CHEMICAL METHODS FOR PRODUCING TAGGED NUCLEOTIDES

• US 20150368710A1
• Filed: 03/23/2015
• Published: 12/24/2015
• Est. Priority Date: 03/24/2014
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University), Genia Technologies, Inc. (Roche Holding AG)
• Inventors: Fuller, Carl W., Kumar, Shiv, Ju, Jingyue, Davis, Randall, Chen, Roger
• Granted Patent: US 10,240,195 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07H 17/02 Heterocyclic radicals containing only nitrogen as ring hetero atoms
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

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.

Claims

1. A tagged nucleotide, comprising a poly-phosphate moiety having a terminal phosphate, 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.

2. The tagged nucleotide of claim 1, wherein the tag is covalently coupled to the terminal phosphate by a 1,2-diazine, or a triazole, or a triazole having the structure:

6. The tagged nucleotide of claim 1, wherein the poly-phosphate moiety is at the 5′

-position of the nucleotide;

wherein the poly-phosphate moiety comprises at least 3 phosphates, or from 4 to 6 phosphates;

wherein the tag comprises nucleotides, oligonucleotides, peptides, polyethylene glycol (PEG), or any combination thereof;

wherein the tag comprises an oligonucleotide;

or wherein the tag comprises an oligonucleotide which comprises at least 7 monomer units, or at least 30 monomer units;

wherein the tag comprises an oligonucleotide and the 5′

-end of the oligonucleotide is covalently coupled to the terminal phosphate of a poly-phosphate moiety;

or wherein the tag comprises an oligonucleotide and the 3′

-end of the oligonucleotide is covalently coupled to the terminal phosphate of a poly-phosphate moiety.

8. The tagged nucleotide of claim 1, wherein the tag comprises oligo-saccharides, carbohydrates, peptide nucleic acids (PNA), vinyl polymers, other water-soluble polymers or any combination thereof;

wherein the tag comprises an oligonucleotide which comprises an unnatural nucleotide;

wherein the tag comprises an oligonucleotide which comprises an unnatural nucleotide comprising a group selected from the group consisting of an L-nucleotide, a 2′

,5′

-linkage, an α

-D-nucleotide, a non-naturally occurring internucleotide linkage, a non-naturally-occurring base, a non-naturally occurring sugar moiety, and any combination thereof;

wherein the tag comprises an oligonucleotide which comprises an unnatural nucleotide comprising a non-naturally occurring base selected from the group consisting of nitropyrrole, nitroindole, nebularine, zebularine, benzene, and benzene derivatives;

or wherein the tag comprises an oligonucleotide which comprises an unnatural nucleotide comprising a non-naturally occurring internucleotide linkage selected from the group consisting of a phosphotriester, phosphorothioate, methylphosphonate, boronophosphate, phosphoramidate, and a morpholino moiety.

13. The tagged nucleotide of claim 6, wherein the tag comprises an oligonucleotide;

and

wherein the 5′

-end of the oligonucleotide is covalently coupled to the terminal phosphate of a poly-phosphate moiety and the oligonucleotide comprises a chemical modification of its 3′

terminus that protects it from exonuclease degradation;

wherein the 5′

-end of the oligonucleotide is covalently coupled to the terminal phosphate of a poly-phosphate moiety, the oligonucleotide comprises a chemical modification of its 3′

terminus that protects it from exonuclease degradation, and the chemical modification of its 3′

terminus is selected from phosphorylation, and covalent coupling with C₃-alkyl to C₁₂-alkyl spacers having terminal hydroxyl groups;

wherein the 3′

-end of the oligonucleotide is covalently coupled to the terminal phosphate of a poly-phosphate moiety and the oligonucleotide comprises a chemical modification of its 5′

terminus that protects it from exonuclease degradation;

or wherein the 3′

-end of the oligonucleotide is covalently coupled to the terminal phosphate of a poly-phosphate moiety, the oligonucleotide comprises a chemical modification of its 5′

terminus that protects it from exonuclease degradation, and the chemical modification of its 5′

terminus is selected from phosphorylation, and covalent coupling with C₃-alkyl to C₁₂-alkyl spacers having terminal hydroxyl groups.

19. The tagged nucleotide of claim 6, wherein the tag comprises an oligonucleotide and the oligonucleotide comprises a cyanine dye moiety, or a cyanine dye moiety which is a Cy3 moiety, or the oligonucleotide and/or linker comprises a spacer moiety comprising an alkyl group of at least 2 carbons to about 12 carbons;

or wherein the oligonucleotide and/or linker comprises a spacer comprising an abasic unit;

or wherein the oligonucleotide and/or linker comprises a spacer selected from the group consisting of idSp, iSp9, iS18, iSpC3, iSpC6, and iSpC12;

or wherein the oligonucleotide and/or linker comprises a spacer selected from the group consisting of pyrolidine, spermine, nitropyrrole, nitroindole, nebularine, benimidazole, benzene, 7-deazapurines, 5-substituted pyrimidine, fluorescein-dT, fluorescein, rhodamine, and ROX;

wherein the tag comprises an oligonucleotide and the oligonucleotide comprises a spacer moiety comprising an alkyl group of at least 2 carbons to about 12 carbons.

