Long lifetime alpha-hemolysin nanopores

Abstract

Described herein are variants of alpha-hemolysin having at least one amino acid substitution at H35G, E111N, M113A, and/or K147N in the mature, wild-type alpha-hemolysin amino acid sequence. In certain examples, the variant may have a substitution at E111S, M113S, T145S, K147S, or L135I in the mature alpha-hemolysin amino acid sequence. The α-hemolysin variants may also include a substitution at H144A and/or a series of glycine residues spanning residues 127 to 131 of the mature, wild-type alpha hemolysin. Also provided are nanopore assemblies including the alpha-hemolysin variants, the assembly having an increased nanopore lifetime. Further, provided are variants that, in addition to providing increased lifetime, provide a decreased time-to-thread. Hence, the variants provided herein both increase nanopore lifetime and improve efficiency and accuracy of DNA sequencing reactions using nanopores comprising the variants.

What is claimed is: 
     
         1 . An α-hemolysin (α-HL) variant of the amino acid sequence set forth as SEQ ID NO: 14, wherein the variant has at least 80%, 90%, 95%, 98%, or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 14 and wherein the variant comprises an amino acid substitution at a position corresponding to any one of E111N/E111S, M113A/M113S, K147N/K147S, T145S, L135I, or combinations thereof of SEQ ID NO: 14. 
     
     
         2 . The α-hemolysin variant of  claim 1 , wherein the variant further comprises an amino acid substitution corresponding to H144A of the amino acid sequence set forth as SEQ ID NO: 14. 
     
     
         3 . The α-hemolysin variant of  claim 1 , wherein the variant further comprises a substitution corresponding to H35G of the amino acid sequence set forth as SEQ ID NO: 14. 
     
     
         4 . The α-hemolysin variant of  claim 1 , wherein the variant further comprises a poly-G substitution corresponding to amino acids 127-129 of the amino acid sequence set forth as SEQ ID NO: 14. 
     
     
         5 . The α-hemolysin variant of  claim 1 , wherein the variant further comprises a substitution corresponding to K131G of the amino acid sequence set forth as SEQ ID NO: 14. 
     
     
         6 . The α-hemolysin variant of  claim 1 , further comprising an amino acid substitution at a position corresponding to any one of V149K, E287R, T109K, or P151K, or combinations thereof of the amino acid sequence set forth as SEQ ID NO: 14. 
     
     
         7 . The α-hemolysin variant of  claim 1 , wherein the variant has a sequence having at least 80%, 90%, 95%, 98%, or more sequence identity to the sequence set forth as SEQ ID NOs: 17, 18, 19, 20, or 22. 
     
     
         8 . A heptameric nanopore assembly comprising at least one α-hemolysin (α-HL) variant according to  claim 1 . 
     
     
         9 . The heptameric nanopore assembly of  claim 8 , wherein the heptameric nanopore assembly comprises seven of the α-hemolysin variants according to  claim 1 . 
     
     
         10 . The heptameric nanopore assembly of  claim 9 , wherein each of the seven α-hemolysin variants is the same variant. 
     
     
         11 . The heptameric nanopore assembly of  claim 8 , further comprising a DNA polymerase that is bound to one of the alpha-hemolysin variants. 
     
     
         12 . The heptameric nanopore assembly of  claim 11 , wherein the variant is bound to the DNA polymerase via an isopeptide bond. 
     
     
         13 . The heptameric nanopore assembly of  claim 11 , wherein the nanopore assembly has an increased lifetime relative to a nanopore assembly consisting of native alpha-hemolysin. 
     
     
         14 . The heptameric nanopore assembly of  claim 13 , wherein the lifetime is increased by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more as compared to a heptameric nanopore assembly consisting of native alpha-hemolysin. 
     
     
         15 . A nucleic acid encoding an α-hemolysin variant according to any one of  claim 1 . 
     
     
         16 . The nucleic acid molecule of  claim 15 , wherein the nucleic acid molecule is derived from  Staphylococcus aureus  (SEQ ID NO: 1). 
     
     
         17 . A vector comprising a nucleic acid encoding an α-hemolysin variant according to  claim 15 . 
     
     
         18 . A host cell transformed with the vector of  claim 17 . 
     
     
         19 . A method of producing an α-hemolysin variant comprising the steps of:
 (a) culturing the host cell according to  claim 18  in a suitable culture medium under suitable conditions to produce the α-hemolysin variant; 
 (b) obtaining said produced α-hemolysin variant. 
 
