MODIFIED OLIGONUCLEOTIDES FOR MISMATCH DISCRIMINATION

• US 20060009628A1
• Filed: 02/28/2001
• Published: 01/12/2006
• Est. Priority Date: 04/03/1998
• Status: Active Grant

• Current Assignee: ELITechGroup Inc. (ELITech Group SAS)
• Original Assignee: Drug Royalty Trust
• Inventors: Vermeulen, Nicolaas M.J., Gall, Alexander A., Dempcy, Robert O., Lokhov, Sergey G., Kutyavin, Igor V., Singer, Michael J., Afonina, Irina A.
• Granted Patent: US 7,045,610 B2
• Time in Patent Office: Days
• US Class Current: 536/23.100
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• 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: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/23 Heterocyclic radicals containing two or more heterocyclic rings condensed among themselves or condensed with a common carbocyclic ring system, not provided for in groups C07H19/14 - C07H19/22
• 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 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

Modified oligonucleotides are provided containing bases selected from unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and optionally have attached minor groove binders and reporter groups.

Claims

1-5. -5. (canceled)

6. A modified oligonucleotide comprising at least two bases selected from the group consisting of unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidine bases, a covalently attached minor groove binder, at least one covalently attached reporter group, wherein said reporter group is a fluorophore, and an attached quencher.

10. A modified oligonucleotide of claim 6 comprising from 4 to 70 bases.

12. A modified oligonucleotide of claim 6, having the formula:

13. A modified oligonucleotide of claim 12, wherein at least one M is a non-natural oligomer-forming sugar.

7-8. -8. (canceled)

9. A modified oligonucleotide comprising at least two bases selected from the group consisting of unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidine bases, comprising from 4 to 70 bases and further comprising an attached fluorophore and a quencher.

11. A modified oligonucleotide comprising at least two bases selected from the group consisting of unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidine bases, wherein at least one base of said at least two bases is an unsubstituted pyrazolo[3,4-d]pyrimidine base having the formula:

14. A modified oligonucleotide comprising at least one 5-substituted pyrimidine base and at least one unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidine base, providing that the pyrimidine base is other than a thiopyrimidine.

15. A modified oligonucleotide of claim 14, further comprising a covalently attached minor groove binder.

16. A modified oligonucleotide of claim 14, further comprising at least one covalently attached reporter group.

17. A modified oligonucleotide of claim 14, further comprising at least one covalently attached quencher.

18. A modified oligonucleotide of claim 16, wherein said reporter group is a fluorophore.

19. A modified oligonucleotide of claim 16, wherein said reporter group is a fluorophore and said modified oligonucleotide further comprises an attached quencher.

20. A modified oligonucleotide of claim 14, comprising from 4 to 70 bases.

21. A modified oligonucleotide of claim 14, comprising from 4 to 70 bases and further comprising an attached minor groove binder.

22. A modified oligonucleotide of claim 14, comprising from 4 to 70 bases and further comprising an attached fluorophore and a quencher.

23. A modified oligonucleotide of claim 15, comprising from 4 to 70 bases and further comprising an attached fluorophore and a quencher.

24. A modified oligonucleotide of claim 14, wherein said at least one 5-substituted pyrimidine base is selected from the group consisting of:

25. A modified oligonucleotide of claim 14, having the formula:

26. A modified oligonucleotide of claim 25, wherein at least one M is a locked oligomer-forming sugar.

27. (canceled)

28. A modified oligonucleotide comprising from about 4 to about 70 bases and an attached minor groove binder, wherein at least one of said bases is replaced by a 5-substituted pyrimidine.

30. A modified oligonucleotide of claim 28, further comprising an attached reporter group.

31. A modified oligonucleotide of claim 30, wherein said reporter group is a fluorophore and said modified oligonucleotide further comprises an attached quencher.

32. A modified oligonucleotide of claim 31, comprising of from about 4 to about 20 bases.

33. A modified oligonucleotide of claim 32, wherein said fluorophore is attached at the 5′

-terminus and said quencher is attached at the 3′

-terminus.

34. A modified oligonucleotide of claim 32, wherein said fluorophore is attached at the 3′

-terminus and said quencher is attached at the 5′

-terminus.

35. A modified oligonucleotide of claim 28, wherein said minor groove binder has a formula selected from the group consisting of:

36. A modified oligonucleotide of claim 30, wherein said reporter group is selected from the group consisting of a resorufin dye, a coumarin dye, a rhodamine dye, a cyanine dye, a BODIPY dye, a fluorescein dye and a pyrene.

