Fluorescent nucleobase conjugates having anionic linkers

• US 6,811,979 B2
• Filed: 10/11/2001
• Issued: 11/02/2004
• Est. Priority Date: 10/11/2000
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Taing, Meng, Menchen, Steven M., Khan, Shaheer H., Rosenblum, Barnett B.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/976,168
• Publication Number:
  US 20020102590A1
• Time in Patent Office: 1,118 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/810, 536/23.1, 536/24.3, 536/26.7, 536/26.8
• US Class Current: 435/6.12
• 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 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: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

Provided are nucleotide-dye conjugates and related compounds in which a dye is linked to a nucleobase directly or indirectly by an anionic linker. The anionic character of the linker is provided by one or more anionic moieties which are present in the linker, such as phosphate, phosphonate, sulfonate, and carboxylate groups. When the dye is a provided as a donor/acceptor dye pair, the anionic linker can be located between the donor and the acceptor, or between the nucleobase and either the donor or acceptor, or both. In one embodiment, conjugates of the invention provide enhanced electrophoretic mobility characteristics to sequencing fragments, e.g., for dideoxy sequencing using labeled terminators.

Claims

1. A conjugate comprising a dye labeled nucleobase of the form:

(1) B-L-D, wherein B is a nucleobase, L is an anionic linker, and D comprises at least one fluorescent dye that comprises a xanthene a rhodamine or a fluorescein, or

(2) B-L1-D1-L2-D2, wherein B is a nucleobase, L1 and L2 axe linkers such that at least one of L1 and L2 is an anionic linker, and D1 and D2 are members of a fluorescent donor/acceptor pair, such that one of D1 and D2 is a donor dye capable of absorbing light at a first wavelength and emitting energy m response thereto, and the other of D1 and D2 is an acceptor dye capable of absorbing energy emitted by the donor dye and fluorescing at a second wavelength in response thereto, and at least one of D1 and D2 comprises a xanthene, a rhodamine or a fluorescein,

wherein L or at least one of L1 and L2 comprises at least one anionic phosphate or anionic phosphonate.

2. The conjugate of claim 1 wherein the dye-labeled nucleobase is of the form B-L-D.

3. The conjugate of claim 2 wherein L comprises a sulfonic acid moiety.

4. The conjugate of claim 2 wherein L comprises a sulfonated benzene moiety.

5. The conjugate of claim 2 wherein L comprises an anionic phosphate moiety.

6. The conjugate of claim 5 wherein the anionic phosphate moiety is a phosphate diester moiety, and the phosphorus atom of the phosphate diester moiety is located in L within a chain of linker atoms that connect B to D.

7. The conjugate of claim 2 wherein L comprises an anionic phosphonate moiety.

8. The conjugate of claim 7 wherein the anionic phosphonate moiety is a phosphonate monoester moiety, and the phosphorus atom of the phosphonate monoester moiety is located in L within a chain of linker atoms that connect B to D.

9. The conjugate of claim 2 wherein L comprises a carboxylic acid moiety.

10. The conjugate of claim 9 wherein the carboxylic acid moiety is a carboxyl benzene moiety.

11. The conjugate of claim 2 wherein L comprises 4 to 20 chain atoms.

12. The conjugate of claim 2 wherein D comprises at least one fluorescein or rhodamine.

13. The conjugate of claim 1 wherein B comprises adenine, 7-deazaadenine, 7-deaza-8-azaadenine, cytosine, guanine, 7-deazaguanine, 7-deaza-8-azaguanine, thymine, uracil, or inosine.

14. The conjugate of claim 1 wherein the labeled nucleobase is of the form B-L1-D1-L2-D2.

15. The conjugate of claim 14 wherein L1 comprises a sulfonic acid moiety.

16. The conjugate of claim 14 wherein L1 comprises a sulfonated benzene moiety.

17. The conjugate of claim 14 wherein L1 comprises an anionic phosphate moiety.

18. The conjugate of claim 17 wherein the anionic phosphate moiety is a phosphate diester moiety, and the phosphorus atom of the phosphate diester moiety is located in L1 within a chain of linker atoms that connect B to D1.

