Designed protein pores as components for biosensors

Abstract

A mutant staphylococcal alpha hemolysin polypeptide containing a heterologous analyte-binding amino acid which assembles into an analyte-responsive heptameric pore assembly in the presence of a wild type staphylococcal alpha hemolysin polypeptide, digital biosensors, and methods of detecting, identifying, and quantifying analytes are described.

1 . A mutant staphylococcal alpha hemolysin polypeptide comprising a heterologous amino acid, wherein said heterologous amino acid binds an analyte and wherein said polypeptide assembles into a heteroheptameric pore assembly in the presence of a plurality of wild type staphylococcal alpha hemolysin polypeptides.  
     
     
         2 . The polypeptide of  claim 1 , wherein said heterologous amino acid occupies a position in a transmembrane channel of said heptameric pore assembly.  
     
     
         3 . The polypeptide of  claim 2 , wherein said heterologous amino acid projects into the lumen of said transmembrane channel.  
     
     
         4 . The polypeptide of  claim 2 , wherein said heterologous amino acid occupies a position in a stem domain of said polypeptide.  
     
     
         5 - 20 . (canceled)  
     
     
         21 . A heptomeric pore assembly comprising a mutated staphylococcal αHL polypeptide (MUT), wherein said MUT is an analyte-binding αHL polypeptide.  
     
     
         22 . The pore assembly of  claim 21 , wherein said pore assembly is a heptamer having the formula WT7-nMUTn, wherein n is greater than zero and less than seven.  
     
     
         23 . The pore assembly of  claim 21 , wherein said analyte-binding αHL polypeptide comprises a heterologous amino acid at a position in a transmembrane channel of said pore assembly, wherein said heterologous amino acid binds a metal.  
     
     
         24 . The pore assembly of  claim 21 , wherein said pore assembly is a heptamer having the formula WT7-nMn, wherein n is greater than zero and less than seven.  
     
     
         25 . A staphylococcal alpha hemolysin (αHL) polypeptide comprising at least two non-consecutive heterologous amino acids in a stem domain of said polypeptide, wherein each of said heterologous amino acids binds an organic molecule, wherein the amino acids occupy two or more of the following positions of SEQ ID NO: 1: 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147 or 149, and wherein said analyte-binding αHL polypeptide is 4H.  
     
     
         26 . (canceled)  
     
     
         27 . The pore assembly of  claim 25 , wherein the pore assembly is a heptamer having the formula WT7-n4Hn.  
     
     
         28 . The pore assembly of  claim 27 , wherein the pore assembly is a heteroheptamer having the formula WT64H1.  
     
     
         29 . A digital biosensor device comprising the pore assembly of  claim 21 .  
     
     
         30 - 34 . (canceled)  
     
     
         35 . A method of detecting the presence of an analyte in a test sample, comprising 
 (a) contacting said sample with the pore assembly of  claim 21;  and    (b) detecting an electrical current in a digital mode through two or more channels, wherein a modulation in current compared to a current measurement in a control sample lacking said analyte indicates the presence of said analyte in said test sample.    
     
     
         36 . A method of detecting the presence of an analyte in a test sample, comprising 
 (a) contacting said sample with the pore assembly of  claim 21;     (b) detecting an electrical current in a digital mode through a single channel, wherein a modulation in current compared to a current measurement in a control sample lacking said analyte indicates the presence of said analyte in said test sample.    
     
     
         37 . The method of  claim 36 , wherein said analyte is a metal ion.  
     
     
         38 . The method of  claim 37 , wherein said metal ion is Zn(II).  
     
     
         39 . The method of  claim 37 , wherein said metal ion is Co(II), Cu(II), Ni(II), or Cd(II).  
     
     
         40 . A method of identifying an unknown analyte in a mixture of analytes comprising, 
 (a) contacting said mixture with the pore assembly of  claim 21;     (b) detecting an electrical current in a digital mode through two or more channels to determine a mixture current signature;    (c) comparing said mixture current signature to a standard current signature of a known analyte, wherein a concurrence of said mixture current signature and said standard current signature indicates the identity of said unknown analyte in said mixture.    
     
     
         41 . The method of  claim 40 , wherein each of said known and unknown analytes is a metal ion.  
     
     
         42 . A method of identifying an analyte in a mixture of analytes comprising, 
 (a) contacting said mixture with the pore assembly of  claim 21;     (b) detecting a single channel current in a digital mode to determine a mixture current signature;    (c) comparing said mixture current signature to a standard current signature of a known analyte, wherein a concurrence of said mixture current signature and said standard current signature indicates the identity of said unknown analyte in said mixture.    
     
     
         43 . The method of  claim 42 , wherein each of said unknown and known analytes is a metal ion.  
     
     
         44 . The method of  claim 43 , wherein said metal ion is Zn(II).  
     
     
         45 . The method of  claim 43 , wherein said metal ion is Co(II), Cu(II), Ni(II), or Cd(II).