37. A modified oligonucleotide of claim 31, wherein said reporter group is selected from the group consisting of resorufin dye, a coumarin dye, a rhodamine dye, a cyanine dye, a BODIPY dye, a fluorescein dye and a pyrene, and said quencher is selected from the group consisting of non-fluorescent quenchers, 1-aza-1,2-diphenylethene derivatives and rhodamine derivatives.

38. A modified oligonucleotide of claim 28, wherein said modified base is selected from the group consisting of

39. A modified oligonucleotide of claim 38, wherein said heterocyclyl and aryl groups are selected from the group consisting of phenyl, tolyl, pyridyl, thiazolyl, imidazolyl, furanyl, oxazolyl, thienyl, pyrrolyl, benzimidazolyl, benzoxazolyl, benzthiazolyl, indolyl, triazinyl, pyrimidinyl and naphthyl.

42. A modified oligonucleotide of claim 39, wherein said minor groove binder has a formula selected from the group consisting of:

135. A modified oligonucleotide of claim 38 wherein X¹ and X² are each independently selected from the group consisting of H, NH₂, and OH.

29. (canceled)

40. A modified oligonucleotide comprising from about 4 to about 70 bases and an attached minor groove binder, wherein at least one of said bases is replaced by a modified base selected from the group consisting of

41. A modified oligonucleotide of claim 40, wherein said heterocyclyl and aryl groups are selected from the group consisting of phenyl, tolyl, pyridyl, thiazolyl, imidazolyl, furanyl, oxazolyl, thienyl, pyrrolyl, benzimidazolyl, benzoxazolyl, benzthiazolyl, indolyl, triazinyl, pyrimidinyl and naphthyl.

43. A modified oligonucleotide of claim 41, wherein said minor groove binder has a formula selected from the group consisting of:

44. A compound having the formula:

45. A compound of claim 44, wherein B is

46. A compound of claim 44, wherein B is

47. A compound of claim 44, wherein B is

48. A compound of claim 44, wherein B is

49. A compound of claim 45, wherein X¹¹ and X¹² are each NH₂.

50. A compound of claim 49, wherein Y¹ is O—

P¹, Y² is a protected hydroxy, Z¹ is H, R¹² is selected from the group consisting of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 4-hydroxy-1-butynyl and 3-(hydroxymethyl)-4-hydroxy-1-butynyl.

51. A compound of claim 50, wherein Y¹ is —

O—

[(2-cyanoethyl) N,N-diisopropylphosphoramidite] and Y² is —

O—

(4,4′

-dimethoxytrityl).

52. A compound of claim 45, wherein X¹¹ is NH₂ and X¹² is H.

53. A compound of claim 52, wherein Y¹ is O—

P¹, Y² is a protected hydroxy, Z¹ is H, R¹² is selected from the group consisting of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 4-hydroxy-1-butynyl and 3-(hydroxymethyl)-4-hydroxy-1-butynyl.

54. A compound of claim 53, wherein Y¹ is —

O-[(2-cyanoethyl) N,N-diisopropylphosphoramidite] and Y² is —

O-(4,4′

-dimethoxytrityl).

55. A compound of claim 46, wherein X¹² is H or NH₂.

56. A compound of claim 55, wherein Y¹ is O—

P¹, Y² is a protected hydroxy, Z¹ is H, R¹² is selected from the group consisting of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 4-hydroxy-1-butynyl and 3-(hydroxymethyl)-4-hydroxy-1-butynyl.

57. A compound of claim 56, wherein X¹ is —

O—

[(2-cyanoethyl) N,N-diisopropylphosphoramidite] and X² is —

O—

(4,4′

-dimethoxytrityl).

58. A compound of claim 47, wherein X¹¹ is NH₂.

59. A compound of claim 58, wherein Y¹ is O—

P¹, Y² is a protected hydroxy, Z¹ is H, R¹¹ is selected from the group consisting of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 4-hydroxy-1-butynyl and 3-(hydroxymethyl)-4-hydroxy-1-butynyl.

60. A compound of claim 59, wherein Y¹ is —

O—

[(2-cyanoethyl) N,N-diisopropylphosphoramidite] and Y² is —

O-(4,4′

-dimethoxytrityl).

61. A compound of claim 48, wherein Y¹ is O—

P¹, Y² is a protected hydroxy, Z¹ is H, and R¹¹ is selected from the group consisting of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 4-hydroxy-1-butynyl and 3-(hydroxymethyl)-4-hydroxy-1-butynyl.

62. A compound of claim 61, wherein Y¹ is —

O—

[(2-cyanoethyl) N,N-diisopropylphosphoramidite] and Y² is —

O—

(4,4′

-dimethoxytrityl).

110. A compound of claim 44 wherein one of Y¹ and Y² is a phosphoramidite group.

111. A compound of claim 110 wherein the other of Y¹ and Y² is a protected hydroxy group.

112. A compound of claim 111 wherein the protected hydroxy group is a 4,4′

-dimethoxytrityloxy group.

