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 . A construct comprising a transmembrane protein pore subunit and a nucleic acid handling enzyme, wherein the subunit is covalently attached to the enzyme, wherein the subunit retains its ability to form a pore and wherein the enzyme retains its ability to handle nucleic acids. 
     
     
         2 . A construct according to  claim 1 , wherein (a) the enzyme is attached to the subunit at more than one point; (b) the enzyme is genetically fused to the subunit; (c) the amino acid sequence of the enzyme is added in frame into the amino acid sequence of the subunit; or (d) the enzyme is chemically fused to the subunit. 
     
     
         3 - 5 . (canceled) 
     
     
         6 . A construct according to  claim 1 , wherein the enzyme is attached to the pore by one or more linkers, optionally amino acid linkers. 
     
     
         7 . (canceled) 
     
     
         8 . A construct according to  claim 1 , wherein the subunit is derived from α-hemolysin (α-HL) or the subunit comprises the sequence shown SEQ ID NO: 2 or a variant thereof. 
     
     
         9 . (canceled) 
     
     
         10 . A construct according to  claim 1 , wherein the nucleic acid handling enzyme is (a) a nuclease and wherein the nuclease is optionally a member of any of the Enzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31; (b) an exonuclease and wherein the enzyme optionally comprises the sequence shown in any one of SEQ ID NOs: 10, 12, 14 and 16 or a variant thereof; (c) is a polymerase and wherein the polymerase is optionally (i) 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 (ii) a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, a DNA-dependent RNA polymerase or an RNA-dependent RNA polymerase; (d) is a helicase and wherein the helicase is optionally (i) a member of any of the Enzyme Classification (EC) groups 3.6.1.- and 2.7.7 or (ii) an ATP-dependent DNA helicase, an ATP-dependent RNA helicase or an ATP-independent RNA helicase; or (e) is a topoisomerase and wherein the helicase is optionally (i) a member of any of the Enzyme Classification (EC) groups 5.99.1.2 and 5.99.1.3 or (ii) a gyrase. 
     
     
         11 - 13 . (canceled) 
     
     
         14 . A construct according to  claim 1 , wherein the construct comprises the sequence shown in any one of SEQ ID NOs: 18, 20, 22, 24, 26, 28 and 30 or a variant thereof. 
     
     
         15 - 19 . (canceled) 
     
     
         20 . A polynucleotide sequence which encodes a construct according to  claim 2 , wherein the polynucleotide optionally comprises the sequence shown in any one of SEQ ID NOs: 17, 19, 21, 23, 25, 27 and 29 or a variant thereof. 
     
     
         21 . (canceled) 
     
     
         22 . A modified pore for use in sequencing nucleic acids, comprising at least one construct comprises a transmembrane protein pore subunit and a nucleic acid handling enzyme, wherein the subunit is covalently attached to the enzyme, wherein the subunit retains its ability to form a pore and wherein the enzyme retains its ability to handle nucleic acids, wherein the pore optionally comprises a construct of claim  4  and six subunits comprising the sequence shown SEQ ID NO: 2 or a variant thereof. 
     
     
         23 . (canceled) 
     
     
         24 . A pore according to  claim 22 , 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; (b) all seven subunits have an arginine at position 113 of SEQ ID NO: 2; and/or (c) the pore comprises a molecular adaptor that facilitates an interaction between the pore and one or more nucleotide(s) and wherein the molecular adaptor is optionally (i) a cyclodextrin or a derivative thereof or (ii) heptakis-6-amino-β-cyclodextrin (am 7 -βCD), 6-monodeoxy-6-monoamino-β-cyclodextrin (am 1 -β CD) or heptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu 7 -βCD). 
     
     
         25 - 28 . (canceled) 
     
     
         29 . A kit for producing a modified pore for use in sequencing nucleic acids, comprising:
 (a) at least one construct comprising a transmembrane protein pore subunit and a nucleic acid handling enzyme, wherein the subunit is covalently attached to the enzyme, wherein the subunit retains its ability to form a pore and wherein the enzyme retains its ability to handle nucleic acids and   the remaining subunits needed to form a pore; or (b) at least one polynucleotide according to  claim 7  and polynucleotide sequences encoding any remaining subunits needed to form a pore.   
     
