Synthesis and compositions of 2'-terminator nucleotides

• US 20050037991A1
• Filed: 06/28/2004
• Published: 02/17/2005
• Est. Priority Date: 06/30/2003
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Bodepudi, Veeraiah, Gelfand, David Harrow, Will, Stephen Gordon
• Granted Patent: US 7,947,817 B2
• Time in Patent Office: Days
• US Class Current: 514/47
• 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 19/16 Purine radicals
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

The invention provides compositions the comprise nucleotides and/or nucleosides having blocking groups at 2′-positions of sugar moieties. Methods of synthesizing these nucleic acids are also provided.

Claims

1. A composition comprising at least one nucleoside and/or at least one nucleotide comprising the formula:

2. The composition of claim 1, wherein B comprises the formula:

3. The composition of claim 1, wherein B comprises the formula:

4. The composition of claim 1, wherein B comprises the formula:

5. The composition of claim 1, wherein B comprises the formula:

6. The composition of claim 1, wherein B comprises the formula:

7. The composition of claim 1, wherein B comprises the formula:

8. The composition of claim 1, wherein B comprises the formula:

9. The composition of claim 1, wherein B comprises the formula:

10. The composition of claim 1, wherein B comprises the formula:

11. The composition of claim 1, wherein B comprises the formula:

12. The composition of claim 1, wherein B comprises the formula:

13. The composition of claim 1, wherein BG is a negatively charged group.

14. The composition of claim 1, wherein BG is a bulky group.

15. The composition of claim 1, wherein BG is selected from the group consisting of:

CN, NO₂, N₃, a silyl group, a halo group, an alcohol group, an ether group, an aldehyde group, an acidic group, an ester group, an amino group, and combinations thereof.

16. The composition of claim 1, wherein BG comprises the formula:

17. The composition of claim 1, wherein BG comprises the formula:

18. The composition of claim 1, wherein B comprises or is attached to the label.

19. The composition of claim 1, wherein a sugar moiety of the nucleoside and/or nucleotide comprises or is attached to the label.

20. The composition of claim 1, wherein BG comprises or is attached to the label.

21. The composition of claim 1, wherein a linker comprises or attaches the label to the nucleoside and/or nucleotide.

22. The composition of claim 1, wherein the nucleotide comprises a 2′

-monophosphate-3′

-hydroxyl-5′

-triphosphate nucleoside.

23. The composition of claim 1, wherein at least one of the nucleotide incorporating biocatalysts is capable of extending a primer nucleic acid to produce an extended primer nucleic acid, incorporating the nucleotide at a terminal end of the extended primer nucleic acid.

24. The composition of claim 1, wherein the nucleotide comprises at least 1 phosphate group attached at the 5′

position.

25. The composition of claim 23, wherein the nucleotide comprises two phosphate groups attached at the 5′

position.

26. The composition of claim 23, wherein the nucleotide comprises three phosphate groups attached at the 5′

position.

27. The composition of claim 1, wherein the label comprises a fluorescent dye, a weakly fluorescent label, a non-fluorescent label, a calorimetric label, a chemiluminescent label, a bioluminescent label, a radioisotope, an antibody, an antigen, biotin, a hapten, or an enzyme.

28. The composition of claim 27, wherein the label is a fluorescent dye selected from the group consisting of:

a rhodamine dye, a fluorescein dye, a halofluorescein dye, a dichlororhodamine dye, an energy transfer dye, a Lucifer dye, Oregon Green, and a cyanine dye.

29. The composition of claim 28, wherein the fluorescent dye is selected from the group consisting of:

JOE, VIC, TET, HEX, FAM, R6G, R110, TAMRA, and ROX.

30. The composition of claim 27, wherein the radioisotope is selected from the group consisting of:

³H, ¹⁴C, ²²Na, ³²P, ³³P ³⁵S, ⁴²K, ⁴⁵Ca, ⁵⁹Fe, ¹²⁵I, and ²⁰³Hg.