21. A kit for sequencing nucleic acid comprising a tagged nucleotide of claim 1.

22. A process for making a tagged nucleotide of claim 1, comprising:

(a) providing (i) a nucleotide comprising a poly-phosphate moiety with a terminal phosphate, wherein the terminal phosphate is coupled to a linker that comprises a first reactive functional group, and (ii) a tag comprising a second reactive functional group,

wherein

(1) the first reactive functional group is selected from the group consisting of a thiol, an imidazole, an amine, an alkyne and a diene, and the second reactive functional group is selected from the group consisting of a maleimide, a haloacetamide, an aldehyde, ester, an isothiocyanate, an isocyanate, a vinyl sulphone, an azide and a tetrazine;

or

(2) the first reactive functional group is selected from the group consisting of a maleimide, a haloacetamide,

an aldehyde, an ester, an isothiocyanate, an isocyanate, a vinyl sulphone, an azide and a tetrazine, and the second reactive functional group is selected from the group consisting of a thiol, an imidazole, an amine, an alkyne and a diene; and

(b) reacting the first reactive functional group with the second reactive functional group to link the nucleotide to the tag.

23. The process of claim 22, wherein the first reactive functional group is selected from the group consisting of a thiol, an imidazole, an amine, an alkyne and a diene, and the second reactive functional group is selected from the group consisting of a maleimide, a haloacetamide, an aldehyde, an ester, an isothiocyanate, an isocyanate, a vinyl sulphone, an azide and a tetrazine;

or

wherein the first reactive functional group is an alkyne and the second reactive functional group is an azide;

or the first reactive functional group is an azide and the second reactive functional group is an alkyne;

or

wherein the first reactive functional group is a cyclooctyne and the second reactive functional group is an azide;

or the first reactive functional group is an azide and the second reactive functional group is a cyclooctyne;

or

wherein the first reactive functional group is selected from the group consisting of a maleimide, a haloacetamide, an aldehyde, an isothiocyanate, an isocyanate, a vinyl sulphone, an azide and a tetrazine, and the second reactive functional group is selected from the group consisting of a thiol, an imidazole, an amine, an alkyne and a diene.

27. The process of claim 22, wherein step (b) is facilitated by a heterogeneous catalyst comprising copper, ruthenium, silver, or any combination thereof;

or

wherein step (b) is not facilitated by a heterogeneous catalyst.

29. The process of claim 22, wherein the tag is selected from the group consisting of the tags listed in Table 5, or the tag is represented by SEQ ID NOS. 17, 18, 22-33, 42-72, 74-82, 86-88, 90, or 94-102;

wherein the tag is DBCO-Cy3, or the tag represented by SEQ ID NOS. 5-16, 19-21, or 34-41;

wherein the tag is represented by SEQ ID NOS. 89, 91-93, or 103-105;

wherein the tag is represented by SEQ ID NOS. 73, 83-85, or 106;

wherein the tagged nucleotide is selected from the group consisting of dG6P-(T₄-Npy₂)₆-C3, dG6P-(T₄-Neb₂)₆-C3, dT6P-dT₆-C7-NH₆-dT₁₈-C3, dT6P-dT₆-Pyrd₆-dT₁₈-C3, dA6P-dT₆-dTNH₆-dT₁₈-C3, dG6P-dT₄-sperm-dT₂₂-C3, dT6P-dT₄-sperm-dSp₃-dT₁₉-C3, dC6P-dT₄-sperm-iFlrT-dT₂₁-C3, dG6P-sperm-dT₃₀-C3, dT6P-Cy3.5-dT₃₀-C3, dT6P-Cy3-Cy3-dT₃₀-C3, dT6P-dT₆-Cy3-dT₂₃-C3, dT6P-dT₁₀-Cy3-dT₁₉-C3, dT6P-Hairpin Block, dA6P-Cy3 T2-Sp18-T₂₂-C3, dT6P-Cy3-dT4-dSp₈-T₁₈-C3, dT6P-Hex-dT₆-dTC2NH₆-dT₁₈-C3, dA6P-Cy3-dT₄-Sp9-T₂₃-C3, dC6P-Cy3-T-dSp₃-T₂₆-C3, dC6P-Cy3-T₄-dSp₃-T₂₃-C3, dC6P-Cy3-T₇-dSp₃-T₂₀-C3, dC6P-Cy3-T₁₀-dSp₃-T₁₇-C3, dC6P-Cy3 T4-iFluorT₃-T23-C3, dC6P-Cy3 T₄-iFluorT-T-iFluorT-T₂₃-C3, Bio-Spermine-dT₃₀-C3, dT6P-dT₃₀-Cy3-C3, dG6P-dT₈-Spermine-dT₂₀-C3, dA6P-Cy3-T₄-iFluorT-T-iFluorT-T₂₃-C3, dT6P-CY3-dT4-Aptamer-dT25-C3, dT6P-Cy3-dT4-12Hairpin-dT25-C3, dT6P-Cy3-dT₅-dSp₃-dT₂₂-C3, dT6P-Cy3-dT₆-dSp₃-dT₂₁-C3, dT6P-Cy3-dT₄-dSp₄-dT₂₂-C3, dT6P-Cy3-dT₄-dSp₅-dT₂₁-C3, dC6P-Cy3-dT₅-SpC12-dT₂₃-C3, dC6P-Cy3-dT₄-SpC6-SpC6-dT₂₄-C3, dC6P-Cy3-dT₄-(SpC3)₃-dT₂₃-C3, dG6P-Cy3-dT₃₀-C3, dT6P-Cy3-dT₂-dSp₈-dT₂₀-C3, dC6P-Cy3-T₃₀-(C₃)₄-PO₄, dC6P-Cy3-T₃₀-PO₄, dC6P-Cy3-T₃₀-C₃-NH₂, dG6Pα

