Tagged nucleotides useful for nanopore detection

Abstract

The present disclosure relates to compounds comprising a negatively-charged polymer moiety which is capable of entering a nanopore and upon entering a nanopore in the presence of positive ions results in an increased flow of the positive ions through the nanopore. The present disclosure provides methods of preparing the compounds and for their use as nanopore-detectable tags, in particular, for nanopore-based nucleic acid detection and sequencing.

What is claimed is: 
     
         1 . A method for determining the sequence of a nucleic acid comprising:
 (a) providing a nanopore sequencing composition comprising:
 (a1) a membrane with a cis side and a trans side, 
 (a2) an electrode on the cis side and the trans side of the membrane, 
 (a3) an electrolyte solution comprising positive ions in contact with both electrodes, 
 (a4) a nanopore comprising a channel extending through the membrane, wherein the channel provide a path through which the positive ions may flow across the membrane, 
 (a5) a set of tagged nucleoside-5′-oligophosphate (N5OP) disposed in the electrolyte solution, wherein at least one of the tagged N5OPs is covalently linked to a tag that comprises a negatively-charged polymer moiety that is capable of increasing the flow of the positive ions through the channel, and 
 (a6) an active polymerase situated adjacent to the nanopore and complexed with a primed strand of the nucleic acid, wherein the active polymerase catalytically incorporates the tagged N5OPs into a complementary strand of the primed nucleic acid, wherein the tag of the N5OP incorporated into the complementary strand enters into the nanopore channel and changes the flow of the positive ions through the channel of the pore; 
   (b) detecting the different flows of positive ions resulting from the entry of the different tags in the nanopore over time and correlating to each of the different compounds incorporated by the polymerase which are complementary to the nucleic acid sequence, and thereby determining the nucleic acid sequence.   
     
     
         2 . The method of  claim 1 , wherein the negatively-charged polymer comprises a covalently linked chain of from 20 to 50 monomer units selected from the monomer unit structures of formula (1a), (1 b), (1c), (1d), (1e), (1f), (1g), (1 h), (1 i), (1 j), (1 k), (1 l), (1 m), (2a), (2b), (2c), (3a), (3b), (3c), and any combination thereof 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         3 . The method of  claim 2 , wherein the monomer units are selected from the group consisting of the structures of formula (2a), (2b), (2c), (3a), (3b), (3c), and any combination thereof. 
     
     
         4 . The method of  claim 1 , wherein the increased flow of the positive ions results in a measured current across the nanopore that is greater than an open channel (O.C.) current. 
     
     
         5 . The method of  claim 4 , wherein the increased flow of the positive ions results in a measured current across the nanopore that is at least 5% greater than O.C. current. 
     
     
         6 . The method of  claim 1 , wherein the negatively-charged polymer moiety has an overall negative charge of from (−25) to (−50). 
     
     
         7 . The method of  claim 1 , wherein the negatively-charged polymer moiety comprises a covalently linked chain of from 25 to 40 monomer units. 
     
     
         8 . The method of  claim 1 , wherein the tagged N50Ps have the structure of formula I:
   N—P-L-T   (I)
   wherein:
 N is a nucleoside; 
 P is an oligophosphate covalently attached to a 5′-O group of the nucleoside, wherein the oligophosphate consists of 3 to 12 phosphate groups; 
 L is a linker covalently attached to a terminal phosphate group of the oligophosphate; and 
 T is the tag, wherein the tag is covalently attached to the linker. 
   
     
     
         9 . The method of  claim 1 , wherein the tagged N5OP comprising the negatively-charged polymer moiety is a compound of structural formula (II) 
       
         
           
           
               
               
           
         
         Wherein,
 Base is selected from adenosine, cytidine, guanosine, thymidine, and uridine; 
 R is selected from H and OH; 
 N is from 1 to 4; 
 Linker is a linker comprising a covalently bonded chain of 2 to 100 atoms; and 
 Tag is the tag. 
 
       
     
     
         10 . The method of  claim 9 , wherein the linker comprises a chemical group selected from the group consisting of: ester, ether, thioether, amine, amide, imide, carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime, triazole, dihydropyridazine, phosphodiester, polyethylene glycol (PEG), and any combination thereof. 
     
