Modified nanopores, compositions comprising the same, and uses thereof

Abstract

Provided herein relate to modified or mutant forms of secretin and compositions comprising the same. In particular, the modified or mutant forms of secretin permits efficient capture and/or translocation of an analyte through the modified or mutant secretin nanopores. Methods for using unmodified secretin or the modified or mutant forms of secretin and compositions, for example, for characterizing an analyte, e.g., a target polynucleotide, are also provided.

1 - 71 . (canceled) 
     
     
         72 . A method of detecting the presence or absence of an analyte in a sample and/or characterising an analyte in a sample, the method comprising contacting a secretin nanopore disposed in a membrane with the sample, applying a voltage gradient across the membrane and detecting ionic current flow through the nanopore. 
     
     
         73 . The method of  claim 72 , wherein the secretin nanopore is a modified secretin nanopore. 
     
     
         74 . The method of  claim 73 , wherein the modified secretin nanopore comprises a lumenal surface defining a lumen that extends through the membrane between a cis-opening and a trans-opening, wherein the lumenal surface comprises one or more amino acid modifications relative to a wild-type secretin nanopore. 
     
     
         75 . The method of  claim 74 , wherein the one or more amino acid modifications comprise a charge-altering modification. 
     
     
         76 . The method of  claim 75 , wherein the charge-altering modification is a substitution of a negatively-charged amino acid with a positively-charged amino acid. 
     
     
         77 . The method of  claim 74 , wherein the one or more amino acid modifications comprise a substitution of a neutral amino acid with a hydrophobic amino acid. 
     
     
         78 . The method of  claim 74 , wherein the cis-opening has a diameter in a range of 60 Å to 120 Å. 
     
     
         79 . The method of  claim 74 , wherein the trans-opening has a diameter in a range of 40 Å to 100 Å. 
     
     
         80 . The method of  claim 74 , wherein the secretin nanopore comprises a constriction having a diameter of about 7.5 Å to 25 Å. 
     
     
         81 . The method of  claim 74 , wherein the secretin is of a type II, type III, or type IV secretion system. 
     
     
         82 . The method of  claim 74 , wherein the secretin is GspD, YscC, InvG, or PilQ. 
     
     
         83 . The method of  claim 74 , wherein the modified secretin nanopore comprises a subunit polypeptide having an amino acid sequence that is at least 95% identical to the amino acid sequence as set forth in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         85 . The method of  claim 74 , wherein the secretin is GspD and the GspD does not comprise a cap gate. 
     
     
         86 . The method of  claim 74 , wherein the secretin is GspD and comprises a central gate that has been modified to replace an amino acid with an amino acid having a smaller side group and/or to replace a negatively charged amino acid with a neutral or positively charged amino acid. 
     
     
         87 . The method of  claim 74 , wherein the secretin is GspD and the modified secretin nanopore comprises a subunit polypeptide having an amino acid sequence that is at least 95% identical to an amino acid sequence as set forth in SEQ ID NO: 36, wherein: (i) one or more of the amino acids from D55 or T56 to T77 are deleted or substituted, one or more of K60, D64, R71 and E73 is substituted with an uncharged amino acid and/or one or more of D55, T56, T77 and K78 is substituted with P; and/or (ii) F156 is substituted with a smaller amino acid, N151 and/or N152 is/are substituted with a smaller amino acid, D153 is substituted with an uncharged amino acid, G137 and G165 are each independently unmodified or substituted with A or V. 
     
     
         88 . The method of  claim 87 , wherein Y63 to R71 are deleted and/or substituted with GSG or SGS, F156 is substituted with A, D153 is substituted with S, and/or N151 and N152 are each independently substituted with G or S. 
     
     
         95 . The method of  claim 74 , wherein the secretin is InvG and the modified secretin nanopore comprises a subunit polypeptide having an amino acid sequence that is at least 95% identical to the amino acid sequence as set forth in SEQ ID NO: 1, wherein the lumenal surface further defines a constriction within the lumen, the constriction having one or more amino acid modifications at amino acids D28, E225, R226, and/or E231 of SEQ ID NO: 1. 
     
     
         96 . The method of  claim 95 , wherein the one or more amino acid modifications of the constriction comprise one or more of the following:
 i. D28N/Q/T/S/G/R/K;   ii. E225N/Q/T/A/S/G/P/H/F/Y/R/K;   iii. R226N/Q/T/A/S/G/P/H/F/Y/K/V;   iv. Deletion of E225;   v. Deletion of R226; and   vi. E231N/Q/T/A/S/G/P/H/R/K.   
     
     
         97 . The method of  claim 74 , wherein the secretin is InvG and the modified secretin nanopore comprises a subunit polypeptide having an amino acid sequence that is at least 95% identical to the amino acid sequence as set forth in SEQ ID NO: 1, wherein the lumenal surface comprises a capture portion having one or more amino acid modifications at amino acids E41, Q45 or E114. 
     
     
         98 . The method of  claim 97 , wherein the capture portion comprises one or more of the following amino acid modifications:
 i. Q45R/K;   ii. E41N/Q/T/S/G/R/K; and   iii. E114N/Q/T/S/G/R/K.