S-Cy3-dT₂-dSp₈-dT₂₀-C3, Rev-P-T₃₀-Cy3-dG6P, Rev-P-T₂₄-dSp₃-T₃-Cy3-dC6P, dT6P-Cy3-dT₄-HP6-dT₂₅-C₃, dA6P-Cy3-dT4-dI6-dT20-C3, dA6P-Cy3-dT4-NitrIndole6-dT20-C3, dA6P-Cy3-dT4-dC6-dT20-C3, dA6P-Cy3-dT4-5IU6-dT20-C3, dA6P-Cy3-dT4-PyrndU6-dT20-C3, dT6P-Cy3-dT₄-(idSP-T)₄-dT₁₈-C₃, dT6P-Cy3-dT₅-(idSP-T)₄-dT₁₇-C3, dT6P-Cy3-dT₄-Propyl₆-dT₂₀-C3, dT6P-Cy3-LdT₃₀-C3, dT6P-Cy3-dT₄-L111-dT₂₆-C3, dT6P-Cy3-dT₄-L 121-dT₂₆-C3, dT6P-Cy3-dT₄-SpC12-SpC12-dT₂₄-C3, dT6P-Cy3-dT₄-(SpC6)₄-dT₂₅-C3, dT6P-Cy3-dT₄-Spermine-dT₂₅-C3, dT6P-Cy3-dT₂-Spermine-dT₂₇-C3, dT6P-Cy3-dT₂-Spermine-Spermine-dT₂₆-C3, dT6P-Cy3-dT₄-Pyrn-dU-TT-Pyrn-dU-dT₂₂-C3, dT6P-Cy3-dT₄-Tmp₆-dT₂₀-C3, dT6P-Cy3-dT₄-Pyrrolidine₆-dT₂₀-C3, dT6P-Pyrrolidine-dT₃₀-C3, dT6P-Pyrrolidine-Pyrrolidine-dT₃₀-C3, and dT6P-Pyrrolidine₃-dT₃₀-C3;

wherein the tagged nucleotide is selected from the group consisting of dT6P-Cy3, dA6P-Cy3, dT6P-Cy3-T₂₅, dA6P-T*₃₀ ODD, dG6P-T₃₀, dT6P-T₆-dSp₈-T₁₆, dC6P-T₆-T*₁₀-T₁₄, dC6P-T₄-dSp₃-T₂₃, dC6P-T₇-dSp₃-T₂₀, dC6P-T₁₀-dSp₃-T₁₇, dC6P-T₁₃-dSp₃-T₁₄, dG6P-T₃₀-C6, dG6P-Cy3-T₃₀-C6, dT6P-T₄-dSp₁₀-T₁₆-C6, dA6P-T₄-Sp18-T₂₂-C3, dA6P-T₄-Sp18₂-T₁₉-C₃, dA6P-T₄-Sp9₂-T₂₂-C3, dC6P-Cy3, dG6P-Cy3, dT6P-T₆-dSp₈-T₁₆-C3, dA6P-Cy3-T₃₀-C6, dT6P-Cy3-T₃₀-C6, dC6P-Cy3-T₃₀-C6, dA6P-Cy3-dT*₃₀ ODD, dA6P-T*₃₀, dA6P-Cy3-T*₃₀, dG6P-Cy3-T₃₀-C3, dG6P-Cy3-T₁₅-C3, dG6P-Cy3-T₂₀-C3, and dG6P-Cy3-T₂₅-C3;

wherein the tagged nucleotide is selected from the group consisting of dT6P-Cy3-dT₃-(SpCl2)₃-dT₂₄-C3, dT6P-Cy3-dT₄-(SpC6)₅-dT₂₃-C3, dT6P-Cy3-dT5-(SpC6)₄-dT₂₄-C3, dT6P-Cy3-dT₂-(SpC6)₅-dT₂₅-C3, dT6P-SpC3-Cy3-dT₃₀-C3, dT6P-SpC3-SpC3-Cy3-dT₃₀-C3, and dT6P-SpC6-Cy3-dT₃₀-C3;

wherein the tagged nucleotide is selected from the group consisting of dT6P-Cy3-dC₃₀-C3, dT6P-Cy3-LdT₄-dSp₃-LdT₂₃-C3, dT6P-Cy3-LdT₄-dSp₈-LdT₁₈-C3, dT6P-Cy3-LdT₄-dI₆-LdT₂₀-C3, and dT6P-Cy3-dT₄ (alpha-dT)₃-dT₂₃-C3;

wherein the tag comprises a chemical modification selected from the group consisting of the chemical modifications listed in Table 6;

or wherein the tagged nucleotide comprises a linker comprising a cyanine dye moiety, or a cyanine dye moiety which is a Cy3 moiety has an improved rate of capture by a polymerase.

