Nucleotide analogues

• US 20050164182A1
• Filed: 11/28/2002
• Published: 07/28/2005
• Est. Priority Date: 11/29/2001
• Status: Abandoned Application

• Current Assignee: GE Healthcare UK Limited (Danaher Corporation)
• Original Assignee: Amersham Biosciences (General Electric Company)
• Inventors: Simmonds, Adrian, Odedra, Raj, Johnson, Karin S, Pickering, Lea
• Application Number:
  US10/496,761
• Publication Number:
  US 20050164182A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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: C09B 11/08 Phthaleins; Phenolphthaleins; Fluorescein
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/117 incorporating modified base

Abstract

The invention relates to nucleosides comprising a reporter moiety which also functions to limit polymerase activity, characterised in that the reporter moiety is attached to the nucleoside through a linkage group cleavable by a hydrolase enzyme wherein the hydrolase enzyme is selected from the group consisting of esterases, phosphatases, peptidases, penicillin amidases, glycosidases and phosphorylases.

Claims

1. A nucleoside comprising a base, a sugar and a reporter moiety which also functions to limit polymerase activity, wherein the reporter moiety is attached to the base of the nucleoside through a linkage group cleavable by a hydrolase enzyme, wherein said hydrolase enzyme is selected from the group consisting of esterases, phosphatases, peptidases, penicillin amidases, glycosidases and phosphorylases.

2. A compound of Formula I

R-L1-L2-L3-L4-L5-BASE-SUGAR 

 

(I)

wherein

R is a reporter moiety

L1 and L5 are optional linkage groups each containing one or more atoms comprising hydrocarbon chains which may also contain other atoms such as n, O and s;

L2 and L4 are optional linkage groups comprising 1 or more amino acid residues; and

L3 is a linkage group that is susceptible to enzymic hydrolysis by a hydrolase enzyme, wherein hydrolytic cleavage may be within the group or adjacent to the group and wherein said hydrolase enzyme is selected from the group consisting of esterases, phosphatases, peptidases, penicillin amidases, glycosidases and phosphorylases.

3. The compound of claim 2, wherein enzymic hydrolysis of the linkage group L3 produces an unstable moiety which undergoes chemical hydrolysis.

4. The compound of claim 2, wherein the base comprises purines or pyrimidines or analogues thereof.

5. The compound of claim 2, wherein the sugar comprises ribose or deoxy-ribose or analogues thereof.

6. The compound of claim 2, wherein a mono-, di- or triphosphate group is attached to the sugar.

7. The compound of claim 6, wherein a triphosphate group is attached to the sugar.

8. The compound of claim 2, wherein the hydrolase is a peptidase selected from the group consisting of subtilisin, proteinase K, elastase, neprilysin, thermolysin, papain, plasmin, trypsin, enterokinase and urokinase.

9. The compound of claim 2, wherein L3 is a peptide selected from the group consisting of alanine-alanine-alanine, alanine-alanine-leucine, glycine-leucine-serine, glycine-serine-alanine-alanine-leucine and glycine-alanine-glycine-leucine.

10. The compound of claim 2, wherein R is a fluorophore, selected from the group consisting of fluoresceins, rhodamines, coumarins, BODIPY®

dyes, phenoxazine dyes, cyanine dyes, acridone dyes and squarate dyes.

15. A set of nucleotides wherein the set contains at least one compound of claim 6.

16. The set of nucleotides of claim 15, which includes the bases A, G, C and T and analogues thereof.

19. A method for nucleic acid molecule sequencing comprising the steps of:

a) immobilising a complex of a primer and a template to a solid phase

b) incubating with a polymerase in the presence of the compound of claim 6.

24. The method of claim 19, wherein the incorporation of the compound is determined by the detection of a single reporter group attached to the compound.

11. A chemical intermediate of Formula II

R-L1-L2-L3-L4-L5 

 

(II)

wherein

R is a reporter moiety

L1 and L5 are optional linkage groups each containing one or more atoms comprising hydrocarbon chains which may also contain other atoms such as N, O and S;

L2 and L4 are optional linkage groups comprising 1 or more amino acid residues, and

L3 is a linkage group that is susceptible to enzymic hydrolysis by a peptidase enzyme.

12. The chemical intermediate of claim 11, wherein L3 is selected from the group consisting of alanine-alanine-alanine, alanine-alanine-leucine, glycine-leucine-serine, glycine-serine-alanine-alanine-leucine and glycine-alanine-glycine-leucine.

13. The chemical intermediate of claim 11, wherein R is selected from the group consisting of fluoresceins, rhodamines, coumarins, BODIPY®

dyes, phenoxazine dyes, cyanine dyes, acridone dyes and squarate dyes.

14. The compound 5-N-[N-(6-Fluorescein-5(and-6) carboxamidohexanoyl)-Gly-Gly-Leu-β

-alanyl]-propargylamino-2′

-deoxyuridine triphosphate.

17-18. -18. (canceled)

20. (canceled)

21. The method of claim 20, wherein the hydrolase enzyme is selected from the group consisting of esterases, phosphatases, peptidases, penicillin amidases, glycosidases and phosphorylases.

22. The method of claim 21, wherein the peptidase is selected from the group consisting of subtilisin, proteinase K, elastase, neprilysin, thermolysin, papain, plasmin, trypsin, enterokinase and urokinase.

23. (canceled)