Methods of enhancing translocation of charged analytes through transmembrane protein pores

Abstract

The invention relates to enhancing translocation of a charged analyte through a transmembrane protein pore. Translocation is enhanced by increasing the net opposing charge of the barrel or channel and/or entrance of the pore. The invention also relates to pores enhanced in accordance with the invention.

1 - 26 . (canceled) 
     
     
         27 . A method of enhancing translocation of a charged analyte through a transmembrane protein pore, comprising:
 (a) increasing the net opposing charge of the barrel or channel and/or entrance of the pore; and   (b) determining whether or not translocation of the analyte through the resulting pore is enhanced.   
     
     
         28 . A method according to  claim 27 , wherein increasing the net opposing charge:
 (a) increases the frequency of translocation of the analyte through the pore;   (b) decreases the threshold voltage for translocation of the analyte through the pore;   (c) decreases the translocation speed of the analyte through the pore; or   (d) decreases the number of non-translocation interactions between the analyte and the pore.   
     
     
         29 . A method according to  claim 27 , wherein the analyte is a polymer or a nucleic acid sequence. 
     
     
         30 . A method according to  claim 27 , wherein the pore comprises:
 (a) seven subunits each comprising SEQ ID NO: 2 or a variant thereof; or   (b) seven subunits each comprising SEQ ID NO: 4 or a variant thereof.   
     
     
         31 . A method according to  claim 27 , wherein the analyte is negatively charged and step (a) involves increasing the net positive charge. 
     
     
         32 . A method according to  claim 31 , wherein the net positive charge is increased by introducing one or more positively charged amino acids into the barrel or channel and/or entrance of the pore. 
     
     
         33 . A method according to  claim 32 , wherein the introducing is carried out by substitution. 
     
     
         34 . A method according to  claim 33 , wherein the one or more positively-charge amino acids are histidine (H), lysine (K) and/or arginine (R). 
     
     
         35 . A method according to  claim 31 , wherein the net positive charge is increased substituting one or more negatively charged amino acids in the barrel or channel and/or entrance of the pore with one or more uncharged amino acids, non-polar amino acids and/or aromatic amino acids. 
     
     
         36 . A method according to  claim 27 , wherein the entrance is the portion of the pore through which the analyte enters the barrel or channel. 
     
     
         37 . A transmembrane protein pore enhanced by a method according to  claim 27 . 
     
     
         38 . A transmembrane protein pore in which the net opposing charge of its barrel or channel and/or entrance has been increased to enhance the translocation of a charged analyte through the pore. 
     
     
         39 . A transmembrane protein pore according to  claim 37  or  38 , wherein the pore comprises:
 (a) at least one subunit comprising SEQ ID NO: 6 or a variant thereof; 
 (b) at least one subunit comprising SEQ ID NO: 8 or a variant thereof; or 
 (c) at least one subunit comprising SEQ ID NO: 10 or a variant thereof. 
 
     
     
         40 . A transmembrane protein pore according to  claim 39 , wherein the pore is a homoheptamer comprising seven subunits of SEQ ID NO: 6, 8 or 10 or a variant thereof. 
     
     
         41 . A polynucleotide encoding a transmembrane protein pore according to  claim 37  or  38 . 
     
     
         42 . A polynucleotide encoding a transmembrane protein pore subunit having the sequence of SEQ ID NO: 6, 8 or 10 or a variant thereof. 
     
     
         43 . A polynucleotide according to  claim 42 , wherein the polynucleotide comprises the sequence shown in SEQ ID NO: 5, 7 or 9 or a variant thereof 
     
     
         44 . A method of determining presence or absence of an analyte in a sample, comprising:
 (a) contacting the sample with a transmembrane protein pore according to  claim 37  or  38  under conditions that allow the analyte, if present, to translocate through and interact with the pore; and   (b) measuring the current passing through the pore during the interaction and thereby determining the presence or absence of the analyte.   
     
     
         45 . A method of sequencing a target nucleic acid sequence, comprising:
 (a) pushing or pulling the target sequence through a transmembrane protein pore according to  claims 37  to  38  so that a proportion of the nucleotides in the target sequence interact with the pore; and   (b) measuring the current passing through the pore during each interaction and thereby determining the sequence of the target sequence.   
     
     
         46 . A kit for sequencing a nucleic acid, comprising a transmembrane protein pore according to  claim 37  or  38  and a nucleic acid handling enzyme.