52. The tagged nucleotide of claim 1, wherein the tag is selected from the group consisting of the tags listed in Table 5, or the tag is represented by SEQ ID NOS. 17, 18, 22-33, 42-72, 74-82, 86-88, 90, or 94-102;

wherein the tag is DBCO-Cy3, or the tag represented by SEQ ID NOS. 5-16, 19-21, or 34-41;

wherein the tag is represented by SEQ ID NOS. 89, 91-93, or 103-105;

wherein the tag is represented by SEQ ID NOS. 73, 83-85, or 106;

wherein the tagged nucleotide is selected from the group consisting of dG6P-(T₄-Npy₂)₆-C3, dG6P-(T₄-Neb₂)₆-C3, dT6P-dT₆-C7-NH₆-dT₁₈-C3, dT6P-dT₆-Pyrd₆-dT₁₈-C3, dA6P-dT₆-dTNH₆-dT₁₈-C3, dG6P-dT₄-sperm-dT₂₂-C3, dT6P-dT₄-sperm-dSp₃-dT₁₉-C3, dC6P-dT₄-sperm-iFlrT-dT₂₁-C3, dG6P-sperm-dT₃₀-C3, dT6P-Cy3.5-dT₃₀-C3, dT6P-Cy3-Cy3-dT₃₀-C3, dT6P-dT₆-Cy3-dT₂₃-C3, dT6P-dT₁₀-Cy3-dT₁₉-C3, dT6P-Hairpin Block, dA6P-Cy3 T2-Sp18-T₂₂-C3, dT6P-Cy3-dT4-dSp₈-T₁₈-C3, dT6P-Hex-dT₆-dTC2NH₆-dT₁₈-C3, dA6P-Cy3-dT₄-Sp9 T₂₃-C3, dC6P-Cy3-T-dSp₃-T₂₆-C3, dC6P-Cy3-T₄-dSp₃-T₂₃-C3, dC6P-Cy3-T₇-dSp₃-T₂₀-C3, dC6P-Cy3-T₁₀-dSp₃-T₁₇-C3, dC6P-Cy3 T₄-iFluorT₃-T23-C3, dC6P-Cy3 T₄-iFluorT-T-iFluorT-T₂₃-C₃, Bio-Spermine-dT₃₀-C3, dT6P-dT₃₀-Cy3-C3, dG6P-dT₈-Spermine-dT₂₀-C3, dA6P-Cy3-T4-iFluorT-T-iFluorT-T₂₃-C3, dT6P-CY3-dT4-Aptamer-dT25-C3, dT6P-Cy3-dT4-12Hairpin-dT25-C3, dT6P-Cy3-dT₅-dSp₃-dT₂₂-C3, dT6P-Cy3-dT₆-dSp₃-dT₂₁-C3, dT6P-Cy3-dT₄-dSp₄-dT₂₂-C3, dT6P-Cy3-dT₄-dSp₅-dT₂₁-C3, dC6P-Cy3-dT₅-SpC12-dT₂₃-C3, dC6P-Cy3-dT₄-SpC6-SpC6-dT₂₄-C₃, dC6P-Cy3-dT₄-(SpC3)₃-dT₂₃-C3, dG6P-Cy3-dT₃₀-C3, dT6P-Cy3-dT₂-dSp₈-dT₂₀-C3, dC6P-Cy3-T₃₀-(C3)₄-PO₄, dC6P-Cy3-T₃₀-PO₄, dC6P-Cy3-T₃₀-C3-NH₂, dG6Pα

S-Cy3-dT₂-dSp₈-dT₂₀-C3, Rev-P-T₃₀-Cy3-dG6P, Rev-P-T₂₄-dSp₃-T₃-Cy3-dC6P, dT6P-Cy3-dT₄-HP6-dT₂₅-C₃, dA6P-Cy3-dT4-dI6-dT20-C3, dA6P-Cy3-dT4-NitrIndole6-dT20-C3, dA6P-Cy3-dT4-dC6-dT20-C3, dA6P-Cy3-dT4-5IU6-dT20-C3, dA6P-Cy3-dT4-PyrndU6-dT20-C3, dT6P-Cy3-dT₄-(idSP-T)₄-dT₁₈-C₃, dT6P-Cy3-dT₅-(idSP-T)₄-dT₁₇-C3, dT6P-Cy3-dT₄-Propyl₆-dT₂₀-C3, dT6P-Cy3-LdT₃₀-C3, dT6P-Cy3-dT4-L111-dT₂₆-C3, dT6P-Cy3-dT₄-L121-dT₂₆-C3, dT6P-Cy3-dT₄-SpC12-SpC12-dT₂₄-C3, dT6P-Cy3-dT₄-(SpC6)₄-dT₂₅-C3, dT6P-Cy3-dT₄-Spermine-dT₂₅-C3, dT6P-Cy3-dT₂-Spermine-dT₂₇-C3, dT6P-Cy3-dT₂-Spermine-Spermine-dT₂₆-C3, dT6P-Cy3-dT₄-Pyrn-dU-TT-Pyrn-dU-dT₂₂-C3, dT6P-Cy3-dT₄-Tmp₆-dT₂₀-C3, dT6P-Cy3-dT₄-Pyrrolidine₆-dT₂₀-C3, dT6P-Pyrrolidine-dT₃₀-C3, dT6P-Pyrrolidine-Pyrrolidine-dT₃₀-C3, and dT6P-Pyrrolidine₃-dT₃₀-C3;

