Enzyme construct

Abstract

The invention relates to methods using constructs comprising a helicase and an additional polynucleotide binding moiety. The helicase is attached to the polynucleotide binding moiety and the construct has the ability to control the movement of a polynucleotide. The constructs can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.

1 . A method of characterising a target polynucleotide, comprising:
 (a) contacting the target polynucleotide with a transmembrane pore and a construct comprising a helicase and an additional polynucleotide binding moiety, wherein the helicase is attached to the polynucleotide binding moiety and the construct has the ability to control the movement of a polynucleotide, such that the construct controls the movement of the target polynucleotide through the pore; and   (b) taking one or more measurements as the polynucleotide moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide.   
     
     
         2 . A method according to  claim 1 , wherein the helicase and the polynucleotide binding moiety are covalently attached, are chemically attached or are genetically fused. 
     
     
         3 . (canceled) 
     
     
         4 . A method according to  claim 1 , wherein the helicase and the polynucleotide binding moiety are attached by one or more linkers or are attached by one or more linkers which are amino acid sequences. 
     
     
         5 .- 34 . (canceled) 
     
     
         35 . A construct comprising two or more helicases, wherein the helicases are attached together and the construct has the ability to control the movement of a polynucleotide. 
     
     
         36 . A construct according to  claim 35 , wherein the two or more helicases in the construct are different from one another, are the same or similar or are the same or similar and attached using the same amino acid residue in each helicase; and/or wherein the two or more helicases are (a) independently selected from superfamilies 1 to 6; (b) monomeric; or (c) independently selected from Hel308 helicases, RecD helicases, TraI helicases, TraI subgroup helicases, XPD helicases and variants thereof. 
     
     
         37 . A construct comprising a helicase and an amino acid sequence comprising SEQ ID NO: 94 or a variant thereof having at least 80% homology to SEQ ID NO: 94 based on amino acid identity over the entire sequence of SEQ ID NO: 94, wherein the helicase is attached to the amino acid sequence and the construct has the ability to control the movement of a polynucleotide. 
     
     
         38 . A construct according to  claim 37 , wherein the construct comprises SEQ ID NO: 90 or a variant thereof having at least 80% homology to SEQ ID NO: 90 based on amino acid identity over the entire sequence of SEQ ID NO: 90. 
     
     
         39 . A polynucleotide sequence which encodes a construct according to  claim 35 , wherein the two or more helicases are genetically fused. 
     
     
         40 . A method of controlling the movement of a polynucleotide, comprising contacting the polynucleotide with a construct according to  claim 35  and thereby controlling the movement of the polynucleotide. 
     
     
         41 . A method of forming a sensor for characterising a target polynucleotide, comprising forming a complex between a pore and a construct as defined in  claim 1  and thereby forming a sensor for characterising the target polynucleotide. 
     
     
         42 . A method according to  claim 41 , wherein the complex is formed by (a) contacting the construct and the pore in the presence of the target polynucleotide and (b) applying a potential, voltage potential or a chemical potential across the pore. 
     
     
         43 . (canceled) 
     
     
         44 . A method according to  claim 42 , wherein the complex is formed by covalently attaching the pore to the construct. 
     
     
         45 . A sensor for characterising a target polynucleotide, comprising a complex between a pore and a construct as defined in  claim 1 . 
     
     
         46 . (canceled) 
     
     
         47 . A kit for characterising a target polynucleotide comprising (a) a pore and (b) a construct as defined in  claim 1 . 
     
     
         48 . A kit according to  claim 47 , wherein the kit further comprises a chip comprising an amphiphilic membrane. 
     
     
         49 . An apparatus for characterising target polynucleotides in a sample, comprising a plurality of pores and a plurality of constructs as defined in  claim 1 . 
     
     
         50 . An apparatus according to  claim 49 , wherein the apparatus comprises:
 (a) a sensor device that is capable of supporting the plurality of pores and being operable to perform polynucleotide characterisation using the pores and constructs; and   at least one reservoir for holding material for performing the characterization; or   (b) a sensor device that is capable of supporting the plurality of pores and being operable to perform polynucleotide characterisation using the pores and constructs;   at least one reservoir for holding material for performing the characterisation;   a fluidics system configured to controllably supply material from the at least one reservoir to the sensor device; and   one or more containers for receiving respective samples, the fluidics system being configured to supply the samples selectively from the one or more containers to the sensor device.   
     
     
         51 . (canceled) 
     
     
         52 . A method of producing a construct according to  claim 35 , comprising attaching two or more helicases together and thereby producing the construct. 
     
     
         53 . A method of producing a construct according to  claim 37 , comprising attaching a helicase to an amino acid sequence comprising SEQ ID NO: 94 or a variant thereof having at least 80% homology to SEQ ID NO: 94 based on amino acid identity over the entire sequence of SEQ ID NO: 94 and thereby producing the construct. 
     
     
         54 . A method according to  claim 52 , wherein the method further comprises determining whether or not the resulting construct is capable of controlling the movement of a polynucleotide. 
     
     
         55 . (canceled)