Enzyme-pore constructs

Abstract

The invention relates to constructs comprising a transmembrane protein pore subunit and a nucleic acid handling enzyme. The pore subunit is covalently attached to the enzyme such that both the subunit and enzyme retain their activity. The constructs can be used to generate transmembrane protein pores having a nucleic acid handling enzyme attached thereto. Such pores are particularly useful for sequencing nucleic acids. The enzyme handles the nucleic acid in such a way that the pore can detect its component nucleotides by stochastic sensing.

We claim: 
     
         1 . A method for detecting the addition of a nucleotide to a nucleic acid strand, comprising:
 (a) contacting the nucleic acid strand with (i) a polymerase and (ii) labelled nucleotides in the presence of a pore such that phosphate labelled species are sequentially released when nucleotides are added to the nucleic acid strand, wherein the phosphate species contain a label specific for each nucleotide; and   (b) detecting the phosphate labelled species using the pore and thereby detecting the addition of the nucleotide to the nucleic acid strand.   
     
     
         2 . A method according to  claim 1 , wherein the pore is attached to the polymerase. 
     
     
         3 . A method according to  claim 2 , wherein the pore is covalently attached to the polymerase. 
     
     
         4 . A method according to  claim 2 , wherein the pore is attached to the polymerase at a site in close proximity to the opening of the barrel of channel of the pore and/or wherein the polymerase is attached to the pore such that its active site is orientated towards the opening of the pore. 
     
     
         5 . A method according to  claim 1 , wherein the phosphate labelled species are detected by contacting the species with the pore so that the species interact with the pore in order and measuring the current passing through the pore during each interaction. 
     
     
         6 . A method according to  claim 1 , wherein the pore is derived from α-hemolysin. 
     
     
         7 . A method according to  claim 6 , wherein the pore comprises seven subunits each having at least 55% homology based on amino acid identity to SEQ ID NO: 2 over its entire length. 
     
     
         8 . A method according to  claim 7 , wherein at least one subunit has a glutamine at position 139 of SEQ ID NO: 2, an arginine at position 113 of SEQ ID NO: 2 or a cysteine at position 119, 121 or 135 of SEQ ID NO: 2. 
     
     
         9 . A method according to  claim 8 , wherein (a) all seven subunits have a glutamine at position 139 of SEQ ID NO: 2 and one of the subunits has a cysteine at position 135 and/or (b) all seven subunits have an arginine at position 113 of SEQ ID NO: 2. 
     
     
         10 . A method according to  claim 1 , wherein the pore comprises a molecular adaptor that facilitates an interaction between the pore and the labelled species. 
     
     
         11 . A method according to  claim 10 , wherein the molecular adaptor is a cyclodextrin or a derivative thereof, heptakis-6-amino-3-cyclodextrin (am 7 -βCD), 6-monodeoxy-6-monoamino-β-cyclodextrin (am 1 -βCD) or heptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu 7 -βCD). 
     
     
         12 . A method according to  claim 1 , wherein the polymerase is (a) a member of any of the Enzyme Classification (EC) groups 2.7.7.6, 2.7.7.7, 2.7.7.19, 2.7.7.48 and 2.7.7.49 or (b) a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, a DNA-dependent RNA polymerase or an RNA-dependent RNA polymerase. 
     
     
         13 . A method according to  claim 1 , wherein the nucleotides are labelled with a fluorescent molecule, a radioisotope, an enzyme, an antibody, an antigen, a polynucleotide or a ligand. 
     
     
         14 . A method for sequencing a nucleic acid strand, comprising:
 (a) contacting the nucleic acid strand with (i) a polymerase and (ii) labelled nucleotides in the presence of a pore such that phosphate labelled species are sequentially released when nucleotides are added to the nucleic acid strand, wherein the phosphate species contain a label specific for each nucleotide;   (b) detecting the phosphate labelled species using the pore and thereby detecting the addition of the nucleotide to the nucleic acid strand; and   (c) using the order of the phosphate labelled species to determine the sequence of the nucleic acid strand.   
     
     
         15 . A kit for detecting the addition of a nucleotide to a nucleic acid strand, comprising a pore, a polymerase and labelled nucleotides.