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.

What is claimed is: 
     
         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 3 , wherein the increase in extension efficiency resulting from the use of the compound as a polymerase substrate is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or more. 
     
     
         5 . The compound of  claim 1 , wherein the detectable signal is selected from a nanopore detectable signal, an optically detectable signal, and a mass spectrometrically detectable signal. 
     
     
         6 . The compound of  claim 1 , wherein the detectable signal is an optically detectable, optionally a signal from a fluorescent moiety. 
     
     
         7 . The compound of  claim 1 , wherein the detectable signal is a nanopore detectable signal and the tag is a molecular moiety capable of entering into, becoming positioned in, being captured by, translocating through, and/or traversing a nanopore, and thereby result in a detectable change in current through the nanopore. 
     
     
         8 . The compound of any one of  claims 1 - 7 , 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. 
   
     
     
         9 . The compound of any one of  claims 1 - 8 , 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. 
 
       
     
     
         10 . The compound of any one of  claims 8 - 9 , wherein m is from 2 to 6, optionally wherein m is from 2 to 3. 
     
     
         11 . The compound of any one of  claims 8 - 9 , 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. 
 
       
     
     
         12 . The compound of any one of  claims 8 - 9 , 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. 
 
       
     
     
         13 . The compound of any one of  claims 1 - 12 , 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 
     
     
         14 . The compound of any one of  claims 1 - 13 , wherein the Tag comprises an oligonucleotide, optionally an oligonucleotide having a structure selected from Table 3, 7, 8, or 10, optionally an oligonucleotide having a sequence selected from SEQ ID NO: 1-109. 
     
     
         15 . The compound of  claim 14 , wherein the oligonucleotide has a monomer unit length of from 15-mer to 45-mer, from 20-mer to 40-mer, from 20-mer to 30-mer, or from 20-mer to 25-mer. 
     
     
         16 . The compound of any one of  claims 1 - 15 , 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. 
     
     
         17 . The compound of any one of  claims 1 - 13 , 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. 
     
     
         18 . The compound of any one of  claims 1 - 17 , wherein the oligophosphate consists of from 3 to 9 phosphate groups, optionally from 4 to 6 phosphate groups, or optionally 6 phosphate groups. 
     
     
         19 . The compound of any one of  claims 1 - 18 , wherein the tag or linker comprises a branched or dendrimeric moiety capable of forming covalent linkages with three or more molecular moieties. 
     
     
         20 . The compound of  claim 17 , 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) 
       
         
           
           
               
               
           
         
       
     
     
         21 . The compound of  claim 20 , 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) 
       
         
           
           
               
               
           
         
       
     
     
         22 . The compound of any one of  claims 1 - 21 , wherein the linker comprises a chemical group selected from the group consisting of: ester, ether, thioether, amine, amide, imide, benzene, benzyl ether, phenol, bis-hydroxyethylbenzene, carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime, triazole, dihydropyridazine, phosphodiester, polyethylene glycol (PEG), and combinations thereof. 
     
     
         23 . The compound of any one of  claims 1 - 21 , wherein the linker comprises a chemical group having structural formula (XVd) or (XVe). 
     
     
         24 . A composition comprising a set of compounds according to any one of  claims 1 - 23 , wherein each compound in the set has a different tag, wherein each different tag causes a different detectable signal. 
     
     
         25 . The composition of  claim 24 , wherein the detectable signal is selected from a nanopore detectable signal, an optically detectable signal, and a mass spectrometrically detectable signal. 
     
     
         26 . The composition of  claim 24 , wherein the different detectable signal is a different blocking current when the tag is situated in a nanopore. 
     
     
         27 . The composition of  claim 24 , wherein at least one of the different tags comprises an oligonucleotide, optionally an oligonucleotide having a structure selected from Table 3, 7, 8, or 10, optionally an oligonucleotide having a sequence selected from SEQ ID NO: 1-109. 
     
     
         28 . The composition of  claim 24 , wherein at least one of the different tags comprises oligonucleotide that has a monomer unit length of from 15-mer to 45-mer, from 20-mer to 40-mer, from 20-mer to 30-mer, or from 20-mer to 25-mer. 
     
     
         29 . The composition of  claim 24 , wherein the set of compounds comprises (dA6P) 2 -dT 5 -(BHEB)-dT 14 -C3; (dC6P) 2 -dT 20 -C3; (dT6P) 2 -dT 4 -(N3CE-dT) 3 -dT 13 -C3; and (dG6P) 2 -dT 6 -(Tmp) 6 -dT 8 -C3. 
     
     
         30 . The composition of  claim 24 , wherein the set of compounds comprises (dA6P) 2 -dT 4 -(idSp-dT) 4 -dT 8 -C3; (dC6P) 2 -dT 20 -C3; (dT6P) 2 -dT 4 -(N3CE-dT) 3 -dT 13 -C3; and (dG6P) 2 -dT 6 -(Tmp) 6 -dT 8 -C3. 
     
     
         31 . The composition of  claim 24 , wherein at least one of the different tags 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. 
     
     
         32 . The composition of  claim 24 , wherein at least one of the different tags 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. 
     
     
         33 . 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.   
     
     
         34 . The method of  claim 33 , wherein the at least two compounds having different tags have blocking currents that differ by at least 10%, at least 25%, at least 50%, or at least 75%. 
     
     
         35 . The method of  claim 33 , wherein each compound in the set of compounds has a different tag, wherein each different tag causes a different detectable signal. 
     
     
         36 . The method of  claim 35 , wherein at least one of the different tags comprises an oligonucleotide, optionally an oligonucleotide having a structure selected from Table 3, 7, 8, or 10, optionally an oligonucleotide having a sequence selected from SEQ ID NO: 1-109. 
     
     
         37 . The method of  claim 35 , wherein at least one of the different tags 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. 
     
     
         38 . The method of  claim 37 , wherein at least one of the different tags comprises oligonucleotide that has a monomer unit length of from 15-mer to 45-mer, from 20-mer to 40-mer, from 20-mer to 30-mer, or from 20-mer to 25-mer. 
     
     
         39 . The method of  claim 33 , wherein the set of compounds comprises (dA6P) 2 -dT 5 -(BHEB)-dT 14 -C3; (dC6P) 2 -dT 20 -C3; (dT6P) 2 -dT4-(N3CE-dT) 3 -dT13-C3; and (dG6P) 2 -dT 6 -(Tmp) 6 -dT 8 -C3. 
     
     
         40 . The method of  claim 33 , wherein the set of compounds comprises (dA6P) 2 -dT 4 -(idSp-dT) 4 -dT 8 -C3; (dC6P) 2 -dT 20 -C3; (dT6P) 2 -dT 4 -(N3CE-dT) 3 -dT 13 -C3; and (dG6P) 2 -dT 6 -(Tmp) 6 -dT 8 -C3. 
     
     
         41 . The method of  claim 35 , wherein at least one of the different tags 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.