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.

1 - 42 . (canceled) 
     
     
         43 . A method for processing a target nucleic acid for analysis, the method comprising:
 (a) providing a transmembrane pore and a membrane, wherein the transmembrane pore is present in the membrane, and wherein a nucleic acid handling enzyme is covalently attached to the transmembrane pore in proximity to a cis opening of the transmembrane pore; and   (b) adding a target nucleic acid to a solution in contact with the cis opening of the transmembrane pore, wherein the target nucleic acid provides a substrate for an enzymatic processing reaction catalyzed by the nucleic acid handling enzyme that results in release of nucleotides, or phosphate species thereof, which enter the transmembrane pore through the cis opening and pass through the transmembrane pore in order of their release.   
     
     
         44 . The method of  claim 43 , wherein the phosphate species comprises a label specific for a nucleotide. 
     
     
         45 . The method of  claim 43 , wherein the enzymatic processing reaction comprises cleaving the target nucleic acid by the nucleic acid handling enzyme to release nucleotides or phosphate species thereof. 
     
     
         46 . The method of  claim 43 , wherein the enzymatic processing reaction comprises releasing phosphate species of nucleotides that are sequentially added to the target nucleic acid by the nucleic acid handling enzyme. 
     
     
         47 . The method of  claim 43 , wherein the nucleic acid handling enzyme is attached to the transmembrane pore through at least one linker. 
     
     
         48 . The method of  claim 47 , wherein the at least one linker comprises a peptide linker. 
     
     
         49 . The method of  claim 47 , wherein the nucleic acid handling enzyme is attached to a subunit of the transmembrane pore. 
     
     
         50 . The method of  claim 43 , wherein the nucleic acid handling enzyme is an exonuclease or a polymerase. 
     
     
         51 . The method of  claim 50 , wherein the polymerase is a DNA polymerase. 
     
     
         52 . The method of  claim 51 , wherein the DNA polymerase is a DNA-dependent DNA polymerase. 
     
     
         53 . The method of  claim 43 , wherein the transmembrane pore is a transmembrane protein pore. 
     
     
         54 . The method of  claim 53 , wherein the transmembrane protein pore is an α-hemolysin pore. 
     
     
         55 . The method of  claim 54 , wherein the α-hemolysin pore comprises a subunit having an amino acid sequence with at least 95% homology to SEQ ID NO: 2. 
     
     
         56 . A method for analyzing a target nucleic acid, the method comprising:
 (a) providing a transmembrane pore and a membrane, wherein the transmembrane pore is present in the membrane, and wherein a nucleic acid handling enzyme is covalently attached to the transmembrane pore in proximity to a cis opening of the transmembrane pore; and   (b) adding a target nucleic acid to a solution in contact with the cis opening of the transmembrane pore, wherein the target nucleic acid provides a substrate for an enzymatic processing reaction, catalyzed by the nucleic acid handling enzyme, that results in release of nucleotides, or phosphate species thereof, which enter the transmembrane pore through the cis opening and pass through the transmembrane pore in order of their release; and   (c) measuring, during application of a potential across the transmembrane pore, an electrical signal across the transmembrane pore as the released nucleotides or phosphate species thereof pass through the transmembrane pore, thereby analyzing the target nucleic acid.   
     
     
         57 . The method of  claim 56 , wherein the electrical signal comprises a current flow through the transmembrane pore. 
     
     
         58 . The method of  claim 56 , wherein the phosphate species comprises a label specific for a nucleotide. 
     
     
         59 . The method of  claim 56 , wherein the enzymatic processing reaction comprises cleaving the target nucleic acid by the nucleic acid handling enzyme to release nucleotides or phosphate species thereof. 
     
     
         60 . The method of  claim 56 , wherein the enzymatic processing reaction comprises releasing phosphate species of nucleotides that are sequentially added to the target nucleic acid by the nucleic acid handling enzyme. 
     
     
         61 . The method of  claim 56 , wherein the nucleic acid handling enzyme is attached to the transmembrane pore through at least one linker. 
     
     
         62 . The method of  claim 61 , wherein the at least one linker comprises a peptide linker. 
     
     
         63 . The method of  claim 61 , wherein the nucleic acid handling enzyme is attached to a subunit of the transmembrane pore. 
     
     
         64 . The method of  claim 56 , wherein the nucleic acid handling enzyme is an exonuclease or a polymerase. 
     
     
         65 . The method of  claim 64 , wherein the polymerase is a DNA polymerase. 
     
     
         66 . The method of  claim 65 , wherein the DNA polymerase is a DNA-dependent DNA polymerase. 
     
     
         67 . The method of  claim 56 , wherein the transmembrane pore is a transmembrane protein pore. 
     
     
         68 . The method of  claim 67 , wherein the transmembrane protein pore is an α-hemolysin pore. 
     
     
         69 . The method of  claim 68 , wherein the α-hemolysin pore comprises a subunit having an amino acid sequence with at least 95% homology to SEQ ID NO: 2.