31. The composition of claim 1, wherein the label comprises at least one mass-modifying group.

32. The composition of claim 31, wherein the mass-modifying group is selected from the group consisting of:

deuterium, F, Cl, Br, I, N₃, XY, CH₃, SPO₄, BH₃, SiY₃, Si(CH₃)₃, Si(CH₃)₂(C₂H₅), Si(CH₃)(C₂H₅)₂, Si(C₂H₅)₃, (CH₂)_nCH₃, (CH₂)_nNY₂, CH₂CONY₂, (CH₂)_nOH, CH₂F, CHF₂, CF₃, and a phosphorothioate group, wherein;

X is O, NH, NY, S, NHC(S), OCO(CH)_nCOO, NHCO(CH₂)_nCOO, OSO₂O, OCO(CH₂)_n, NHC(S)NH, OCO(CH₂)_nS, OCO(CH₂)S, NC₄O₂H₂S, OPO(O-alkyl), or OP(O-alkyl);

n is an integer from 1 to 20 inclusive; and

,

Y is H, deuterium, an alkyl group, an alkoxy group, an aryl group, a polyoxymethylene group, a monoalkylated polyoxymethylene group, a polyethylene imine group, a polyamide group, a polyester group, an alkylated silyl group, a heterooligo, a polyaminoacid, a heterooligo/polyaminoacid group, or a polyethylene glycol group.

33. The composition of claim 1, wherein a polymer comprises the nucleoside and/or nucleotide.

34. The composition of claim 33, wherein the polymer comprises an oligodeoxynucleotide, an oligoribonucleotide, a polydeoxynucleotide, a polyribonucleotide, an aptamer, a DNA-RNA hybrid, an antisense nucleic acid, an RNAi, a molecular beacon, a nucleic acid probe, a nucleic acid primer, a PNA, a PNA-DNA conjugate, or a PNA-RNA conjugate.

35. The composition of claim 1, further comprising one or more extendible nucleotides.

36. The composition of claim 35, wherein the extendible nucleotides comprise ribonucleotides and/or deoxyribonucleotides.

37. The composition of claim 35, wherein the non-extendible nucleotide and the extendible nucleotides are present in a molar ratio of 1:

1 or less.

38. A method of producing a labeled, non-extendible nucleotide, the method comprising:

attaching at least one phosphate group to a 5′

-position of a sugar moiety of a nucleoside;

attaching at least one blocking group to a 2′

-position of the sugar moiety of the nucleoside; and

,

attaching at least one label to the sugar moiety, the blocking group, and/or a base of the nucleoside, thereby producing the labeled, non-extendible nucleotide, which nucleotide is non-extendible by one or more nucleotide incorporating biocatalysts selected from the group consisting of;

G46E E678G CS5 DNA polymerase, a G46E L329A E678G CS5 DNA polymerase, G46E E678G CS6 DNA polymerase, Δ

ZO5R polymerase, E615G Taq DNA polymerase, Thermus flavus polymerase, Tma-25 polymerase, Tma-30 polymerase, Tth DNA polymerase, Thermus specie SPS-17 polymerase, E615G Taq polymerase, Thermus ZO5R polymerase, T7 DNA polymerase, Kornberg DNA polymerase I, Klenow DNA polymerase, Taq DNA polymerase, Micrococcal DNA polymerase, alpha DNA polymerase, reverse transcriptase, AMV reverse transcriptase, M-MuLV reverse transcriptase, DNA polymerase, RNA polymerase, E. coli RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, T4 DNA polymerase, T7 RNA polymerase, RNA polymerase II, terminal transferase, polynucleotide phosphorylase, and ribonucleotide incorporating DNA polymerase.

39. The method of claim 38, wherein the labeled, non-extendible nucleotide is produced specifically or selectively.

40. The method of claim 38, wherein the blocking group is a negatively charged group.

41. The method of claim 38, wherein the blocking group is a bulky group.

42. The method of claim 38, wherein the blocking group is selected from the group consisting of:

CN, NO₂, N₃, a silyl group, a halo group, an alcohol group, an ether group, an aldehyde group, an acidic group, an ester group, an amino group, and combinations thereof.