     
     
         20 . A method for detecting a target molecule, comprising:
 (a) providing a chip comprising a heptameric nanopore according to  claim 8  in a membrane that is disposed adjacent or in proximity to a sensing electrode;   (b) directing a nucleic acid molecule through said nanopore, wherein said nucleic acid molecule is associated with a reporter molecule, wherein said nucleic acid molecule comprises an address region and a probe region, wherein said reporter molecule is associated with said nucleic acid molecule at said probe region, and wherein said reporter molecule is coupled to a target molecule;   (c) sequencing said address region while said nucleic acid molecule is directed through said nanopore to determine a nucleic acid sequence of said address region; and   (d) identifying, with the aid of a computer processor, said target molecule based upon a nucleic acid sequence of said address region determined in (c).   
     
     
         21 . A heptameric nanopore assembly for nucleic acid sequencing, the heptameric nanopore assembly comprising:
 seven α-hemolysin monomers, wherein each monomer comprises an amino acid sequence having at least 80%, 90%, 95%, 98%, or more sequence identity to the amino acid sequence set forth as SEQ ID NOS: 17, 18, 19, 20, or 22;   a DNA polymerase, wherein the DNA polymerase is bound to at least one of the seven α-hemolysin monomers;   wherein the nanopore assembly has an increased lifetime relative to a nanopore consisting of native α-hemolysin.   
     
     
         22 . The heptameric nanopore assembly of  claim 21 , further comprising a V149K substitution of the amino acid sequence set forth as SEQ ID NOS: 17, 18, 19, 20, or 22 wherein the V149K substitution decreases Time-To-Thread. 
     
     
         23 . The α-hemolysin variant of  claim 1 , wherein the variant further comprises at least two amino acid substitutions selected from the group consisting of: a substitution corresponding to H144A of the amino acid sequence set forth as SEQ ID NO: 14; a substitution corresponding to H35G of the amino acid sequence set forth as SEQ ID NO: 14; a poly-G substitution corresponding to amino acids 127-129 of the amino acid sequence set forth as SEQ ID NO: 14; a substitution corresponding to K131G of the amino acid sequence set forth as SEQ ID NO: 14; and a substitution at a position corresponding to any one of V149K, E287R, T109K, or P151K of the amino acid sequence set forth as SEQ ID NO: 14. 
     
     
         24 . The α-hemolysin variant of  claim 1 , wherein the variant further comprises at least three amino acid substitutions selected from the group consisting of: a substitution corresponding to H144A of the amino acid sequence set forth as SEQ ID NO: 14; a substitution corresponding to H35G of the amino acid sequence set forth as SEQ ID NO: 14; a poly-G substitution corresponding to amino acids 127-129 of the amino acid sequence set forth as SEQ ID NO: 14; a substitution corresponding to K131G of the amino acid sequence set forth as SEQ ID NO: 14; and a substitution at a position corresponding to any one of V149K, E287R, T109K, or P151K of the amino acid sequence set forth as SEQ ID NO: 14. 
     
     
         25 . The α-hemolysin variant of  claim 1 , wherein the variant further comprises at least four amino acid substitutions selected from the group consisting of: a substitution corresponding to H144A of the amino acid sequence set forth as SEQ ID NO: 14; a substitution corresponding to H35G of the amino acid sequence set forth as SEQ ID NO: 14; a poly-G substitution corresponding to amino acids 127-129 of the amino acid sequence set forth as SEQ ID NO: 14; a substitution corresponding to K131G of the amino acid sequence set forth as SEQ ID NO: 14; and a substitution at a position corresponding to any one of V149K, E287R, T109K, or P151K of the amino acid sequence set forth as SEQ ID NO: 14. 
     
     
         26 . The α-hemolysin variant of  claim 1 , wherein the variant further comprises at least five amino acid substitutions selected from the group consisting of: a substitution corresponding to H144A of the amino acid sequence set forth as SEQ ID NO: 14; a substitution corresponding to H35G of the amino acid sequence set forth as SEQ ID NO: 14; a poly-G substitution corresponding to amino acids 127-129 of the amino acid sequence set forth as SEQ ID NO: 14; a substitution corresponding to K131G of the amino acid sequence set forth as SEQ ID NO: 14; and a substitution at a position corresponding to any one of V149K, E287R, T109K, or P151K of the amino acid sequence set forth as SEQ ID NO: 14.