Tagged multi-nucleotides useful for nucleic acid sequencing

Abstract

The present disclosure relates to tagged multi-nucleotide compounds, which comprise a single tag moiety covalently linked to a plurality of nucleoside-5′-oligophosphate moieties. As disclosed herein, these tagged multi-nucleotide compounds have improved characteristics as polymerase substrates and can be used in a range of nucleic acid detection and sequencing methods, including nanopore sequencing-by-synthesis.

1 . A compound comprising a single tag covalently linked to a plurality of nucleoside-5′-oligophosphate moieties, wherein the tag is a molecular moiety capable of producing a detectable signal, and each nucleoside-5′-oligophosphate moiety is capable of being a substrate for a polymerase. 
     
     
         2 . The compound of  claim 1 , wherein the compound comprises the single tag covalently linked to from 2 to 12 nucleoside-5′-oligophosphate moieties, optionally from 2 to 6 nucleoside-5′-oligophosphate moieties. 
     
     
         3 . The compound of  claim 1 , wherein the compound when used as a polymerase substrate results in increased extension efficiency relative to a compound comprising the single tag covalently linked to a single nucleoside-5′-oligophosphate. 
     
     
         4 . The compound of  claim 1 , wherein the compound has structural formula (I)
   [N-P-L] m -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; 
 m is from 2 to 12 and indicates the number of N-P-L moieties; and 
 T is a tag covalently attached the N-P-L moieties, wherein the tag is a molecular moiety capable of producing a detectable signal. 
   
     
     
         5 . The compound of  claim 1 , wherein the compound has 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; 
 m is from 2 to 12; and 
 Tag is a molecular moiety which is capable of producing a detectable signal. 
 
       
     
     
         6 . The compound of  claim 5 , wherein the compound has structural formula (IIIa), (IIIb), or (IIIc): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         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 a molecular moiety capable of producing a detectable signal. 
 
       
     
     
         7 . The compound of  claim 5 , wherein the compound has structural formula (IIId), (IIIe), or (IIIf): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         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 a molecular moiety capable of producing a detectable signal. 
 
       
     
     
         8 . The compound  claim 1 , wherein the Tag comprises a molecular moiety selected from the group consisting of a polyethylene-glycol (PEG) oligomer, an organic dye moiety, an oligonucleotide (comprising natural and/or non-natural analog monomer units), a polypeptide (comprising natural and/or non-natural analog monomer units), and an oligomeric moiety comprising a combination of any of these 
     
     
         9 . The compound of  claim 1 , wherein the Tag comprises an oligonucleotide, optionally an oligonucleotide having a structure selected from Table 3, 7, or 9, optionally an oligonucleotide having a sequence selected from SEQ ID NO: 1-109. 
     
     
         10 . The compound of  claim 1 , wherein the Tag comprises a polymeric structure, optionally a polymeric structure comprising at least one monomer unit resulting from the reaction of an amidite reagent selected from Table 4. 
     
     
         11 . The compound of  claim 1 , wherein the Tag comprises a polypeptide, optionally a polypeptide having a structure selected from Table 5, optionally a polypeptide having a sequence selected from SEQ ID NO: 110-123. 
     
     
         12 . The compound of  claim 1 , wherein the tag or linker comprises a branched or dendrimeric moiety capable of forming covalent linkages with three or more molecular moieties. 
     
     
         13 . The compound of  claim 12 , wherein the branched or dendrimeric moiety is a doubler linker, optionally wherein the doubler linker results from the reaction of an amidite reagent of compound ( 19 ) 
       
         
           
           
               
               
           
         
       
     
     
         14 . The compound of  claim 12 , wherein the branched or dendrimeric moiety is a trebler linker, optionally wherein the trebler linker results from the reaction of an amidite reagent of compound ( 20 ) 
       
         
           
           
               
               
           
         
       
     
     
         15 . A composition comprising a set of compounds according to  claim 1 , wherein each compound in the set has a different tag, wherein each different tag causes a different detectable signal. 
     
     
         16 . A method for determining the sequence of a nucleic acid comprising:
 (a) providing a nanopore sequencing composition comprising: a membrane, an electrode on the cis side and the trans side of the membrane, a nanopore with its pore extending through the membrane, an electrolyte solution in contact with both electrodes, an active polymerase situated adjacent to the nanopore, and a primer strand complexed with the polymerase;   (b) contacting the nanopore sequencing composition with (i) a strand of the nucleic acid; and (ii) a set of compounds each comprising a single tag covalently linked to a plurality of nucleoside-5′-oligophosphate moieties, wherein the tag is a molecular moiety capable of producing a detectable signal, and each nucleoside-5′-oligophosphate moiety is capable of being a substrate for a polymerase, and each member of the set of compounds has a different tag that produces a different blocking current and/or dwell time when the tag is situated in a nanopore; and   (c) detecting the different blocking currents and/or different dwell times of the tags over time and correlating to each of the different tags the different compounds incorporated by the polymerase which are complementary to the nucleic acid sequence, and thereby determining the nucleic acid sequence.