Labelled nucleotides
Nucleosides and nucleotides are disclosed that are linked to detectable labels via a cleavable linker group.
1. A nucleotide or nucleoside molecule having a ribose or deoxyribose sugar moiety and a base linked to a detectable label via a cleavable linker, wherein the sugar moiety comprises a protecting group attached via a 3′ oxygen atom, and wherein said protecting group comprises an azido group that can be modified or removed to expose a 3′ OH group.
2. The molecule of claim 1 , wherein the base is a purine, or a pyrimidine.
3. The molecule of claim 1 , wherein the base is a deazapurine.
4. The molecule of claim 1 , which is a deoxyribonucleotide triphosphate.
5. The molecule of claim 1 , wherein the detectable label is a fluorophore.
6. The molecule of claim 1 , wherein the cleavable linker is acid labile, photolabile or comprises a disulphide linkage.
7. The molecule of claim 1 , wherein the cleavable linker and the protecting group are cleavable under identical conditions.
8. The molecule of claim 1 , wherein the protecting group comprises azidomethyl (CH 2 N 3 ).
9. A kit comprising a plurality of nucleotides, each having a ribose or deoxyribose sugar moiety and a base linked to a detectable label via a cleavable linker, wherein the sugar moiety comprises a protecting group attached via the 3′ oxygen atom, and said protecting group comprises an azido group that can be modified or removed to expose a 3′ OH group, and wherein the detectable label linked to each nucleotide can be distinguished upon detection from the detectable labels used for structurally different nucleotides.
10. The kit of claim 9 , wherein the cleavable linker and the protecting group are cleavable under identical conditions.
11. The kit of claim 9 , wherein the protecting group comprises azidomethyl (CH 2 N 3 ).
12. The kit of claim 9 , further comprising an enzyme and buffers appropriate for the action of the enzyme.
13. The kit of claim 12 , wherein the enzyme is a terminal transferase, a polymerase or a reverse transcriptase.
14. The molecule of claim 4 , wherein the detectable label is a fluorophore.
15. The molecule of claim 14 , wherein the protecting group comprises azidomethyl (CH 2 N 3 ).
16. The molecule of claim 15 , wherein said protecting group consists of azidomethyl bound via the carbon atom of the azidomethyl group to the 3′ oxygen atom, and wherein the azidomethyl protecting group can be removed to expose a 3′ OH group.
17. The molecule of claim 16 , wherein the cleavable linker and the protecting group are cleavable under identical conditions.
18. The molecule of claim 17 , wherein the base is a deazapurine.
19. The molecule of claim 18 , wherein the deazapurine is a 7-deazaadenine, and wherein said cleavable linker is bound to the 7-deazaadenine at the 7-position.
20. The molecule of claim 18 , wherein the deazapurine is a 7-deazaguanine, and wherein said cleavable linker is bound to the 7-deazaguanine at the 7-position.
21. The molecule of claim 17 , wherein the base is a cytosine, and wherein said cleavable linker is bound to the cytosine at the 5-position.
22. The molecule of claim 17 , wherein the base is a uracil, and wherein said cleavable linker is bound to the uracil at the 5-position.
23. The molecule of claim 19 , wherein the cleavable linker comprises a disulfide moiety.
24. The molecule of claim 20 , wherein the cleavable linker comprises a disulfide moiety.
25. The molecule of claim 21 , wherein the cleavable linker comprises a disulfide moiety.
26. The molecule of claim 22 , wherein the cleavable linker comprises a disulfide moiety.
27. The molecule of claim 19 , wherein the cleavable linker comprises an azido moiety.
28. The molecule of claim 20 , wherein the cleavable linker comprises an azido moiety.
29. The molecule of claim 21 , wherein the cleavable linker comprises an azido moiety.
30. The molecule of claim 22 , wherein the cleavable linker comprises an azido moiety.
31. A nucleotide molecule, wherein:
the nucleotide molecule comprises an azidomethyl moiety attached to the 3′ oxygen atom such that the azidomethyl moiety prevents addition of a nucleotide to the 3′ oxygen atom,
the nucleotide molecule comprises a 2′ deoxyribose, wherein said 2′ deoxyribose comprises a triphosphate at the 5′ position,
the nucleotide molecule comprises a 7-deazaadenine base,
the 7-deazaadenine base of the nucleotide molecule is substituted with a linker at the 7-deaza position,
the linker is bound to a fluorophore,
the linker comprises a cleavable moiety located between the 7-deazaadenine base and the fluorophore,
the cleavable moiety comprises a disulfide moiety or an azido moiety,
the cleavable moiety and the azidomethyl moiety are cleavable chemically under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the nucleotide molecule and part of the linker remains attached to the 7-deazaadenine base, and
upon cleavage of the azidomethyl moiety, a 3-OH group is formed and the formation of the 3-OH group allows addition of a nucleotide to the 3′ oxygen atom.