43. The method of claim 38, wherein the blocking group comprises the formula:

44. The method of claim 38, wherein the blocking group comprises the formula:

45. The method of claim 38, wherein a linker attaches the label to at least one of:

the sugar moiety, the blocking group, or the base of the nucleoside.

46. The method of claim 38, wherein at least one of the nucleotide incorporating biocatalysts is capable of extending a primer nucleic acid to produce an extended primer nucleic acid, incorporating the nucleotide at a terminal end of the extended primer nucleic acid.

47. The labeled, non-extendible nucleotide produced by the method of claim 38.

48. The method of claim 38, wherein the label comprises a fluorescent dye, a weakly fluorescent label, a non-fluorescent label, a colorimetric label, a chemiluminescent label, a bioluminescent label, a radioisotope, an antibody, an antigen, biotin, a hapten, or an enzyme.

49. The method of claim 48, wherein the label is a fluorescent dye selected from the group consisting of:

a rhodamine dye, a fluorescein dye, a halofluorescein dye, a dichlororhodamine dye, an energy transfer dye, a Lucifer dye, Oregon Green, and a cyanine dye.

50. The method of claim 49, wherein the fluorescent dye is selected from the group consisting of:

JOE, VIC, TET, HEX, FAM, R6G, R110, TAMRA, and ROX.

51. The method of claim 48, wherein the radioisotope is selected from the group consisting of:

³H, ¹⁴C, ²²Na, ³²P, ³³P, ³⁵S, ⁴²K, ⁴⁵ Ca, ⁵⁹Fe, ¹²⁵I, and ²⁰³Hg.

52. The method of claim 38, wherein the label comprises at least one mass-modifying group.

53. The method of claim 52, wherein the mass-modifying group is selected from the group consisting of:

deuterium, F, Cl, Br, I, S, N₃, XY, CH₃, SPO₄, BH₃, SiY₃, Si(CH₃)₃, Si(CH₃)₂(C₂H₅), Si(CH₃)(C₂H₅)₂, Si(C₂H₅)₃, (CH₂)_nCH₃, (CH₂)_nNY₂, CH₂CONY₂, (CH₂)_nOH, CH₂F, CHF₂, CF₃, and a phosphorothioate group, wherein;

X is O, NH, NY, S, NHC(S), OCO(CH)_nCOO, NHCO(CH₂)_nCOO, OSO₂₀, OCO(CH₂)_n, NHC(S)NH, OCO(CH₂)_nS, OCO(CH₂)S, NC₄O₂H₂S, OPO(O-alkyl), or OP(O-alkyl);

n is an integer from 1 to 20 inclusive; and

,

Y is H, deuterium, an alkyl group, an alkoxy group, an aryl group, a polyoxymethylene group, a monoalkylated polyoxymethylene group, a polyethylene imine group, a polyamide group, a polyester group, an alkylated silyl group, a heterooligo, a polyaminoacid, a heterooligo/polyaminoacid group, or a polyethylene glycol group.

54. A method of producing a 2′

-monophosphate nucleoside, the method comprising reacting a nucleotide comprising the formula;

55. The method of claim 54, wherein the nucleotide comprises two phosphate groups.

56. The method of claim 54, wherein the nucleotide comprises three phosphate groups.

57. The method of claim 54, wherein B comprises the formula:

58. The nucleoside of claim 54, wherein B comprises the formula:

59. The method of claim 54, wherein B comprises the formula:

60. The method of claim 54, wherein B comprises the formula:

61. The method of claim 54, wherein B comprises the formula:

62. The method of claim 54, wherein B comprises the formula:

63. The method of claim 54, wherein B comprises the formula:

64. The method of claim 54, wherein B comprises the formula:

65. The method of claim 54, wherein B comprises the formula:

66. The method of claim 54, wherein B comprises the formula:

67. The method of claim 54, wherein B comprises the formula:

68. The method of claim 54, wherein the nucleotide and the trisodium trimetaphosphate are reacted in an alkaline solution at a pH of about 14.