wherein the tagged nucleotide is selected from the group consisting of dT6P-Cy3, dA6P-Cy3, dT6P-Cy3-T₂₅, dA6P-T*₃₀ ODD, dG6P-T₃₀, dT6P-T₆-dSp₈-T₁₆, dC6P-T₆-T*₁₀-T₁₄, dC6P-T₄-dSp₃-T₂₃, dC6P-T₇-dSp₃-T₂₀, dC6P-T₁₀-dSp₃-T₁₇, dC6P-T₁₃-dSp₃-T₁₄, dG6P-T₃₀-C6, dG6P-Cy3-T₃₀-C6, dT6P-T₄-dSp₁₀-T₁₆-C6, dA6P-T₄-Sp18-T₂₂-C3, dA6P-T₄-Sp18₂-T₁₉-C₃, dA6P-T₄-Sp9₂-T₂₂-C3, dC6P-Cy3, dG6P-Cy3, dT6P-T₆-dSp₈-T₁₆-C3, dA6P-Cy3-T₃₀-C6, dT6P-Cy3-T₃₀-C6, dC6P-Cy3-T₃₀-C6, dA6P-Cy3-dT*₃₀ ODD, dA6P-T*₃₀, dA6P-Cy3-T*₃₀, dG6P-Cy3-T₃₀-C3, dG6P-Cy3-T₁₅-C3, dG6P-Cy3-T₂₀-C3, and dG6P-Cy3-T₂₅-C3;

wherein the tagged nucleotide is selected from the group consisting of dT6P-Cy3-dT₃-(SpC12)₃-dT₂₄-C3, dT6P-Cy3-dT₄-(SpC6)₅-dT₂₃-C3, dT6P-Cy3-dT5-(SpC6)₄-dT₂₄-C3, dT6P-Cy3-dT₂-(SpC6)₅-dT₂₅-C3, dT6P-SpC3-Cy3-dT₃₀-C3, dT6P-SpC3-SpC3-Cy3-dT₃₀-C3, and dT6P-SpC6-Cy3-dT₃₀-C3;

wherein the tagged nucleotide is selected from the group consisting of dT6P-Cy3-dC₃₀-C3, dT6P-Cy3-LdT₄-dSp₃-LdT₂₃-C3, dT6P-Cy3-LdT₄-dSp₈-LdT₁₈-C3, dT6P-Cy3-LdT₄-dI₆-LdT₂₀-C3, and dT6P-Cy3-dT₄ (alpha-dT)₃-dT₂₃-C3;

wherein the tag comprises a chemical modification selected from the group consisting of the chemical modifications listed in Table 6;

or wherein the tagged nucleotide comprises a linker comprising a cyanine dye moiety, or a cyanine dye moiety which is a Cy3 moiety has an improved rate of capture by a polymerase.

3-5. -5. (canceled)

7. (canceled)

9-12. -12. (canceled)

14-18. -18. (canceled)

20. (canceled)

24-26. -26. (canceled)

28. (canceled)

30-33. -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.

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;

wherein each polyphosphate moiety comprises at least 3 phosphates;

wherein each polyphosphate moiety comprises from 4-6 phosphates;

or wherein each tag comprises nucleotides, oligonucleotides, peptides, polyethylene glycol (PEG), 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, or by a triazole wherein each triazole is formed by a reaction between an azide and an alkyne, or by a triazole having the structure:

45. The method of claim 34, wherein each tag is selected from the group consisting of the tags listed in Table 5, or each tag is represented by SEQ ID NOS. 17, 18, 22-33, 42-72, 74-82, 86-88, 90, or 94-102, or wherein each tag is DBCO-Cy3, or the tag represented by SEQ ID NOS. 5-16, 19-21, or 34-41;

or wherein each tag is represented by SEQ ID NOS. 89, 91-93, or 103-105;

or wherein each tag is represented by SEQ ID NOS. 73, 83-85, or 106;

or wherein each tag comprises a chemical modification selected from the group consisting of the chemical modifications listed in Table 6, or wherein each tag comprises oligo-saccharides, carbohydrates, peptide nucleic acids (PNA), vinyl polymers, other water-soluble polymers or any combination thereof;

or wherein each tagged nucleotide is selected from the group consisting of dG6P-(T₄-NPy₂)₆-C3, dG6P-(T₄-Neb₂)₆-C3, dT6P-dT₆-C7-NH₆-dT₁₈-C3, dT6P-dT₆-Pyrd₆-dT₁₈-C3, dA6P-dT₆-dTNH₆-dT₁₈-C3, dG6P-dT₄-sperm-dT₂₂-C3, dT6P-dT₄-sperm-dSp₃-dT₁₉-C3, dC6P-dT₄-sperm-iFlrT-dT₂₁-C3, dG6P-sperm-dT₃₀-C3, dT6P-Cy3.5-dT₃₀-C3, dT6P-Cy3-Cy3-dT₃₀-C3, dT6P-dT₆-Cy3-dT₂₃-C₃, dT6P-dT₁₀-Cy3-dT₁₉-C3, dT6P-Hairpin Block, dA6P-Cy3 T2-Sp18-T₂₂-C3, dT6P-Cy3-dT4-dSp₈-T₁₈-C3, dT6P-Hex-dT₆-dTC2NH₆-dT₁₈-C3, dA6P-Cy3-dT₄-Sp9-T₂₃-C3, dC6P-Cy3-T-dSp₃-T₂₆-C3, dC6P-Cy3-T₄-dSp₃-T₂₃-C3, dC6P-Cy3-T₇-dSp₃-T₂₀-C3, dC6P-Cy3-T₁₀-dSp₃-T₁₇-C3, dC6P-Cy3 T4-iFluorT₃-T23-C3, dC6P-Cy3 T₄-iFluorT-T-iFluorT-T₂₃-C3, Bio-Spermine-dT₃₀-C3, dT6P-dT₃₀-Cy3-C3, dG6P-dT₈-Spermine-dT₂₀-C3, dA6P-Cy3-T₄-iFluorT-T-iFluorT-T23-C3, dT6P-CY3-dT4-Aptamer-dT25-C3, dT6P-Cy3-dT4-12Hairpin-dT25-C3, dT6P-Cy3-dT₅-dSp₃-dT₂₂-C3, dT6P-Cy3-dT₆-dSp₃-dT₂₁-C3, dT6P-Cy3-dT₄-dSp₄-dT₂₂-C3, dT6P-Cy3-dT₄-dSp₅-dT₂₁-C3, dC6P-Cy3-dT₅-SpC12-dT₂₃-C₃, dC6P-Cy3-dT₄-SpC6-SpC6-dT₂₄-C₃, dC6P-Cy3-dT₄-(SpC3)₃-dT₂₃-C3, dG6P-Cy3-dT₃₀-C3, dT6P-Cy3-dT₂-dSp₈-dT₂₀-C3, dC6P-Cy3-T₃₀-(C₃)₄-PO₄, dC6P-Cy3-T₃₀-PO₄, dC6P-Cy3-T₃₀-C₃-NH₂, dG6Pα