32. The nucleotide molecule of claim 31 , wherein the cleavable moiety comprises a disulfide moiety.
33. The nucleotide molecule of claim 31 , wherein the cleavable moiety comprises an azido moiety.
34. A 7-substituted 7-deaza-2′-deoxy-adenosine triphosphate nucleotide molecule, wherein:
the 3′-O is substituted with an azidomethyl moiety that prevents addition of a nucleotide to the 3′-O,
the 7-deazaadenine base of the molecule is linked to a fluorophore via a cleavable linker at the 7-deaza position of the base,
the linker comprises a cleavable moiety located between the 7-deazaadenine base and the fluorophore,
the cleavable linker and the azidomethyl moiety are cleavable in a chemical reaction under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the molecule and part of the linker remains attached to the 7-deazaadenine base, and
upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′-O.
35. A nucleotide molecule having the structure:
wherein:
X is triphosphate,
R 1 is —OCH 2 N 3 ,
R 2 is hydrogen,
Linker is a cleavable linker comprising a cleavable moiety,
Label is a fluorophore, and
upon cleavage of the cleavable moiety, Label is removed and part of Linker remains attached to the nucleobase.
36. The nucleotide molecule of claim 35 , wherein the cleavable moiety and the —CH 2 N 3 of R 1 are cleavable in a chemical reaction under identical conditions.
37. The nucleotide molecule of claim 36 , wherein the cleavable moiety comprises a disulfide moiety.
38. The nucleotide molecule of claim 36 , wherein the cleavable moiety comprises an azido moiety.
39. A nucleotide molecule, wherein:
the nucleotide molecule comprises an azidomethyl moiety attached to the 3′ oxygen atom such that the azidomethyl moiety prevents addition of a nucleotide to the 3′ oxygen atom,
the nucleotide molecule comprises a 2′ deoxyribose, wherein said 2′ deoxyribose comprises a triphosphate at the 5′ position,
the nucleotide molecule comprises a 7-deazaguanine base,
the 7-deazaguanine base of the nucleotide molecule is substituted with a linker at the 7-deaza position,
the linker is bound to a fluorophore,
the linker comprises a cleavable moiety located between the 7-deazaguanine base and the fluorophore,
the cleavable moiety comprises a disulfide moiety or an azido moiety,
the cleavable moiety and the azidomethyl moiety are cleavable chemically under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the nucleotide molecule and part of the linker remains attached to the 7-deazaguanine base, and
upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′ oxygen atom.
40. The nucleotide molecule of claim 39 , wherein the cleavable moiety comprises a disulfide moiety.
41. The nucleotide molecule of claim 39 , wherein the cleavable moiety comprises an azido moiety.
42. A 7-substituted 7-deaza-2′-deoxy-guanosine triphosphate nucleotide molecule, wherein:
the 3′-O is substituted with an azidomethyl moiety that prevents addition of a nucleotide to the 3′-O,
the 7-deazaguanine base of the molecule is linked to a fluorophore via a cleavable linker at the 7-deaza position of the base,
the linker comprises a cleavable moiety located between the 7-deazaguanine base and the fluorophore,
the cleavable linker and the azidomethyl moiety are cleavable in a chemical reaction under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the molecule and part of the linker remains attached to the 7-deazaguanine base, and
upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′-O.
43. A nucleotide molecule having the structure:
wherein:
X is triphosphate,
R 1 is —OCH 2 N 3 ,
R 2 is hydrogen,
Linker is a cleavable linker comprising a cleavable moiety,
Label is a fluorophore, and
upon cleavage of the cleavable moiety, Label is removed and part of Linker remains attached to the nucleobase.
44. The nucleotide molecule of claim 43 , wherein the cleavable moiety and CH 2 N 3 of the —CH 2 N 3 of R 1 are cleavable in a chemical reaction under identical conditions.
