Modified nucleotides

• US 9,388,464 B2
• Filed: 08/07/2015
• Issued: 07/12/2016
• Est. Priority Date: 08/23/2002
• Status: Expired due to Term

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Milton, John, Wu, Xiaolin, Smith, Mark, Brennan, Joseph, Barnes, Colin, Liu, Xiaohai, Ruediger, Silke
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/821,548
• Publication Number:
  US 20160032378A1
• Time in Patent Office: 340 Days
• Field of Search: 435/6.1, 435/91.1
• US Class Current: 1/1
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention provides modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3′-OH blocking group covalently attached thereto, such that the 3′ carbon atom has attached a group of the structure —O—Z wherein Z is any of —C(R′)2-O—R″, —C(R′)2-N(R″)2, —C(R′)2-N(H)R″, —C(R′)2-S—R″ and —C(R′)2-F, wherein each R″ is or is part of a removable protecting group; each R′ is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group; or (R′)2 represents an alkylidene group of formula ═C(R′″)2 wherein each R′″ may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and wherein said molecule may be reacted to yield an intermediate in which each R″ is exchanged for H or, where Z is —C(R′)2-F, the F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3′OH; with the proviso that where Z is —C(R′)2-S—R″, both R′ groups are not H.

Claims

1. A method of controlling the incorporation of a nucleotide having a ribose or deoxyribose sugar moiety, and said nucleotide is complementary to a second nucleotide in a target single-stranded polynucleotide in a synthesis or sequencing reaction, comprising

incorporating into the growing complementary polynucleotide said nucleotide, the incorporation of said nucleotide preventing or blocking introduction of subsequent nucleoside or nucleotide molecules into said growing complementary polynucleotide;

wherein a protecting group is attached to the 3′

or the 2′

oxygen atom of the ribose or deoxyribose sugar moiety of said nucleotide; and

said nucleotide comprises a detectable label linked by a linker; and

reacting the nucleotide with a water-soluble phosphine to cleave the linker;

wherein said detectable label is a fluorophore; and

wherein said water-soluble phosphine also cleaves said protecting group.

2. The method of claim 1, wherein said linker contains a disulfide linkage.

3. The method of claim 1, wherein said linker is a phosphine-cleavable azide-containing linker.

4. The method of claim 1, further comprising repeating said incorporating and reacting steps one or more times, thereby incorporating multiple nucleotides into the growing complementary polynucleotide.

5. The method of claim 1, wherein said incorporating comprises contacting the growing complementary polynucleotide with a polymerase.

6. The method of claim 1, wherein said protecting group is attached to the 3′

oxygen atom of the ribose or deoxyribose sugar moiety of said nucleotide.

7. The method of claim 1, wherein said protecting group is attached to the 2′

oxygen atom of the ribose or deoxyribose sugar moiety of said nucleotide.

8. A method of controlling the incorporation of a nucleotide having a ribose or deoxyribose sugar moiety, and said nucleotide is complementary to a second nucleotide in a target single-stranded polynucleotide in a synthesis or sequencing reaction, comprising

incorporating said nucleotide into the growing complementary polynucleotide with a polymerase, the incorporation of said nucleotide preventing or blocking introduction of subsequent nucleoside or nucleotide molecules into said growing complementary polynucleotide;

wherein a protecting group is attached to the 3′

oxygen atom of the ribose or deoxyribose sugar moiety of said nucleotide; and

said nucleotide comprises a detectable label linked by a linker; and

reacting the nucleotide with a water-soluble phosphine to cleave the linker;

wherein said detectable label is a fluorophore; and

wherein said water-soluble phosphine also cleaves said protecting group.

9. The method of claim 8, wherein said linker contains a disulfide linkage.

10. The method of claim 8, wherein said linker is a phosphine-cleavable azide-containing linker.

11. The method of claim 8, further comprising repeating said incorporating and reacting steps one or more times, thereby incorporating multiple nucleotides into the growing complementary polynucleotide.