HK1211310B - Msp nanopores and related methods - Google Patents

Description

MSP nanopores and related methods

This application is a divisional application of Chinese invention patent application No. 200980142855.5, filed on September 22, 2009, with the invention name “MSP Nanopores and Related Methods”.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 61/098,938, filed September 22, 2008, which is incorporated herein by reference in its entirety.

Statement of Government Licensing Rights

This invention was made with government support under Grant No. 5R21HG004145 awarded by the National Institutes of Health. The government has certain rights in this invention.

background

Established DNA sequencing techniques require large amounts of DNA and several lengthy steps to construct just a few dozen bases of a full-length sequence. This information must then be assembled in a "shotgun" fashion (a process that is nonlinearly dependent on the size of the genome and the length of the fragments from which the full-length genome is constructed). These steps are expensive and time-consuming, especially when sequencing mammalian genomes.

Overview

Provided herein are methods comprising applying an electric field to a Mycobacterium smegmatis porin (Msp) porin having a vestibule and a constriction zone defining a tunnel, wherein the Msp porin is positioned between a first conductive liquid medium and a second conductive liquid medium.

Also provided are methods for improving conductance through the tunnel of an Msp porin comprising removing, adding, or substituting at least one amino acid in the vestibule or constriction region of a wild-type Msp porin.

Also provided is a system comprising an Msp porin having a vestibule and a constriction region defining a channel, wherein the channel is located between a first liquid medium and a second liquid medium, wherein at least one of the liquid media comprises an analyte, and wherein the system is effective for detecting a property of the analyte.

Also provided is a system comprising an Msp porin having a vestibule and a constriction region defining a channel, wherein the channel is located in a lipid bilayer between a first liquid medium and a second liquid medium, and wherein the only point of liquid communication between the first and second liquid media exists in the channel.

Also provided are mutant Msp porins. For example, mutant Mycobacterium smegmatis porin A (MspA) is provided, comprising a vestibule and a constriction defining a channel, and at least a first mutant MspA monomer comprising a mutation at position 93 and a mutation at position 90, position 91, or positions 90 and 91. Also provided are mutant MspA porins comprising a vestibule having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and a constriction having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm, wherein the vestibule and constriction together define a channel, and further comprising at least a first mutant MspA paralog or homolog monomer. Also provided are mutant MspA paralogs or homologs comprising a vestibule having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and a constriction having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm, wherein the vestibule and constriction together define a channel.

Methods for generating mutant Msp porins are described. For example, provided herein are methods for generating mutant MspA porins comprising modifying a wild-type MspA monomer at position 93 and at position 90, position 91, or positions 90 and 91. Also provided are methods for generating mutant MspA porins having a vestibule and a constriction region defining a channel, comprising deleting, adding, or substituting any amino acid in the vestibule or constriction region of a wild-type MspA paralog or homolog monomer such that the resulting mutant MspA porin is capable of translocating an analyte through the channel upon application of an electric field.

Also provided is a method comprising translocating an analyte through a channel of a Mycobacterium smegmatis porin (Msp) porin without the use of an electric field.

Nucleic acid sequences are provided herein. Optionally, the nucleic acid sequence may comprise a first and a second nucleotide sequence, wherein the first nucleotide sequence encodes a first Msp monomer sequence and the second nucleotide sequence encodes a second Msp monomer sequence. The nucleic acid sequence may also comprise a third nucleotide sequence encoding an amino acid linker sequence. Optionally, the nucleic acid sequence further comprises a third or more nucleotide sequence encoding a third or more Msp monomer sequence. For example, the nucleic acid sequence may further comprise a 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequence. The 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences encode the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th Msp monomer sequence, and the nucleic acid sequence further comprises a 9th nucleotide sequence encoding an amino acid linker sequence. Msp porins comprising two or more single-chain Msp are also provided.

Also provided are polypeptides encoded by the nucleic acids described herein. Also provided are vectors comprising the polypeptides described herein. Also provided are cultured cells or progeny thereof transfected with any of the vectors described herein, wherein the cells are capable of expressing an Msp porin or an Msp porin monomer. Also provided are Mycobacterium smegmatis strains comprising any of the vectors described herein.

Also provided are mutant bacterial strains capable of inducible expression of Msp monomers, comprising: (a) a deletion of wild-type MspA; (b) a deletion of wild-type MspC; (c) a deletion of wild-type MspD; and (d) a vector comprising an inducible promoter operably linked to an Msp monomer nucleic acid sequence.

Also provided is a method for producing a single-chain Msp porin, comprising: (a) transforming a mutant bacterial strain with a vector comprising a nucleic acid sequence capable of encoding a single-chain Msp porin; and optionally (b) purifying the single-chain Msp porin from the bacteria. The mutant strain may comprise a deletion of wild-type MspA, wild-type MspB, wild-type MspC, and wild-type MspD, and a vector comprising an inducible promoter operably linked to the Msp nucleic acid sequence. The mutant strain may be transformed with a vector comprising a nucleic acid sequence capable of encoding a single-chain Msp porin.

Also provided are methods for using Mps porins, such as single-chain Msp porins. For example, the methods can include generating a lipid bilayer having a first side and a second side, adding an Msp porin, such as a purified single-chain Msp porin, to the first side of the lipid bilayer, applying a positive potential to the second side of the bilayer, translocating an experimental nucleic acid sequence or polypeptide sequence through the Msp porin, measuring a blocking current resulting from translocation of the sequence through the Msp porin, and comparing the experimental blocking current to a blocking current standard to identify the experimental sequence.

Summary of the Figures

The above-described aspects and many of the attendant advantages will become more readily understood as they become better appreciated with reference to the following detailed description when taken in conjunction with the following drawings.

Figure 1 shows the structure and charge distribution of the wild-type MspA (WTMspA) porin. At pH 8, acidic residues are expected to be predominantly negatively charged and basic residues to be predominantly positively charged. The positions and residues of the mutations are indicated by arrows and labels. See Faller et al., Science, 303:1189 (2004).

Figure 2 shows the results of channel formation activity and single-channel conductance measurements of WTMspA, mutant D90N/D91N/D93N (M1MspA, also known as M1-NNN), and mutant D90N/D91N/D93N/D118R/E139K/D134R (M2MspA, also known as M2-NNN) porins. The left panel shows the change in bilayer conductance over time when the MspA porin is present in a solution (1 M KCl, 20°C) in a water bath bilayer. The gradual increase in conductance is interpreted as insertion of the MspA porin into the bilayer. The right panel is a histogram of the size of these conductance steps. The histograms for the WTMspA, M1MspA, and M2MspA porins summarize 40 insertions from three replicates, 144 insertions from three replicates, and 169 insertions from five replicates, respectively.

Figures 3A and 3B show the spontaneous blocking behavior of the WTMspA porin. Figure 3A is a schematic diagram of the experiment. Figure 3B shows representative ion current signals observed for the WTMspA porin at 60 mV (left) and 100 mV (right) in the absence of DNA. The intervals of negative current correspond to the reversal of the applied voltage, which is generally required to reestablish unblocked ion current levels.

Figure 4 shows the expression of mutant MspA monomers in the electrophoresis gel. Crude extract (13 μL) was added to each lane. The gel was stained with Coomassie blue. Lane 1: protein molecular weight standard; Lane 2: WTMspA; Lane 3: no MspA; Lane 4: mutant M1MspA; Lane 5: mutant D90N/D91N/D93N/D118R; Lane 6: mutant D90N/D91N/D93N/D118R/E139R; Lane 7: mutant D90N/D91N/D93N/D118R/E139K; Lane 8: mutant M2MspA. The mutants in lanes 5 to 7 were constructed, extracted, and measured to ensure that expression and channel formation activity were maintained for each consecutive amino acid replacement. The schematic diagram above the gel schematically shows the approximate position and polarity of the amino acids mutated in this experiment.

Figures 5A-5C show detection of ssDNA hairpin constructs using the M1MspA porin. Figure 5A is a schematic diagram of the experiment. Figure 5B shows representative ion current signals observed for the M1MspA porin at 180 and 140 mV in the absence of DNA and in the presence of 8 μM hp08 (SEQ ID NO: 4) hairpin DNA. Figure 5C shows the numbered blockades from the traces in Figure 5B on an expanded time scale.

Figure 6 shows the characteristics of deep blockage from hairpin constructs in the M1MspA porin. The coordinates of each point give the duration and average current of one deep blockage. Black and gray data were acquired at 140 and 180 mV, respectively. For each data set, the log10 pattern of the deep blockage dwell time tD is plotted. The diagram on the right shows the sequence of each hairpin construct: hp08 (5' GCTGTTGC TCTCTC GCAACAGC A ₅₀ 3') (SEQ ID NO: 4), hp10 (5' GCTCTGTTGC TCTCTC GCAACAGAGC A ₅₀ 3') (SEQ ID NO: 5), and hp12 (5' GCTGTCTGTTGC TCTCTC GCAACAGACAGC A ₅₀ -3') (SEQ ID NO: 6).

Figure 7 is a graph showing the distribution of partial blockade dwell times for hp08 (SEQ ID NO: 4) in the MlMspA porin. The distribution fits a single exponential model very well. The partial blockade at 180 mV has a time constant approximately 3 times longer than at 140 mV.

Fig. 8 provides the detailed appearance of the residence time distribution of hairpin construct deep obstruction in M1MspA porin. The left frame shows the logarithmic binary plot (staircase plot) of the probability distribution with log10 of residence time (x) and the residence time histogram of the corresponding kernel smooth density estimate. The maximum value tD of these smooth density estimates is used to parameterize the residence time distribution. The vertical line shows the tD value. The right frame shows the survival probability curve derived from the residence time data (solid line) and the single decaying exponential (single decaying exponential), and the time constant is set to the tD value (dashed line) of each data group. The data obviously deviate from the single exponential variation morphology (simple exponential behavior). However, it is reasonable to perform a qualitative comparison between the tD value and the exponential time constant for other observations (Kasianowicz et al., Proc. Natl Acad. Sci. USA, 93:13770 (1996)) because these two parameters reflect the similar aspects of the residence time distribution.

Figures 9A-9G show data obtained from a transbilayer probe experiment. Figure 9A shows an animation of the molecular configurations: (1) an unblocked pore; (2) filamentous ssDNA with neutravidin (nA) blocking translocation of the nA–ssDNA complex; (3) target DNA hybridized to the nA–ssDNA dissociated under a negative voltage; and (4) the nA–ssDNA complex ejected from the pore at a voltage dependent on target DNA hybridization. Figure 9B is a time series of applied voltages. Current blockage triggered a change from a capture voltage of 180 mV to a holding voltage of 40 mV after a delay of approximately 200 ms. The holding voltage was maintained for 5 seconds to allow hybridization and then decreased toward a negative voltage. Figures 9C and 9D show current time series indicating ejection of nA–ssDNA under negative and positive voltages, respectively. A large current spike is generated due to the transient voltage change and spontaneous pore closure at a large negative voltage. Figures 9E-9G are histograms of exit voltage (Vexit). Figure 9E shows an experiment in which the probe 5'- _{C6A54 -} CTCTATTCTTATCTC-3' (SEQ ID NO:7) was complementary to the target ssDNA molecule 5'-GAGATAAGAATAGAG-3' (SEQ ID NO:9). Figure 9F shows the same well as in Figure 9E, but using the probe 5'- _{C6A54 -} CACACACACACACAC-3' (SEQ ID NO:8), which is not complementary to the target DNA. Figure 9G shows results from a separate control using the same probe (SEQ ID NO:7) as in Figure 9E, but without target DNA in the trans compartment. Significant negative Vexit events were observed only in Figure 9E, where the probe (SEQ ID NO:7) was complementary to the target. The rare occurrence of negative Vexit events in Figures 9F and 9G excludes the possibility that most of the negative Vexit in Figure 9E is caused by nonspecific probe-target binding or by binding of the probe to the well.

Figures 10A-10C compare dT50 (SEQ ID NO: 32) homopolymer blockade of M1MspA and M2MspA porins. Figure 10A is a schematic diagram of the experiment. Figure 10B shows representative ion current signals observed for M1MspA porins using 8 μM dT50 (left) and M2MspA porins using 2 μM dT50 (right). Figure 10C shows the numbered blockades from the traces in Figure 10B on an enlarged time scale.

Figure 11 shows the statistical characteristics of dT50 (SEQ ID NO: 32) blockade in the M2MspA porin. Comparison of average structures at the beginning and end of blockade. The graph was generated by overlaying events from the aligned data files at the beginning of the event (left) and the end of the event (right). The trend of blockade ending with a brief downward deflection of ionic current and the increase in this trend with increasing voltage are shown.

Figure 12A shows a histogram of blockade current levels in the M1MspA porin blocked by DNA constructs. The DNA constructs are, from top to bottom: 3'-A ₄₇ AAC-hp-5' (SEQ ID NO: 14); 3'-A ₄₇ ACA-hp-5' (SEQ ID NO: 33); 3'-A ₄₇ CAA-hp-5' (SEQ ID NO: 13); 3'-C ₅₀ -hp-5' (SEQ ID NO: 16); 3'-A ₅₀ -hp-5' (SEQ ID NO: 10). Figure 12B shows a plot of current levels versus the position of a single C, scaled for the difference between poly-C (= 1.0) and poly-A (= 0.0) levels. Gaussian fit indicates that the recognition position for a single C is 1.7 ± 0.8 nucleotides (nt) from the end of the hairpin.

Figure 13 shows a current histogram of a number of DNA blocking M1-NNN MspA (also known as M1MspA) porins. The DNA constructs are, from top to bottom: 3'- _C50 -hp-5' (SEQ ID NO: 16); 3'- _A50 -hp-5' (SEQ ID NO: 10); 3'- _T47TTT -hp-5' (SEQ ID NO: 17); 3'- _A47AAT -hp-5' (SEQ ID NO: 34); 3'- _A47ATA -hp-5' (SEQ ID NO: 35); 3'- _A47TAA -hp-5' (SEQ ID NO: 36); 3'- _C47CCA -hp-5' (SEQ ID NO: 37); 3'- _C47CAC -hp-5' (SEQ ID NO: 38); 3'- _C47ACC -hp-5' (SEQ ID NO: 39). Each construct or mixture is shown on the left. The number of events in each histogram is shown on the right. Top panel: "Calibration mixture" (poly-A-hp and poly-C-hp). Figures 2-5: Single T bases in poly-T-hp and poly-A background. Bottom three panels: Single A bases in poly-A background. Poly-A-hp is included in the reference mixture (small peak at 19.5%). All data were acquired at 180 mV.

Figure 14 shows that the DNA tails do not affect the recognition properties. The legend is related to Figure 13. Two heterogeneous tails ('ran1' (SEQ ID NO:51) and 'ran2' (SEQ ID NO:52), each 47 bases) are attached to the trinucleotide and hairpin. The middle figure shows the current histogram generated when a mixture of A50-hp DNA (SEQ ID NO:10) and ran1-C3-hp DNA is applied to the pore, which serves as a reference point for the other figures. The current levels are the same as those of the A50 or C50 tails. All data are at 180mV.

Figures 15A and 15B show characterization data for the M2-QQN porin, another mutant MspA porin. Figure 15A shows the expression levels of this mutant. All proteins were expressed in ML16 Mycobacterium smegmatis strains. 10 μl of a 0.5% octylpolyoxyethylene crude extract was added to each well. Lane 1: WTMspA; Lane 2: Background (pMS2, empty vector); Lane 3: M2-QQN (pML866). Figure 15B shows the current traces of the M2-QQN porin in a diphytanoylphosphatidylcholine lipid bilayer recorded in 1 M KCl. Approximately 70 pg of protein was added to the bilayer chamber. Approximately 100 pores from four membranes were analyzed in the lipid bilayer experiment. The main conductance of the M2-QQN porin was 2.4 nanoseconds (nS).

Figure 16 shows blockade current histograms for three different mutant MspA porins exposed to a mixture of hairpin DNAs: hp-T50 (SEQ ID NO: 17), hp-C50 (SEQ ID NO: 16), and hp-A50 (SEQ ID NO: 10). In each case, the current was normalized to the open state current shown on the right for each mutant. hp-C50 and hp-A50 were used as a mixture, while T50 was used alone.

FIG17 shows the probability of deep current blockade for two mutant MspA porins. The probability of an event lasting longer than t is shown. Circles represent the M2-QQN porin, and crosses represent the M2-NNN porin. The voltages applied across the bilayer were 100, 120, and 140 mV. Data were normalized to the total number of events in each recording.

Figure 18 shows an alignment of the MspA, MspB, MspC, and MspD monomers of Mycobacterium smegmatis. The first ATG or GTG codon of the open reading frame is considered the presumed start codon. Protein numbering begins with the first amino acid in the mature portion. The amino acid sequence of the MspA monomer is SEQ ID NO:28, the amino acid sequence of the MspB monomer is SEQ ID NO:29, the amino acid sequence of the MspC monomer is SEQ ID NO:30, and the amino acid sequence of the MspD monomer is SEQ ID NO:31.

FIG. 19 is an image of a gel showing the deletion of each porin gene in the M. smegmatis porin-quadruple mutant ML59.

Figure 20 shows a Western blot showing expression of the Msp porin in M. smegmatis and M. smegmatis porin mutants. Lane 1 is a 1:10 dilution of a protein extract of wild-type M. smegmatis, lane 2 is mutant MN01 (ΔmspA), lane 3 is mutant ML10 (ΔmspAC), lane 4 is mutant ML16 (ΔmspACD), and lane 5 is mutant ML180 (ΔmspABCD).

Figures 21A and 21B show plasmid maps for the construction of quadruple porin mutants. Hyg: hygromycin resistance gene; ColE1: E. coli replication origin. Figure 21A is an integration plasmid map for MspA expression. AmiC, A, D, S are required for acetamide-inducible expression of MspA. attP: chromosomal attachment site of bacteriophage L5; int: L5 integrase; FRT: Flp recombinase site. Figure 21B is a plasmid map of the MspB deletion vector. MspBup, MspBdown: upstream and downstream regions of MspB; loxP: Cre recombination site; SacB: sucrose 6-fructosyltransferase; XylE: catechol-2,3-dioxygenase; Gfp2+: green fluorescent protein; tsPAL5000: temperature-sensitive replication origin of mycobacteria.

FIG22 is an image of a Coomassie blue-stained gel showing inducible expression of MspA monomers in M. smegmatis.

FIG. 23 is an image showing growth of the Msp quadruple mutant ML705 on Middlebrook 7H10 agar plates.

FIG24 is a photograph showing the growth rate of ML705 in rich liquid culture.

Figure 25 is a Western blot image showing the expression of MspA monomers in the quadruple mutant ML705 after induction with acetamide. Lane 1 is wild-type Mycobacterium smegmatis, lane 2 is the quadruple mutant strain ML705 using acetamide, lane 3 is the quadruple msp mutant strain ML705 without acetamide, and lane 4 is the triple mutant strain ML16. Proteins were detected using a polyclonal antibody against MspA.

Figures 26A-26D show the structure and channel activity of single-chain MspA nanopore dimer. Figure 26A is an image of the molecular model of the single-chain nanopore MspA dimer. Figure 26B shows the schematic diagram of the single-chain MspA nanopore dimer (scMspA) gene construct. The amino acid linker region (GGGGS) ₃ (SEQ ID NO:3) is amplified. Also shown is the DNA sequence of the amino acid linker (5'-GGCGGTGGCGGTAGCGGCGGTGGCGGTAGCGGCGGT GGCGGTAGC-3') (SEQ ID NO:19). Figure 26C is an image of a Western blot showing that the scMspA nanopore dimer is expressed in Mycobacterium smegmatis. Lane 1 is a molecular weight standard (M), lane 2 is wild-type Mycobacterium smegmatis (WT Msmeg), lane 3 is ML16 strain without scMspA gene construct (ML16), lane 4 is ML16 strain with wild-type MspA gene construct (WTMspA), and lane 5 is ML16 strain with scMspA nanopore dimer gene construct (scMspA). Figure 26D shows the current trace of the scMspA nanopore dimer.

Figure 27 shows a schematic diagram of the transport of dC58 (SEQ ID NO: 40) ssDNA through the wild-type MspA porin. DNA transport consists of the following steps: a) start of the simulation; b) and c) DNA conformational changes before and after rapid forward movement; and d) DNA attachment to the surface of the MspA porin.

Figure 28 is a graph showing the cumulative ionic current for the dC58 (SEQ ID NO: 40) ssDNA transport of Figure 27. Transport was performed at a transmembrane bias of 1.2V.

Figure 29 shows the design of the single-chain MspA (scMspA) nanopore octamer sequence. The scMspA octamer is composed of the following: a wild-type MspA gene monomer, an MspA1 monomer, an MspA2 monomer, an MspA3 monomer, an MspA4 monomer, an MspA5 monomer, an MspA6 monomer, and an MspA7 monomer. PacI and HindIII restriction sites flank the scMspA nanopore octamer sequence. X1-X14 are unique restriction sites flanking the individual monomer sequences. The black line connecting each monomer represents the (GGGGS) ₃ (SEQ ID NO: 3) connector.

FIG30 shows the constriction region (rectangular box) of a wild-type MspA monomer and monomers of various MspA paralogs and homologs.

FIG31 shows a histogram of blockade current levels in M1MspA blocked by DNA constructs. The DNA constructs are, from top to bottom: 3'-A ₄₀ AAAAAAAAAA-hp-5' (SEQ ID NO: 10); 3'-A ₄₀ CCCCAAAAAA-hp-5' (SEQ ID NO: 11); 3'-A ₄₀ AAACCCCAAA-hp-5' (SEQ ID NO: 12); 3'-A ₄₀ AAAAAAACAA-hp-5' (SEQ ID NO: 13); 3'-A ₄₀ AAAAAAAAAC-hp-5' (SEQ ID NO: 14); 3'-A ₄₀ AAAAAACCCC-hp-5' (SEQ ID NO: 15); 3'-C40CCCCCCCCCC-hp-5' (SEQ ID NO: 16); 3'-T ₄₀ TTTTTTTTTT-hp-5' (SEQ ID NO: 17); 3'-A ₄₀ AAAAAAAGGG-hp-5' (SEQ ID NO: 18).

Detailed Description of the Invention

Provided herein are methods comprising applying an electric field to a Mycobacterium smegmatis porin (Msp) porin having a vestibule and a constriction region defining a channel, wherein the Msp porin is located between a first conductive liquid medium and a second conductive liquid medium. Optionally, the first and second liquid conductive media are the same. Optionally, the first and second liquid conductive media are different. The Msp porin can be any Msp porin described herein. For example, the Msp porin can be selected from a wild-type MspA porin, a mutant MspA porin, a wild-type MspA paralog or homolog porin, and a mutant MspA paralog or homolog porin.

In any of the embodiments herein, the Msp porin may further comprise a molecular motor. The molecular motor may be capable of moving the analyte into or through the channel at a translocation velocity or average translocation velocity that is less than the translocation velocity or average translocation velocity of the analyte into or through the channel by electrophoresis in the absence of the molecular motor. Thus, in any of the embodiments herein comprising applying an electric field, the electric field may be sufficient to cause the analyte to be translocated through the channel by electrophoresis.

Any liquid medium described herein, such as a conductive liquid medium, may contain an analyte. The analyte may be any analyte described herein. Embodiments herein may also include detecting the analyte in, for example, a method comprising measuring an ionic current when the analyte interacts with an Msp porin channel to provide a current pattern, wherein the presence of a blockage in the current pattern indicates the presence of the analyte.