     
         11 . The method of  claim 1 , wherein the tagged N5OP comprising the negatively-charged polymer moiety is a compound of structural formula (III) 
       
         
           
           
               
               
           
         
         Wherein,
 Base is selected from adenosine, cytidine, guanosine, thymidine, and uridine; 
 R is selected from H and OH; 
 N is from 1 to 4; 
 L B -X-L A  is the linker, wherein (a) L A  and L B  each independently comprises a chemical moiety selected from the group consisting of: linear (C 1 -C 12 ) alkyl, linear (C 1 -C 12 ) alkene, linear (C 1 -C 12 ) alkyne, ester, ether, thioether, amine, amide, imide, carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime, triazole, dihydropyridazine, phosphodiester, polyethylene glycol (PEG), and combinations thereof; and (b) X comprises a chemical moiety selected from the group consisting of ester, ether, thioether, amine, amide, imide, carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime, triazole, and dihydropyridazine; and 
 Tag is the tag. 
 
       
     
     
         12 . The method of  claim 1 , wherein the tagged N5OP comprising the negatively-charged polymer moiety is a compound of structural formula (IIIa) 
       
         
           
           
               
               
           
         
         wherein,
 Base is selected from adenosine, cytidine, guanosine, thymidine, and uridine; 
 R is selected from H and OH; 
 N is from 1 to 4; 
 P is from 2 to 10; and 
 Tag is the tag comprising the negatively-charged polymer moiety. 
 
       
     
     
         13 . The method of  claim 12 , wherein R=H, n=4, and p=5. 
     
     
         14 . a composition having the structure of formula I:
   N—P-L-T   (I)
   wherein:
 N is a nucleoside; 
 P is an oligophosphate covalently attached to a 5′-O group of the nucleoside, wherein the oligophosphate consists of 3 to 12 phosphate groups; 
 L is a linker covalently attached to a terminal phosphate group of the oligophosphate; and 
 T is a tag covalently attached to the linker, wherein the tag comprises a negatively-charged polymer moiety which is capable of entering a nanopore and upon entering a nanopore in the presence of positive ions results in an increased flow of the positive ions through the nanopore. 
   
     
     
         15 . The composition of  claim 14 , wherein the composition is a compound of structural formula (II) 
       
         
           
           
               
               
           
         
         wherein,
 Base is selected from adenosine, cytidine, guanosine, thymidine, and uridine; 
 R is selected from H and OH; 
 N is from 1 to 4; 
 Linker is a linker comprising a covalently bonded chain of 2 to 100 atoms; and 
 Tag is the tag. 
 
       
     
     
         16 . The composition of  claim 14 , wherein the linker comprises a chemical group selected from the group consisting of: ester, ether, thioether, amine, amide, imide, carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime, triazole, dihydropyridazine, phosphodiester, polyethylene glycol (PEG), and any combination thereof. 
     
     
         17 . The composition of  claim 14 , wherein the composition is a compound of structural formula (III) 
       
         
           
           
               
               
           
         
         wherein,
 Base is selected from adenosine, cytidine, guanosine, thymidine, and uridine; 
 R is selected from H and OH; 
 N is from 1 to 4; 
 L B -X-L A  is the linker, wherein (a) L A  and L B  each independently comprises a chemical moiety selected from the group consisting of: linear (C 1 -C 12 ) alkyl, linear (C 1 -C 12 ) alkene, linear (C 1 -C 12 ) alkyne, ester, ether, thioether, amine, amide, imide, carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime, triazole, dihydropyridazine, phosphodiester, polyethylene glycol (PEG), and combinations thereof; and (b) X comprises a chemical moiety selected from the group consisting of ester, ether, thioether, amine, amide, imide, carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime, triazole, and dihydropyridazine; and 
 Tag is the tag. 
 
       
     
     
         18 . The composition of  claim 14 , wherein the composition is a compound of structural formula (IIIa) 
       
         
           
           
               
               
           
         
         Wherein,
 Base is selected from adenosine, cytidine, guanosine, thymidine, and uridine; 
 R is selected from H and OH; 
 N is from 1 to 4; 
 P is from 2 to 10; and 
 Tag is the tag comprising the negatively-charged polymer moiety. 
 
       
     
     
         19 . The composition of  claim 18 , wherein R=H, n=4, and p=5. 
     
     
         20 . The composition of  claim 14 , wherein the negatively-charged polymer comprises a covalently linked chain of from 20 to 50 monomer units selected from the monomer unit structures of formula (2a), (2b), (2c), (3a), (3b), (3c), and any combination thereof