Chemical methods for producing tagged nucleotides

Patent metadata

Field	Value
Publication number	US-10240195-B2
Application number	US-201514666124-A
Country	US
Kind code	B2
Filing date	Mar 23, 2015
Priority date	Mar 24, 2014
Publication date	Mar 26, 2019
Grant date	Mar 26, 2019

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.

First claim

What is claimed is:

1. A tagged nucleotide, comprising a poly-phosphate moiety having a terminal phosphate at the 5′ position of the nucleotide, 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 the tag comprises an oligonucleotide, wherein:
(i) the oligonucleotide comprises a non-naturally occurring internucleotide linkage;
(ii) the 5′-end of the oligonucleotide is covalently coupled to the terminal phosphate and the 3′ terminus of the oligonucleotide has a chemical modification which protects it from exonuclease degradation, or the 3′-end of the oligonucleotide is covalently coupled to the terminal phosphate and the 5′ terminus of the oligonucleotide has a chemical modification which protects it from exonuclease degradation; or
(iii) the oligonucleotide comprises at least one abasic spacer (dSp).

2. The tagged nucleotide of claim 1 , wherein the tag is covalently coupled to the terminal phosphate at the 5′ position of the nucleotide by a 1,2-diazine or a triazole having 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; or
a triazole having 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.

3. The tagged nucleotide of claim 1 , wherein the poly-phosphate moiety comprises at least 3phosphates, or from 4 to 6 phosphates; wherein the tag further comprises nucleotides, peptides, polyethylene glycol (PEG), or any combination thereof; wherein the oligonucleotide of the tag comprises at least 7 monomer units, or at least 30 monomer units; wherein the 5′-end of the oligonucleotide of the tag is covalently coupled to the terminal phosphate; or wherein the 3′-end of the oligonucleotide of the tag is covalently coupled to the terminal phosphate.

4. The tagged nucleotide of claim 3 ,
wherein the 5′-end of the oligonucleotide of the tag is covalently coupled to the terminal phosphate and the oligonucleotide comprises a chemical modification of its 3′ terminus that protects it from exonuclease degradation; wherein the 5′-end of the oligonucleotide of the tag is covalently coupled to the terminal phosphate and 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 C 3 -alkyl to C 12 -alkyl spacers having terminal hydroxyl groups; wherein the 3′-end of the oligonucleotide of the tag is covalently coupled to the terminal phosphate 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 of the tag is covalently coupled to the terminal and 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 C 3 -alkyl to C 12 -alkyl spacers having terminal hydroxyl groups.

5. The tagged nucleotide of claim 3 , wherein the oligonucleotide of the tag comprises a cyanine dye moiety, or a cyanine dye moiety which is a Cy3 moiety; wherein the oligonucleotide of the tag comprises a spacer moiety comprising an alkyl group of at least 2 carbons to about 12 carbons; wherein the oligonucleotide of the tag comprises a spacer comprising an abasic unit; wherein the oligonucleotide of the tag comprises a spacer selected from the group consisting of idSp, iSp9, iSp18, iSpC3, iSpC6, and iSpC12; or wherein the oligonucleotide of the tag 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.

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

7. The tagged nucleotide of claim 1 , wherein the tag is represented by SEQ ID NOS. 109-114, 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 represented by SEQ ID NOS. 6, 8-16, 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-(T 4 -Npy2) 6 -C3, dG6P-(T 4 -Neb2) 6 -C3, dT6P-dT 6 -C7-NH 6 -dT 18 -C3, dT6P-dT 6 -Pyrd 6 -dT 18 -C3, dA6P-dT 6 -dTNH 6 -dT 18 -C3, dG6P-dT 4 -sperm-dT 22 -C3, dT6P-dT 4 -sperm-dSp 3 -dT 19 -C3, dC6P-dT 4 -sperm-iFlrT-dT 21 -C3, dG6P-sperm-dT 30 -C3, dT6P-Cy3.5-dT 30 -C3, dT6P-Cy3-Cy3-dT 30 -C3, dT6P -dT 6 -Cy3-dT 23 -C3, dT6P-dT 10 -Cy3-dT 19 -C3, dT6P -Hairpin Block, dA6P-Cy3-T2-Sp18-T 22 -C3, dT6P-Cy3-dT4-dSp 8 -T 18 -C3, dT6P-Hex-dT 6 -dTC2NH 6 -dT 18 -C3, dA6P-Cy3-dT 4 -Sp9-T 23 -C3, dC6P-Cy3-T-dSp 3 -T 26- C3, dC6P-Cy3-T 4 -dSp 3 -T 23- C3, dC6P-Cy3-T 7 -dSp 3 -T 20- C3, dC6P-Cy3-T 10 -dSp 3 -T 17- C3, dC6P-Cy3-T 4 -iFluorT 3 -T23-C3, dC6P-Cy3 T 4 -iFluorT-T-iFluorT-T 23 -C3, Bio-Spermine-dT 30 -C3, dT6P-dT 30 -Cy3-C3, dG6P-dT 8 -Spermine-dT 20 -C3, dA6P-Cy3-T 4 -iFluorT-T-iFluorT-T 23 -C3, dT6P-CY3-dT4-Aptamer-dT25-C3, dT6P-Cy3-dT4-12Hairpin-dT25-C3, dT6P-Cy3-dT 5 -dSp 3 -dT 22 -C3, dT6P-Cy3-dT 6 -dSp 3 -dT 21 -C3, dT6P-Cy3-dT 4 -dSp 4 -dT 22 -C3, dT6P-Cy3-dT 4 -dSp 5 -dT 21 -C3, dC6P-Cy3-dT 5 -SpC12-dT 23 -C3, dC6P-Cy3-dT 4 -SpC6-SpC6-dT 24 -C 3 , dC6P-Cy3-dT 4 -(SpC3) 3 -dT 23 -C3, dG6P-Cy3-dT 30 -C3, dT6P-Cy3-dT 2 -dSp 8 -dT 20 -C3, dC6P-Cy3-T 30 -(C 3 ) 4 -PO 4 , dC6P-Cy3-T 30 -PO 4 , dC6P-Cy3-T 30 -C 3 -NH 2 , dG6PαS-Cy3-dT 2 -dSp 8 -dT 20 -C3, Rev-P-T 30 -Cy3-dG6P, Rev-P-T 24 -dSp 3 -T 3 -Cy3-dC6P, dT6P-Cy3-dT 4 -HP6-dT 25 -C 3 , 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-dT 4 -(idSP-T) 4 -dT 18 -C 3 , dT6P-Cy3-dT 5 -(idSP-T) 4 -dT 17 -C3, dT6P-Cy3-dT 4 -Propyl 6 -dT 20 -C3, dT6P-Cy3-LdT 30 -C3, dT6P-Cy3-dT 4 -L111-dT 26 -C3, dT6P-Cy3-dT 4 -L121-dT 26 -C3, dT6P-Cy3-dT 4 -SpC12-SpC12-dT 24 -C3, dT6P-Cy3-dT 4 -(SpC6) 4 -dT 25 -C3, dT6P

Assignees

• Fuller Carl W
• Kumar Shiv
• Ju Jingyue
• Davis Randall
• Chen Roger
• Univ Columbia
• Genia Tech Inc

Inventors

• Fuller Carl W
• Kumar Shiv
• Ju Jingyue
• Davis Randall
• Chen Roger

Classifications

• C07H19/10

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C07H19/20

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C07H17/02 Primary

  Heterocyclic radicals containing only nitrogen as ring hetero atoms · CPC title