Chemical methods for producing tagged nucleotides

Abstract

This disclosure provides systems and methods for attaching nanopore-detectable tags to nucleotides. The disclosure also provides methods for sequencing nucleic acids using the disclosed tagged nucleotides.

1 - 33 . (canceled) 
     
     
         34 . A method for determining the nucleotide sequence of a single-stranded nucleic acid comprising:
 (a) contacting the single-stranded nucleic acid, wherein the single-stranded DNA is in an electrolyte solution which is in contact with a nanopore in a membrane and wherein the single-stranded nucleic acid has a primer hybridized to a portion thereof, with a nucleic acid polymerase and at least four tagged nucleotides under conditions permitting the nucleic acid polymerase to catalyze incorporation of one of the tagged nucleotides into the primer if it is complementary to the nucleotide residue of the single-stranded nucleic acid which is immediately 5′ to a nucleotide residue of the single-stranded nucleic acid hybridized to the 3′ terminal nucleotide residue of the primer, so as to form a nucleic acid extension product, wherein each of the at least four tagged nucleotides comprises a poly-phosphate moiety having a terminal phosphate, a base which is adenine, guanine, cytosine, thymine, or uracil, or a derivative of each thereof, and a tag covalently coupled to the terminal phosphate of the nucleotide by a triazole, a 1,2-diazine, a disulfide, a secondary amine, a hydrazone, a thio-acetamide, or a maleimide-thioadduct, wherein (i) the type of base in each tagged nucleotide is different from the type of base in each of the other three tagged nucleotides, and (ii) either the number of phosphates in the poly-phosphate moiety of each tagged nucleotide is different from the number of phosphates in the poly-phosphate moiety of the other three tagged nucleotides, or the number of phosphates in the poly-phosphate moiety of each tagged nucleotide is the same and the type of tag on each tagged nucleotide is different from the type of tag on each of the other three tagged nucleotides, wherein incorporation of the tagged nucleotide results in release of a polyphosphate having the tag attached thereto;   (b) determining which tagged nucleotide has been incorporated into the primer to form a nucleic acid extension product in step (a) by applying a voltage across the membrane and measuring an electronic change across the nanopore resulting from the polyphosphate having the tag attached thereto generated in step (a) entering into, becoming positioned in, and/or translocating through the nanopore, wherein the electronic change is different for each different number of phosphates in the poly-phosphate moiety, or for each different type of tag, as appropriate, thereby identifying the nucleotide residue in the single-stranded nucleic acid complementary to the incorporated tagged nucleotide; and   (c) iteratively performing steps (a) and (b) for each nucleotide residue of the single-stranded nucleic acid being sequenced, wherein in each iteration of step (a) the tagged nucleotide is incorporated into the nucleic acid extension product resulting from the previous iteration of step (a) if it is complementary to the nucleotide residue of the single-stranded nucleic acid which is immediately 5′ to a nucleotide residue of the single-stranded nucleic acid hybridized to the 3′ terminal nucleotide residue of the nucleic acid extension product,   
       thereby determining the nucleotide sequence of the single-stranded nucleic acid. 
     
     
         35 . (canceled) 
     
     
         36 . The method of  claim 34 , wherein the nucleic acid is DNA and the nucleic acid polymerase is a DNA polymerase, or wherein the nucleic acid is RNA and the nucleic acid polymerase is a reverse transcriptase. 
     
     
         37 . The method of  claim 34 , wherein the number of phosphates in the poly-phosphate moiety of each tagged nucleotide is the same and the type of tag on each tagged nucleotide is different from the type of tag on each of the other three tagged nucleotides. 
     
     
         38 . The method of  claim 34 , wherein each tag comprises nucleotides, oligonucleotides, peptides, polyethylene glycol (PEG), oligo-saccharides, carbohydrates, peptide nucleic acids (PNA), vinyl polymers, other water-soluble polymers or any combination thereof. 
     
     
         39 . The method of  claim 34 , wherein each tag is covalently coupled to the terminal phosphate by a triazole, or by a 1,2-diazine. 
     