S-Cy3-dT₂-dSp₈-dT₂₀-C3, Rev-P-T₃₀-Cy3-dG6P, Rev-P-T₂₄-dSp₃-T₃-Cy3-dC6P, dT6P-Cy3-dT₄-HP6-dT₂₅-C₃, dA6P-Cy3-dT4-dI6-dT20-C3, dA6P-Cy3-dT4-NitrIndole6-dT20-C3, dA6P-Cy3-dT4-dC6-dT20-C3, dA6P-Cy3-dT4-5IU6-dT20-C3, dA6P-Cy3-dT4-PyrndU6-dT20-C3, dT6P-Cy3-dT₄-(idSP-T)₄-dT₁₈-C₃, dT6P-Cy3-dT₅-(idSP-T)₄-dT₁₇-C3, dT6P-Cy3-dT₄-Propyl₆-dT₂₀-C3, dT6P-Cy3-LdT₃₀-C3, dT6P-Cy3-dT₄-L111-dT₂₆-C3, dT6P-Cy3-dT₄-L121-dT₂₆-C3, dT6P-Cy3-dT₄-SpC12-SpC12-dT₂₄-C3, dT6P-Cy3-dT₄-(SpC6)₄-dT₂₅-C3, dT6P-Cy3-dT₄-Spermine-dT₂₅-C3, dT6P-Cy3-dT2-Spermine-dT₂₇-C3, dT6P-Cy3-dT₂-Spermine-Spermine-dT₂₆-C3, dT6P-Cy3-dT₄-Pyrn-dU-TT-Pyrn-dU-dT₂₂-C3, dT6P-Cy3-dT₄-Tmp₆-dT₂₀-C3, dT6P-Cy3-dT₄-Pyrrolidine₆-dT₂₀-C3, dT6P-Pyrrolidine-dT₃₀-C3, dT6P-Pyrrolidine-Pyrrolidine-dT₃₀-C3, and dT6P-Pyrrolidine₃-dT₃₀-C3;

wherein each tagged nucleotide is selected from the group consisting of dT6P-Cy3, dA6P-Cy3, dT6P-Cy3-T₂₅, dA6P-T*₃₀ ODD, dG6P-T₃₀, dT6P-T₆-dSp₈-T₁₆, dC6P-T₆T*₁₀-T₁₄, dC6P-T₄-dSp₃-T₂₃, dC6P-T₇-dSp₃-T₂₀, dC6P-T₁₀-dSp₈-T₁₇, dC6P-T₁₃-dSp₃-T₁₄, dG6P-T₃₀-C6, dG6P-Cy3-T₃₀-C6, dT6P-T₄-dSP₁₀-T₁₆-C6, dA6P-T₄-Sp18-T₂₂-C3, dA6P-T₄-Sp18₂-T₁₉-C₃, dA6P-T₄-Sp9₂-T₂₂-C3, dC6P-Cy3, dG6P-Cy3, dT6P-T₆-dSp₈-T₁₆-C3, dA6P-Cy3-T₃₀-C6, dT6P-Cy3-T₃₀-C6, dC6P-Cy3-T₃₀-C₆, dA6P-Cy3-dT*₃₀ ODD, dA6P-T*₃₀, dA6P-Cy3-T*₃₀, dG6P-Cy3-T₃₀-C3, dG6P-Cy3-T₁₅-C3, dG6P-Cy3-T₂₀-C3, and dG6P-Cy3-T₂₅-C3;

wherein each tagged nucleotide is selected from the group consisting of dT6P-Cy3-dT₃₀-(SpC12)₃-dT₂₄-C3, dT6P-Cy3-dT₄-(SpC6)₅-dT₂₃-C3, dT6P-Cy3-dT5-(SpC6)₄-dT₂₄-C3, dT6P-Cy3-dT₂-(SpC6)₅-dT₂₅-C3, dT6P-SpC3-Cy3-dT₃₀-C3, dT6P-SpC3-Spc3-Cy3-dT₃₀-C3, and dT6P-SpC6-Cy3-dT₃₀-C3;