Optionally, the Msp porin is a mutant MspA or mutant MspA paralog or homolog porin, and the translocation rate or average translocation rate of the analyte through the porin channel is less than or greater than the translocation rate or average translocation rate of the analyte through the channel of wild-type MspA or a wild-type MspA paralog or homolog porin.

In any of the embodiments herein, the analyte may have a translocation velocity or average translocation velocity through the channel of less than 0.5 nm/μs. Optionally, the analyte may have a translocation velocity or average translocation velocity through the channel of less than 0.05 nm/μs.

Any of the Msp porins described herein may be contained in a lipid bilayer. In such embodiments herein or any other embodiment, the Msp porin may have a cis side and a trans side. Optionally, the analyte is translocated from the cis side through the channel to the trans side by electrophoresis or other means. Optionally, the analyte is translocated from the trans side through the channel to the cis side by electrophoresis or other means. Optionally, the analyte is driven from the cis side or the trans side into the channel by electrophoresis or other means and remains in the channel or then retreats back to the cis side or the trans side, respectively.

Any of the embodiments herein may further comprise identifying the analyte.Such methods may comprise comparing the current pattern obtained for the unknown analyte with a known current pattern obtained under the same conditions using a known analyte.

In any of the embodiments herein, the analyte can be a nucleotide, a nucleic acid, an amino acid, a peptide, a protein, a polymer, a drug, an ion, a pollutant, a nanoscale object, or a biological warfare agent. Optionally, the analyte is a polymer such as a protein, a peptide, or a nucleic acid. Optionally, the analyte is a nucleic acid. Optionally, the nucleic acid has an average translocation velocity through the channel of less than 1 nucleotide/μs. Optionally, the nucleic acid has a translocation velocity or average translocation velocity through the channel of less than 0.1 nucleotide/μs. The nucleic acid can be ssDNA, dsDNA, RNA, or a combination thereof.

Embodiments herein may include distinguishing at least a first unit within a polymer from at least a second unit within the polymer. The distinguishing may include measuring ionic currents generated when the first and second units are respectively translocated through a channel to generate first and second current patterns, respectively, wherein the first and second current patterns are different from each other.

Any of the embodiments herein may further comprise determining the sequence of the polymer. Sequencing may comprise measuring the ionic current pattern or optical signal as each unit of the polymer is separately translocated through the channel to provide a current pattern associated with each unit, and comparing each current pattern with a current pattern of a known unit obtained under the same conditions to determine the sequence of the polymer.

Any embodiment herein may also include determining the concentration, size, molecular weight, shape or orientation of an analyte, or any combination thereof. Any liquid medium described herein, such as a conductive liquid medium, may contain a variety of analytes. Any analyte described herein may include optical beads or magnetic beads.

Any of the Msp porin proteins discussed herein can also be further identified as mutant MspA porin proteins. The mutant MspA porin can include: a vestibule and a constriction region defining a channel, and at least a first mutant MspA monomer comprising a mutation at position 93, position 91, position 90, or any combination thereof. The mutant MspA porin can comprise mutations at positions 93 and 91, positions 93 and 90, positions 91 and 90, or positions 93, 90, and 91. Optionally, the mutant MspA porin comprises one or more mutations at any of the following amino acid positions: 88, 105, 108, 118, 134, or 139, or any other mutations described herein.

In any of the embodiments herein, the diameter of the mutant MspA porin or mutant MspA paralog or homolog may be smaller than the diameter of the constriction of the corresponding wild-type MspA porin or wild-type MspA paralog or homolog. The mutant MspA porin or mutant MspA paralog or homolog may have a mutation in the vestibule or constriction that allows an analyte to electrophoretically translocate or otherwise pass through the channel of the mutant MspA porin or mutant MspA paralog or homolog at a rate or average translocation rate that is less than the rate or average translocation rate of the analyte when translocated through the channel of the wild-type Msp porin or wild-type MspA paralog or homolog.

A mutated Msp porin, such as a mutated MspA porin or a mutated MspA paralog or homolog porin, may comprise a neutral constriction zone. A mutated Msp porin, such as a mutated MspA porin or a mutated MspA paralog or homolog porin, may have a conductance through the channel that is higher (e.g., 2-fold higher) than the conductance through the channel of its corresponding wild-type Msp porin. A mutated Msp porin (e.g., a mutated MspA porin or a mutated MspA paralog or mutated porin) may comprise a conductance through the channel that is lower than the conductance through the channel of its corresponding wild-type Msp porin.

Any of the Msp porins discussed herein can comprise a vestibule having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and a constriction having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm, wherein the vestibule and constriction together define a channel. Also provided herein are mutant MspA porins comprising a vestibule having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and a constriction having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm, wherein the vestibule and constriction together define a channel, and further comprising at least a first mutant MspA paralog or homolog monomer.

The diameter of the constriction zone of a mutant Msp porin (e.g., a mutant MspA porin or a mutant MspA paralog or homolog) can be smaller than the diameter of the constriction zone of its corresponding wild-type Msp porin (e.g., a wild-type MspA porin or a wild-type MspA paralog or homolog). A mutant Msp porin (e.g., a mutant MspA porin or a mutant MspA paralog or homolog) can comprise a mutation in the vestibule or constriction zone that allows an analyte to be translocated, electrophoretically or otherwise, through the channel of the porin at a rate or average translocation rate that is less than the rate or average translocation rate of the analyte when translocated through the channel of its corresponding wild-type Msp porin (e.g., a wild-type MspA porin, a wild-type MspA paralog or homolog).

Optionally, the Msp porin is encoded in whole or in part by a nucleic acid sequence encoding a partial or complete single-chain Msp porin, wherein the nucleic acid sequence comprises: (a) a first and a second nucleotide sequence, 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 connector sequence. The monomer sequence can be any monomer sequence described herein. Optionally, the first and second Msp monomer sequences are independently selected from wild-type MspA monomer, wild-type MspB monomer, wild-type MspC monomer, wild-type MspD monomer and a mutant thereof. Optionally, the first Msp monomer sequence comprises a wild-type MspA monomer or a mutant thereof. Optionally, the first Msp monomer sequence comprises a mutated MspA monomer.

In any embodiment herein, the Msp porin may be encoded in whole or in part by a nucleic acid sequence encoding a partial or complete single-chain Msp porin, wherein the nucleic acid sequence comprises: (a) the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences or any subset thereof, wherein the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences encode the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th Msp monomer sequences, respectively; and (b) a 9th nucleotide sequence encoding an amino acid linker sequence. Thus, the porin may comprise one or more partial single-chain Msp porins that hybridize, dimerize, trimerize, etc. with other Msp monomers or other partial single-chain Msp porins. Alternatively, the complete single-chain Msp porin may form a porin without being bound to other Msp elements. In any of the embodiments herein, for example, the Msp porin can be encoded by a nucleic acid sequence encoding a complete single-chain Msp porin, wherein the nucleic acid sequence comprises: (a) a 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, and 8th nucleotide sequence, wherein the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, and 8th nucleotide sequence encodes a 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, and 8th Msp monomer sequence, respectively; and (b)

The 9th nucleotide sequence encoding the amino acid linker sequence. Each Msp monomer may comprise a wild-type MspA monomer or a mutant thereof. Optionally, at least one Msp monomer comprises a wild-type MspA monomer or a mutant thereof. Thus, the porin can be fully encoded.

In any of the embodiments herein, the Msp monomer can be a wild-type MspA paralog or homolog, such as MspA/Msmeg0965, MspB/Msmeg0520, MspC/Msmeg5483, MspD/Msmeg6057, MppA, PorM1, PorM2, PorM1, Mmcs4296, Mmcs4297, Mmcs3857, Mmcs4382, Mmcs4383, Mjls3843, Mjls3857, Mjls3931, Mjls4674, Mjls4675, Mjls 4677, Map3123c, Mav3943, Mvan1836, Mvan4117, Mvan4839, Mvan4840, Mvan5016, Mvan5017, Mvan5768, MUL_2391, Mflv1734, Mflv1 735, Mflv2295, Mflv1891, MCH4691c, MCH4689c, MCH4690c, MAB1080, MAB1081, MAB2800, RHA1ro08561, RHA1ro04074, and RHA1ro03127.

Also provided herein are methods for modifying the conductance of a channel through an Msp porin comprising removing, adding, or substituting at least one amino acid in the vestibule or constriction of a wild-type Msp porin. For example, the method may include increasing the conductance. The method may include decreasing the conductance.

Also provided are methods comprising translocating an analyte through a channel of an Msp porin without applying an electric field. In this embodiment or any other embodiment herein, the Msp porin may further comprise a molecular motor. The Msp porin may be any Msp porin described herein, such as a wild-type MspA porin, a mutant MspA porin, a wild-type MspA paralog or homolog porin, and a mutant MspA paralog or homolog porin. The Msp porin may be encoded by a nucleic acid sequence encoding a single-chain Msp porin.

Also provided are systems comprising an Msp porin having a vestibule and a constriction defining a channel, wherein the channel is located between a first liquid medium and a second liquid medium, wherein at least one of the liquid media comprises an analyte, and wherein the system is effective for detecting a property of the analyte. The system can be effective for detecting a property of any analyte, including subjecting the Msp porin to an electric field so that the analyte interacts with the Msp porin. The system is effective for detecting a property of an analyte, including subjecting the Msp porin to an electric field so that the analyte is electrophoretically translocated through the channel of the Msp porin. Also provided are systems comprising an Msp porin having a vestibule and a constriction defining a channel, wherein the channel is located in a lipid bilayer between the first liquid medium and the second liquid medium, and wherein the only point of liquid communication between the first and second liquid media is in the channel. In addition, any Msp porin described herein can be included in any system described herein.

The first and second liquid media can be the same or different, and either or both liquid media can contain one or more of the following: a salt, a detergent, or a buffer. In fact, any liquid medium described herein can contain one or more of the following: a salt, a detergent, or a buffer. Optionally, at least one liquid medium is conductive. Optionally, at least one liquid medium is non-conductive. Any liquid medium described herein can contain a substance that changes viscosity or a substance that changes rate. The liquid medium can contain any analyte described herein. The properties of the analyte can be electrical, chemical, or physical properties.

The Msp porin may be comprised in a lipid bilayer in the system or any other embodiment described herein.The system may comprise a plurality of Msp porins.

The system may comprise any Msp porin described herein, such as a wild-type MspA porin, a mutant MspA porin, a wild-type MspA paralog or homolog porin, or a mutant MspA paralog or homolog porin. Optionally, the Msp porin is further identified as a mutant MspA porin. The system may comprise a mutant Msp porin comprising a vestibule and a constriction defining a channel and at least a first mutant MspA monomer (which comprises a mutation at position 93 and a mutation at position 90, position 91, or positions 90 and 91). The mutant Msp porin included in the system may comprise a vestibule having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and a constriction having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm, wherein the vestibule and constriction together define a channel. The mutant MspA porin may also comprise at least a first mutant MspA paralog or homolog monomer. The Msp porin included in the system can be encoded by a nucleic acid sequence encoding a single-chain Msp porin.

The Msp porin included in the system may further comprise a molecular motor. The molecular motor in the system herein or any other embodiment may be capable of moving an analyte into or through the channel at a translocation velocity or average translocation velocity that is less than the translocation velocity or average translocation velocity of the analyte into or through the channel in the absence of the molecular motor.

Any of the systems described herein may further include a patch clamp amplifier or a data acquisition device.The system may further include one or more temperature regulation devices in communication with the first liquid medium, the second liquid medium, or both.

Any of the systems described herein are effective for translocating an analyte through an Msp porin channel, electrophoretically or otherwise.

Also provided are Msp porins comprising an antechamber having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and a constriction having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm, wherein the antechamber and constriction together define a channel. Also provided are mutant Msp porins comprising an antechamber having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and a constriction having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm, wherein the antechamber and constriction together define a channel. Also provided are mutant MspA porins comprising an antechamber having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and a constriction having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm, wherein the antechamber and constriction together define a channel. Also provide the MspA paralogue or homologue porin of sudden change, it comprises the antechamber with the length of about 2 to about 6nm and the diameter of about 2 to about 6nm, and the constriction zone with the length of about 0.3 to about 3nm and the diameter of about 0.3 to about 3nm, wherein said antechamber defines passage together with constriction zone.The MspA paralogue or homologue of any sudden change described herein can also comprise the MspA paralogue or homologue monomer of at least the first sudden change.Also provide the MspA porin of sudden change, it comprises the antechamber with the length of about 2 to about 6nm and the diameter of about 2 to about 6nm, and the constriction zone with the length of about 0.3 to about 3nm and the diameter of about 0.3 to about 3nm, wherein said antechamber defines passage together with constriction zone, and also comprises the MspA paralogue or homologue monomer of at least the first sudden change.Any of these porins can be used for any embodiment herein.

Also provided are mutant MspA porins comprising a vestibule and a constriction region defining a channel, and at least a first mutant MspA monomer comprising a mutation at position 93 and a mutation at position 90, position 91, or positions 90 and 91. This mutant MspA porin and any other mutant Msp porin or MspA porin described herein can be used in any of the embodiments described herein. The mutant MspA porin can comprise mutations at positions 93 and 90. The mutant MspA porin can comprise mutations at positions 93 and 91. The mutant MspA porin can comprise mutations at positions 93, 91, and 90. The mutant MspA porin can comprise any other mutation described herein.

The diameter of the constriction of the mutant MspA porin can be smaller than the diameter of the constriction of the corresponding wild-type MspA porin. The MspA porin can have a mutation in the vestibule or constriction that allows the analyte to be translocated through the mutant's channel by electrophoresis or other means at a translocation rate or average translocation rate that is less than the translocation rate or average translocation rate of the analyte when translocating through the channel of the wild-type Msp porin. The MspA porin can have a mutation in the vestibule or constriction that allows the analyte to be translocated through the channel by electrophoresis, for example, 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 nucleotides, nucleic acids, amino acids, peptides, proteins, polymers, drugs, ions, biological warfare agents, pollutants, nanoscale objects, or combinations or clusters thereof. Optionally, the analyte is further determined to be a nucleic acid. The nucleic acid can be translocated through the channel by electrophoresis or other means at an average translocation velocity of less than 1 nucleotide/μs or less than 0.1 nucleotide/μs. The nucleic acid can be further identified as ssDNA, dsDNA, RNA, or a combination thereof.

The analyte in any of the embodiments herein may also include magnetic beads. Magnetic beads may be further defined as streptavidin-coated magnetic beads. The analyte may also include optical beads. Any analyte described herein may be ionic or neutral. The analyte may include biotin.

Any Msp porin described herein, such as the MspA porin of mutation, can comprise 2 to 15 identical or different Msp monomers. Optionally, the Msp porin, such as the MspA porin of mutation, comprises 7 to 9 identical or different Msp monomers. Optionally, at least the second monomer is selected from the MspA monomer of wild-type MspA monomer, the MspA monomer of the second mutation, the wild-type MspA paralogue or homologue monomer and the MspA paralogue or homologue monomer of mutation, wherein the MspA monomer of the second mutation can be identical or different with the MspA monomer of the first mutation. Optionally, the second monomer is a wild-type MspA paralogue or homologue monomer. The wild-type MspA paralogue or homologue monomer can be a wild-type MspB monomer. The MspA monomer can comprise one or more mutations at any of the following amino acid positions: 88, 105, 108, 118, 134 or 139. An MspA monomer may comprise one or more of the following mutations: L88W, D90K/N/Q/R, D91N/Q, D93N, I105W, N108W, D118R, D134R, or E139K. An MspA monomer may comprise the following mutations: D90N/D91N/D93N. An MspA monomer may comprise the following mutations: D90N/D91N/D93N/D118R/D134R/E139K. An MspA monomer may comprise the following mutations: D90Q/D91Q/D93N. An MspA monomer may comprise the following mutations: D90Q/D91Q/D93N/D118R/D134R/E139K. An MspA monomer may comprise the following mutations: D90(K,R)/D91N/D93N. The MspA monomer may comprise the following mutations: (L88, I105)W/D91Q/D93N. The MspA monomer may comprise the following mutations: I105W/N108W. In addition, the MspA monomer may comprise any other mutation described herein.

In any of the embodiments herein, the mutant Msp porin, e.g., a mutant MspA porin or a mutant MspA paralog or homolog, may comprise at least one additional positively charged amino acid compared to the vestibule or constriction region of the wild-type Msp porin, respectively; may comprise at least one additional negatively charged amino acid compared to the vestibule or constriction region of the wild-type MspA porin, respectively; may comprise at least one less positively charged amino acid compared to the vestibule or constriction region of the wild-type MspA porin, respectively; or may comprise at least one less negatively charged amino acid compared to the vestibule or constriction region of the wild-type MspA porin, respectively.

Optionally, each positively charged amino acid in the vestibule and constriction region of the wild-type Msp porin can be replaced with a negatively charged amino acid, and each negatively charged amino acid is the same or different; or each negatively charged amino acid in the vestibule and constriction region of the wild-type Msp porin can be replaced with a positively charged amino acid, and each positively charged amino acid is the same or different.

Optionally, the vestibule or constriction region of the mutant Msp porin comprises a greater number of positively charged residues than the positively charged residues in the vestibule or constriction region, respectively, of the wild-type Msp porin; or the vestibule or constriction region comprises a greater number of negatively charged residues than the negatively charged residues in the vestibule or constriction region, respectively, of the wild-type Msp porin; or at least one positively charged amino acid in the vestibule or constriction region of the wild-type Msp porin (e.g., a wild-type MspA porin or a wild-type MspA paralog or homolog porin) is deleted or substituted with a negatively charged amino acid; or at least one negatively charged amino acid in the vestibule or constriction region of the wild-type Msp porin is deleted or substituted with a positively charged amino acid.

At least one amino acid in the vestibule or constriction region of a wild-type Msp porin (e.g., a wild-type MspA porin or a wild-type MspA paralog or homolog porin) can be replaced with an amino acid having a sterically larger side chain, an amino acid having a sterically smaller side chain, an amino acid having a more polar side chain, an amino acid having a less polar side chain, or an amino acid having a more hydrophobic side chain, an amino acid having a less hydrophobic side chain.

In any of the embodiments herein, at least one amino acid in the vestibule or constriction region of the mutant Msp porin can include an unnatural amino acid or a chemically modified amino acid.

Any of the Msp porins described herein may comprise deletions, additions, or substitutions of one or more periplasmic loops.

As described herein, any Msp porin, such as a mutant MspA porin, can also comprise a molecular motor. Any molecular motor described herein can be capable of moving an analyte into or through a channel at a translocation velocity or average translocation velocity that is less than the translocation velocity or average translocation velocity of the analyte when translocated into or through the channel in the absence of the molecular motor. In any of the embodiments herein, the molecular motor can be an enzyme, such as a polymerase, an exonuclease, or a Klenow fragment.

Also provided are methods for producing the Msp porins described herein. Thus, methods for producing mutant MspA porins comprising at least one mutant MspA monomer are provided, the methods comprising modifying a wild-type MspA monomer at positions 93 and 90, position 91, or positions 90 and 91. The methods may comprise modifying a wild-type MspA monomer at positions 93 and 90. The methods may comprise modifying a wild-type MspA monomer at positions 93 and 91. The methods may comprise modifying a wild-type MspA monomer at positions 93, 91, and 90. The methods may further or alternatively comprise modifying a wild-type MspA monomer at any one or more of the following positions: 88, 105, 108, 118, 134, or 139, or performing any other modifications described herein. The mutant MspA porins that can be produced by the methods described herein may include any of the mutations or porin properties described herein. For example, the mutant MspA may include a neutral constriction zone. The mutant MspA porin can also include at least one Msp monomer, such as a wild-type MspA monomer, a mutant MspA monomer, a wild-type MspA paralog or homolog, or a second mutant MspA paralog or homolog monomer. The conductance through the channel of the mutant MspA porin can be higher, such as 1-fold higher, than the conductance through the channel of its corresponding wild-type MspA porin.

Any mutant Msp porin described herein, such as a mutant MspA porin or a mutant MspA paralog or homolog porin, may comprise one or more mutant MspB, mutant MspC, or mutant MspD monomers, or combinations thereof.

Also provided are methods for generating mutant MspA porins having a vestibule and a constriction region defining a channel, comprising deleting, adding, or substituting any amino acid within the vestibule and constriction region of a wild-type MspA paralog or homolog monomer, such that the resulting mutant MspA porin is capable of translocating an analyte through the channel upon application of an electric field. The mutant MspA porin can be any type described herein.

Also provided are nucleic acid sequences encoding the Msp porins described herein. For example, nucleic acid sequences encoding mutant MspA porins or mutant MspA paralogs or homologs are provided. Also provided are vectors comprising the nucleic acid sequences described herein, for example, vectors comprising nucleic acid sequences encoding mutant MspA porins or mutant MspA paralogs or homologs. Any of the vectors described herein may further comprise a promoter sequence. Any of the vectors described herein may further comprise a constitutive promoter. The constitutive promoter may include the _psmyc promoter. The promoter may include an inducible promoter. The inducible promoter may include an acetamide-inducible promoter.

Also provided are cultured cells transfected with any of the vectors described herein, or progeny thereof, wherein the cells are capable of expressing an Msp porin, such as a mutant MspA porin or a mutant MspA paralog or homolog.

Also provided are Mycobacterium smegmatis strains comprising any of the vectors described herein. Also provided are Mycobacterium smegmatis strains that are free of endogenous porins, which may comprise any of the vectors described herein. "Free" means that endogenous porins cannot be detected on immunoblot using an appropriate Msp-specific antiserum, or that contain less than 1% endogenous porins.

Also provided are vectors comprising a nucleic acid sequence encoding a wild-type Msp monomer, wherein the nucleic acid sequence is effectively controlled by an inducible promoter. The vector can be an integrating vector. Also provided are cultured cells or their progeny transfected with the vector, wherein the cells are capable of expressing the wild-type Msp porin. Also provided are Mycobacterium smegmatis strains comprising the vector.

Also provided is a nucleic acid sequence encoding a partial or complete single-chain Msp porin described herein.Described nucleic acid sequence may include, for example: (a) a first and a second nucleotide sequence, wherein the first nucleotide sequence encodes the first Msp monomer sequence and the second nucleotide sequence encodes the second Msp monomer sequence; and (b) a third nucleotide sequence encoding an amino acid connector sequence.Described first and second Msp monomer sequences may be independently selected from wild-type MspA monomer, the MspA monomer of mutation, wild-type MspA paralogue or homologue monomer and the MspA paralogue or homologue monomer of mutation.Described first Msp monomer sequence comprises wild-type MspA monomer or its mutant.Optionally, described first Msp monomer sequence comprises the MspA monomer of mutation. The first Msp monomer sequence may comprise one or more mutations selected from the group consisting of an A to P substitution at amino acid 138, an E to A or K substitution at amino acid 139, a D to K or R or Q substitution at amino acid 90, a D to N or Q substitution at amino acid 91, a D to N substitution at amino acid 93, an L to W substitution at amino acid 88, an I to W substitution at amino acid 105, an N to W substitution at amino acid 108, a D to R substitution at amino acid 118, and a D to R substitution at amino acid 134. Indeed, any of the Msp monomers described herein may comprise any such substitutions.

