concentration, size, molecular weight, shape or orientation of the analyte, or any combination thereof.

62. The method of claim 57, further comprising determining the concentration, size, molecular weight, shape or orientation of the analyte, or any combination thereof.

63. The method according to any one of claims 49, 51-56 and 58, further comprising determining the concentration, size, molecular weight, shape or orientation of the analyte, or any combination thereof.

64. The method according to claim 46 or 47, wherein at least one of the first or second conductive media comprises a variety of different analytes.

65. The method of claim 48, wherein at least one of the first or second conductive media comprises a variety of different analytes.

66. The method of claim 50, wherein at least one of the first or second conductive media comprises a plurality of different analytes.

67. The method of claim 57, wherein at least one of the first or second conductive media comprises a plurality of different analytes.

68. The method of claim 59, wherein at least one of the first or second conductive media comprises a variety of different analytes.

69. The method of claim 63, wherein at least one of the first or second conductive media comprises a plurality of different analytes.

70. The method according to any one of claims 49, 51-56, 58 and 60-62, wherein at least one of the first or second conductive media comprises a variety of different analytes.

71. The method according to claim 46 or 47, wherein the Msp is located in a lipid bilayer between a first conductive medium and a second conductive medium.

72. The method of claim 49, wherein the Msp is located in a lipid bilayer between a first conductive medium and a second conductive medium.

73. The method of claim 51, wherein the Msp is located in a lipid bilayer between a first conductive medium and a second conductive medium.

74. The method of claim 57, wherein the Msp is located in a lipid bilayer between a first conductive medium and a second conductive medium.

75. The method of claim 59, wherein the Msp is located in a lipid bilayer between a first conductive medium and a second conductive medium.

76. The method of claim 61, wherein the Msp is located in a lipid bilayer between a first conductive medium and a second conductive medium.

77. The method of claim 62, wherein the Msp is located in a lipid bilayer between a first conductive medium and a second conductive medium.

78. The method of claim 70, wherein the Msp is located in a lipid bilayer between a first conductive medium and a second conductive medium.

79. The method according to any one of claims 49, 51-56, 58, 60-62 and 65-69, wherein the Msp is located in a lipid bilayer between a first conductive medium and a second conductive medium.

80. The method of claim 79, wherein the Msp is contacted with lipids prior to being located in the lipid bilayer.

81. The method of claim 79, wherein the Msp is refolded in a buffer containing a refolding agent and a detergent prior to being located in the lipid bilayer.

82. A method for increasing the number of Msp insertions in a lipid bilayer, comprising: (a) Contacting the Msp according to claim 2 with lipids to form a lipid-associated Msp; and (b) Insert the lipid-associated Msp from step (a) into the lipid bilayer.

83. The method of claim 82, wherein the contacting step comprises inserting the Msp into the lipid bilayer and disrupting the lipid bilayer to form a lipid-associated Msp.

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