45. The nucleotide molecule of claim 44 , wherein the cleavable moiety comprises a disulfide moiety.
46. The nucleotide molecule of claim 44 , wherein the cleavable moiety comprises an azido moiety.
47. A nucleotide molecule, wherein:
the nucleotide molecule comprises an azidomethyl moiety attached to the 3′ oxygen atom such that the azidomethyl moiety prevents addition of a nucleotide to the 3′ oxygen atom,
the nucleotide molecule comprises a 2′ deoxyribose, wherein said 2′ deoxyribose comprises a triphosphate at the 5′ position,
the nucleotide molecule comprises a cytosine base,
the cytosine base of the nucleotide molecule is substituted with a linker at the 5 position of the base,
the linker is bound to a fluorophore,
the linker comprises a cleavable moiety located between the cytosine base and the fluorophore,
the cleavable moiety comprises a disulfide moiety or an azido moiety,
the cleavable moiety and the azidomethyl moiety are cleavable chemically under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the nucleotide molecule and part of the linker remains attached to the cytosine base, and
upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′ oxygen atom.
48. The nucleotide molecule of claim 47 , wherein the cleavable moiety comprises a disulfide moiety.
49. The nucleotide molecule of claim 47 , wherein the cleavable moiety comprises an azido moiety.
50. A 5-substituted 2′-deoxy-cytidine triphosphate nucleotide molecule, wherein:
the 3′-O is substituted with an azidomethyl moiety that prevents addition of a nucleotide to the 3′-O,
the cytosine base of the molecule is linked to a fluorophore via a cleavable linker at the 5 position of the base,
the linker comprises a cleavable moiety located between the cytosine base and the fluorophore,
the cleavable linker and the azidomethyl moiety are cleavable in a chemical reaction under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the molecule and part of the linker remains attached to the cytosine base, and
upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′-O.
51. A nucleotide molecule having the structure:
wherein:
X is triphosphate,
R 1 is —OCH 2 N 3 ,
R 2 is hydrogen,
Linker is a cleavable linker comprising a cleavable moiety,
Label is a fluorophore, and
upon cleavage of the cleavable moiety, Label is removed and part of Linker remains attached to the nucleobase.
52. The nucleotide molecule of claim 51 , wherein the cleavable moiety and the —CH 2 N 3 of R 1 are cleavable in a chemical reaction under identical conditions.
53. The nucleotide molecule of claim 52 , wherein the cleavable moiety comprises a disulfide moiety.
54. The nucleotide molecule of claim 52 , wherein the cleavable moiety comprises an azido moiety.
55. A nucleotide molecule, wherein:
the nucleotide molecule comprises an azidomethyl moiety attached to the 3′ oxygen atom such that the azidomethyl moiety prevents addition of a nucleotide to the 3′ oxygen atom,
the nucleotide molecule comprises a 2′ deoxyribose, wherein said 2′ deoxyribose comprises a triphosphate at the 5′ position,
the nucleotide molecule comprises a uracil base,
the uracil base of the nucleotide molecule is substituted with a linker at the 5 position of the base,
the linker is bound to a fluorophore,
the linker comprises a cleavable moiety located between the uracil base and the fluorophore,
the cleavable moiety comprises a disulfide moiety or an azido moiety,
the cleavable moiety and the azidomethyl moiety are cleavable chemically under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the nucleotide molecule and part of the linker remains attached to the uracil base, and
upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′ oxygen atom.
56. The nucleotide molecule of claim 55 , wherein the cleavable moiety comprises a disulfide moiety.
57. The nucleotide molecule of claim 55 , wherein the cleavable moiety comprises an azido moiety.
58. A 5-substituted 2′-deoxy-uridine triphosphate nucleotide molecule, wherein:
the 3′-O is substituted with an azidomethyl moiety that prevents addition of a nucleotide to the 3′-O,
the uracil base of the molecule is linked to a fluorophore via a cleavable linker at the 5 position of the base,
the linker comprises a cleavable moiety located between the uracil base and the fluorophore,
the cleavable linker and the azidomethyl moiety are cleavable in a chemical reaction under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the molecule and part of the linker remains attached to the uracil base, and
upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′-O.
59. A nucleotide molecule having the structure:
wherein:
X is triphosphate,
R 1 is —OCH 2 N 3 ,
R 2 is hydrogen,
Linker is a cleavable linker comprising a cleavable moiety,
Label is a fluorophore, and
upon cleavage of the cleavable moiety, Label is removed and part of Linker remains attached to the nucleobase.
60. The nucleotide molecule of claim 59 , wherein the cleavable moiety and the —CH 2 N 3 of R 1 are cleavable in a chemical reaction under identical conditions.
61. The nucleotide molecule of claim 60 , wherein the cleavable moiety comprises a disulfide moiety.
62. The nucleotide molecule of claim 60 , wherein the cleavable moiety comprises an azido moiety.