Optionally, the mutant MspA monomer comprises an A to P substitution at amino acid 138, an E to A substitution at amino acid 139, or a combination thereof; a D to K or R substitution at amino acid 90, a D to N substitution at amino acid 91, a D to N substitution at amino acid 93, or any combination thereof; a D to Q substitution at amino acid 90, a D to Q substitution at amino acid 91, a D to N substitution at amino acid 93, or any combination thereof; an L to W substitution at amino acid 88, an I to W substitution at amino acid 105, a D to Q substitution at amino acid 91, a D to N substitution at amino acid 93, or any combination thereof; an I to W substitution at amino acid 105, an N to W substitution at amino acid 108, or a combination thereof; or a D to R substitution at amino acid 118, an E to K substitution at amino acid 139, a D to R substitution at amino acid 134, or any combination thereof.

Any Msp porin may comprise a first, second or more Msp monomer sequence, including a wild-type MspA paralog or a mutant thereof, wherein the paralog or mutant thereof is a wild-type MspB monomer or a mutant thereof. One or more Msp monomer sequences may comprise SEQ ID NO: 1, SEQ ID NO: 2 or a combination thereof. Optionally, the second Msp monomer sequence comprises a mutated MspB monomer. Optionally, the first Msp monomer sequence comprises a wild-type MspA monomer or a mutant thereof and the second Msp monomer sequence comprises a wild-type MspB monomer or a mutant thereof. Optionally, the first Msp monomer sequence comprises SEQ ID NO: 1 and the second Msp monomer sequence comprises SEQ ID NO: 2.

Amino acid linker sequences are described herein. In any of the embodiments herein, the amino acid linker sequence can, for example, comprise 10 to 20 amino acids. For example, the amino acid linker comprises 15 amino acids. Optionally, the amino acid linker sequence comprises the (GGGGS) ₃ (SEQ ID NO: 3) peptide sequence.

The present invention also encompasses polypeptides encoded by any of the nucleic acid sequences described herein.

Also provided is a nucleic acid sequence encoding a partial or complete single-chain Msp porin, wherein the nucleic acid sequence comprises: (a) the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences or any subset thereof, wherein the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences encode the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th Msp monomer sequences, respectively; and (b) a 9th nucleotide sequence encoding an amino acid connector sequence. The first and second Msp monomer sequences can be independently selected from wild-type Msp monomers, mutated Msp monomers, wild-type MspA paralogs or homologs monomers, and mutated MspA paralogs or homologs monomers. Each Msp monomer can comprise a wild-type MspA monomer or a mutant thereof. Optionally, at least one Msp monomer comprises a wild-type MspA monomer or a mutant thereof. Optionally, at least one Msp monomer comprises a mutated MspA monomer. The mutated Msp monomer sequence can comprise any mutation described herein. For example, one or more of the mutations are selected from an A to P substitution on amino acid 138, an E to A or K substitution on amino acid 139, a D to K or R or Q substitution on amino acid 90, a D to N or Q substitution on amino acid 91, a D to N substitution on amino acid 93, an L to W substitution on amino acid 88, an I to W substitution on amino acid 105, an N to W substitution on amino acid 108, a D to R substitution on amino acid 118, and a D to R substitution on amino acid 134. Each Msp monomer sequence may comprise SEQ ID NO: 1. Optionally, at least one Msp monomer sequence comprises SEQ ID NO: 1. Optionally, at least one Msp monomer sequence comprises a wild-type MspA paralog or a mutant thereof, wherein the MspA paralog or a mutant thereof is a wild-type MspB monomer or a mutant thereof. Optionally, at least one Msp monomer sequence comprises SEQ ID NO: 2. Optionally, at least one Msp monomer sequence comprises a mutated MspB monomer. Optionally, at least one Msp monomer sequence comprises a wild-type MspA monomer or a mutant thereof and at least one Msp monomer sequence comprises a wild-type MspB monomer or a mutant thereof. Optionally, at least one Msp monomer sequence comprises SEQ ID NO:1 and at least one Msp monomer sequence comprises SEQ ID NO:2. Also provided are polypeptides encoded by any of the aforementioned nucleic acid sequences. Also provided are vectors comprising any of the aforementioned nucleic acid sequences. The vectors may further comprise a promoter sequence. The promoter may comprise a constitutive promoter. The constitutive promoter may comprise a _psmyc promoter. The promoter may comprise an inducible promoter. The inducible promoter may comprise an acetamide inducible promoter.

Also provided are mutant bacterial strains capable of inducible expression of Msp, comprising: (a) a deletion of wild-type MspA; (b) a deletion of wild-type MspC; (c) a deletion of wild-type MspD; and (d) a vector comprising an inducible promoter operatively linked to an Msp monomer nucleic acid sequence. The bacterial strain may also include Mycobacterium smegmatis strain ML16. The Msp nucleic acid may encode a wild-type MspA monomer or a wild-type MspA paralog or homolog monomer. The Msp nucleic acid may encode an Msp monomer selected from a wild-type MspA monomer, a wild-type MspC monomer, and a wild-type MspD monomer. Optionally, the Msp nucleic acid encodes a wild-type MspA monomer. The inducible promoter may comprise an acetamide-inducible promoter. The bacterial strain may also comprise a deletion of wild-type MspB. The bacterial strain may also comprise a vector described herein, for example, a vector comprising a constitutive promoter operatively linked to a nucleic acid sequence encoding an Msp porin or monomer. Msp can be a wild-type MspA porin or monomer or a wild-type MspA paralog or homolog porin or monomer. The Msp porin or monomer can be selected from a wild-type MspA porin or monomer, a wild-type MspB porin or monomer, a wild-type MspC porin or monomer, and a wild-type MspD porin or monomer. Optionally, the Msp porin or monomer is a wild-type MspA porin or monomer.

The bacterial strain may also comprise a vector comprising a nucleic acid encoding a complete or partial single-chain Msp porin, wherein the nucleic acid comprises: (a) a first and a second nucleotide sequence, 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. The bacterial strain may also comprise a vector comprising a nucleic acid encoding a complete or partial single-chain Msp porin, wherein the nucleic acid comprises: (a) the first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences or any subset 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.

Also provided is a method for producing a complete or partial single-chain Msp porin, the method comprising: (a) transforming a bacterial strain described herein with a vector containing a nucleic acid sequence capable of encoding a complete or partial single-chain Msp porin; and (b) purifying the complete or partial single-chain Msp porin from the bacteria. The vector may comprise a nucleic acid sequence encoding a complete or partial single-chain Msp porin, wherein the nucleic acid sequence comprises: (a) a first and a second nucleotide sequence, 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. The vector may comprise a nucleic acid sequence encoding a complete or partial single-chain Msp porin, wherein the nucleic acid sequence comprises: (a) the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences or any subset thereof, wherein the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences encode the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th Msp monomer sequences, respectively; and (b) a 9th nucleotide sequence encoding an amino acid connector sequence. The Msp monomer sequence can be independently selected from a wild-type MspA monomer, a mutated MspA monomer, a wild-type MspA paralog or homolog monomer, and a mutated MspA paralog or homolog monomer. For example, the Msp monomer sequence is a wild-type MspA monomer.

"Mycobacterium smegmatis porin (Msp)" or "Msp porin" refers to a multimeric complex composed of two or more Msp monomers. Msp monomers are encoded by genes in Mycobacterium smegmatis. Mycobacterium smegmatis has four identified Msp genes, referred to as MspA, MspB, MspC and MspD. The Msp porin can, for example, be composed of a wild-type MspA monomer, a mutated MspA monomer, a wild-type MspA paralog or homolog monomer, or a mutated MspA paralog or homolog monomer. Optionally, the Msp porin is a single-chain Msp porin or a multimer of several single-chain Msp porins. A single-chain Msp porin can, for example, comprise a multimer of two or more Msp monomers (e.g., 8 monomers) connected by one or more amino acid linker peptides. A partially single-chain Msp porin refers to a single-chain multimeric complex that must dimerize, trimerize, etc. to form a porin. A completely single-chain Msp porin refers to a porin that forms without dimerization, trimerization, etc. to form a single-chain multimeric complex of the porin.

The Msp porin in any embodiment herein can be any Msp porin described herein, such as a wild-type MspA porin, a mutant MspA porin, a wild-type MspA paralog or homolog porin, or a mutant MspA paralog or homolog porin. The Msp porin can be encoded by a nucleic acid sequence encoding a single-chain Msp porin. Any Msp porin herein can comprise any Msp monomer described herein, such as a mutant Msp monomer.

Nutrients pass through wild-type porins in mycobacteria. Wild-type MspA porin, wild-type MspB porin, wild-type MspC porin, and wild-type MspD porin are examples of wild-type channel-forming porins. Msp porins can be further identified as any Msp porin described herein, including paralogs, homologs, mutants, and single-chain porins.

A "mutated MspA porin" is a multimeric complex that is at least or at most 70, 75, 80, 85, 90, 95, 98, or 99% or more, or any range therebetween, but less than 100%, identical to its corresponding wild-type MspA porin and retains channel-forming ability. The mutated MspA porin can be a recombinant protein. Optionally, the mutated MspA porin is a porin that has a mutation in the constriction or vestibule of the wild-type MspA porin. Optionally, the mutation can be present at the edge or exterior of the periplasmic loop of the wild-type MspA porin. The mutated MspA porin can be used in any of the embodiments described herein.

Exemplary wild-type MspA paralogs and homologs are provided in Table 1. Wild-type MspA paralogs are provided, including wild-type MspB, wild-type MspC, and wild-type MspD. "Paralogs," as defined herein, are genes with similar structure and function from the same bacterial species. "Homologs," as defined herein, are genes with similar structure and evolutionary origin from another bacterial species. For example, wild-type MspA homologs are provided, including MppA, PorM1, PorM2, PorM1, and Mmcs4296.

A "mutated MspA paralog or homolog porin" is a multimeric complex that has at least or at most 70, 75, 80, 85, 90, 95, 98, or 99% or more, or any range therebetween, but less than 100%, identity to its corresponding wild-type MspA paralog or homolog porin and retains channel-forming ability. The mutated MspA paralog or homolog porin can be a recombinant protein. Optionally, the mutated MspA paralog or homolog porin is a porin that has a mutation in the constriction or vestibule of the wild-type MspA paralog or homolog porin. Optionally, the mutation may be present at the edge or exterior of the periplasmic loop of the wild-type MspA paralog or homolog porin. Any mutated MspA paralog or homolog porin can be used in any embodiment described herein and can comprise any mutation described herein.

The Msp porin may comprise two or more Msp monomers. An "Msp monomer" is a protein monomer that is a wild-type MspA monomer, a mutant MspA monomer, a wild-type MspA paralog or homolog monomer, or a mutant MspA paralog or homolog monomer, and that maintains channel-forming ability when combined with one or more other Msp monomers. Any Msp porin described herein may comprise one or more any Msp monomers described herein. Any Msp porin may comprise, for example, 2 to 15 Msp monomers, each of which may be the same or different.

"MspA monomer of mutation" refers to an Msp monomer that has at least or at most 70, 75, 80, 85, 90, 95, 98 or 99% or more, or any range therebetween, but is less than 100% identity to a wild-type MspA monomer and that maintains the ability to form a channel when combined with one or more other Msp monomers. Optionally, the MspA monomer of mutation is further defined as comprising a mutation in the sequence portion that promotes the formation of the vestibule or constriction zone of a fully formed channel-forming porin. The Msp monomer of mutation can be, for example, a recombinant protein. The MspA monomer of mutation can comprise any mutation described herein.

" the MspA paralogue of sudden change or homologue monomer " refer to and wild-type MspA paralogue or homologue monomer have at least or at the most 70,75,80,85,90,95,98 or 99% or more, or can be from any scope therebetween, but be less than 100% homogeneity and keep the MspA paralogue or homologue monomer of passage forming ability.Optionally, the MspA paralogue of sudden change or homologue monomer are further defined as comprising sudden change at this part of sequence, and described part promotes the formation of the vestibule and/or constriction zone of fully formed passage formation porin. The MspA paralogue of sudden change or homologue monomer can for example be recombinant protein. The MspA paralogue of any sudden change or homologue monomer can optionally be used for any embodiment herein.

The Msp porin can be expressed as a combination of two or more wild-type MspA monomers, mutant MspA monomers, wild-type MspA paralogs or homologs monomers or mutant MspA paralogs or homologs monomers. Thus, the Msp porin can be or can include a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, etc. For example, the Msp porin can include a combination of a wild-type MspA monomer and a wild-type MspB monomer. The Msp porin can include 1 to 15 monomers, each of which is the same or different. In fact, any Msp porin described herein can include at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 monomers, or a number that can be from any range therebetween, each of which is the same or different. For example, the Msp porin can include one or more identical or different mutant MspA monomers. As another example, an Msp porin can comprise at least one mutant MspA monomer and at least one MspA paralog or homolog monomer.

As defined above, a single-chain Msp porin comprises two or more Msp monomers connected by one or more amino acid linker peptides. A single-chain Msp porin comprising two Msp monomers (wherein the Msp monomers are connected by an amino acid linker sequence) can be referred to as a single-chain Msp porin dimer. A single-chain Msp porin comprising eight Msp monomers (wherein the Msp monomers are connected by an amino acid linker sequence) can be referred to as a single-chain Msp porin octamer. A single-chain Msp porin can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more Msp monomers connected by an amino acid linker sequence or can be derived from any range of Msp monomers therein. Optionally, the single-chain Msp porin may, for example, include two or more single-chain Msp porin dimers, two or more single-chain Msp porin trimers, two or more single-chain Msp porin tetramers, two or more single-chain Msp porin pentamers, one or more single-chain Msp porin hexamers, one or more single-chain Msp porin heptamers, one or more single-chain Msp porin octamers, or a combination thereof. For example, a single-chain Msp porin may include one single-chain Msp porin dimer and two single-chain Msp porin trimers. As another example, a single-chain Msp porin may include one single-chain Msp porin tetramer and two single-chain Msp porin dimers.

The wild-type single-chain Msp porin is composed of wild-type Msp monomers. Optionally, one or more mutations in the single-chain Msp porin are present in the vestibule or constriction region of the single-chain Msp porin. The mutated single-chain Msp porin has at least one mutation (e.g., deletion, substitution, or addition) in the amino acid sequence of the periplasmic loop, vestibule, or constriction region compared to the wild-type single-chain Msp. Single-chain polymers can also form porins, wherein each single chain comprises 2, 3, 4, 5, 6, 7, or more Msp monomers.

Provided herein is the nucleotide sequence of coding Msp monomeric sequence and its mutant.For the MspA monomeric sequence of the mutation listed above, it is mature wild-type MspA monomeric sequence (SEQ ID NO:1) with reference to the MspA sequence.Each nucleotide sequence in the nucleotide sequence provided herein can for example comprise the MspA monomeric sequence of mutation.The limiting examples of the MspA sequence of mutation is provided in Table 7.Optionally, the MspA of mutation comprises the A on amino acid 138 to P displacement, the E on amino acid 139 to A displacement or its combination.Optionally, the MspA of mutation comprises the D on amino acid 90 to K or R displacement, the D on amino acid 91 to N displacement, the D on amino acid 93 to N displacement or its any combination.Optionally, the MspA of mutation comprises the D on amino acid 90 to Q displacement, the D on amino acid 91 to Q displacement, the D on amino acid 93 to N displacement or its any combination. Optionally, the mutated MspA comprises an L to W substitution at amino acid 88, an I to W substitution at amino acid 105, a D to Q substitution at amino acid 91, a D to N substitution at amino acid 93, or any combination thereof. Optionally, the mutated MspA comprises an I to W substitution at amino acid 105, an N to W substitution at amino acid 108, or a combination thereof; Optionally, the mutated MspA comprises a D to R substitution at amino acid 118, an E to K substitution at amino acid 139, a D to R substitution at amino acid 134, or any combination thereof. For the mutated MspB monomer sequences listed below, the reference MspB sequence is the mature wild-type MspB monomer sequence (SEQ ID NO: 2). Optionally, the mutated MspB comprises a D to K or R substitution at amino acid 90, a D to N substitution at amino acid 91, a D to N substitution at amino acid 93, or any combination thereof.

The monomeric sequence of the wild-type Msp discussed herein is disclosed in the GenBank at pubmed.gov on the World Wide Web, and this type of sequence and other sequences and each subsequence or fragment therein are incorporated herein by reference in their entirety. For example, the monomeric nucleotide and amino acid sequence of wild-type MspA can be found in GenBank accession numbers AJ001442 and CAB56052, respectively. The monomeric nucleotide and amino acid sequence of wild-type MspB can be found in GenBank accession numbers NC_008596.1 (from nucleotides 600086 to 600730) and YP_884932.1, respectively. The monomeric nucleotide and amino acid sequence of wild-type MspC can be found in GenBank accession numbers AJ299735 and CAC82509, respectively. The monomeric nucleotide and amino acid sequence of wild-type MspD can be found in GenBank accession numbers AJ300774 and CAC83628, respectively. Thus, provided are nucleotide sequences of MspA, MspB, MspC, and MspD monomers comprising a nucleotide sequence that is at least about 70, 75, 80, 85, 90, 95, 98, 99% or greater, or any range therebetween, identical to the nucleotide sequence of the aforementioned GenBank Accession No. Also provided are amino acid sequences of MspA, MspB, MspC, and MspD monomers ( FIG. 18 ), comprising an amino acid sequence that is at least about 70, 75, 80, 85, 90, 95, 98, 99% or greater, or any range therebetween, identical to the sequence of the aforementioned GenBank Accession No.

Also provide the aminoacid sequence of MspA paralogue and homologous monomer, described MspA paralogue and homologous monomer comprise and wild-type MspA paralogue or homologous monomer at least about 70,75,80,85,90,95,98,99% or larger, or can be derived from the aminoacid sequence of the homology in any scope thereof.Wild-type MspA paralogue and homologous monomer are well known in the art.Table 1 provides the non-limiting list of this type of paralogue and homologue:

Table 1. Wild-type MspA and wild-type MspA paralogs and homologous monomers

Only proteins with significant amino acid similarity over the full length of the protein were included.Data were obtained using the PSI-Blast algorithm (BLOSUM62 matrix) using the NIH GenBank database available on the World Wide Web at ncbi.nlm.nih.gov/blast/Blast.cgi.

n.d.: "undetermined"

*Stahl et al., Mol. Microbiol. 40:451 (2001)

**Dorner et al., Biochim. Biophys. Acta. 1667:47-55 (2004)

The peptides, polypeptides, monomers, polymers, proteins, etc. described herein can be further modified and changed as long as the desired function is maintained or enhanced. It should be understood that any known modification and derivative of the genes and proteins disclosed herein or a method for the modification and derivative that may be produced is by determining the modification and derivative based on the identity with a specific known sequence. Specifically disclosed are polypeptides having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identity with wild-type MspA and wild-type MspA paralogs or homologs (e.g., wild-type MspB, wild-type MspC, wild-type MspD, MppA, PorM1, Mmcs4296) and mutants provided herein.

Those skilled in the art will readily understand the method for measuring the identity of two polypeptides. For example, identity can be calculated after aligning two sequences (so that identity is at its highest level). For example, in order to measure the "percentage identity" of two amino acid sequences or two nucleic acid sequences, sequences are aligned for optimal comparison purposes (for example, for optimal comparison with a second amino acid or nucleic acid sequence, a gap can be introduced into the first amino acid or nucleic acid sequence). The amino acid residues or nucleotides positioned at the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percentage identity between two sequences is a function of the number of identical positions shared by each sequence (i.e., the total number of the number/position of identical positions (e.g., overlapping positions) x 100 of percentage identity = identical positions). In one embodiment, the two sequences are identical in length.

There are several methods for determining percent identity. One method can determine percent identity in the following manner. The target nucleic acid or amino acid sequence is compared to the identified nucleic acid or amino acid sequence using the BLAST 2 sequence (Bl2seq) program from a standalone version of BLASTZ, including BLASTN version 2.0.14 and BLASTP version 2.0.14. A standalone version of BLASTZ can be obtained from the U.S. government's National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov). Instructions for explaining how to use the Bl2seq program can be found in the help file that comes with BLASTZ.

Bl2seq uses the BLASTN or BLASTP algorithm to compare two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the following options can be set: set -i to the file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt); set -j to the file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); set -p to blastn; set -o to any desired file name (e.g., C:\output.txt); set -q to -1; set -r to 2; and keep all other options at their default settings. The following command will generate an output file containing the comparison between the two sequences: C:\Bl2seq -i c:\seq1.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2. If the target sequence shares homology with any portion of the identified sequence, the specified output file will provide such regions of homology as aligned sequences. If the target sequence does not share homology with any portion of the identified sequences, then the specified output file will not provide aligned sequences.

Once aligned, length is determined by counting the number of consecutive nucleotides in the alignment of the target sequence with the sequence from the identified sequence starting at any matching position and ending at any other matching position. A matching position is any position in the target sequence and the identified sequence that provide the same nucleotide. Gaps provided in the target sequence are not counted because gaps are not nucleotides. Similarly, gaps provided in the identified sequence are not counted because target sequence nucleotides are counted, not nucleotides from the identified sequence.

The percent identity over a particular length range can be determined by counting the number of positions that are matched over the length range and dividing that number by the length, and then multiplying the resulting value by 100. For example, if (1) a 50 nucleotide target sequence is compared to a sequence encoding wild-type MspA, (2) the Bl2seq program provides 45 nucleotides from the target sequence that align with a region of the sequence encoding wild-type MspA, where the first and last nucleotides of the 45 nucleotide region are matched, and (3) the number of matches over the 45 aligned nucleotide range is 40, then the 50 nucleotide target sequence comprises a length of 45 nucleotides and the percent identity over the length range is 89 (i.e., 40/45 x 100 = 89).

Another method for calculating identity can be performed by published algorithms. Optimal alignment of sequences for comparison can be performed using the local identity algorithm of Smith and Waterman, Adv. Appl. Math 2:482 (1981), using the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), using the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), using computerized versions of such algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or using visual inspection.

The same type of identity for nucleic acids can be obtained, for example, using algorithms disclosed in, for example, Zuker, Science 244:48-52 (1989); Jaeger et al., Proc. Natl. Acad. Sci. USA 86:7706-10 (1989); Jaeger et al., Methods Enzymol. 183:281-306 (1989), which are incorporated herein by reference for at least substantial relevance to nucleic acid alignments. It will be understood that generally any of the methods described can be used, and that in some cases the results of different such methods may differ, but one skilled in the art understands that if identity is found using at least one of such methods, then the sequence is considered to have the claimed identity and is disclosed herein.

Also disclosed are nucleic acids encoding the protein sequences disclosed herein, as well as variants and fragments thereof. Such sequences include all degenerate sequences related to a particular protein sequence, i.e., all nucleic acids having a sequence encoding a particular protein sequence, as well as all nucleic acids encoding disclosed variants and derivatives of said protein sequence, including degenerate nucleic acids. Thus, although each specific nucleic acid sequence may not be set forth herein, it should be understood that each sequence is in fact disclosed and described herein by the disclosed protein sequence.

Fragments and partial sequences of Msp porins or monomers can be used in the methods described herein. With respect to all peptides, polypeptides, and proteins, including fragments thereof, it will be understood that additional modifications in the amino acid sequence of the Msp polypeptides disclosed herein can be made without altering the properties or function of the peptides, polypeptides, and proteins. It will be understood that the only limitation to these modifications is practicality, and they must include the functional elements necessary for the relevant embodiments (e.g., channel forming ability). Such modifications include conservative amino acid substitutions and are described in more detail below.

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 inserts into the bilayer, as illustrated in Example 2 below: If the protein inserts into the bilayer, then the porin is a channel-forming protein. Typically, channel formation can be detected by observing discontinuous changes in conductance. See, for example, FIG2 , Example 2, and Niederweis et al., Mol. Microbiol. 33:933 (1999). Bilayers are described herein.

