Nucleotide analogues comprising a reporter moiety and a polymerase enzyme blocking moiety

• US 20030194722A1
• Filed: 03/24/2003
• Published: 10/16/2003
• Est. Priority Date: 06/01/2000
• Status: Active Grant

• Current Assignee: Amersham Biosciences UK Ltd (GE Healthcare Technologies, Inc.)
• Original Assignee: Amersham Biosciences UK Ltd (GE Healthcare Technologies, Inc.)
• Inventors: Simmonds, Adrian, Pickering, Lee, Odedra, Raj
• Granted Patent: US 7,078,499 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

Abstract

Nucleotides comprising a reporter moiety and a polymerase enzyme blocking moiety in which the reporter moiety does not also act as a polymerase enzyme blocking moiety are described. Also described are compounds of Formula (I): wherein W is a phosphate group, B is a base, Y is a linker comprising an enzyme-cleavable group, R² is a reporter moiety, R³ is selected from H or OH, Z and Z′ are selected from H, OH, or a group X—R¹, wherein X is a linker comprising an enzyme-cleavable group and R¹ is a polymerase enzyme blocking group, provided that at least one of Z and Z′ is X—R¹.

Claims

1. A nucleotide comprising a reporter moiety and a polymerase enzyme blocking moiety characterised in that the reporter moiety does not also act as a polymerase enzyme blocking moiety.

2. A compound of Formula I

3. A compound as claimed in claim 2 wherein W is a triphosphate.

4. A compound as claimed in any of claims 2 or 3 wherein B is selected from the bases A, C, G and T or their analogues.

5. A compound as claimed in any of claims 2 to 4 wherein enzyme-cleavable groups, X and Y, are the same.

6. A compound as claimed in any of claims 2 to 5 wherein X and Y are groups cleavable by enzymes selected from esterases, phosphatases, peptidases, amidases, glycosidases or phosphorylases.

7. A compound as claimed in any of claims 2 to 6 wherein X and/or Y is a group cleavable by an amidase.

8. A compound as claimed in any of claims 2 to 7 wherein R² is a fluorophore, preferably selected from fluoresceins, rhodamines, coumarins, BODIPY™

dyes, cyanine dyes and squarate dyes.

9. A compound as claimed in any of claims 2 to 8 wherein R¹ is selected from CH₃, glycyl or leucyl groups.

10. A compound as claimed in any of claims 2 to 9 wherein Z′

is X—

R¹ and, preferably, Z is OH.

11. A compound as claimed in any of claims 2 to 9 wherein R¹ is not a reporter moiety.

12. A chemical intermediate selected from the group consisting of:

4′

-C-(Glycylaminomethyl)thymidine triphosphate;

4′

-C-(N-trifluoroacetylaminomethyl)thymidine triphosphate;

4′

-C-(Aminomethyl)thymidine triphosphate;

5-(N-Trifluoroacetyl)propargylamino-4′

-C-(acetylaminomethyl)-2′

-deoxyuridine;

4′

-C-(N-Acetylglycylaminomethyl)thymidine;

4′

-C-(N-Leucylaminomethyl)thymidine;

4′

-C-(N-Glycylaminomethyl)thymidine (shown in FIG.

     8);

N-{α

-[4′

-methyloxythyimidyl]phenyl}phenylacetamide triphosphate;

N-[α

-(3′

-O-thymidyl)-phenyl)]-phenylacetamide triphosphate, N-{α

-[3′

-O-(5-N-(α

-methyloxy-N′

-trifluoroacetylaminopropyl benzamide) phenylacetamide-2′

-deoxyuridyl]-phenyl}-phenylacetamide and a compound of formula;

13. A set of nucleotides characterised in that the set contains at least one compound of Formula I.

14. A set of nucleotides as claimed in claim 13 comprising each of the four natural bases A, G, C and T (or their analogues).

15. A set of nucleotides as claimed in any of claims 13 or 14 further comprising at least two compounds of Formula I having different bases, B, and characterised in that each compound of Formula I has a different reporter moiety, R².

16. A set of nucleotides as claimed in any of claims 13 to 15 comprising four compounds of Formula I characterised in that each compound has a different base, B, such that each of the bases A, G, C and T, or analogues thereof, are present and each of the four compounds of Formula I has a reporter moiety which is distinguishable from all the reporter moiety of each of the compounds of Formula I having the other three bases.

17. 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 a compound of Formula I.

18. A method as claimed in claim 17 further comprising the steps of:

c) detecting the incorporation of a compound of Formula I

d) incubating in the presence of enzyme under suitable conditions for enzymatic cleavage of the enzyme-cleavable groups X and Y.

19. A method as claimed in claim 18 further comprising:

e) repeating steps a)-d).

20. A method as claimed in claim 18 or 19 wherein the enzyme in step d) is an amidase.