     
         40 . The method of  claim 34 , wherein each poly-phosphate moiety comprises at least 3 phosphates or from 4 to 6 phosphates. 
     
     
         41 . (canceled) 
     
     
         42 . The method of  claim 39 , wherein each tag is covalently coupled to the terminal phosphate by a triazole, and each triazole is formed by a reaction between an azide and an alkyne. 
     
     
         43 . The method of  claim 39 , wherein each triazole has the structure: 
       
         
           
           
               
               
           
         
         wherein R 1  comprises the tag, and R 2  comprises the nucleotide; or 
         wherein R 1  comprises the nucleotide, and R 2  comprises the tag. 
       
     
     
         44 . The method of  claim 39 , wherein each triazole has the structure: 
       
         
           
           
               
               
           
         
         wherein R 1  and R 3  combine to form a cyclic moiety; and 
         wherein R 1  and R 3  combined comprise a tag, and R 2  comprises a nucleotide; or 
         wherein R 1  and R 3  combined comprise a nucleotide, and R 2  comprises a tag. 
       
     
     
         45 . (canceled) 
     
     
         46 . The method of  claim 34 , wherein each tagged nucleotide comprises a cyanine dye moiety in a linker connecting the tag to the nucleotide, and the tagged nucleotide has an improved rate of capture by a polymerase compared to a tagged nucleotide without a cyanine dye moiety. 
     
     
         47 . The method of  claim 34 , wherein the four tagged nucleotides are dA6P-Cy3-T 4 -FldT-T-FldT-T 23 -C3, dT6P-Cy3-T 2 -dSp 8 -T 20 -C 3 , dG6P-Cy3-T 30 -C 6 , and dC6P-Cy3-T 4 -dSp 3 -T 23 -C 3 . 
     
     
         48 . The method of  claim 34 , wherein the poly-phosphate moiety is at the 5′-position of the nucleotide, or wherein the tag comprises an oligonucleotide. 
     
     
         49 . The method of  claim 34 , wherein the oligonucleotide of the tag comprises at least 7 monomer units, or at least 30 monomer units. 
     
     
         50 . The method of  claim 34 , wherein the oligonucleotide comprises an unnatural nucleotide, wherein the unnatural nucleotide comprises a group selected from the group consisting of an L-nucleotide, a 2′,5′-linkage, an -D-nucleotide, a non-naturally occurring internucleotide linkage, a non-naturally-occurring base, a non-naturally occurring sugar moiety, and any combination thereof, or the unnatural nucleotide comprises a non-naturally occurring base selected from the group consisting of nitropyrrole, nitroindole, nebularine, zebularine, benzene, and benzene derivatives, or the unnatural nucleotide comprises a non-naturally occurring internucleotide linkage selected from the group consisting of a phosphotriester, phosphorothioate, methylphosphonate, boronophosphate, phosphoramidate, and a morpholino moiety. 
     
     
         51 . The method of  claim 48 , wherein the 5′-end of the oligonucleotide is covalently coupled to the terminal phosphate of a poly-phosphate moiety. 
     
     
         52 . The method of  claim 51 , wherein the oligonucleotide comprises a chemical modification of its 3′ terminus that protects it from exonuclease degradation. 
     
     
         53 . The method of  claim 52 , wherein the chemical modification of its 3′ terminus is selected from phosphorylation, and covalent coupling with a C 3 -alkyl to C 12 -alkyl spacers. 
     
     
         54 . The method of  claim 48 , wherein the 3′-end of the oligonucleotide is covalently coupled to the terminal phosphate of a poly-phosphate moiety. 
     
     
         55 . The method of  claim 54 , wherein the oligonucleotide comprises a chemical modification of its 5′ terminus that protects it from exonuclease degradation. 
     
     
         56 . The method of  claim 55 , wherein the chemical modification of its 5′ terminus is selected from phosphorylation, and covalent coupling with a C 3 -alkyl to C 12 -alkyl spacers.