As indicated above, Msp porins are generally capable of being inserted into lipid bilayers or other membranes, each of which is well known in the art. Examples of inserting mutant MspA porins into lipid bilayers are explained herein; this technique is equally applicable to other Msp porins. In addition, U.S. Patent No. 6,746,594 (incorporated herein by reference) describes a variety of lipid bilayers and membranes, including inorganic materials, that can be used with the Msp porins described herein. The methods, devices, and techniques described in U.S. Patent No. 6,267,872 (incorporated herein by reference in its entirety) can also be used with the Msp porins described herein.

In addition, more than one Msp porin may be included in the lipid bilayer. For example, the lipid bilayer may contain 2, 3, 4, 5, 10, 20, 200, 2000, or more Msp porins. Optionally, any number of Msp porins in the range of 2 to 1010 may be used in the methods described herein. Such multiple Msp porins may be present in the form of clusters of Msp porins. Clusters may be randomly assembled or may adopt a specific pattern. As used herein, "cluster" refers to molecules that come together and move as a unit (but are not covalently bound to each other).

Optionally, the Msp porin does not gate spontaneously. "Gating" refers to a spontaneous change in the conductance of the channel through the protein that is typically short-lived (e.g., lasting as short as 1-10 milliseconds to as much as a second). Long-duration gating events can generally be reversed by changing polarity. In most cases, the probability of gating can be increased by applying a higher voltage. Gating and the degree of conduction through the channel are highly variable between Msp porins, depending on, for example, the composition of the vestibule and constriction and the properties of the liquid medium in which the protein is immersed. Typically, the protein's conductivity decreases during gating, and as a result, the conductance may cease permanently (i.e., the channel may close permanently), such that the process is irreversible. Optionally, gating refers to the conductance of the channel through the protein spontaneously changing to less than 75% of its open-state current.

Various conditions, such as light and the liquid medium contacting the Msp porin (including its pH, buffer composition, detergent composition, and temperature), can temporarily or permanently affect the behavior of the Msp porin, particularly its conductance through the channel and the movement of analytes relative to the channel.

Of particular relevance is the geometry of the channels of the Msp porins, particularly the MspA porins. The geometry of the Msp porins can provide improved spatial resolution. In addition, the wild-type MspA porin is very stable and retains channel-forming activity after exposure to any pH and after extraction at extreme temperatures (e.g., up to 100° C. for up to 30 minutes and incubation at up to 80° C. for up to 15 minutes). The polypeptides can be tested for their desired activities using the in vitro assays described herein.

Specifically for the MspA porin, optionally, the MspA porin is an octamer composed of eight 184 amino acid MspA monomers. One or more mutations can be generated in one or more amino acid MspA monomers of the wild-type MspA porin to generate a mutant MspA porin. Furthermore, the MspA porin can have fewer than or more than eight monomers, any one or more of which can contain a mutation.

In addition, the wild-type MspA porin contains a periplasmic loop consisting of 13 amino acids and located just adjacent to the constriction. See Huff et al., J. Biol. Chem. 284:10223 (2009). Wild-type MspB, C, and D porins also contain a periplasmic loop. One or more mutations may be present in the periplasmic loop of a wild-type Msp porin, thereby generating a mutant Msp porin. For example, deletions of up to all 13 amino acids may occur in the periplasmic loop of a wild-type MspA porin. Generally, deletions in the periplasmic loop do not affect the channel-forming ability of an Msp porin.

The Msp porin or Msp monomer can also be modified chemically or biologically. For example, the Msp porin or Msp monomer can be modified with chemicals to generate disulfide bridges, which are known to those skilled in the art.

The Msp porin may comprise a nucleotide binding site. As used herein, a "nucleotide binding site" refers to a location in the Msp porin where a nucleotide remains in contact with or on an amino acid for a longer period than due to diffusion (e.g., longer than 1 picosecond or 1 nanosecond). Molecular dynamics calculations can be used to estimate such short resting times.

The "vestibule" refers to the conical portion of the interior of the Msp porin, whose diameter generally decreases from one end to the other along the central axis, with the narrowest part of the vestibule connected to the constriction. The vestibule may also be referred to as a "goblet." For an example of the vestibule of the wild-type MspA porin, see Figure 1. Together, the vestibule and the constriction define the channel of the Msp porin.

When referring to the diameter of the antechamber, it is understood that because the antechamber is conical in shape, the diameter varies along the path of the central axis, with the diameter being larger at one end than the other. The diameter can range from about 2 nm to about 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 therebetween. The length range of central axis can be about 2nm to about 6nm.Optionally, 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 6.0nm, or can be from any scope therebetween.When mentioning " diameter " in this article, diameter can be determined by measuring center to center distance or atomic surface to surface distance.

The "constriction" is the narrowest part of the channel of the Msp porin in terms of diameter, which is connected to the vestibule. The constriction of the wild-type MspA porin is shown in Figure 1 (labeled "inner constriction"). 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 therebetween. 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 in any range therebetween.

A "neutral constriction" refers to a constriction comprising amino acid side chains that generally exhibit no net charge when immersed in an aqueous solution. The pH of the liquid medium (e.g., a buffered aqueous solution) in contact with the constriction can affect whether the constriction is characterized as neutral.

The "channel" refers to the central hollow portion of the Msp defined by the vestibule and constriction, through which gases, liquids, ions, or analytes can pass.

As used herein, "front side" refers to the side of an Msp channel through which analytes enter the channel or across whose surface analytes move.

As used herein, "reverse side" refers to the side of the Msp channel through which the analyte (or fragment thereof) exits the channel or the surface through which the analyte does not move.

As used herein, "translocation of an analyte by electrophoresis" and grammatical variations thereof refers to applying an electric field to an Msp porin in contact with (e.g., immersed in) one or more solutions so that current flows through an Msp porin channel. The electric field moves the analyte so that it interacts with the channel. "Interacting" means that the analyte moves into and, optionally, through the channel, where "through an Msp channel" (or "translocation") means entering one side of the channel and moving toward and out of the other side of the channel.

In particular, any analyte discussed herein can be translocated through the Msp porin channel by electrophoresis or other means in any embodiment described herein. In this regard, it is expressly mentioned that unless otherwise specified, any embodiment herein that includes translocation can refer to electrophoretic translocation or non-electrophoretic translocation. Optionally, methods that do not use electrophoretic translocation are also contemplated.

"Liquid media" include aqueous, organic-aqueous, and purely organic liquid 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. Descriptions and examples of such media, including conductive liquid media, are provided in, for example, U.S. Patent 7,189,503, which is incorporated herein by reference in its entirety. Salts, detergents, or buffers can be added to such media. Such reagents can be used to change the pH or ionic strength of the liquid medium. Viscosity-altering substances such as glycerol or various polymers (e.g., polyvinyl pyrrolidone, polyethylene glycol, polyvinyl alcohol, cellulosic polymers), and mixtures thereof, can be included in the liquid medium. Methods for measuring viscosity are well known in the art. Any reagent that can be added to the liquid medium can also change the speed of the analyte being studied. Thus, the speed-altering reagent can be a salt, detergent, buffer, a viscosity-altering substance, or any other substance added to the liquid medium that can increase or decrease the speed of the analyte.

Generally, the analyte used in this article is soluble or partially soluble in at least one liquid medium contacted with Msp described herein.Any analyte can be used herein, including for example nucleotides, nucleic acids, amino acids, peptides, proteins, polymers, drugs, ions, biological warfare agents, pollutants, nanoscale objects or any other molecule including one or a combination of such analytes. Analyte can be the clustering of molecules, because the clustering is considered to be analyte as a whole. Generally, the size of analyte will not be so large that it cannot enter the channel of Msp: in other words, classical analytes will be smaller than the opening of the channel of Msp in size. However, the analyte larger than the opening of channel can be used, and whether the method described herein can be used to determine analyte is too large so that it cannot enter channel. Optionally, the molecular weight of analyte is less than 1 million Da. Optionally, the analyte has a molecular weight of about, up to 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, 300 ,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 within any range therebetween.

Protein modification includes amino acid sequence modification. The modification of the amino acid sequence can be naturally produced as an allelic variant (for example, due to genetic polymorphism), can be produced due to environmental influences (for example, due to exposure to ultraviolet radiation), or can be produced due to human intervention (for example, by mutagenesis of cloned DNA sequences), such as induced point mutants, deletions, insertions and substitution mutants. Such modifications can lead to changes in the amino acid sequence, provide silent mutations, change restriction sites or provide other specific mutations. Amino acid sequence modifications generally fall into one or more of the following three categories: substitution, insertion or deletion modifications. Insertion includes insertion within the sequence of amino acids and/or terminal fusions and single or multiple amino acid residues. Insertion will generally be an insertion smaller than amino or carboxyl terminal fusions, for example, an insertion of 1 to 4 residues. Deletion can be characterized by removing one or more amino acid residues from the protein sequence. Generally, deletion is no more than about 2 to 6 residues at any one position within the protein molecule. Amino acid substitutions are typically substitutions of a single residue, but can occur at many different positions at once; insertions are typically insertions of about 1 to 10 amino acid residues; deletions can range from 1 to 30 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., deletions of 2 residues or insertions of 2 residues. Substitutions, deletions, insertions, or any combination thereof can be combined to obtain the final construct. Mutations may or may not occur in sequences outside the reading frame and may or may not produce complementary regions that can form mRNA secondary structure. Substitutional modifications are modifications in which at least one residue has been removed and a different residue inserted in its place.

Modifications can be made using known methods, including substitutions of specific amino acids. For example, modifications can be made by site-directed mutagenesis of nucleotides in the DNA encoding the protein, thereby generating DNA encoding the modification, which is then expressed in recombinant cell culture. Techniques for generating substitution mutations at predetermined positions in DNA with a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis.

One or more mutations in the Msp porin may be present in the vestibule or constriction region of the protein. Optionally, the mutant Msp porin has at least one difference (e.g., deletion, substitution, addition) in the amino acid sequence of its periplasmic loop, vestibule, or constriction region compared to the wild-type Msp porin.

As used herein, " amino acid " refers to any amino acid in the 20 kinds of naturally occurring amino acids found in protein, naturally occurring amino acid whose D-stereoisomer (for example, D-threonine), non-natural amino acids and chemically modified amino acid.Each type in these amino acid types is not mutually exclusive.Alpha-amino acid comprises the carbon atom bonded to amino, carboxyl, hydrogen atom and the unique group called " side chain ".Naturally occurring amino acid whose side chain is well known in the art and includes for example hydrogen (for example, as in glycine), alkyl (for example, as in alanine, valine, leucine, isoleucine, proline), substituted alkyl (for example, as in threonine, serine, methionine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine and lysine), aralkyl (for example, as in phenylalanine and tryptophan), substituted aralkyl (for example, as in tyrosine) and heteroaralkyl (for example, as in histidine).

The following abbreviations are used for the 20 naturally occurring amino acids: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamate (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

Unnatural amino acids (i.e., amino acids not naturally found in proteins) are also known in the art, as shown, 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 referred to herein as unnatural amino acids. The following table shows non-limiting examples of unnatural amino acids referred to herein.

Table 2. Exemplary unnatural amino acids

abbreviation. amino acids abbreviation. amino acids Aad 2-aminoadipic acid EtAsn N-ethylasparagine Baad 3-Aminoadipic acid Hyl Hydroxylysine Bala β-Alanine, β-aminopropionic acid AHyl allo-hydroxylysine Abu 2-aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu 4-aminobutyric acid, piperidinic acid 4Hyp 4-Hydroxyproline Acp 6-aminohexanoic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid AIle Allo-isoleucine Aib 2-aminoisobutyric acid MeGly N-methylglycine, sarcosine Baib 3-Aminoisobutyric acid MeiIle N-methylisoleucine Apm 2-aminopimelanoic acid MeLys 6-N-methyllysine Dbu 2,4-Diaminobutyric acid MeVal N-methylvaline

abbreviation. amino acids abbreviation. amino acids Des Desmosin Nva Norvaline Dpm 2,2'-Diaminopimelane Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGly N-ethylglycine

As used herein, "chemically modified amino acid" refers to an amino acid whose side chain has been chemically modified. For example, the side chain can be modified to include a signal-generating moiety such as a fluorophore or a radiolabel. The side chain can be modified to include a novel functional group such as a sulfhydryl, carboxylic acid, or amino group. Post-translationally modified amino acids are also included in the definition of chemically modified amino acids.

Amino acids, and more specifically their side chains, can be characterized by their chemical characteristics. For example, amino acid side chains can be positively charged, negatively charged, or neutral. The pH of a solution affects the charge properties of certain side chains, as is known to those skilled in the art. Non-limiting examples of positively charged side chains include histidine, arginine, and lysine. Non-limiting examples of negatively charged side chains include aspartic acid and glutamic acid. Non-limiting examples of neutral side chains include glycine, alanine, phenylalanine, valine, leucine, isoleucine, cysteine, asparagine, glutamine, serine, threonine, tyrosine, methionine, proline, and tryptophan.

Side chain stereochemistry can also be used to characterize amino acids. Atomic diameter tables can help determine whether one side chain is larger than another. Computer models can also help with this determination.

Amino acids can be characterized by the polarity of their side chains. Polar side chains (which are generally more hydrophilic than non-polar side chains) include, for example, the side chains of serine, threonine, tyrosine, cysteine, asparagine, and glutamine. Non-polar side chains (which are generally more hydrophobic than polar side chains) include, for example, the side chains of glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. The polarity of the side chain can be determined using conventional techniques known in the art involving atomic electronegativity determination and three-dimensional structure estimation of the side chain. The hydrophobicity/hydrophilicity of the side chain can also be compared using conventional techniques known in the art, for example, comparing the octanol/water partition coefficient of each amino acid. See Sangster, In: Octanol-Water Partition Coefficients: Fundamentals and Physical Chemistry, Wiley Series in Solution Chemistry, Chichester: John Wiley & Sons Ltd., 2: 178 pages (1997).

The following table provides non-limiting examples of amino acid properties that can help one skilled in the art determine how to select amino acids for modification of the Msp porins or monomers described herein.

Table 3. Amino acid properties

aThis column represents the tendency of amino acids to be buried in the interior of proteins (defined as <5% of residues accessible to solvent) and is based on the structures of nine proteins (a total of approximately 2000 individual residues studied, of which 587 (29%) were buried). The numerical values indicate how often each amino acid is found buried relative to the total number of residues of that amino acid present in the protein. The values in parentheses indicate the number of residues where that amino acid is found buried relative to the total number of buried residues in the protein. Data from Schien, BioTechnology 8:308 (1990); for other calculation methods with similar results, see Janin, Nature 277:491 (1979); and Rose et al., Science 229:834 (1985).

b Average volume of buried residues (Vr), calculated from the surface area of the side chains. Richards, Annu. Rev. Biophys. Bioeng. 6: 151 (1977); Baumann, Protein Eng. 2: 329 (1989).

c Data from Darby N.J. and Creighton T.E. Protein structure. In In focus (ed. D. Rickwood), p. 4. IRL Press, Oxford, United Kingdom (1993).

d Total accessible surface area (ASA) of the amino acid side chains of residue X in a Gly-X-Gly tripeptide with the backbone in an extended conformation. Miller et al., J. Mol. Biol. 196:641 (1987).

The values shown in e represent the average rank of amino acids according to their frequency of occurrence at each sequence level for 38 published hydrophobicity scales. Trinquier and Sanejouand, Protein Eng.11:153 (1998). Although most of these hydrophobicity scales are derived from experimental measurements of the chemical behavior or physicochemical properties of isolated amino acids (e.g., solubility in water, distribution between water and organic solvents, chromatographic translocation, or the effect on surface tension), several "operational" hydrophobicity scales based on known environmental characteristics of amino acids in proteins, such as their solubility accessibility or their tendency to occupy the protein core (based on the position of the residues in the tertiary structure, as observed using x-ray crystallography or NMR) are also included. Lower ranks represent the most hydrophobic amino acids, and higher values represent the most hydrophilic amino acids. For comparison purposes, the hydrophobicity scale of Radzicka and Wolfenden, Biochem.27:1664 (1988) is shown in brackets. This scale is derived from the measured hydration potentials of amino acids based on their free energies of transfer from the gas phase to cyclohexane, 1-octanol, and neutral aqueous solutions.

Alternatively, the hydropathic index of amino acids can be considered. Based on their hydrophobicity and/or charge characteristics, each amino acid has been assigned a hydropathic index, which is: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) and/or arginine (-4.5). The importance of the hydropathic amino acid index in conferring interactive biological function on proteins is generally known in the art. It is known that certain amino acids can be substituted for other amino acids having similar hydropathic indices and/or scores and/or still retain similar biological function. When changes are made based on the hydropathic index, the hydropathic index of the substituted amino acid can be within ±2, within ±1, or within ±0.5.

It is also understood in the art that similar amino acid substitutions can be effectively made based on hydrophilicity. As described in U.S. Patent No. 4,554,101 (incorporated herein by reference), the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based on similar hydropathicity values, it is contemplated that amino acids whose hydropathic indices are within ±2, within ±1, or within ±0.5 are substituted.

Any mutated Msp porin or monomer may comprise conservative amino acid substitutions compared to the wild-type Msp porin or monomer. Any such substitution mutation is conservative if the substitution has minimal disruption to the biochemical properties of the protein. Non-limiting examples of mutations introduced to replace conservative amino acid residues include: substitution of positively charged residues (e.g., H, K, and R) with positively charged residues; substitution of negatively charged residues (e.g., D and E) with negatively charged residues; substitution of neutral polar residues (e.g., C, G, N, Q, S, T, and Y) with neutral polar residues; and substitution of neutral non-polar residues (e.g., A, F, I, L, M, P, V, and W) with neutral non-polar residues. Conservative substitutions may be made according to Table 4 below. Non-conservative substitutions (e.g., substitution of proline for glycine) may also be made.

Table 4: Exemplary amino acid substitutions

amino acids Replacement Ala Ser, Gly, Cys Arg Lys, Gln, Met, Ile Asn Gln, His, Glu, Asp Asp Glu, Asn, Gln Cys Ser, Met, Thr Gln Asn, Lys, Glu, Asp Glu Asp, Asn, Gln Gly Pro, Ala His Asn, Gln Ile Leu, Val, Met Leu Ile, Val, Met Lys Arg, Gln, Met, Ile Met Leu, Ile, Val Phe Met, Leu, Tyr, Trp, His Ser Thr, Met, Cys Thr Ser, Met, Val Trp Tyr, Phe Tyr Trp, Phe, His Val Ile, Leu, Met

As used herein, a "peptide" refers to two or more amino acids linked together by an amide bond (i.e., a "peptide bond"). A peptide comprises up to or including 50 amino acids. A peptide can be linear or cyclic. A peptide can be α, β, γ, δ, or higher, or mixed. A peptide can comprise any mixture of amino acids as defined herein, for example, any combination of D, L, α, β, γ, δ, or higher amino acids.

As used herein, "protein" refers to an amino acid sequence having 51 or more amino acids.

As used herein, "polymer" refers to a molecule comprising two or more linear units (also referred to as "mers"), each of which can be the same or different. Non-limiting examples of polymers include nucleic acids, peptides, and proteins, as well as various hydrocarbon polymers (e.g., polyethylene, polystyrene) and functionalized hydrocarbon polymers in which the backbone of the polymer comprises a carbon chain (e.g., polyvinyl chloride, polymethacrylate). Polymers include copolymers, block copolymers, and branched polymers such as star polymers and dendrimers.

Methods for determining the sequence of polymers using Msp porins are described herein. In addition, sequencing methods can be performed in a manner similar to that described in U.S. Patent 7,189,503 (incorporated herein by reference in its entirety). See also U.S. Patent 6,015,714, incorporated herein by reference in its entirety. More than one read can be performed with such sequencing methods to improve accuracy. Methods for analyzing the characteristics (e.g., size, length, concentration, identity) of polymers and identifying discrete units (or "mers") of polymers are also discussed in the '503 patent and can be used with the present Msp porins. In fact, the Msp porins can be used with any of the methods discussed in the '503 patent.

Currently, several types of observable signals are being developed as readout mechanisms in nanopore sequencing and analyte detection. The most direct and most developed readout method initially proposed relies on an ionic "blockade current" or "copassing current" that is uniquely determined by the identity of the nucleotide or other analyte occupying the narrowest constriction in the pore. This method is called "blockade current nanopore sequencing" or BCNX. Blockade current detection and characterization of nucleic acids has been exemplified in the protein pore α-hemolysin (αHL) and solid-state nanopores. Blockade current detection and characterization have been shown to provide a wealth of information about the structure of DNA passing through or retained in the nanopore in different contexts.

In general, "blockade" is evidenced by a change in the ion current that is clearly distinguishable from noise fluctuations and is typically associated with the presence of an analyte molecule at the central opening of the pore. The intensity of the blockage depends on the type of analyte present. More specifically, "blockade" refers to an interval in which the ion current drops to a threshold value of about 5-100% below the unblocked current level, remains at that point for at least 1.0 μs, and then spontaneously returns to the unblocked level. For example, the ion current may drop to a threshold value of less than about, at least about, or at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or any range therebetween. If the average current of the unblocked signal just before or after the blockage deviates from the typical unblocked level by more than twice the square root noise value of the unblocked signal, then the blockage is discarded. "Deep blockade" was defined as the interval where the ion current dropped to less than 50% of the unblocked level. "Partial blockade" was defined as the interval where the current remained between 80% and 50% of the unblocked level.

As used herein, the term "subject" refers to a living mammalian organism such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. Optionally, the patient or subject is a primate. Non-limiting examples of human subjects are adults, adolescents, infants, and fetuses.

The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer present in single-stranded or double-stranded form, and unless otherwise limited, includes known analogs of natural nucleotides hybridized to nucleic acids in a manner similar to naturally occurring nucleotides, such as peptide nucleic acids (PNAs) and thiophosphate DNA. Unless otherwise noted, a specific nucleic acid sequence includes its complementary sequence. Nucleotide includes but is 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 (thiothymidine triphosphate), pyrrolo-pyrimidine triphosphate and 2-thiocytidine and the α-thiotriphosphates (alphathiotriphosphates) of all nucleotides mentioned above and the 2'-O-methyl ribonucleoside triphosphates of all the above bases. Modified bases include, but are not limited to, 5-Br-UTP, 5-Br-dUTP, 5-F-UTP, 5-F-dUTP, 5-propynyl dCTP, and 5-propynyl-dUTP.

As used herein, "drug" refers to any substance that can alter a biological process in a subject. A drug can be designed for or used in the diagnosis, treatment, or prevention of a disease, disorder, syndrome, or other health affliction in a subject. A drug can be recreational in nature, i.e., used only to alter a biological process and not for or used in the diagnosis, treatment, or prevention of a disease, disorder, syndrome, or other health affliction in a subject. Biological products, i.e., substances produced by biological mechanisms involving recombinant DNA technology, are also included in the term "drug." Drugs include, for example, antibacterial drugs, anti-inflammatory drugs, anticoagulants, antivirals, antihypertensive drugs, antidepressants, antimicrobials, analgesics, anesthetics, beta-blockers, bisphosphonates, chemotherapeutic drugs, contrast agents, fertility medications, hallucinogens, hormones, narcotics, opiates, sedatives, statins, steroids, and vasodilators. Non-limiting examples of drugs can also be found in the Merck Index. Examples of antibacterial drugs used to treat tuberculosis include isoniazid, rifampicin, pyrazinamide, and ethambutol.