wherein each tagged nucleotide is selected from the group consisting of dT6P-Cy3-dC₃₀-C3, dT6P-Cy3-LdT₄-dSp₃-LdT₂₃-C3, dT6P-Cy3-LdT₄-dSp₈-LdT₁₈-C3, dT6P-Cy3-LdT₄-dI₆-LdT₂₀-C3, and dT6P-Cy3-dT₄ (alpha-dT)₃-dT₂₃-C3;

or 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, or wherein the four tagged nucleotides are dA6P-Cy3-T₄-FldT-T-FldT-T₂₃-C3, dT6P-Cy3-T₂-dSp₈-T₂₀-C3, dG6P-Cy3-T₃₀-C₆, and dC6P-Cy3-T₄-dSp₃-T₂₃-C₃.

35. 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 nucleic acid 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, a nucleic acid polymerase and a tagged nucleotide under conditions permitting the nucleic acid polymerase to catalyze incorporation of the tagged nucleotide 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 DNA extension product, wherein the tagged nucleotide 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 incorporation of a tagged nucleotide results in release of a polyphosphate having the tag attached thereto;

and if a tagged nucleotide is not incorporated, iteratively repeating the contacting with a different tagged nucleotide until a tagged nucleotide is incorporated, with the proviso that (1) the type of base in each tagged nucleotide is different from the type of base in each of the other three tagged nucleotides, and (2) 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,

(b) determining if 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 value of n, 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 DNA.

48. The method of claim 35, 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.

49. The method of claim 35, 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;

wherein each polyphosphate moiety comprises at least 3 phosphates;

wherein each polyphosphate moiety comprises from 4-6 phosphates;

or wherein each tag comprises nucleotides, oligonucleotides, peptides, polyethylene glycol (PEG), or any combination thereof.

50. The method of claim 35, wherein each tag is covalently coupled to the terminal phosphate by a triazole, or by a 1,2-diazine, or by a triazole wherein each triazole is formed by a reaction between an azide and an alkyne, or by a triazole having the structure:

51. The method of claim 35, wherein each tag is selected from the group consisting of the tags listed in Table 5, or wherein each tag is represented by SEQ ID NOS. 17, 18, 22-33, 42-72, 74-82, 86-88, 90, or 94-102, or wherein each tag is DBCO-Cy3, or the tag represented by SEQ ID NOS. 5-16, 19-21, or 34-41;

or wherein each tag is represented by SEQ ID NOS. 89, 91-93, or 103-105;

or wherein each tag is represented by SEQ ID NOS. 73, 83-85, or 106;

or wherein each tag comprises a chemical modification selected from the group consisting of the chemical modifications listed in Table 6, or wherein each tag comprises oligo-saccharides, carbohydrates, peptide nucleic acids (PNA), vinyl polymers, other water-soluble polymers or any combination thereof;

or wherein each tagged nucleotide is selected from the group consisting of dG6P-(T₄-Npy₂)₆-C3, dG6P-(T₄-Neb₂)₆-C3, dT6P-dT₆-C7-NH₆-dT₁₈-C3, dT6P-dT₆-Pyrd₆-dT₁₈-C3, dA6P-dT₆-dTNH₆-dT₁₈-C3, dG6P-dT₄-sperm-dT₂₂-C3, dT6P-dT₄-sperm-dSp₃-dT₁₉-C3, dC6P-dT₄-sperm-iFlrT-dT₂₁-C3, dG6P-sperm-dT₃₀-C3, dT6P-Cy3.5-dT₃₀-C3, dT6P-Cy3-Cy3-dT₃₀-C3, dT6P-dT₆-Cy3-dT₂₃-C3, dT6P-dT₁₀-Cy3-dT₁₉-C3, dT6P-Hairpin Block, dA6P-Cy3 T2-Sp18-T₂₂-C3, dT6P-Cy3-dT4-dSp₈-T₁₈-C3, dT6P-Hex-dT₆-dTC2NH₆-dT₁₈-C3, dA6P-Cy3-dT₄-Sp9-T₂₃-C3, dC6P-Cy3-T-dSp₃-T₂₆-C3, dC6P-Cy3 T₄-dSp₃-T₂₃-C3, dC6P-Cy3-T₇-dSp₃-T₂₀-C3, dC6P-Cy3-T₁₀-dSp₃-T₁₇-C3, dC6P-Cy3 T₄-iFluorT₃-T23-C3, dC6P-Cy3 T₄-iFluorT-T-iFluorT-T₂₃-C3, Bio-Spermine-dT₃₀-C3, dT6P-dT₃₀-Cy3-C3, dG6P-dT₈-Spermine-dT₂₀-C3, dA6P-Cy3-T₄-iFluorT-T-iFluorT-T₂₃-C3, dT6P-CY3-dT4-Aptamer-dT25-C3, dT6P-Cy3-dT4-12Hairpin-dT25-C3, dT6P-Cy3-dT₅-dSp₃-dT₂₂-C3, dT6P-Cy3-dT₆-dSp₃-dT₂₁-C3, dT6P-Cy3-dT₄-dSp₄-dT₂₂-C3, dT6P-Cy3-dT₄-dSp₅-dT₂₁-C3, dC6P-Cy3-dT₅-SpC12-dT₂₃-C3, dC6P-Cy3-dT₄-SpC6-SpC6-dT₂₄-C₃, dC6P-Cy3-dT₄-(SpC3)₃-dT₂₃-C3, dG6P-Cy3-dT₃₀-C3, dT6P-Cy3-dT₂-dSp₈-dT₂₀-C3, dC6P-Cy3-T₃₀-(C₃)₄-PO₄, dC6P-Cy3-T₃₀-PO₄, dC6P-Cy3-T₃₀-C₃-NH₂, dG6Pα