     
         30 . A kit according to  claim 29 , wherein the kit comprises:
 (a) a construct comprising a transmembrane protein pore subunit and a nucleic acid handling enzyme, wherein the subunit is covalently attached to the enzyme, wherein the subunit retains its ability to form a pore, wherein the enzyme retains its ability to handle nucleic acids, and wherein the subunit is derived from α-hemolysin (α-HL) or the subunit comprises the sequence shown SEQ ID NO: 2 or a variant thereof and   six subunits each comprising the sequence shown in SEQ ID NO: 2 or a variant thereof.   
     
     
         31 - 32 . (canceled) 
     
     
         33 . A method of producing a construct according to  claim 1 , comprising:
 (a) covalently attaching a nucleic acid handling enzyme to a transmembrane protein pore subunit; and   (b) determining whether or not the resulting construct is capable of forming a pore and handling nucleic acids, wherein the enzyme is optionally attached to the subunit before the subunit forms part of a pore (post expression modification) or after the subunit has formed part of a pore (post oligomerisation modification) and/or wherein step (a) optionally comprises:   (i) providing a polynucleotide that encodes the construct, wherein the polynucleotide optionally comprises the sequence shown in any one of SEQ ID NOs: 17, 19, 21, 23, 25, 27 and 29 or a variant thereof; and   (ii) expressing the polynucleotide sequence.   
     
     
         34 - 35 . (canceled) 
     
     
         36 . A method of producing a modified pore according to  claim 22 , comprising:
 (a) covalently attaching a nucleic acid handling enzyme to a transmembrane protein pore; and   (b) determining whether or not the resulting pore is capable of handling nucleic acids and detecting nucleotides; or comprising:   (a) allowing at least one construct to form a pore with other suitable subunits, wherein the construct comprises a transmembrane protein pore subunit and a nucleic acid handling enzyme, wherein the subunit is covalently attached to the enzyme, wherein the subunit retains its ability to form a pore and wherein the enzyme retains its ability to handle nucleic acids; and   (b) determining whether or not the resulting pore is capable of handling nucleic acids and detecting nucleotides.   
     
     
         37 . (canceled) 
     
     
         38 . A method of purifying a transmembrane pore comprising at least one construct according to  claim 1 , comprising:
 (a) providing the at least one construct and the other subunits required to form the pore;   (b) oligomerising the at least one construct and other subunits on synthetic lipid vesicles; and   (c) contacting the vesicles with a non-ionic surfactant; and   (d) recovering the oligomerised pore, wherein the synthetic vesicles optionally comprise 30% cholesterol, 30% phosphatidylcholine (PC), 20% phosphatidylethanolamine (PE), 10% sphingomyelin (SM) and 10% phosphatidylserine (PS) and/or wherein the non-ionic surfactant is optionally an Octyl Glucoside (OG) or DoDecyl Maltoside (DDM) detergent.   
     
     
         39 - 40 . (canceled) 
     
     
         41 . A method of sequencing a target nucleic acid sequence, comprising:
 (a) contacting the target sequence with a pore according to  claim 24 , which comprises an exonuclease, such that the exonuclease digests an individual nucleotide from one end of the target sequence;   (b) contacting the nucleotide with the pore so that the nucleotide interacts with the adaptor;   (c) measuring the current passing through the pore during the interaction and thereby determining the identity of the nucleotide; and   (d) repeating steps (a) to (c) at the same end of the target sequence and thereby determining the sequence of the target sequence.   
     
     
         42 . A method of sequencing a target nucleic acid sequence, comprising:
 (a) contacting the target sequence with a pore according to  claim 22  so that the enzyme pushes or pulls the target sequence through the pore and a proportion of the nucleotides in the target sequence interacts with the pore; and   (b) measuring the current passing through the pore during each interaction and thereby determining the sequence of the target sequence.   
     
     
         43 . A kit according to  claim 29 , wherein the kit comprises a polynucleotide encoding a construct comprising a transmembrane protein pore subunit and a nucleic acid handling enzyme, wherein the subunit is covalently attached to the enzyme, wherein the subunit retains its ability to form a pore and wherein the enzyme retains its ability to handle nucleic acids, and six polynucleotides each encoding a subunit comprising the sequence shown in SEQ ID NO: 2 or a variant thereof.