Methods using drugs as analytes can also include drug screening. For example, Msp porins can be used to study the uptake of drugs into cells or organisms by observing ion current blockage. Constriction zones and/or vestibules of specific Msp porins with different sizes, electrostatic properties, and chemical properties can be constructed to approximately mimic the desired pathways for drugs to enter or exit cells or organisms. Such methods can greatly accelerate drug screening and drug design. Such studies have been performed using other porins, such as those described by Pagel et al., J. Bacteriology 189:8593 (2007).

As used herein, "biological warfare agent" refers to any organism or any naturally occurring, bioengineered, or synthetic component of any such microorganism that is capable of causing death or disease in plants or animals (including humans), or deterioration of food or water supplies, or environmental degradation. Non-limiting examples include Ebola virus, Marburg virus, Bacillus anthracis and Clostridium botulinum, Variola major, Variola minor, anthrax, and ricin.

As used herein, "pollutants" refer to substances that pollute 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 zinc, copper, or mercury (e.g., methylmercury).

The analyte may be a "nanoscale object," which is an object that is smaller than 100 nm in two of its dimensions.

Beads that can be used include magnetic beads and optical beads. For example, streptavidin-coated magnetic beads can be used to apply a force opposite to the electrostatic force that pulls DNA through the Msp porin channel. In this latter technique, the magnetic beads are attached to biotinylated DNA, and a force comparable to the electrostatic driving force (about 10 pN) can be applied using a strong magnetic field gradient. See, Gosse and Croquette, Biophys. J. 82: 3314 (2002). In this way, the blocking current readout will not be affected, but the force on the DNA can be independently controlled. Tens or hundreds of complete independent readouts of each DNA are then linked and assembled to reconstruct the accurate DNA sequence.

Optical beads manipulated by "optical tweezers" are also known in the art, and such methods can be applied to the Msp porins described herein. Optical tweezers are a common tool for generating forces on nanoscale objects. The analyte is attached to one end of the bead, while the other end can be inserted into the channel of the porin. Optical tweezers are used to control and measure the position and force of the bead. Such methods control the passage of the analyte into the channel and allow greater control over the readout of the analyte, such as the readout of polymer units. For a description of such methods in the context of artificial nanopores, see, for example, Trepagnier et al., Nano Lett. 7:2824 (2007). U.S. Patent No. 5,795,782 (incorporated herein by reference) also discusses the use of optical tweezers.

Fluorescence resonance energy transfer (FRET) is a well-known technique that 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 porin. The analyte is then labeled with a matching FRET-donor or FRET-acceptor. When the matching FRET-donor and FRET acceptor are within a certain distance, energy transfer can occur. The resulting signal can be used for analytical purposes instead of, or in addition to, the methods described herein using ionic current. Thus, methods for detection, identification, or sequencing can include FRET technology.

Other optical methods that can be used include introducing optically active molecules into the interior of the Msp porin (e.g., vestibule or constriction). External light can be used to influence the interior of the protein: such methods can be used to influence the translocation rate of the analyte or can allow the analyte to enter or exit the channel, thereby providing controlled passage of the analyte. Alternatively, an optical pulse focused on the pore can be used to heat the pore to influence the way it interacts with the analyte. Such control can be very fast because the heat from the small volume of the focal point can be dissipated quickly. Methods for controlling the translocation rate of the analyte can thus use such optically active molecules or optical pulses.

Manipulation of translocation velocity can also be achieved by attaching an object to one end of the analyte, which then interacts with the Msp porin at its other end. The object can be a bead (e.g., a polystyrene bead), a cell, a macromolecule such as streptavidin, neutravidin, DNA, or a nanoscale object. The object can then be placed in a fluid flow, where it can be subjected to passive viscous drag.

"Molecular motors" are well known in the art and refer to molecules (e.g., enzymes) that physically interact with an analyte, such as a polymer (e.g., a polynucleotide) and are capable of physically moving the analyte relative to a fixed location (e.g., the vestibule, constriction, or channel of an Msp porin). However, without wishing to be bound by theory, molecular motors utilize chemical energy to generate mechanical force. Molecular motors can interact with each unit (or "mer") of a polymer in a sequential manner. Non-limiting examples of molecular motors include DNA polymerases, RNA polymerase helicases, ribosomes, and exonucleases. Non-enzymatic motors are also known, such as viral motors that package DNA. See Smith et al., Nature 413:748 (2001). A variety of molecular motors and the desired properties of such motors are described in U.S. Patent 7,238,485, which is incorporated herein by reference in its entirety. Molecular motors can be deposited on the cis or trans side of an Msp porin and optionally can be immobilized, such as described by the '485 patent. Molecular motors can be incorporated into Msp porins using the methods described in the '485 patent. Similarly, the systems and devices described in the '485 patent can also be used with the Msp porins described herein. In fact, any of the embodiments discussed in the '485 patent can be applied using Msp porins, as described herein. Molecular motors are also described, 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).

Molecular motors are generally used to regulate the rate or translocation speed of an analyte when interacting with an Msp porin. Any Msp protein described herein may comprise a molecular motor. Optionally, a molecular motor is applied to reduce the rate at which an analyte enters the Msp porin channel, or to reduce the translocation speed of an analyte translocated through an Msp porin channel. Optionally, the translocation speed or average translocation speed is less than 0.5 nm/μs. Optionally, the translocation speed or average translocation speed is less than 0.05 nm/μs. Optionally, the translocation speed or average translocation speed is less than 1 nucleotide/μs. Optionally, the translocation speed or average translocation speed is less than 0.1 nucleotide/μs. Optionally, the rate of movement of the analyte is in the range of greater than 0 Hz to 2000 Hz. Here, the rate refers to the number of subunits (or "polymers") of a structured polymer that advance in 1 second (Hz). Optionally, the range is between about 50 and 1500 Hz, 100 and 1500 Hz, or 350 and 1500 Hz. Optionally, the rate of motion is about, at most about, or at least about 25, 75, 100, 150, 200, 250, 300, 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 therebetween. During the characterization process, the rate can be controlled by using a molecular motor that moves the analyte at a substantially constant rate, at least for a period of time. Furthermore, the range of motion rates can depend on the molecular motor. For example, for RNA polymerases, the range can be 350 to 1500 Hz; for DNA polymerases, the range can be 75 to 1500 Hz; and for ribosomes, helicases, and exonucleases, the range can be 50 to 1500 Hz.

Recording and detection techniques that can be used with the methods described herein. In addition, U.S. Patents 5,795,782 and 7,189,503 (incorporated herein by reference in their entirety) also describe recording methods and instruments that can be used with Msp porins, as well as methods for optimizing conductivity readings. U.S. Patent 6,746,594 (incorporated herein by reference in its entirety) describes supports for thin films containing nanopores and methods of using supports that can be used with the Msp porins described herein.

Also provided in addition is a vector comprising any nucleic acid described herein. As used herein, a vector can comprise a nucleic acid molecule encoding a single-stranded Msp nanopore (e.g., a single-stranded Msp dimer or a single-stranded Msp octamer), wherein the nucleic acid molecule is effectively connected to an expression control sequence. Suitable vector backbones include, for example, the conventionally used backbones in this area, such as plasmids, artificial chromosomes BAC or PAC. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clonetech (Pal Alto, CA), Stratagene (La Jolla, CA) and Invitrogen/Life Technologies (Carlsbad, CA). Vectors typically comprise one or more regulatory regions. Regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription initiation sites, terminator sequences, polyadenylation sequences and introns.

In another aspect, cultured cells transfected with a vector comprising a nucleic acid as described herein are provided. In this regard, cells are successfully transfected with the vector when the transcription machinery of the intact cell contacts the nucleic acid template to produce mRNA. Protocols for facilitating transfection of vectors into cells are well known in the art.

Provided herein are progeny of cultured cells stably transfected with the above-described vectors. Such progeny may contain copies of the vector without undergoing a transfection protocol and may be capable of transcribing nucleic acids contained in the vector under the control of expression control sequences. Techniques for producing large quantities of polypeptides using cultured cells transfected with expression vectors are well known in the art. See, for example, Wang, H., et al., J. Virology 81:12785 (2007).

Also provided herein are mutant bacterial strains capable of inducible expression of Msp. The mutant bacterial strain comprises a deletion of wild-type MspA, a deletion of wild-type MspC, a deletion of wild-type MspD, and a vector comprising an inducible promoter effectively connected to an Msp monomer nucleic acid sequence. Optionally, the mutant bacterial strain comprises Mycobacterium smegmatis strain ML16. Optionally, the Msp monomer nucleic acid sequence encodes an Msp monomer selected from wild-type MspA monomer, wild-type MspC monomer, wild-type MspD monomer, and a mutant thereof. Optionally, the inducible promoter comprises an acetamide-inducible promoter.

Optionally, the mutant bacterial strain further comprises a deletion of wild-type MspB. The mutant bacterial strain comprising a deletion of wild-type MspB may further comprise a vector having a constitutive promoter operably linked to a nucleic acid sequence encoding an Msp porin or monomer. Optionally, the Msp porin or monomer is selected from wild-type MspA, wild-type MspC, wild-type MspD, and mutants thereof. Optionally, the vector comprises any nucleic acid described herein.

Also provided are methods for producing complete or partial single-chain Msp porins. The methods include transforming a mutant bacterial strain. The mutant strain comprises a deletion of wild-type MspA, wild-type MspB, wild-type MspC, and wild-type MspD, and a vector comprising an inducible promoter operatively linked to an Msp monomer nucleic acid sequence. The mutant strain is transformed with a vector comprising a nucleic acid sequence encoding a single-chain Msp porin. The single-chain Msp porin is then purified from the bacteria. Optionally, the single-chain Msp porin comprises a single-chain MspA porin. Optionally, the vector comprises any nucleic acid described herein.

Also provided are methods for determining nucleic acid or polypeptide sequences using a single-chain Msp porin. The method comprises generating a lipid bilayer comprising a first and a second side, adding a purified Msp porin to the first side of the lipid bilayer, applying a positive voltage to the second side of the lipid bilayer, translocating the test nucleic acid or polypeptide sequence through the single-chain Msp porin, comparing the test blocking current to a blocking current standard, and determining the test sequence. Optionally, the single-chain Msp porin comprises a wild-type MspA monomer or a mutant thereof. Optionally, the Msp monomer comprises an MspA paralog or homolog monomer selected from Table 1.

Unless explicitly stated that the alternatives are mutually exclusive, the use of the term "or" in the claims is intended to mean "and/or," although the present disclosure supports a definition that indicates both the alternative and "and/or."

Throughout this application, the term "about" is used to indicate that a numerical value includes the standard deviation of error for the device or method being used to determine the value. In any embodiment discussed in the context of a numerical value used in conjunction with the term "about", it is expressly intended that the term about can be omitted.

Under long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in a claim or specification, mean one or more unless expressly stated otherwise.

The present invention discloses materials, compositions, and components that can be used in conjunction with the methods and compositions disclosed herein, for use in their preparation as products thereof, and such and other materials are disclosed herein, and it should be understood that when combinations, subgroups, interactions, classes, etc. of such materials are disclosed, while not explicitly disclosing every different individual and collective combination and permutation of such compounds, these are still explicitly contemplated and described herein. For example, if a method is disclosed and discussed, and a number of modifications that can be made to a number of molecules included in the method are discussed (including the method), then unless explicitly stated to the contrary, all combinations and permutations and possible modifications of the method are explicitly contemplated. Similarly, any subgroup or combination of such methods and modifications is also explicitly contemplated and disclosed. This concept applies to all aspects of the present disclosure, including but not limited to steps in the methods using the disclosed compositions. Thus, if there are many additional steps that can be performed, it should be understood that each of such additional steps can be performed using any particular method step or combination of method steps in the disclosed method, and each such combination or subgroup of combinations is explicitly contemplated and should be considered disclosed. It is therefore contemplated that any embodiment discussed in this specification can be applied to any method, compound, protein, porin, peptide, polypeptide, multimer, monomer, nucleic acid, vector, strain, cultured cell, system or composition disclosed herein, and vice versa. For example, any protein described herein can be used in any method disclosed herein.

The publications cited herein and the materials for which they are cited are hereby incorporated by reference in their entirety.

The following examples are provided to illustrate, but not to limit, the materials disclosed herein.

Example

Example 1: Materials and methods of Examples 1 to 7

Homogeneous ssDNA oligonucleotides _dA50 , _dC50 , and _dT50 (SEQ ID NO: 10, SEQ ID NO: 16, and SEQ ID NO: 17, respectively) and hairpin constructs hp08 (5' GCTGTTGC TCTCTC GCAACAGC A50 3') (SEQ ID NO: 4), hp10 (5' GCTCTGTTGC TCTCTC GCAACAGAGC _A50 3') (SEQ ID NO: 5), and hp12 (5' GCTGTCTGTTGC TCTCTC GCAACAGACAGC _{A50 3} ') (SEQ ID NO: 6) were synthesized by Integrated DNA Technologies , (IDT; Coralville, IA).

Bacterial strains and growth conditions. All bacterial strains used in this study are listed in Table 5. Mycobacteria were cultured at 37°C in Middlebrook 7H9 liquid medium (Difco) supplemented with 0.2% glycerol, 0.05% Tween or on Middlebrook 7H10 agarose (Difco) supplemented with 0.2% glycerol. Escherichia coli DH5α was used for all cloning experiments and was routinely cultured in Luria-Bertani (LB) medium at 37°C. Hygromycin was used at concentrations of 200 μg/mL (for E. coli) and 50 μg/mL (for Mycobacterium smegmatis).

Table 5. Strains and plasmids.

Annotation: HygR indicates resistance to hygromycin. MspA, mspC, and mspD are porin genes of Mycobacterium smegmatis.

Site-directed mutagenesis of mspA. Using site-directed mutagenesis, M1MspA and M2MspA mutant monomers were constructed in a step-by-step manner using a combined chain reaction (CCR) as described by Bi and Stambrook, Nucl. Acids Res. 25: 2949 (1997). Plasmid pMN016 contains a psmyc-mspA transcriptional fusion (Stephan et al., Mol. Microbiol. 58: 714 (2005)) and was used as a template. Oligonucleotides psmyc1 and pMS-seq1 (Table 6) were used as forward and reverse primers for CCR, respectively. Three subsequent mutations were introduced into mspA to construct the m1mspA gene. Three additional mutations were introduced into m1mspA to generate m2mspA. All plasmids were verified by sequencing the entire mspA gene before transformation into triple porin mutant M. smegmatis ML16 (Stephan et al., Mol. Microbiol. 58:714 (2005)) for protein production.

Table 6. Oligonucleotides

The codons that were changed to introduce the MspA mutation are underlined.

Single channel experiments. Bilayers were generated using diphytanoyl-PA and diphytanoyl-PC lipids prepared in equal or unequal proportions, and pores of approximately 20 μm in diameter were formed across the horizontal plane in Teflon as described in (Akeson et al., Biophys. J. 77:3227 (1999)). MspA porin was added to one side (cis side) of the bilayer at a concentration of approximately 2.5 ng/mL. The cis side was grounded, and a positive voltage was applied to the reverse side of the bilayer. An Axopatch-1B patch clamp amplifier (Axon Instruments) was used to apply voltage across the bilayer and measure the ionic current flowing through the pore. The analog signal was low-pass filtered at 50 kHz using a 4-pole Bessel filter. The filtered signal was digitized at 250 kHz. Data acquisition was controlled using custom software written in LabWindows/CVI (National Instruments). All experiments were performed at 21 ± 2 °C in 1 M KCl, 10 mM Hepes/KOH buffered at pH 8.

Data Analysis. Data analysis was performed using custom software written in Matlab (The MathWorks; Natick, MA). Blockades were defined as intervals in which the ion current dropped below a threshold of 80% of the unblocked current level, remained at that point for at least 12 μs, and then spontaneously returned to the unblocked level. Blockades were rejected if the average current of the unblocked signal just before or after the block deviated from the typical unblocked level by more than twice the square root noise value of the unblocked signal. Blockades were also rejected if they occurred within 26 μs of another blockade. Deep blockades were defined as intervals in which the ion current dropped below 50% of the unblocked level. Intervals in which the current remained between 80% and 50% of the unblocked level were defined as "partial blockades." Each event was parameterized using the dwell time and average current of its constituent part and depth subintervals.

The tD value used to parameterize the hairpin deep blockade dwell time distribution was estimated as the peak of the probability density distribution of the logarithm to base 10 of the dwell time ( FIG8 ). The distribution was estimated using the Matlab kernel smoothing density estimator using a normal kernel function and a width of 0.15. The cross-double layer data were analyzed by detecting abrupt changes in conductance from less than 1 nS to greater than 1 nS. The voltage at which these changes occurred was recorded and then summarized in the histograms shown in FIG9E-9G .

In all experiments, the wells were oriented with the "inlet" (Figure 1) exposed to the cis compartment of the device.

All hairpin data presented in Figures 5-8 are derived from data collected for the same long-lived M1MspA porin. The homopolymer data presented in Example 5 were obtained using a different long-lived M1MspA porin than the hairpin data described, but there is quantitative agreement between the extended hairpin data sets collected for the two pores.

Example 2: Blocking characteristics of wild-type MspA (WTMspA) porin with and without analyte

Purification of MspA Porin. The MspA porin was selectively extracted from M. smegmatis and subsequently purified by anion exchange and gel filtration chromatography as described (Heinz and Niederweis, Anal. Biochem. 285: 113 (2000); Heinz et al., Methods Mol. Biol. 228: 139 (2003)).

Consistent with previous results (Niederweis et al., Mol. Microbiol. 33:933 (1999)), the purified protein showed high channel-forming activity, with a peak conductance of 4.9 nS in 1.0 M KCl at approximately 20°C (Figure 2). The cis compartment was grounded and a positive voltage was applied to the trans compartment (Figure 3A). Above approximately 60 mV, the WTMspA porin showed frequent spontaneous blockades of ionic current in the absence of ssDNA (Figure 3). Some spontaneous blockades were transient, while others required a voltage reversal to reestablish unblocked current levels. Despite this behavior, intervals of stable unblocked signal persisted for tens of seconds for voltages up to approximately 100 mV (Figure 3). Addition of approximately 2–8 μM dC50 (SEQ ID NO:48) ssDNA to the cis compartment did not result in a significant enhancement or change in these blocking characteristics. Above approximately 100 mV, spontaneous blockades were so frequent that ssDNA detection experiments could not be performed.

One explanation for the apparent absence of ssDNA interaction with the WTMspA porin is the high density of negative charges in the pore ( FIG1 ). Electrostatic interactions with the negatively charged interior of the channel may inhibit DNA entry into the pore. To address this issue, the aspartic acid residues in the constriction region were replaced with asparagine ( FIG1 ). The resulting MspA mutant D90N/D91N/D93N (M1MspA) porin is discussed in Example 3.

Example 3: Blocking characteristics of the MspA mutant M1MspA porin with and without analyte

experiment

As noted in Example 2, electrostatic interactions between ssDNA and the channel of the WTMspA porin may affect the translocation of ssDNA through the pore. The MspA mutant D90N/D91N/D93N (M1MspA, also known as M1-NNN) was designed to test this theory. The M1MspA porin was expressed and purified from the Mycobacterium smegmatis strain ML16, which lacks most endogenous porins (Stephan et al., Mol. Microbiol. 58:714 (2005)). The expression level and channel-forming activity of the M1MspA porin ( FIG4 ) were similar to those of the WTMspA porin, however, the conductance was reduced by 1/2 to 1/3 ( FIG2 ). Furthermore, the frequency of spontaneous blockages was dramatically reduced in the M1MspA porin, making it possible to perform DNA detection experiments at voltages as high as 180 mV and higher ( FIG5 ).

ssDNA hairpin constructs were used to study DNA interactions with the M1MspA porin. Each construct contained a 50-nt poly-dA overhang at the 3' end, a dsDNA duplex region of variable length (8, 10, and 20 bp for constructs hp08 (SEQ ID NO: 4), hp10 (SEQ ID NO: 5), and hp12 (SEQ ID NO: 6), respectively), and a 6-nt loop (Figure 6). At 180 mV, addition of approximately 8 μM hp08 ssDNA to the cis compartment increased the rate of transient ion current blockades from 0.1–0.6 blockades per second to 20–50 blockades per second (Figure 5). The blockade rate was proportional to DNA concentration and strongly voltage-dependent, with a 20-mV decrease in applied voltage reducing the blockade rate by approximately one-third. Blockades long enough to be well resolved were either partial blockades, where the ion current was reduced to 80% to 50% of the unblocked level, or deep blockades, where the ion current was reduced to less than 50% of the unblocked level (Figure 5C). It is very rare to show blockage of both partial and deep sub-regions simultaneously. Partial blockage lasts for tens to hundreds of microseconds, and its residence time increases with increasing voltage (Fig. 5C and 7). Deep blockage lasts for hundreds of microseconds to hundreds of milliseconds, and its residence time decreases with increasing voltage (Fig. 6 and 7). These trends were observed in experiments using all three hairpins.

analyze

Similar to similar signals observed with αHL (Butler et al., Biophys. J. 93:3229-40 (2007)), partial blockage is interpreted as DNA entering the vestibule of the M1MspA porin without filamenting the single-stranded segment through the channel constriction. For this mechanism, a modest decrease in ionic current is expected. Without wishing to be bound by theory, the increase in dwell time with increasing voltage ( FIG. 7 ) is most likely due to the increasing electrostatic barrier preventing the DNA molecule from escaping from the vestibule back to the cis compartment. This interpretation of the dwell time can be understood within a kinetic framework in which the decay of the polymer from the vestibule occurs through two first-order processes: escape from the applied voltage gradient and filamentation of one end through the constriction. The lifetime is therefore the inverse of the sum of the rate constants of these processes. If (i) the escape rate constant decreases with increasing voltage and (ii) its decrease exceeds any change in the filamentation rate constant, then the lifetime will increase with increasing voltage.

For deep blockade, the significant decrease in dwell time with increasing voltage is inconsistent with any process involving DNA escape back into the cis compartment. The extent of the ionic current reduction and the voltage dependence of the dwell time are consistent with a process in which a single-stranded polydA segment is driven through an approximately 1-nm diameter constriction until an approximately 2.2-nm diameter DNA duplex reaches the constriction and stops translocation (Figure 5A). The hairpin construct maintains its filamentous configuration until the DNA duplex melts (Vercoutere et al., Nat. Biotech. 19:248-52 (2001); Sauer-Budge et al., Phys. Rev. Lett. 90:238101 (2003); Mathe et al., Biophys. J. 87:3205-12 (2004)) or a conformational rearrangement of the constriction zone of the M1MspA porin allows translocation to complete. Without being bound by theory, a mechanism of unwinding by translocation seems most plausible because passage of the dsDNA helix requires the constriction to roughly double in diameter, disrupting the hydrogen bonds flanking the β-barrel of the constriction (Faller et al., Science 303:1189 (2004)) and potentially exposing hydrophobic regions of the protein and the interior of the bilayer to water.