S-Cy3-dT₂-dSp₈-dT₂₀-C3, Rev-P-T₃₀-Cy3-dG6P, Rev-P-T₂₄-dSp₃-T₃-Cy3-dC6P, dT6P-Cy3-dT₄-HP6-dT₂₀-C3, dA6P-Cy3-dT4-dI6-dT20-C3, dA6P-Cy3-dT4-NitrIndole6-dT20-C3, dA6P-Cy3-dT4-dC6-dT20-C3, dA6P-Cy3-dT4-5IU6-dT20-C3, dA6P-Cy3-dT4-PyrndU6-dT20-C3, dT6P-Cy3-dT₄-(idSP-T)₄-dT₁₈-C₃, dT6P-Cy3-dT₅-(idSP-T)₄-dT₁₇-C3, dT6P-Cy3-dT₄-Propyl₆-dT₂₀-C3, dT6P-Cy3-LdT₃₀-C3, dT6P-Cy3-dT₄-L111-dT₂₆-C3, dT6P-Cy3-dT₄-L121-dT₂₆-C3, dT6P-Cy3-dT₄-SpC12-SpC12-dT₂₄-C3, dT6P-Cy3-dT₄-(SpC6)₄-dT₂₀-C3, dT6P-Cy3-dT₄-Spermine-dT₂₅-C3, dT6P-Cy3-dT2-Spermine-dT₂₇-C3, dT6P-Cy3-dT₂-Spermine-Spermine-dT₂₆-C3, dT6P-Cy3-dT₄-Pyrn-dU-TT-Pyrn-dU-dT₂₂-C3, dT6P-Cy3-dT₄-Tmp₆-dT₂₀-C3, dT6P-Cy3-dT₄-Pyrrolidine₆-dT₂₀-C3, dT6P-Pyrrolidine-dT₃₀-C3, dT6P-Pyrrolidine-Pyrrolidine-dT₃₀-C3, and dT6P-Pyrrolidine₃-dT₃₀-C3, wherein each tagged nucleotide is selected from the group consisting of dT6P-Cy3, dA6P-Cy3, dT6P-Cy3-T₂₅, dA6P-T*₃₀ ODD, dG6P-T₃₀, dT6P-T₆-dSp₈-T₁₆, dC6P-T₆-T*₁₀-T₁₄, dC6P-T₄-dSp₃-T₂₃, dC6P-T₇-dSp₃-T₂₀, dC6P-T₁₀-dSp₃-T₁₇, dC6P-T₁₃-dSp₃-T₁₄, dG6P-T₃₀-C6 dG6P-Cy3-T₃₀-C6, dT6P-T₄-dSp₁₀-T₁₆-C6, dA6P-T₄-Sp18-T₂₂-C3, dA6P-T₄-Sp18₂-T₁₉-C₃, dA6P-T₄-Sp9₂-T₂₂-C3, dC6P-Cy3, dG6P-Cy3, dT6P-T₆-dSp₈-T₁₆-C3, dA6P-Cy3-T₃₀-C6, dT6P-Cy3-T₃₀-C₆, dC6P-Cy3-T₃₀-C6, dA6P-Cy3-dT*₃₀ ODD, dA6P-T*₃₀, dA6P-Cy3-T*₃₀, dG6P-Cy3-T₃₀-C3, dG6P-Cy3-T₁₅-C3, dG6P-Cy3-T₂₀-C3, and dG6P-Cy3-T₂₅-C3;

wherein each tagged nucleotide is selected from the group consisting of dT6P-Cy3-dT₃-(SpC12)₃-dT₂₄-C3, dT6P-Cy3-dT₄-(SpC6)₅-dT₂₃-C3, dT6P-Cy3-dT5-(SpC6)₄-dT₂₄-C3, dT6P-Cy3-dT₂-(SpC6)₅-dT₂₅-C3, dT6P-SpC3-Cy3-dT₃₀-C3, dT6P-SpC3-SpC3-Cy3-dT₃₀-C3, and dT6P-SpC6-Cy3-dT₃₀-C3;

wherein each tagged nucleotide is selected from the group consisting of dT6P-Cy3-dC₃₀-C3, dT6P-Cy3-LdT₄-dSp₃-LdT₂₃-C3, dT6P-Cy3-LdT₄-dSp₈-LdT₁₈-C3, dT6P-Cy3-LdT₄-dI₆-LdT₂₀-C3, and dT6P-Cy3-dT₄ (alpha-dT)₃-dT₂₃-C3;

or 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, or wherein the four tagged nucleotides are dA6P-Cy3-T₄-FldT-T-FldT-T₂₃-C3, dT6P-Cy3-T₂-dSp₈-T₂₀-C₃ dG6P-Cy3-T₃₀-C₆, and dC6P-Cy3 T₄-dSp₃-T₂₃-C₃.

38. (canceled)

40-44. -44. (canceled)

46. (canceled)

47. (canceled)