Labeled nucleotide analogs

• US 9,464,107 B2
• Filed: 02/03/2014
• Issued: 10/11/2016
• Est. Priority Date: 03/13/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Wegener, Jeffrey, Korlach, Jonas
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/171,391
• Publication Number:
  US 20140186825A1
• Time in Patent Office: 981 Days
• Field of Search: 536/26.6, 435/6.1, 435/91.1
• US Class Current: 1/1
• 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: 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Labeled reactant compositions, and particularly labeled nucleic acid reaction compositions that include structural components including double-stranded nucleic acids. In some embodiments, the structural components maintain potentially damaging labeling components sufficiently distal from the reactant portion of the molecule such that damaging effects of the label group on other reaction components, such as enzymes, are reduced, minimized and/or eliminated.

Claims

1. A labeled nucleotide analog comprising:

a label component,

a linker group comprising a double-stranded nucleic acid, and

a nucleoside polyphosphate portion,

wherein the linker group comprising the double-stranded nucleic acid is coupled to the label component and is coupled to the nucleoside polyphosphate portion through a phosphate of the nucleoside polyphosphate portion.

2. The labeled nucleotide analog of claim 1 wherein the nucleoside polyphosphate portion has three to seven phosphates.

3. The labeled nucleotide analog of claim 1 wherein the label component comprises a fluorophore.

4. The labeled nucleotide analog of claim 1 wherein the label component is coupled to one strand of the double-stranded nucleic acid, and the nucleotide polyphosphate portion is coupled to the other strand of the double-stranded nucleic acid.

5. The labeled nucleotide analog of claim 1 wherein the double-stranded nucleic acid comprise a single molecule with complementary portions in which the molecule self-hybridizes to form a hairpin loop structure, and wherein the label component is attached to the hairpin loop structure.

6. The labeled nucleotide analog of claim 1 wherein the linker group comprises an aliphatic linker group.

7. The labeled nucleotide analog of claim 1 wherein the linker group comprises thioether linkages, peptide bonds, or maleimide-spacer-NHS linkers.

8. The labeled nucleotide analog of claim 1 wherein the functional distance between the nucleoside polyphosphate portion and the label component is at least 1 nm.

9. The labeled nucleotide analog of claim 1 wherein the functional distance between the nucleoside polyphosphate portion and the label component is at least 2 nm.

10. The labeled nucleotide analog of claim 1 wherein the double-stranded nucleic acid comprises DNA.

11. A labeled nucleotide analog comprising a structure of formula I, II or III,

12. The labeled nucleotide analog of claim 11 wherein the label component comprises a fluorophore.

13. The labeled nucleotide analog of claim 11 wherein the linker group comprises an aliphatic linker group.

14. The labeled nucleotide analog of claim 11 wherein the linker group comprises thioether linkages, peptide bonds, or maleimide-spacer-NHS linkers.

15. The labeled nucleotide analog of claim 11 wherein the functional distance between the nucleoside polyphosphate portion and the label component is at least 1 nm.

16. The labeled nucleotide analog of claim 11 wherein the functional distance between the nucleoside polyphosphate portion and the label component is at least 2 nm.

17. A composition for sequencing, comprising a polymerase enzyme and a nucleotide analog substrate for the polymerase enzyme, the nucleotide analog substrate comprising:

a label component,

a linker group comprising a double-stranded nucleic acid, and a

nucleoside polyphosphate portion,

wherein the linker group comprising the double-stranded nucleic acid is coupled to the label component and is coupled to the nucleoside polyphosphate portion through a phosphate of the nucleoside polyphosphate portion.

18. The composition of claim 17 wherein the nucleoside polyphosphate portion has three to seven phosphates.

19. The composition of claim 17 wherein the label component comprises a fluorophore.

20. The composition of claim 17 wherein the label component is coupled to one strand of the double-stranded nucleic acid, and the nucleotide polyphosphate portion is coupled to the other strand of the double-stranded nucleic acid.

21. The composition of claim 17 wherein the double-stranded nucleic acid comprise a single molecule with complementary portions in which the molecule self-hybridizes to form a hairpin loop structure, and wherein the label component is attached to the hairpin loop structure.