The deep blockage of hairpins in the M1MspA porin has a very broad dwell time distribution that cannot be fully described by a simple exponential or sum of exponentials ( FIG. 8 ). To parameterize the distribution, the logarithmic model of the deep blockade dwell times, tD , corresponding to the dwell times with the highest blockade density in FIG. 6 was used ( FIG. 8 ). hp08 had the shortest tD for all voltages. Below 160 mV, hp10 and hp12 had similar tD s. However, above 160 mV, hp10 consistently had a longer tD than hp12. These observations differ slightly from those from αHL, where the hairpin blockade dwell time distribution was modeled using a single exponential and hairpins with larger standard free energies of formation consistently produced longer deep blockades ( Vercoutere et al., Nat. Biotechnol. 19:248 (2001); Mathe et al., Biophys. J. 87:3205 (2004)). Assuming that deep blockade results from translocation with duplex melting as the rate-limiting step, this process should be 10- to 100-fold slower in the M1MspA porin than in αHL (Mathe et al., Biophys. J. 87:3205 (2004)). Interestingly, the hp10 blockade persisted longer than the hp12 blockade. In six replicate experiments using hp10 at 180 mV, an average unblocked current level of 340 ± 7 pA and an average tD of 9 ± 1 ms (mean ± SEM) were observed in each of the six replicates.

Example 4: Trans-bilayer detection using M1MspA porin

theory.

To obtain direct evidence of DNA translocation through MspA, a transmembrane detection technique, illustrated in FIG9 and developed by Nakane et al. (Nakane et al., Biophys. J. 87:615 (2004)), was used. An ssDNA probe molecule with a bulky anchor complex at one end was electrophoretically driven into the nanopore. The free ssDNA end passed through the pore into the trans compartment until the anchor terminated translocation. If the trans compartment contained a short ssDNA target molecule complementary to the end of the ssDNA probe, the probe and target could hybridize. If hybridization occurred, the probe was locked in a threaded configuration until a sufficiently negative voltage was applied to cause the probe to dissociate from the target and expel into the cis compartment. If hybridization did not occur due to random causes or because the probe end was not complementary to the target, or if there was no target molecule in the trans compartment, then no negative voltage was required for the probe to retreat to the cis compartment. The presence of a blockage that could only be cleared by a sufficiently negative voltage was evidence that the ssDNA probe had passed through the nanopore to the trans compartment and hybridized with the target DNA.

experiment.

A probe molecule was constructed containing a 75-nt-long ssDNA molecule linked to a neutravidin (nA) anchor at its biotinylated 5′ end and a heterogeneous 15-nt-long complementary sequence at its 3′ end. nA was obtained from Invitrogen (Carlsbad, CA). Two different 5′-biotinylated ssDNA constructs, 5′-bt-dC6dA54d(CTCTATTCTTATCTC)-3′ (SEQ ID NO: 7) and 5′-bt-dC6dA54d(CACACACACACACAC)-3′ (SEQ ID NO: 8), were synthesized by IDT. nA and the ssDNA construct were mixed in a 1:1 ratio in an experimental 1 M KCl buffer at a concentration of 50 μM and stored at −20° C. until immediately used. A 15-nt target DNA, 3′-GAGATAAGAATAGAG-5′ (SEQ ID NO:9), was synthesized by IDT, suspended in the assay buffer, and stored at -20°C until immediately used. The cis compartment was preloaded with approximately 100 μM target DNA, and the trans compartment was priming with DNA-free buffer. After bilayer formation, the cis compartment was priming to remove any target DNA that had diffused through the pore. Once a stable M1MspA porin was established, the nA–ssDNA complex was added to the trans compartment to a final concentration of approximately 1 μM. Custom experimental control software written in LabWindows was used to continuously monitor the current and apply the appropriate voltage.

When the probe molecule was driven from the cis compartment into the pore with 180 mV, an ambiguous deep current blockade was observed. For the cross-double layer experiment, the probe molecule was captured using 180 mV. After a short delay to ensure that the ssDNA passed through the M1MspA porin as much as possible, the voltage was reduced to 40 mV and held at this level for 5 seconds to allow one of the 15-nt long target ssDNAs to anneal to the complementary end of the probe. The voltage was then decreased at a rate of 130 mV/s. For each event, the probe exit voltage Vexit was determined as the voltage at which a large and sharp increase in conductance was observed as the voltage gradually decreased (Figures 9C and 9D).

Transbilayer data were analyzed by detecting abrupt changes in conductance from less than 1 to greater than 1 nS. The voltage at which these changes occurred was recorded and summarized in the histograms shown in Figures 9E-9G. For additional information on data analysis, see Materials and Methods in Example 1.

analyze.

Histograms of Vexit from experiments using three different probe/target combinations are shown in Figure 9. When the probe DNA was complementary to the target DNA (Figure 9E), a significant number of Vexit values were negative, indicating probe/target hybridization. Similar negative Vexit populations were observed in six replicate experiments using complementary probe/target molecules. In five replicate experiments in which the ssDNA 3' end was not complementary to the target molecule (Figure 9F) and in one experiment without target DNA (Figure 9G), few negative Vexit values were observed. Complementary and non-complementary probe/target combinations were used for two different nanopores. Data for one of these pores are shown in Figures 10E and 10F. These data provide clear and direct evidence that ssDNA can pass through the M1MspA porin, confirming the hypothesis that the deep blockage observed in Figure 5 is indeed caused by ssDNA translocation through the M1MspA porin.

Example 5: MspA mutant M1MspA porin and linear homogeneous ssDNA

The interaction between the M1MspA porin and linear homogeneous ssDNA 50-mers was also studied. At 180 mV, the addition of approximately 8 μM dT50 to the cis compartment resulted in approximately 5 blockages per second ( FIG. 10 ), an approximately 20-fold increase in the blockage rate in the absence of dT50 (SEQ ID NO: 32). Most of these blockages were shorter than 30 μs, which was too short to discern the internal structure or estimate the depth of the blockage. Experiments using dA50 (SEQ ID NO: 49) and dC50 (SEQ ID NO: 48) produced similar results. The short duration of the observed blockages suggests that the translocation of these linear homogeneous ssDNA 50-mers is typically shorter than 30 μs. The blockages also coincide with the polymer's short trip into the antechamber (ending with escape back into the cis compartment). Although both translocation and escape can occur in experiments using linear ssDNA 50-mers, it is impossible to estimate the relative frequencies of these two processes.

Example 6: Blocking characteristics of the MspA mutant M2MspA porin with and without analyte

To further examine the effect of charge in the MspA porin on its DNA analysis ability, three additional mutations were made to the M1MspA porin, replacing negatively charged residues in the vestibule and around the entrance with positively charged residues ( FIG1 ). The resulting mutant D90N/D91N/D93N/D118R/D134R/E139K (M2MspA) porin showed similar expression levels ( FIG4 ) and channel formation activity to the WTMspA ( FIG2 ) porin.

Like the M1MspA porin, the M2MspA porin has a smaller conductance than the WTMspA porin ( FIG. 2 ) and exhibits minimal spontaneous blockade for voltages up to 180 mV or higher. At 180 mV, addition of 2 μM dT50 (SEQ ID NO: 32) to the cis compartment resulted in a blockade rate of approximately 25 blockades per second ( FIG. 10B ). A partial blockade of approximately 100 μs, ending with a clear downward spike, was a common blocking pattern ( FIG. 10C ). Both the duration of the partial blockade and its tendency to end with a downward spike increased with increasing voltage ( FIG. 11 ). These trends are consistent with a process in which the polymer enters the antechamber and remains there, thereby generating a partial blockade until one end enters a strong electric field, constricts, and initiates translocation. This mechanism accurately explains the similar partial-to-deep blockade pattern observed with αHL (Butler et al., Biophys. J. 93: 3229 (2007)). The short duration of the downward spike suggests that translocation of the linear ssDNA 50-mer through the M2MspA porin is shorter than approximately 30 μs. Partial blockades that do not end with a downward spike are interpreted as escape back to the cis compartment or translocation shorter than approximately 10 μs, which is too short to be observed in these experiments.

Example 7: Comparison of properties of M1 and M2MspA mutant porins and αHL

A key similarity between the M1MspA and M2MspA porins is that the translocation of linear ssDNA 50-mers appears too rapid to produce deep blockages with discernible structures. Without being bound by theory, this observation suggests that the constriction, which is identical for both mutants, is the region that primarily determines the rate at which linear ssDNA molecules translocate through the MspA porin. Comparison of the MspA translocation velocities of approximately 2 to 10 bases/μs for the M1MspA and M2MspA porins with the approximately 0.5–1 base/μs observed with αHL (Meller et al., Proc. Natl Acad. Sci. USA 97:1079 (2000); Butler et al., Biophys. J. 93:3229 (2007)) supports the concept that details of channel geometry and composition play a primary role in determining translocation speed.

The large difference in translocation speed in the case of the MspA porin and αHL may be due to the width of the channel region flanking the constriction. If interactions between DNA and the channel walls slow down the passage of DNA (Slonkina and Kolomeisky, J. Chem. Phys. 118:7112-8 (2003)), then slower translocation would be expected in αHL, where the 10 to 20 bases that are highly confined in the constriction and transmembrane region are forced to interact with the channel walls. In the MspA porin, only 2 to 4 bases in the constriction are forced to contact the protein. The distribution of charge in the constriction is another significant difference between αHL and the M1 and M2 MspA mutant porins. The αHL constriction is formed by the side chains of E111, K147, and M113 (Song et al., Science 274:1859 (1996)), which forces the negatively charged ssDNA backbone into close proximity with seven positively and seven negatively charged residues. The lack of charged residues in the constriction of the M1 and M2MspA mutant porins may also be responsible for the faster translocation rate compared to αHL.

Further comparison of the homopolymer blocking characteristics between the two MspA mutant porins helps to understand how the arrangement of charged residues in the channel affects its interaction with DNA. For a given ssDNA concentration, the blocking rate of the M2MspA porin is about 20 times that of the M1MspA porin (Figure 10B). At as low as about 80mV, the M2MspA porin also shows easily observable blockages, while for the M1MspA porin, almost no blockage is seen below about 140mV. Finally, the partial blockage for the M2MspA porin is at least about 100 times longer than that for the M1MspA porin (Figure 9C). These trends are consistent with a simple electrostatic model, in which the positively charged residues in the M2MspA porin all promote ssDNA entry into the vestibule and inhibit the ssDNA molecule from escaping from the vestibule back to the cis compartment. These observations show that the appropriate placement of charged residues provides a simple method to essentially tailor the interaction between the MspA porin and DNA.

Example 8: M1MspA porin recognizes single nucleotides in DNA held in the pore via the hairpin (hp) segment

Experiments using the M1MspA porin and poly-A DNA strands (i) with a single C embedded in a poly-A background and (ii) with a single T embedded in a poly-A background were performed as described in Example 3. As noted above, the hairpin retained the DNA construct in the MspA porin constriction long enough to obtain a well-defined current signature.

Figure 12A shows the current histograms generated by single Cs at positions 1, 2, and 3 after the hairpin, as well as a mixture of poly-A and poly-C. The current histograms for each position are very different and show a "recognition site" near position 2. For a more quantitative description, the peak of the current distribution was scaled according to the current difference found for poly-C and poly-A (Figure 12B). Gaussian fitting shows that the recognition position of the MspA porin for a single C is 1.7 nucleotides (nt) from the position of the hairpin. The length of the recognition position (constriction length) is comparable to the width of the Gaussian (1.6 nt, approximately 1.5 nt long).

A single T embedded in a poly-A DNA hairpin construct. Experiments using a single T in poly-A DNA were performed in a similar manner, focusing only on the first three positions adjacent to the hairpin ( FIG. 13 , panels 2-4). Specificity was equally impressive, but in this case, the greatest sensitivity was exhibited near position 1. The position of a single T can be resolved much better than a single position. Without being bound by theory, the inventors speculate that the difference in positional recognition compared to a C in poly-A is actually caused by the DNA itself, which contributes to the electrostatic environment that forms the constriction. Data for a single A in a background of C are shown in the bottom three panels of FIG. 13 . Although a single A produces a current blockade feature that is only weakly distinguishable from the poly-C background, the current distribution is narrow enough to resolve a single A. The optimal position for A in a poly-C chain appears to be near position 2, similar to that of a single C in an A chain.

The composition of the DNA tail outside position 3 does not affect the base recognition properties. Poly-A DNA forms a secondary structure, and the data difference between the C in the poly-A background and the A in the poly-C background can be attributed to the interruption of the secondary structure (rigidity) of the poly-A tail. Measurements were made using a 47-base long heterogeneous sequence after the first 3 positions occupied by either A or C trinucleotides. It was found that the current level could not be distinguished from that of pure A50 and C50 tails, thereby indicating that the tail secondary structure or composition does not affect the current blockade (Figure 14).

An additional series of experiments were performed to (1) assess the ability of the M1MspA porin to discriminate between different nucleotides and (2) assess the location and length (spatial resolution) of the regions to which the porin is sensitive. In these experiments, different DNA constructs were used with ssDNA chains of 50 nucleotides attached to a 14 base pair hairpin segment to prevent immediate translocation. The data are summarized in Figure 31. dA ₅₀ (SEQ ID NO:49) and dC ₅₀ (SEQ ID NO:48) produced significantly different blocking currents. Next, a series of constructs were tested in which the recognition site was isolated to the first four bases after the hairpin. These constructs had ssDNA sequences after the hairpin of dC ₄ dA ₄₆ (SEQ ID NO:15), dA ₃ dC ₄ dA ₄₃ (SEQ ID NO:12), and dA ₆ dC ₄ dA ₄₀ (SEQ ID NO:11). dC ₄ dA ₄₆ showed a blocking current distribution almost identical to dC ₅₀ , while dA ₃ dC ₄ dA ₄₃ and dA ₆ dC ₄ dA ₄₀ blocked currents similar to dA _50. This narrowed the recognition site to the first three nucleotides after the hairpin. The constructs were then tested using a single dC at different positions within a poly-dA background. Hp-dC ₁ dA ₄₉ (dC at position 1) (SEQ ID NO: 14) blocked current at a level intermediate between the poly-dA and poly-dC values. Construct dA ₂ dC ₃ dA ₄₇ (dC at position 3) (SEQ ID NO: 50) blocked current at a level intermediate between the poly-dA and poly-dC values, but closer to poly-dA. Poly-dT ₅₀ (SEQ ID NO:32) produced the smallest current block, while hp-dG ₃ dA ₄₇ (SEQ ID NO:18) produced a current intermediate between poly-dC and poly-dA. Blockade currents for poly-dC, poly-dA, and poly-dT were measured in different mutants (D90/91Q+D93N+D118/134R+E139K) and were distinguishable from each other. These data suggest that the M1MspA porin has recognition capabilities and a relatively short recognition site. Furthermore, given that the hairpin is blocked cis-laterally to the constriction, the recognition site appears to be located within the constriction.

Example 9: Construction and characterization of mutant MspA M1-QQN and M2-QQN porins

In another set of experiments designed to slow DNA translocation through the MspA porin channel, two additional mutants were generated. In a similar manner to M1-NNN (or M1MspA) described above, one mutant (designated M1-QQN) was generated by replacing the amino acids at positions 90 and 91 of the wild-type MspA monomer with glutamine and the amino acid at position 90 with asparagine. Regarding M2-QQN, the constriction zone of the pore was reduced by introducing bulkier glutamines at positions 90 and 91 in the context of the M2MspA mutant (see Example 6; D90Q+D91Q+D93N+D118R+E139K+D134R). This was expressed in the M. smegmatis ML16 mutant described in Examples 1 and 3 above. The amount of M2-QQN porin in detergent extracts was as high as that of the WTMspA porin ( FIG. 15A ), indicating that the new mutations did not affect pore expression. Lipid bilayer experiments showed that the M2-QQN porin formed stable open pores, similar to the WTMspA porin ( Figure 15B ). Its pore-forming activity was similar to that of the WTMspA porin. The single-channel conductance of the M2-QQN porin (2.4 nS) was higher than that of its parental M2 (1.4 nS).

The QQN mutant also distinguishes between A, C, and T bases. Similar to the properties of the M1MspA mutant porin (also known as the M1-NNN mutant), the QQN mutant exhibits well-resolved current levels using homopolymeric hp chains, but the relative spacing between levels is different in the M1-QQN porin. For each pore, data were collected for hairpins with A50, T50, and C50 tails (SEQ ID NO:49, SEQ ID NO:32, and SEQ ID NO:48, respectively). The blockade current was plotted as a fraction of the unblocked open pore current ( FIG16 ). In each case, poly-T blocked more readily than poly-C, which in turn blocked more readily than poly-A. Each peak was well resolved from the others. In the QQN porin, the average poly-A and poly-C current levels were not as well separated as in the M1-NNN porin, but in the M1-NNN porin, poly-T and poly-C were better separated. Surprisingly, the relative levels of poly-T blockade were strikingly different in the two QQN mutant porins. The two mutants differ only in the displacement of the edge domain away from the constriction. Without being bound by theory, this may be due to an interaction between the edge domain and the anchoring hairpin.

The QQN mutant porin appears to slow DNA translocation through MspA. The primary motivation for constructing the QQN mutant was to slow DNA passage. Translocation of a heterogeneous 100nt ssDNA segment (without an anchoring hairpin) was recorded as the duration of the deeply blocked state. The presence curve ( FIG17 ) shows a portion of the blocking event that lasted longer than time t. During the first approximately 100 μs, the NNN mutant decayed significantly faster than the mutant with the QQN constriction. These data are consistent with increased resistance to translocation by QQN.

Example 10: Construction of a quadruple Msp deletion mutant of Mycobacterium smegmatis

To prepare the MspA porin, the protein was selectively extracted from a mutant strain of Mycobacterium smegmatis ML16, which contains only one of the four Msp genes (MspB) (the others are MspA, MspC, and MspD). This method exploits the extreme thermostability of MspA by boiling M. smegmatis cells in 0.5% n-octylpolyoxyethylene olefin (OPOE), a nonionic detergent, to yield an MspA porin with minimal contamination by other proteins (Heinz and Niederweis, Anal. Biochem. 285:113-20 (2000)). However, background expression of MspB was still detectable in immunoblots using Msp-specific antiserum (Stephan et al., Mol. Microbiol. 58:714-30 (2005)), suggesting that mixed MspA/MspB oligomers can form and contribute to the pore heterogeneity observed in pore reconstitution experiments. Therefore, one goal was to construct a strain of M. smegmatis that lacks endogenous porins. Since M. smegmatis requires porin activity for survival, a loxP-flanked MspA expression cassette was integrated into the chromosomal attB site for the mycobacteriophage L5 of the porin triple mutant ML16.

This restored the expression of the MspA monomer in strain ML56 to half of the wild-type level. Next, the suicide vector pMN247 was used to replace the MspB gene using the hyg gene connected by FRT-flank in a two-step strategy as described by (Stephan et al., Gene 343:181-190 (2004)). After utilizing the Flp recombinase to excise the hyg gene, the porin quadruple mutant strain ML59 (ΔMspA ΔMspB ΔMspC ΔMspD attB::loxP-MspA-loxP) was obtained. The deletion of the MspB gene was confirmed by Southern blot hybridization. PCR proved that each of the four original Msp genes did not exist (Figure 19). Excision of the loxP-MspA-loxP box resulted in small viable clones, one of which (ML180) was examined in more detail. Using the same high temperature method to extract protein from ML180 cells, Western analysis showed that ML180 cells do not express Msp porins, and there are no reconstitution events in lipid bilayer experiments after adding 20 μg of protein (Figure 20). These results, taken together, show that a Mycobacterium smegmatis porin mutant lacking all four Msp porins has been generated. However, it was not possible to detect expression of MspA monomers using the MspA expression vector, which is most likely due to unknown secondary mutations. Therefore, this Mycobacterium smegmatis strain cannot be used for expression of MspA pores engineered for DNA translocation.

Example 11: Construction of the Mycobacterium smegmatis quadruple Msp deletion mutant ML705 using inducible expression of MspA

To isolate wild-type and mutant MspA porins, the Mycobacterium smegmatis ML16 strain (ΔMspA, ΔMspC, ΔMspD) is currently used. However, background expression of MspB complicates the interpretation of translocation experiments. Therefore, it is necessary to construct an M. smegmatis strain lacking all four Msp genes to improve single-well experiments. To achieve this, the MspA gene under the control of an acetamide-inducible promoter was integrated into the L5attB site of M. smegmatis ML16, resulting in the removal of the MspB gene by allelic exchange. Therefore, in the presence of acetamide, expression of MspA rescues the growth of the M. smegmatis quadruple mutant.

To achieve this, the integrative plasmid pML967 was constructed, which contains the MspA gene under the control of an acetamide-inducible promoter ( FIG. 21A ). The MspB deletion vector pML1611 ( FIG. 21B ) was also constructed, which contains two reporter genes, gfp and xylE, as markers for integration and allelic replacement.

After the MspA monomer expression plasmid pML967 was integrated into Mycobacterium smegmatis ML16, strain ML341 (ML16, attP::pML967) was obtained. The hygromycin resistance gene was removed from the strain by temporarily expressing the Flp recombinase (Song et al., Mycobacteria protocols (2008)) from the plasmid pML2005 described previously, thereby producing strain ML343 (ML341, attP::p _acet -MspA). In order to check the functionality of the integrated MspA gene monomer, MspA was extracted from uninduced and induced cells with detergent. Figure 22 shows that after adding 2% acetamide, MspA was expressed from the integrated construct at 20% of the wild-type level. This MspA monomer level is sufficient to enable Mycobacterium smegmatis to survive. There was very low background expression of Msp porin in uninduced cells (Figure 22), indicating that the expression system is regulated.

The MspB deletion vector pML1611 was then transformed into ML343. Transformants were plated on Middlebrook 7H10 agar plates containing 10% sucrose for direct selection of double-crossover candidates. Several colonies were obtained that displayed the presence of GFP by green fluorescence when illuminated with blue light and in the absence of XylE. Colony PCR from one of these clones confirmed the absence of the MspB gene and the construction of a viable Msp quadruple mutant. This strain was designated ML378. The ML378 strain was transformed with the pCreSacB1 plasmid to remove the gfp-hyg expression cassette. After subsequent negative selection, several colonies were obtained and examined by colony PCR. One of the eight untagged porin quadruple mutants of Mycobacterium smegmatis, designated ML705, was further characterized.

To examine whether MspA monomers compensate for the phenotype of the quadruple mutant, MspA expression plasmid pMN016 was transformed into ML705. Figure 24 shows that the growth of ML705 on 7H10 agar plates was dramatically reduced; however, expression of MspA from pMN016 completely restored the growth of ML705 to wild-type levels (Figure 23). These results demonstrate that no secondary mutations cause the growth defect and that MspA monomers can be expressed to produce MspA porins in the Msp quadruple mutant ML705.

The growth of the porin quadruple mutant ML705 in Middlebrook 7H9 medium was much slower than that of wild-type M. smegmatis and significantly slower than that of the porin triple mutant ML16 (Figure 24). Adding 2% acetamide to induce expression of the MspA gene monomer at the L5 site and expression of MspA on plasmid pMN016 restored the growth rate to wild-type levels (Figure 24). The growth of ML705 on plates and in liquid culture was slower than that of the triple mutant, indicating that ML705 has fewer porins in the outer membrane than the Msp triple mutant ML16. This hypothesis was verified in Western blotting (Figure 25). The amount of MspA monomer was less than 5% of the amount of MspA monomer in wild-type (wt) M. smegmatis and 50% less than the amount of MspA monomer in the triple mutant. Figure 25 also shows that when 2% acetamide was added, we could induce MspA to 25% of the wild-type level.