113. A compound of claim 110 wherein the phosphoramidite group is —

O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite.

114. A compound of claim 112 wherein the phosphoramidite group is —

O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite.

115. A compound of claim 110 wherein B is

116. A compound of claim 115 wherein X¹¹ and X¹² are protected amino groups.

117. A compound of claim 116 wherein one of Y¹ and Y² is —

O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite and the other of Y¹ and Y² is a 4,4′

-dimethoxytrityloxy group.

118. A compound of claim 116 wherein one or both of the protected amino groups comprises a benzoyl analogue protecting group.

119. A compound of claim 110 wherein B is

120. A compound of claim 119 wherein X¹² is a protected amino group.

121. A compound of claim 120 wherein X¹² is —

N═

CH—

N(CH₃)₂.

122. A compound of claim 121 wherein one of Y¹ and Y² is —

O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite and the other of Y¹ and Y² is a 4,4′

-dimethoxytrityloxy group.

123. A compound of claim 110 wherein B is

124. A compound of claim 123 wherein the phosphoramidite group is —

O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite.

125. A compound of claim 110 wherein B is

126. A compound of claim 125 wherein the phosphoramidite group is —

O—

(2-cyanoethyl)-N,N-diisopropylphosphoramidite.

127. A compound of claim 110 wherein B is

128. A compound of claim 127 wherein the other of Y¹ and Y² is a protected hydroxy group.

129. A compound of claim 128 wherein the phosphoramidite group is —

O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite.

130. A compound of claim 129 wherein the other of Y¹ and Y² is a 4,4′

-dimethoxytrityloxy group.

131. A compound of claim 128 wherein one or both of the protected amino groups comprises a benzoyl analogue protecting group.

132. A compound of claim 110 wherein Z¹ is hydrogen, Y¹ is OH and Y² is OH.

133. A compound of claim 110 wherein Z¹ and Z² are combined to form a five-to-seven-membered ring.

63. An oligonucleotide having the formula:

64. An oligonucleotide of claim 63, wherein n is an integer of from 4 to 30.

65. An oligonucleotide of claim 63, wherein at least one B is

66. An oligonucleotide of claim 63, wherein at least one B is

67. An oligonucleotide of claim 63, wherein at least one B is

68. An oligonucleotide of claim 63, wherein at least one B is

69. An oligonucleotide of claim 63, wherein W¹ is a minor groove binder-linking group moiety.

70. An oligonucleotide of claim 63, wherein W¹ is a minor groove binder-linking group moiety having an attached reporter group.

71. An oligonucleotide of claim 63, wherein W² is a minor groove binder-linking group moiety.

72. An oligonucleotide of claim 63, wherein W² is a minor groove binder-linking group moiety having an attached reporter group.

73. An oligonucleotide of claim 63, wherein at least one Z³ is NH.

134. An oligonucleotide of claim 63 wherein Z¹ and Z² on one or more of the same furanose rings are combined to form a five- to seven-membered ring.

74. A modified oligonucleotide array, said array comprising a solid support and a plurality of attached oligonucleotides, wherein at least 50% of the oligonucleotides in said array contain a modified base selected from the group consisting of unsubstituted pyrazolo[3,4-d]pyrimidines, 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines.

75. A modified oligonucleotide array of claim 74, wherein said attached oligonucleotides have T_ms within about 2°

C. of each other and basepair lengths within about 2 bases of each other.

76. A modified oligonucleotide array of claim 74, wherein said attached oligonucleotides have T_ms within about 1°

C. of each other and basepair lengths within about 2 bases of each other.

77. A modified oligonucleotide array of claim 74, wherein said array comprises from about 10 to about 10,000 attached oligonucleotides, each having T_ms within about 2°

C. of each other and basepair lengths within about 2 bases of each other.

78. A modified oligonucleotide array of claim 74, wherein said array comprises from about 10 to about 10,000 attached oligonucleotides, a portion of said attached oligonucleotides having a covalently attached minor groove binder.

79. (canceled)

80. A composition comprising a plurality of modified oligonucleotides having at least one base selected from the group consisting of unsubstituted pyrazolo[3,4-d]pyrimidines, 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and further comprising a minor groove binder and an attached fluorophore wherein each of said plurality of oligonucleotides comprises an attached fluorophore and an attached quencher.

81. A composition of claim 80, wherein each of said modified oligonucleotides are from 4 to 30 bases in length and have T_ms that are within about 2°

C. of each other.

82. A composition of claim 81, wherein said plurality is from about 6 to about 100.

83-109. -109. (canceled)