69. The method of claim 54, wherein the nucleotide and the trisodium trimetaphosphate are reacted at room temperature.

70. The 2′

-monophosphate nucleoside produced by the method of claim 54.

71. The method of claim 54, wherein the 2′

-monophosphate nucleoside comprises a 2′

-monophosphate purine nucleoside that is produced in a mixture comprising a 3′

-monophosphate purine nucleoside and the method further comprises separating the 2′

-monophosphate purine nucleoside from the 3′

-monophosphate purine nucleoside.

72. The method of claim 71, wherein the 2′

-monophosphate purine nucleoside and the 3′

-monophosphate purine nucleoside are separated from each other using liquid chromatography.

73. The method of claim 54, further comprising labeling the 2′

-monophosphate nucleoside with at least one label.

74. The method of claim 73, wherein the label is attached to B of the 2′

-monophosphate nucleoside.

75. The method of claim 73, wherein the label is attached to the sugar moiety of the 2′

-monophosphate nucleoside.

76. The method of claim 73, wherein the label is attached to the 2′

-monophosphate of the 2′

-monophosphate nucleoside.

77. The method of claim 73, wherein a linker attaches the label to the 2′

-monophosphate nucleoside.

78. The method of claim 73, wherein the label comprises a fluorescent dye, a weakly fluorescent label, a non-fluorescent label, a colorimetric label, a chemiluminescent label, a bioluminescent label, an antibody, an antigen, biotin, a hapten, or an enzyme.

79. The method of claim 78, wherein the label is a fluorescent dye selected from the group consisting of:

a rhodamine dye, a fluorescein dye, a halofluorescein dye, a dichlororhodamine dye, an energy transfer dye, a Lucifer dye, Oregon Green, and a cyanine dye.

80. The method of claim 79, wherein the fluorescent dye is selected from the group consisting of:

JOE, VIC, TET, HEX, FAM, R6G, R110, TAMRA, and ROX.

81. The method of claim 73, wherein the label comprises a radioisotope.

82. The method of claim 81, wherein the radioisotope is selected from the group consisting of:

³H, ¹⁴C, ²²Na, ³²P, ³³P, ³⁵S, ⁴²K, ⁴⁵Ca, ⁵⁹Fe, ¹²⁵I, and ²⁰³Hg.

83. The method of claim 73, wherein the label comprises at least one mass-modifying group.

84. The method of claim 83, wherein the mass-modifying group is selected from the group consisting of:

deuterium, F, Cl, Br, I, N₃, XY, CH₃, SPO₄, BH₃, SiY₃, Si(CH₃)₃, Si(CH₃)₂(C₂H₅), Si(CH₃)(C₂H₅)₂, Si(C₂H₅)₃, (CH₂)_nCH₃, (CH₂)_nNY₂, CH₂CONY₂, (CH₂)_nOH, CH₂F, CHF₂, CF₃, and a phosphorothioate group, wherein;

X is O, NH, NY, S, NHC(S), OCO(CH)_nCOO, NHCO(CH₂)_nCOO, OSO₂O, OCO(CH₂)_n, NHC(S)NH, OCO(CH₂)_nS, OCO(CH₂)S, NC₄O₂H₂S, OPO(O-alkyl), or OP(O-alkyl);

n is an integer from 1 to 20 inclusive; and

,

Y is H, deuterium, an alkyl group, an alkoxy group, an aryl group, a polyoxymethylene group, a monoalkylated polyoxymethylene group, a polyethylene imine group, a polyamide group, a polyester group, an alkylated silyl group, a heterooligo, a polyaminoacid, a heterooligo/polyaminoacid group, or a polyethylene glycol group.