The above experiments show that an Msp quadruple mutant (M1705) has been constructed that can grow to temporarily produce wild-type MspA monomers in the presence of acetamide. The ML705 strain is then transformed with a plasmid containing an expression cassette for a wild-type or mutant MspA monomer or a wild-type or mutant single-chain Msp porin. The generation of the wild-type MspA monomer can be shut off by washing away acetamide and transferring the cells to a culture medium without acetamide. This causes the generation of wild-type or mutant MspA monomers or the generation of wild-type or mutant single-chain Msp porin that is less contaminated with wild-type MspA. Therefore, ML705 is suitable for producing wild-type and mutant MspA porin for any purpose.

Example 12: Construction of single-chain MspA porin dimers

Single-stranded DNA is not rotationally symmetric. Therefore, it is advantageous to have an asymmetric hole for sequencing purposes. In order to combine the excellent sequencing capabilities of the MspA porin with the enhanced ability to adapt the vestibule and constriction properties to DNA sequencing, a single-stranded MspA nanopore will be constructed. The MspA chain ends are linked together in the MspA porin dimer (Figure 26A), which can be connected by a short peptide linker. To test this idea, the MspA gene monomer is fused to the MspB gene monomer, which encodes a protein with only 2 changes (A138P, E139A) compared to the wild-type MspA monomer (Stahl et al., Mol.Microbiol.40:451 (2001)). The (GGGGS) ₃ (SEQ ID NO: 3) peptide (Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879 (1988)), commonly used to link proteins, was used to link the C-terminus of the MspA monomer to the N-terminus of the MspB monomer, which lacks a signal peptide ( FIG. 26B ). The resulting MspA-MspB porin dimer was placed under the control of the constitutive _psmyc promoter in plasmid pML870 and then expressed in Mycobacterium smegmatis ML16. The protein was purified using standard heat extraction methods. Although the expression level of the single-chain MspA porin dimer was lower than that of the wild-type MspA porin ( FIG. 26C ), the channel activity of the two porins was similar ( FIG. 26D ). Analysis of current recordings showed that the single-channel conductance of the pore formed by the MspA dimer was 2.6 nS. This result shows that the linker segment does not impair the folding or function of the Msp pore.

Example 13: Construction of single-chain MspA porin

To combine the excellent sequencing capabilities of MspA with the increased ability to adapt the vestibule and constriction properties to DNA sequencing, a single-stranded MspA porin octamer was constructed that allows the optimal properties of the vestibule and constriction to be used for DNA sequencing. The MspA chain ends were linked together in the MspA porin by a short peptide linker. The carboxyl terminus of the preceding MspA monomer was linked to the amino terminus of the subsequent MspA monomer lacking a signal peptide using the (GGGGS) ₃ (SEQ ID NO: 3) peptide.

To generate vectors containing the MspA porin sequence, each MspA monomer sequence was flanked by unique restriction sites to allow for mutation of any individual monomer. The entire MspA porin sequence was flanked by PacI and HindIII restriction sites. Restriction sites between MspA monomer sequences include: BamHI, ClaI, EcoRV, HpaI, KpnI, MluI, NdeI, NheI, PstI, ScaI, SpeI, XbaI, NotI, and SphI (Figure 31). To generate the MspA porin sequence, each MspA sequence was assembled in steps using unique restriction sites to form dimers, tetramers, and octamers of single-stranded MspA. To avoid recombination issues in generating single-chain MspA multimers, seven MspA genes (SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27) were synthesized using different codon usage, i.e., the genes encode identical amino acid sequences, but the DNA sequences are altered compared to the original MspA gene nucleotide sequence (SEQ ID NO: 20). To generate the MspA porin sequence, the first Msp monomer must contain the leader sequence shown in Figure 18 (e.g., amino acids 1 to 27 of SEQ ID NO: 28). Each of the seven Msp monomer sequences following the first Msp monomer sequence can contain SEQ ID NO: 1 or a mutation of SEQ ID NO: 1 selected from the mutations listed in Table 7. The expression vector pML2604 is the parent vector containing the MspA porin sequence cloned into Pad and HindIII restriction sites. pML2604 was transformed into the quadruple porin mutant, and the expression level and oligomeric state of the MspA porin were examined by Western blotting of native and denatured proteins. The channel activity of the MspA porin was examined by lipid bilayer assay.

Table 7: MspA mutants

The present invention specifically relates to the following embodiments:

1. A method comprising applying an electric field to a Mycobacterium smegmatis porin (Msp) having a vestibule and a constriction region defining a channel, wherein the Msp porin is located between a first conductive liquid medium and a second conductive liquid medium.

2. The method of embodiment 1, wherein the Msp porin is selected from the group consisting of a wild-type MspA porin, a mutant MspA porin, a wild-type MspA paralog or homolog porin, and a mutant MspA paralog or homolog porin.

3. The method of embodiment 1 or embodiment 2, wherein the Msp porin further comprises a molecular motor, wherein the molecular motor is capable of moving the analyte into or through the channel at an average translocation velocity that is less than the average translocation velocity of the analyte when translocated into or through the channel by electrophoresis in the absence of the molecular motor.

4. The method of any one of embodiments 1 to 3, wherein at least one conductive liquid medium contains an analyte.

5. The method of embodiment 3 or embodiment 4, further comprising detecting an analyte in a method comprising measuring an ionic current to provide a current pattern when the analyte interacts with the channel, wherein the presence of a blockade in the current pattern is indicative of the presence of the analyte.

6. The method of any one of embodiments 3 to 5, wherein the applied electric field is sufficient to cause electrophoretic transport of the analyte through the channel.

7. The method of any one of embodiments 3 to 6, wherein the Msp porin is a mutant MspA or a mutant MspA paralog or homolog porin, and the average translocation velocity of the analyte through the channel is less than the average translocation velocity of the analyte through the channel of a wild-type MspA or a wild-type MspA paralog or homolog porin.

8. The method of any one of embodiments 3 to 6, wherein the Msp porin is a mutant MspA or a mutant MspA paralog or homolog porin, and the average translocation velocity of the analyte through the channel is greater than the average translocation velocity of the analyte through the channel of a wild-type MspA or a wild-type MspA paralog or homolog porin.

9. The method of any one of embodiments 3 to 8, wherein the average translocation velocity of the analyte through the channel is less than 0.5 nm/μs.

10. The method of any one of embodiments 3 to 9, further comprising identifying the analyte.

11. The method of embodiment 10, wherein identifying the analyte comprises comparing the current pattern to a known current pattern obtained under the same conditions using a known analyte.

12. The method of any one of embodiments 3 to 11, wherein the analyte is a nucleotide, a nucleic acid, an amino acid, a peptide, a protein, a polymer, a drug, an ion, a pollutant, a nanoscale object, or a biological warfare agent.

13. The method of any one of embodiments 3 to 12, wherein the analyte is a polymer.

14. The method of embodiment 13, wherein the polymer is a protein, a peptide, or a nucleic acid.

15. The method of embodiment 14, wherein the polymer is a nucleic acid.

16. The method of embodiment 15, wherein the average translocation velocity of the nucleic acid through the channel is less than 1 nucleotide/μs.

17. The method of embodiment 15 or embodiment 16, wherein the nucleic acid is ssDNA, dsDNA, RNA, or a combination thereof.

18. The method of any one of embodiments 13 to 17, further comprising distinguishing at least a first unit within the polymer from at least a second unit within the polymer, wherein the distinguishing comprises measuring ionic currents generated when the first and second units are respectively translocated through the channel to generate first and second current patterns, respectively, wherein the first and second current patterns are different from each other.

19. The method of any one of embodiments 13 to 18, further comprising determining the sequence of the polymer.

20. The method of embodiment 19, wherein sequencing comprises measuring the ionic current or optical signal as each unit of the polymer is separately translocated through the channel to provide a current pattern associated with each unit, and comparing each current pattern with a current pattern of a known unit obtained under the same conditions to determine the sequence of the polymer.

21. The method of any one of embodiments 3 to 20, further comprising determining the concentration, size, molecular weight, shape or orientation of the analyte, or any combination thereof.

22. The method of any one of embodiments 1 to 21, wherein the at least one conductive liquid medium comprises a plurality of analytes.

23. The method of any one of embodiments 1 to 22, wherein the Msp porin is further defined as a mutant MspA porin.

24. The method of embodiment 23, wherein the mutant MspA porin comprises:

Antechambers and constrictions defining the passageway, and

At least a first mutant MspA monomer comprises a mutation at position 93 and a mutation at position 90, a mutation at position 91, or a mutation at positions 90 and 91.

25. The method of embodiment 23 or embodiment 24, wherein the diameter of the constriction zone of the mutant MspA porin is smaller than the diameter of the constriction zone of the wild-type MspA porin.

26. The method of any one of embodiments 23 to 25, wherein the mutant MspA porin comprises a mutation in the vestibule or constriction that allows the average translocation rate of an analyte by electrophoretic translocation through the channel of the mutant to be less than the average translocation rate of an analyte by electrophoretic translocation through the channel of the wild-type MspA porin.

27. The method of any one of embodiments 24 to 26, wherein the first mutant MspA monomer further comprises one or more mutations at any of the following amino acid positions: 88, 105, 108, 118, 134, or 139.

28. The method of any one of embodiments 23 to 27, wherein the mutant MspA porin comprises a neutral constriction region.

29. The method of any one of embodiments 23 to 28, wherein the conductance of the channel through the mutant MspA porin is higher than the conductance of the channel through its corresponding wild-type MspA porin.

30. The method of embodiment 23, wherein the mutant MspA porin comprises

a vestibule having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and

a constriction having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm,

wherein the vestibule and constriction together define a passageway, and

Also included is at least a first mutant MspA paralog or homolog monomer.

31. The method of embodiment 30, wherein the diameter of the constriction zone of the mutant MspA porin is smaller than the diameter of the constriction zone of its corresponding wild-type MspA porin.

32. The method of embodiment 30 or embodiment 31, wherein the mutant MspA porin comprises a mutation in the vestibule or constriction that allows an average translocation rate of an analyte by electrophoretic translocation through the channel of the mutant to be less than an average translocation rate of an analyte by electrophoretic translocation through the channel of its corresponding wild-type MspA porin.

33. The method of any one of embodiments 30 to 32, wherein the conductance of the channel through the mutant MspA porin is higher than the conductance of the channel through its corresponding wild-type MspA porin.

34. The method of any one of embodiments 1, 3 to 6, or 9 to 22, wherein the Msp porin is further defined as an Msp porin comprising

a vestibule having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and

a constriction having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm,

The vestibule and the constriction zone together define a passageway.

35. The method of any one of embodiments 1, 3 to 6, or 9 to 22, wherein the Msp porin is encoded by a nucleic acid sequence encoding a single-chain Msp porin, wherein the nucleic acid sequence comprises:

(a) a first and a second nucleotide sequence, 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.

36. The method of embodiment 35, wherein the first and second Msp monomer sequences are independently selected from a wild-type MspA monomer, a wild-type MspB monomer, a wild-type MspC monomer, a wild-type MspD monomer, and mutants thereof.

37. The method of embodiment 35, wherein the first Msp monomer sequence comprises a wild-type MspA monomer or a mutant thereof.

38. The method of embodiment 37, wherein the first Msp monomer sequence comprises a mutant MspA monomer.

39. The method of any one of embodiments 1, 3 to 6, or 9 to 22, wherein the Msp porin is encoded by a nucleic acid sequence encoding a single-chain Msp porin, wherein the nucleic acid sequence comprises:

(a) the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences, wherein the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences encode the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th Msp monomer sequences, respectively; and

(b) The ninth nucleotide sequence encoding the amino acid linker sequence.

40. The method of embodiment 39, wherein each Msp monomer comprises a wild-type MspA monomer or a mutant thereof.

41. The method of embodiment 39, wherein at least one Msp monomer comprises a wild-type MspA monomer or a mutant thereof.

42. The method of any one of embodiments 35 to 41, wherein the Msp monomer is a wild-type MspA paralog or homolog.

43. The method of embodiment 42, wherein the wild-type MspA paralog or homolog is selected from the group consisting of MspA/Msmeg0965, MspB/Msmeg0520, MspC/Msmeg5483, MspD/Msmeg6057, MppA, PorM1, PorM2, PorM1, Mmcs4296, Mmcs4297, Mmcs3857, Mmcs4382, Mmcs4383, Mjls3843, Mjls3857, Mjls3931, Mjls4674, Mjls4675, Mjls4676, 7. Map3123c, Mav3943, Mvan1836, Mvan4117, Mvan4839, Mvan4840, Mvan5016, Mvan5017, Mvan5768, MUL_2391, Mflv1734, Mflv173 5. Mflv2295, Mflv1891, MCH4691c, MCH4689c, MCH4690c, MAB1080, MAB1081, MAB2800, RHA1ro08561, RHA1ro04074 and RHA1ro03127.

44. A method of altering the conductance of a channel through a Mycobacterium smegmatis porin (Msp) comprising removing, adding or substituting at least one amino acid in the vestibule or constriction of a wild-type Msp porin.

45. A system comprising a Mycobacterium smegmatis porin (Msp) having a vestibule and a constriction region defining a channel, wherein the channel is located between a first liquid medium and a second liquid medium, wherein at least one of the liquid media comprises an analyte, and wherein the system is effective for detecting a property of the analyte.

46. The system of embodiment 45, wherein the system is effective for translocating the analyte through the channel.

47. The system of embodiment 45, wherein the system is effective for detecting a property of an analyte comprising subjecting the Msp porin to an electric field such that the analyte interacts with the Msp porin.

48. The method of any one of embodiments 45 to 47, wherein the system is effective for detecting a property of an analyte comprising subjecting the Msp porin to an electric field such that the analyte is electrophoretically translocated through a channel of the Msp porin.

49. The system of any one of embodiments 45 to 48, wherein the property is an electrical property, a chemical property, or a physical property of the analyte.

50. The system of any one of embodiments 45 to 49, wherein the Msp porin is contained in a lipid bilayer.

51. The system of any one of embodiments 45 to 50, further defined as comprising a plurality of Msp porins.

52. The system of any one of embodiments 45 to 51, wherein the Msp porin is selected from a wild-type MspA porin, a mutant MspA porin, a wild-type MspA paralog or homolog porin, or a mutant MspA paralog or homolog porin.

53. The system of any one of embodiments 45 to 52, wherein the Msp porin is further defined as a mutant MspA porin.

54. The system of embodiment 53, wherein the mutant MspA porin comprises

Antechambers and constrictions defining the passageway, and

At least a first mutant MspA monomer comprises a mutation at position 93 and a mutation at position 90, a mutation at position 91, or a mutation at positions 90 and 91.

55. The system of embodiment 53, wherein the mutant MspA porin comprises

a vestibule having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and

a constriction having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm,

wherein the vestibule and constriction together define a passageway, and

Also included is at least a first mutant MspA paralog or homolog monomer.

56. The system of any one of embodiments 45 to 51, wherein the Msp porin is further defined as an Msp porin comprising

a vestibule having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and

a constriction having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm,

The vestibule and the constriction zone together define a passageway.

57. The system of embodiment 52, wherein the Msp is a wild-type MspA paralog or homolog porin.

58. The system of embodiment 57, wherein the wild-type MspA paralog or homolog porin is selected from the group consisting of MspA/Msmeg0965, MspB/Msmeg0520, MspC/Msmeg5483, MspD/Msmeg6057, MppA, PorM1, PorM2, PorM1, Mmcs4296, Mmcs4297, Mmcs3857, Mmcs4382, Mmcs4383, Mjls3843, Mjls3857, Mjls3931, Mjls4674, Mjls4675, Mjls4676, 677, Map3123c, Mav3943, Mvan1836, Mvan4117, Mvan4839, Mvan4840, Mvan5016, Mvan5017, Mvan5768, MUL_2391, Mflv1734, Mflv17 35. Mflv2295, Mflv1891, MCH4691c, MCH4689c, MCH4690c, MAB1080, MAB1081, MAB2800, RHA1ro08561, RHA1ro04074 and RHA1ro03127.

59. The system of any one of embodiments 45 to 51, wherein the Msp porin is encoded by a nucleic acid sequence encoding a single-chain Msp porin, wherein the nucleic acid sequence comprises:

(a) a first and a second nucleotide sequence, 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.

60. The method of any one of embodiments 45 to 51, wherein the Msp porin is encoded by a nucleic acid sequence encoding a single-chain Msp porin, wherein the nucleic acid sequence comprises:

(a) the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences, wherein the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences encode the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th Msp monomer sequences, respectively; and

(b) The ninth nucleotide sequence encoding the amino acid linker sequence.

61. The system of any one of embodiments 45 to 60, wherein the Msp porin further comprises a molecular motor, wherein the molecular motor is capable of moving the analyte into or through the channel at an average translocation velocity that is less than the average translocation velocity of the analyte into or through the channel in the absence of the molecular motor.

62. The system of any one of embodiments 45 to 61, further comprising a patch clamp amplifier.

63. The system of any one of embodiments 45 to 62, further comprising a data acquisition device.

64. The system of any one of embodiments 45 to 63, further comprising one or more temperature regulating devices in communication with the first liquid medium, the second liquid medium, or both.

65. A system comprising a Mycobacterium smegmatis porin (Msp) having a vestibule and a constriction region defining a channel, wherein the channel is located in a lipid bilayer between a first liquid medium and a second liquid medium, and wherein the only point of liquid communication between the first and second liquid media occurs in the channel.

66. A mutant Mycobacterium smegmatis porin A (MspA) porin comprising

Antechambers and constrictions defining the passageway, and

At least a first mutant MspA monomer comprises a mutation at position 93 and a mutation at position 90, a mutation at position 91, or a mutation at positions 90 and 91.

67. The mutant MspA porin of embodiment 66, comprising mutations at positions 93 and 90.

68. The mutant MspA porin of embodiment 66, comprising mutations at positions 93 and 91.

69. The mutant MspA porin of embodiment 66, comprising mutations at positions 93, 91 and 90.

70. The mutant MspA porin of embodiment 66, wherein the diameter of the constriction zone of the mutant MspA porin is smaller than the diameter of the constriction zone of the wild-type MspA porin.

71. The mutant MspA porin of any one of embodiments 66 to 70, wherein the MspA porin has a mutation in the vestibule or constriction that allows an analyte to translocate through the channel of the mutant at an average translocation velocity that is less than the average translocation velocity of the analyte through the channel of the wild-type Msp porin.

72. The mutant MspA porin of any one of embodiments 66 to 70, wherein the MspA porin has a mutation in the vestibule or constriction that allows an analyte to be electrophoretically translocated through the channel of the mutant at an average translocation velocity that is less than the average translocation velocity of the analyte to be electrophoretically translocated through the channel of the wild-type Msp porin.

73. The mutant MspA porin of any one of embodiments 66 to 72, wherein the MspA porin has a mutation in the vestibule or constriction that allows translocation of an analyte through the channel at an average translocation velocity of less than 0.5 nm/μs.

74. The mutant MspA porin of any one of embodiments 71 to 73, wherein the analyte is selected from the group consisting of nucleotides, nucleic acids, amino acids, peptides, proteins, polymers, drugs, ions, biological warfare agents, pollutants, nanoscale objects, or combinations or clusters thereof.

75. The mutant MspA porin of embodiment 74, wherein the analyte is further defined as a nucleic acid.

76. The mutant MspA porin of embodiment 75, wherein the nucleic acid translocates through the channel at an average translocation velocity of less than 1 nucleotide/μs.

77. The mutant MspA porin of embodiment 75 or embodiment 76, wherein the nucleic acid is further defined as ssDNA, dsDNA, RNA, or a combination thereof.

78. The mutant MspA porin of any one of embodiments 66 to 77, wherein the mutant MspA porin comprises 2 to 15 Msp monomers that are the same or different.

79. The mutant MspA porin of embodiment 78, wherein the mutant MspA porin comprises 7 to 9 Msp monomers that are the same or different.

80. The mutant MspA porin of any one of embodiments 66 to 79, further comprising at least a second monomer selected from a wild-type MspA monomer, a second mutant MspA monomer, a wild-type MspA paralog or homolog monomer, and a mutant MspA paralog or homolog monomer, wherein the second mutant MspA monomer may be the same as or different from the first mutant MspA monomer.

81. The mutant MspA porin of embodiment 80, wherein the second monomer is a wild-type MspA paralog or homolog monomer.

82. The mutant MspA porin of embodiment 81, wherein the wild-type MspA paralog or homolog monomer is a wild-type MspB monomer.

83. The mutant MspA porin of any one of embodiments 66 to 82, wherein the first mutant MspA monomer further comprises one or more mutations at any of the following amino acid positions: 88, 105, 108, 118, 134, or 139.

84. The mutant MspA porin of any one of embodiments 66 to 82, wherein the first mutant MspA monomer comprises one or more of the following mutations: L88W, D90K/N/Q/R, D91N/Q, D93N, I105W, N108W, D118R, D134R, or E139K.

85. The mutant MspA porin of any one of embodiments 66 to 82, wherein the first mutant MspA monomer comprises the following mutations: D90N/D91N/D93N.

86. The mutant MspA porin of any one of embodiments 66 to 82, wherein the first mutant MspA monomer comprises the following mutations: D90N/D91N/D93N/D118R/D134R/E139K.

87. The mutant MspA porin of any one of embodiments 66 to 82, wherein the first mutant MspA monomer comprises the following mutations: D90Q/D91Q/D93N.

88. The mutant MspA porin of any one of embodiments 66 to 82, wherein the first mutant MspA monomer comprises the following mutations: D90Q/D91Q/D93N/D118R/D134R/E139K.

89. The mutant MspA porin of any one of embodiments 66 to 82, wherein the first mutant MspA monomer comprises the following mutations: D90(K,R)/D91N/D93N.

90. The mutant MspA porin of any one of embodiments 66 to 82, wherein the first mutant MspA monomer comprises the following mutations: (L88, I105)W/D91Q/D93N.

91. The mutant MspA porin of any one of embodiments 66 to 82, wherein the first mutant MspA monomer comprises the following mutations: I105W/N108W.

92. The mutant MspA porin of any one of embodiments 66 to 91, further comprising a deletion of one or more periplasmic loops.

93. The mutant MspA porin of any one of embodiments 66 to 92, wherein the conductance through the channel is higher than the conductance through the channel of its corresponding wild-type MspA porin.

94. The mutant MspA porin of any one of embodiments 66 to 93, further comprising a molecular motor, wherein the molecular motor is capable of moving an analyte into or through the channel at an average translocation velocity that is less than the average translocation velocity of the analyte into or through the channel in the absence of the molecular motor.

95. The mutant MspA porin of any one of embodiments 66 to 94, further comprising a molecular motor, wherein the molecular motor is capable of moving an analyte into or through the channel at an average translocation velocity that is less than the average translocation velocity of the analyte into or through the channel by electrophoresis in the absence of the molecular motor.

96. The mutant MspA porin of embodiment 94 or embodiment 95, wherein the molecular motor is an enzyme.

97. The mutant MspA porin of embodiment 96, wherein the enzyme is a polymerase, an exonuclease, or a Klenow fragment.

98. A mutant Mycobacterium smegmatis porin A (MspA) porin comprising

a vestibule having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and

a constriction having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm,

wherein the vestibule and constriction together define a passageway, and

Also included is at least a first mutant MspA paralog or homolog monomer.

99. The mutant MspA porin of embodiment 98, wherein the diameter of the constriction zone of the mutant MspA porin is smaller than the diameter of the constriction zone of its corresponding wild-type MspA porin.

100. The mutant MspA porin of embodiment 98 or embodiment 99, having a mutation in the vestibule or constriction region, wherein the mutation allows an analyte to translocate through the channel of the mutant at an average translocation velocity that is less than the average translocation velocity of the analyte through the channel of its corresponding wild-type MspA porin.

101. The mutant MspA porin of any one of embodiments 98 to 100, comprising a mutation in the vestibule or constriction that allows electrophoretic translocation of an analyte through the channel of the mutant at an average translocation velocity that is less than the average translocation velocity of the analyte through the channel of its corresponding wild-type MspA porin.

102. The mutant MspA porin of any one of embodiments 98 to 101, having a mutation in the vestibule or constriction that allows the analyte to traverse the channel at an average translocation velocity of less than 0.5 nm/μs.

103. The mutant MspA porin of any one of embodiments 101 to 102, wherein the analyte is further defined as a nucleotide, a nucleic acid, an amino acid, a peptide, a protein, a polymer, a drug, an ion, a biowarfare agent, a pollutant, a nanoscale object, or a combination or cluster thereof.

104. The mutant MspA porin of embodiment 103, wherein the analyte is further defined as a nucleic acid.

105. The mutant MspA porin of embodiment 104, wherein the nucleic acid translocates through the channel at an average translocation velocity of less than 1 nucleotide/μs.

106. The mutant MspA porin of embodiment 104 or embodiment 105, wherein the nucleic acid is further defined as ssDNA, dsDNA, RNA, or a combination thereof.

107. The mutant MspA porin of any one of embodiments 98 to 106, wherein the mutant MspA porin further comprises at least one Msp monomer.

108. The mutant MspA porin of embodiment 107, wherein the Msp monomer is selected from a wild-type MspA monomer, a mutant MspA monomer, a wild-type MspA paralog or homolog, or a second mutant MspA paralog or homolog monomer.

109. The mutant MspA porin of any one of embodiments 98 to 108, further comprising a deletion of one or more periplasmic loops.

110. The mutant MspA porin of any one of embodiments 98 to 109, comprising a mutation in the vestibule or constriction.

111. The mutant MspA porin of any one of embodiments 98 to 110, wherein the conductance through the channel is higher than the conductance through the channel of its corresponding wild-type MspA porin.

112. The mutant MspA porin of any one of embodiments 98 to 111, further comprising a molecular motor, wherein the molecular motor is capable of moving an analyte into or through the channel at an average translocation velocity that is less than the average translocation velocity of the analyte when translocated through or through the channel in the absence of the molecular motor.

113. The mutant MspA porin of any one of embodiments 98 to 112, further comprising a molecular motor, wherein the molecular motor is capable of moving an analyte into or through the channel at an average translocation velocity that is less than the average translocation velocity of the analyte into or through the channel by electrophoresis in the absence of the molecular motor.

114. The mutant MspA porin of embodiment 112 or embodiment 113, wherein the molecular motor is an enzyme.

115. The mutant MspA porin of embodiment 114, wherein the enzyme is a polymerase, an exonuclease, or a Klenow fragment.

116. A mutant Mycobacterium smegmatis porin A (MspA) paralog or homolog comprising

a vestibule having a length of about 2 to about 6 nm and a diameter of about 2 to about 6 nm, and

a constriction having a length of about 0.3 to about 3 nm and a diameter of about 0.3 to about 3 nm,

The vestibule and the constriction zone together define a passageway.

117. The mutant MspA paralog or homolog of embodiment 116, further comprising at least a first mutant MspA paralog or homolog monomer.

118. A method of making a mutant Mycobacterium smegmatis Porin A (MspA) porin comprising at least one mutant MspA monomer, the method comprising modifying a wild-type MspA monomer at positions 93 and 90, position 91, or positions 90 and 91.

119. A method for preparing a mutant Mycobacterium smegmatis porin A (MspA) porin having a vestibule and a constriction region defining a channel, comprising deleting, adding or substituting any amino acid in the vestibule or constriction region of a wild-type MspA paralog or homolog monomer such that the resulting mutant MspA porin is capable of translocating an analyte through the channel upon application of an electric field.

120. A method comprising translocating an analyte through a channel of a Mycobacterium smegmatis porin (Msp) porin in the absence of an applied electric field.

121. The method of embodiment 120, wherein the Msp porin further comprises a molecular motor.

122. The method of embodiment 120 or embodiment 121, wherein the Msp porin is selected from the group consisting of a wild-type MspA porin, a mutant MspA porin, a wild-type MspA paralog or homolog porin, and a mutant MspA paralog or homolog porin.

123. The method of embodiment 120 or embodiment 121, wherein the Msp porin is encoded by a nucleic acid sequence encoding a single-chain Msp porin, wherein the nucleic acid sequence comprises:

(a) a first and a second nucleotide sequence, 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.

124. The method of embodiment 120 or embodiment 121, wherein the Msp porin is encoded by a nucleic acid sequence encoding a single-chain Msp porin, wherein the nucleic acid sequence comprises:

(a) the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences, wherein the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences encode the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th Msp monomer sequences, respectively; and

(b) The ninth nucleotide sequence encoding the amino acid linker sequence.

125. The method of any one of embodiments 120 to 124, wherein the analyte comprises optical beads or magnetic beads.

126. A nucleic acid sequence encoding a single-chain Msp porin, wherein the nucleic acid sequence comprises:

(a) a first and a second nucleotide sequence, 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.

127. The nucleic acid sequence of embodiment 126, wherein the first and second Msp monomer sequences are independently selected from a wild-type MspA monomer, a mutant MspA monomer, a wild-type MspA paralog or homolog monomer, and a mutant MspA paralog or homolog monomer.

128. The nucleic acid sequence of embodiment 126 or embodiment 127, wherein the first Msp monomer sequence comprises a wild-type MspA monomer or a mutant thereof.

129. The nucleic acid sequence of embodiment 128, wherein the Msp monomer sequence comprises a mutant MspA monomer.

130. The nucleic acid sequence of embodiment 128, wherein the first Msp monomer sequence comprises one or more mutations selected from the following: an A to P substitution on amino acid 138, an E to A or K substitution on amino acid 139, a D to K or R or Q substitution on amino acid 90, a D to N or Q substitution on amino acid 91, a D to N substitution on amino acid 93, an L to W substitution on amino acid 88, an I to W substitution on amino acid 105, an N to W substitution on amino acid 108, a D to R substitution on amino acid 118, and a D to R substitution on amino acid 134.

131. The nucleic acid sequence of embodiment 130, wherein the mutant MspA monomer comprises an A to P substitution at amino acid 138, an E to A substitution at amino acid 139, or a combination thereof.

132. The nucleic acid sequence of embodiment 130, wherein the mutant MspA monomer comprises a D to K or R substitution at amino acid 90, a D to N substitution at amino acid 91, a D to N substitution at amino acid 93, or any combination thereof.

133. The nucleic acid sequence of embodiment 130, wherein the mutant MspA monomer comprises a D to Q substitution at amino acid 90, a D to Q substitution at amino acid 91, a D to N substitution at amino acid 93, or any combination thereof.

134. The nucleic acid sequence of embodiment 130, wherein the mutant MspA monomer comprises an L to W substitution at amino acid 88, an I to W substitution at amino acid 105, a D to Q substitution at amino acid 91, a D to N substitution at amino acid 93, or any combination thereof.

135. The nucleic acid sequence of embodiment 130, wherein the mutant MspA monomer comprises an I to W substitution at amino acid 105, an N to W substitution at amino acid 108, or a combination thereof.

136. The nucleic acid sequence of embodiment 130, wherein the mutant MspA monomer comprises a D to R substitution at amino acid 118, an E to K substitution at amino acid 139, a D to R substitution at amino acid 134, or any combination thereof.

137. The nucleic acid sequence of embodiment 126, wherein the first MspA monomer sequence comprises SEQ ID NO: 1.

138. The nucleic acid sequence of embodiment 126, wherein the second MspA monomer sequence comprises a wild-type MspA paralog or a mutant thereof, wherein the paralog or mutant thereof is a wild-type MspB monomer or a mutant thereof.

139. The nucleic acid sequence of embodiment 138, wherein the second MspA monomer sequence comprises SEQ ID NO:2.

140. The nucleic acid sequence of embodiment 138, wherein the second MspA monomer sequence comprises a mutant MspB monomer.

141. The nucleic acid sequence of embodiment 126, wherein the first MspA monomer sequence comprises a wild-type MspA monomer or a mutant thereof, and the second Msp monomer comprises a wild-type MspB monomer or a mutant thereof.

142. The nucleic acid sequence of embodiment 126, wherein the first MspA monomer sequence comprises SEQ ID NO: 1 and the second Msp monomer comprises SEQ ID NO: 2.

143. The nucleic acid sequence of any one of embodiments 126 to 142, wherein the amino acid linker sequence comprises 10 to 20 amino acids.

144. The nucleic acid sequence of embodiment 143, wherein the amino acid linker sequence comprises 15 amino acids.

145. The nucleic acid sequence of embodiment 144, wherein the amino acid linker sequence comprises a (GGGGS) ₃ (SEQ ID NO: 3) peptide sequence.

146. A polypeptide encoded by the nucleic acid sequence of one of embodiments 126 to 145.

147. A vector comprising the nucleic acid sequence of one of embodiments 126 to 145.

148. The vector of embodiment 147, wherein the vector further comprises a promoter sequence.

149. The vector of embodiment 148, wherein the promoter comprises a constitutive promoter.

150. The vector of embodiment 149, wherein the constitutive promoter comprises a _psmyc promoter.

151. The vector of embodiment 148, wherein the promoter comprises an inducible promoter.

152. The vector of embodiment 151, wherein the inducible promoter comprises an acetamide-inducible promoter.

153. A nucleic acid sequence encoding a single-chain Msp porin, wherein the nucleic acid sequence comprises:

(a) the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences, wherein the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences encode the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th Msp monomer sequences, respectively; and

(b) The ninth nucleotide sequence encoding the amino acid linker sequence.

154. The nucleic acid sequence of embodiment 153, wherein the first and second Msp monomer sequences are independently selected from a wild-type Msp monomer, a mutant Msp monomer, a wild-type MspA paralog or homolog monomer, and a mutant MspA paralog or homolog monomer.

155. The nucleic acid sequence of embodiment 153, wherein each Msp monomer comprises a wild-type MspA monomer or a mutant thereof.

156. The nucleic acid sequence of embodiment 153, wherein at least one Msp monomer comprises a wild-type MspA monomer or a mutant thereof.

157. The nucleic acid sequence of embodiment 155 or embodiment 156, wherein at least one Msp monomer comprises a mutant MspA monomer.

158. The nucleic acid sequence of embodiment 157, wherein the mutant Msp monomer sequence comprises one or more mutations selected from the following: an A to P substitution on amino acid 138, an E to A or K substitution on amino acid 139, a D to K or R or Q substitution on amino acid 90, a D to N or Q substitution on amino acid 91, a D to N substitution on amino acid 93, an L to W substitution on amino acid 88, an I to W substitution on amino acid 105, an N to W substitution on amino acid 108, a D to R substitution on amino acid 118, and a D to R substitution on amino acid 134.

159. The nucleic acid sequence of embodiment 158, wherein the mutant MspA monomer comprises an A to P substitution at amino acid 138, an E to A substitution at amino acid 139, or a combination thereof.

160. The nucleic acid sequence of embodiment 158, wherein the mutant MspA monomer comprises a D to K or R substitution at amino acid 90, a D to N substitution at amino acid 91, a D to N substitution at amino acid 93, or any combination thereof.

161. The nucleic acid sequence of embodiment 158, wherein the mutant MspA monomer comprises a D to Q substitution at amino acid 90, a D to Q substitution at amino acid 91, a D to N substitution at amino acid 93, or any combination thereof.

162. The nucleic acid sequence of embodiment 158, wherein the mutant MspA monomer comprises an L to W substitution at amino acid 88, an I to W substitution at amino acid 105, a D to Q substitution at amino acid 91, a D to N substitution at amino acid 93, or any combination thereof.

163. The nucleic acid sequence of embodiment 158, wherein the mutant MspA monomer comprises an I to W substitution at amino acid 105, an N to W substitution at amino acid 108, or a combination thereof.

164. The nucleic acid sequence of embodiment 158, wherein the mutant MspA monomer comprises a D to R substitution at amino acid 118, an E to K substitution at amino acid 139, a D to R substitution at amino acid 134, or any combination thereof.

165. The nucleic acid sequence of embodiment 153, wherein each Msp monomer sequence comprises SEQ ID NO:1.

166. The nucleic acid sequence of embodiment 153, wherein at least one Msp monomer sequence comprises SEQ ID NO:1.

167. The nucleic acid sequence of embodiment 153, wherein at least one Msp monomer sequence comprises a wild-type MspA paralog or a mutant thereof, wherein the MspA paralog or a mutant thereof is a wild-type MspB monomer or a mutant thereof.

168. The nucleic acid sequence of embodiment 166 or embodiment 167, wherein at least one Msp monomer sequence comprises SEQ ID NO:2.

169. The nucleic acid sequence of any one of embodiments 166 to 168, wherein at least one Msp monomer sequence comprises a mutant MspB monomer.

170. The nucleic acid sequence of embodiment 153, wherein at least one Msp monomer sequence comprises a wild-type MspA monomer or a mutant thereof, and at least one Msp monomer sequence comprises a wild-type MspB monomer or a mutant thereof.

171. The nucleic acid sequence of embodiment 153, wherein at least one Msp monomer sequence comprises SEQ ID NO:1, and at least one Msp monomer sequence comprises SEQ ID NO:2.

172. The nucleic acid sequence of any one of embodiments 153 to 171, wherein the amino acid linker sequence comprises 10 to 20 amino acids.

173. The nucleic acid sequence of embodiment 172, wherein the amino acid linker sequence comprises 15 amino acids.

174. The nucleic acid sequence of embodiment 173, wherein the amino acid linker sequence comprises a (GGGGS) ₃ (SEQ IDNO: 3) peptide sequence.

175. A polypeptide encoded by the nucleic acid sequence of one of embodiments 66 to 115 or 153 to 174.

176. A vector comprising the nucleic acid sequence of one of embodiments 66 to 115 or 153 to 174.

177. The vector of embodiment 176, wherein the vector further comprises a promoter sequence.

178. The vector of embodiment 177, wherein the promoter comprises a constitutive promoter.

179. The vector of embodiment 178, wherein the constitutive promoter comprises a _psmyc promoter.

180. The vector of embodiment 179, wherein the promoter comprises an inducible promoter.

181. The promoter of embodiment 180, wherein the inducible promoter comprises an acetamide-inducible promoter.

182. A cultured cell or progeny thereof transfected with a vector according to any one of embodiments 176 to 181, wherein the cell is capable of expressing the Msp porin or Msp porin monomer.

183. A Mycobacterium smegmatis strain comprising the vector of one of embodiments 176 to 181.

184. A mutant bacterial strain capable of inducibly expressing an Msp monomer, the bacterial strain comprising:

(a) Deletion of wild-type MspA;

(b) deletion of wild-type MspC;

(c) deletion of wild-type MspD; and

(d) A vector comprising an inducible promoter operably linked to an Msp monomer nucleic acid sequence.

185. The bacterial strain of embodiment 184, wherein the bacterial strain comprises Mycobacterium smegmatis strain ML16.

186. The bacterial strain of embodiment 184, wherein the Msp nucleic acid encodes a wild-type MspA monomer or a wild-type MspA paralog or homolog monomer.

187. The bacterial strain of embodiment 184, wherein the Msp nucleic acid encodes an Msp monomer selected from the group consisting of a wild-type MspA monomer, a wild-type MspC monomer, and a wild-type MspD monomer.

188. The bacterial strain of embodiment 187, wherein the Msp nucleic acid encodes a wild-type MspA monomer.

189. The bacterial strain of embodiment 184, wherein the inducible promoter comprises an acetamide-inducible promoter.

190. The bacterial strain of embodiment 184, further comprising a deletion of wild-type MspB.

191. The bacterial strain of embodiment 190, further comprising a vector comprising a constitutive promoter operably linked to a nucleic acid sequence encoding an Msp porin or monomer.

192. The bacterial strain of embodiment 191, wherein the Msp is a wild-type MspA porin or monomer or a wild-type MspA paralog or homolog porin or monomer.

193. The bacterial strain of embodiment 191, wherein the Msp porin or monomer is selected from the group consisting of a wild-type MspA porin or monomer, a wild-type MspB porin or monomer, a wild-type MspC porin or monomer, and a wild-type MspD porin or monomer.

194. The bacterial strain of embodiment 193, wherein the Msp porin or monomer is a wild-type MspA porin or monomer.

195. The bacterial strain of embodiment 190, further comprising a vector comprising a nucleic acid encoding a single-chain Msp porin, wherein the nucleic acid comprises:

(a) a first and a second nucleotide sequence, 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.

196. The bacterial strain of embodiment 190, further comprising a vector comprising a nucleic acid encoding a single-chain Msp porin, wherein the nucleic acid comprises:

(a) the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences, wherein the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences encode the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th Msp monomer sequences, respectively; and

(b) The ninth nucleotide sequence encoding the amino acid linker sequence.

197. A method for producing a single-chain Msp porin, the method comprising:

(a) transforming the bacterial strain of embodiment 190 with a vector comprising a nucleic acid sequence capable of encoding a single-chain Msp porin; and

(b) Purification of single-chain Msp porins from bacteria.

198. The method of embodiment 197, wherein the vector comprises a nucleic acid sequence encoding a single-chain Msp porin, wherein the nucleic acid sequence comprises:

(a) a first and a second nucleotide sequence, 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.

199. The method of embodiment 197, wherein the vector comprises a nucleic acid sequence encoding a single-chain Msp porin, wherein the nucleic acid sequence comprises:

(a) the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences, wherein the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th nucleotide sequences encode the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th Msp monomer sequences, respectively; and

(b) The ninth nucleotide sequence encoding the amino acid linker.

200. The method of embodiment 198 or embodiment 199, wherein the Msp monomer sequences are independently selected from a wild-type MspA monomer, a mutant MspA monomer, a wild-type MspA paralog or homolog monomer, and a mutant MspA paralog or homolog monomer.

201. The method of any one of embodiments 198 to 200, wherein the Msp monomer sequence is a wild-type MspA monomer.

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Although exemplary embodiments have been illustrated and described herein, it will be understood that various changes can be made therein without departing from the spirit and scope of what is described herein.

Claims (40)

1. Mycobacterium smegmatis porin (Msp) comprising a precordial and constricted regions defining a channel, wherein the Msp comprises a mutant MspA monomer containing a mutation at position 93, and mutations at positions 90, 91, or both, wherein the mutation at position 90 is D90N or D90Q, and/or the mutation at position 91 is D91N or D91Q, and wherein, compared to the wild-type MspA monomer, negatively charged amino acids at positions 118, 134, and 139 are replaced by positively charged amino acids. 2. The Msp of claim 1, wherein the mutation MspA comprises mutations at positions 90, 91, and 93. 3. The Msp of claim 1, wherein the mutation MspA comprises mutations at positions 90, 91 and 93, wherein the mutation at position 90 is D90N, the mutation at position 91 is D91N, and the mutation at position 93 is D93N. 4. The Msp of claim 3, wherein the mutation at position 118 is D118R. 5. The Msp of claim 4, wherein the mutation at position 139 is E139K or E139R. 6. The Msp of claim 5, wherein the mutation at position 118 is D118R, the mutation at position 134 is D134R, and the mutation at position 139 is E139K. 7. The Msp of claim 3, wherein the mutation MspA comprises the mutation at position 126, wherein the mutation at position 126 is Q126R. 8. The Msp of claim 1, wherein the mutation MspA comprises a mutation at position 88. 9. The Msp of claim 1, wherein the mutation MspA comprises mutations at positions 90, 91 and 93, wherein the mutation at position 90 is D90Q, the mutation at position 91 is D91Q, and the mutation at position 93 is D93N. 10. The Msp of claim 9, wherein the mutation at position 118 is D118R, the mutation at position 134 is D134R, and the mutation at position 139 is E139K. 11. Mycobacterium smegmatis porin (Msp) comprising a precordial and constricted regions defining a channel, wherein the Msp contains a mutant MspA, the mutant MspA comprising mutations at positions 90, 91 and 93, wherein the mutation at position 90 is D90N, the mutation at position 91 is D91N and the mutation at position 93 is D93N. 12. Mycobacterium smegmatis porin (Msp) comprising a precordial and constricted regions defining a channel, wherein the Msp comprises a mutant MspA comprising mutations at positions 90, 91, and 93, wherein the mutation at position 90 is D90Q, the mutation at position 91 is D91Q, and the mutation at position 93 is D93N, and wherein a positively charged amino acid replaces a negatively charged amino acid at positions 118, 134, and 139 of the wild-type Msp. 13. The Msp of claim 12, further comprising mutations at positions 118, 139, and 134, wherein the mutation at position 118 is D118R, the mutation at position 134 is D134R, the mutation at position 139 is E139K, and wherein a positively charged amino acid replaces a negatively charged amino acid at positions 118, 134, and 139 of the wild-type Msp. 14. The Msp of any one of claims 1-13, used for detecting the presence of an analyte. 15. The Msp of claim 14, wherein the analyte is a nucleic acid. 16. The Msp of claim 15, wherein the nucleic acid is DNA. 17. The Msp of claim 16, wherein the DNA is single-stranded DNA. 18. The Msp of claim 17, wherein the single-stranded DNA comprises a hairpin. 19. The Msp of any one of claims 1-13, used for analyzing nucleic acids. 20. The Msp of claim 19, wherein the nucleic acid is DNA. 21. The Msp of claim 20, wherein the DNA is single-stranded DNA. 22. The Msp of claim 21, wherein the single-stranded DNA comprises a hairpin. 23. A system comprising the Msp of any one of claims 1-13, wherein the channel of the Msp 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 effective for detecting the analyte. 24. The system of claim 23, comprising means for applying an electric field sufficient to transpose the analyte from the first conductive liquid medium to the second conductive liquid medium via liquid communication through Msp. 25. The system of claim 24, comprising a device for measuring the current across Msp. 26. The system of claim 23, comprising means for grounding the first conductive liquid medium and applying a positive voltage to the second conductive liquid medium. 27. The system of claim 23, wherein the analyte is a polymer. 28. The system of claim 27, wherein the polymer is a nucleic acid. 29. The system of claim 28, wherein the nucleic acid is DNA. 30. The system of claim 29, wherein the DNA is single-stranded DNA. 31. The system of claim 28, wherein the nucleic acid is RNA. 32. The system of claim 28, wherein the system is used for nucleic acid sequencing. 33. The system of claim 28, wherein the system further comprises a molecular engine, wherein the molecular engine is capable of moving the analyte into or through the channel at an average transposition velocity less than the average transposition velocity of the analyte into or through the channel in the absence of the molecular engine. 34. The system of claim 33, wherein the molecular engine is a helicase. 35. The system of claim 33, wherein the molecular engine is a polymerase. 36. The nucleic acid encoding the Msp of any one of claims 1-13. 37. A vector comprising a promoter operatively linked to the nucleic acid sequence of claim 36. 38. A mutant Mycobacterium smegmatis bacterium capable of expressing Msp, said bacterium comprising: (a) Deficiency of wild-type MspA; (b) The absence of wild-type MspC; (c) The absence of wild-type MspD; (d) The carrier of claim 37. 39. The mutant Mycobacterium smegmatis bacterium of claim 38, wherein the bacterium is Mycobacterium smegmatis ML16 strain. 40. Mycobacterium smegmatis porin that can be obtained from the mutant Mycobacterium smegmatis bacteria of claim 38 or 39.