19. The conjugate of claim 14 wherein L1 comprises an anionic phosphonate moiety.

20. The conjugate of claim 19 wherein the anionic phosphonate moiety is a phosphonate monoester moiety, and the phosphorus atom of the phosphonate monoester moiety is located in L1 within a chain of linker atoms that connect B to D1.

21. The conjugate of claim 14 wherein L1 comprises a carboxylic acid moiety.

22. The conjugate of claim 21 wherein the carboxylic acid moiety is a carboxy benzene moiety.

23. The conjugate of claim 14 wherein L1 comprises z4 to 20 linker chain atoms.

24. The conjugate of claim 14 wherein L2 is not an anionic linker.

25. The conjugate of claim 24 wherein D1-L2-D2 comprises structure (a), (b) or (c) below:

(a) -D1-R₂₁Z₁C(O)R₂₂R₂₈-D2—

(b) -D1-R₂₈R₂₂C(O)Z₁R₂₁-D2—

(c) -D1-R₂₈R₂₂R₂₈-D2—

wherein;

R₂₁ is C₁-C₅ alkyldiyl,

Z₁ is NH, S, or O,

R₂₂ is ethenediyl, ethynediyl, 1,3-dienediyl, diynediyl, 1,3-diynediyl, a 5- or 6-membered ring having at least one unsaturated bond or a fused ring structure, and

R₂₈ is a bond or spacer group.

26. The conjugate of claim 25, wherein D1 is a donor dye and D2 is an acceptor dye.

27. The conjugate of claim 25 wherein R₂₂ is a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.

28. The conjugate of claim 25 wherein R₂₈ is of the form R₂₉Z₂C(O), wherein R₂₉ is C₁-C₅ alkyldiyl, and Z₂ is NH, S, or O.

29. The conjugate of claim 25 wherein R₂₁ is CH₂, Z₁ is NH, R₂₂ is phena-1,4-diyl, and R₂₉ is CH₂, and Z₂ is NH.

30. The conjugate of claim 25 wherein L2 comprises up to 20 linker chain atoms.

31. The conjugate of claim 24 wherein D1 is a donor dye and D2 is an acceptor dye.

32. The conjugate of claim 14 wherein L2 comprises a sulfonic acid moiety.

33. The conjugate of claim 14 wherein L2 comprises a sulfonated benzene moiety.

34. The conjugate of claim 14 wherein L2 comprises an anionic phosphate moiety.

35. The conjugate of claim 34 wherein the anionic phosphate moiety is a phosphate diester moiety, and the phosphorus atom of the phosphate diester moiety is located in L2 within a chain of linker atoms that connect D1 to D2.

36. The conjugate of claim 14 wherein L2 comprises an anionic phosphonate moiety.

37. The conjugate of claim 36 wherein the anionic phosphonate moiety is a phosphonate monoester moiety, and the phosphorus atom of the phosphonate monoester moiety is located in L2 within a chain of linker atoms that connect D1 to D2.

38. The conjugate of claim 14 wherein L2 comprises a carboxylic acid moiety.

39. The conjugate of claim 38 wherein the carboxylic acid moiety is a carboxy benzene moiety.

40. The conjugate of claim 14 wherein L2 comprises up to 20 linker chain atoms.

41. The conjugate of claim 14 wherein D1 is a donor dye and D2 is an acceptor dye.

42. The conjugate of claim 14 wherein L2 is an anionic linker and L1 is not an anionic linker.

43. The conjugate of claim 42 wherein L2 comprises a sulfonic acid moiety.

44. The conjugate of claim 42 wherein L2 comprises a sulfonated benzene moiety.

45. The conjugate of claim 42 wherein L2 comprises an anionic phosphate moiety.

46. The conjugate of claim 45 wherein the anionic phosphate moiety is a phosphate diester moiety, and the phosphorus atom of the phosphate diester moiety is located in L2 within a chain of linker atoms that connect D1 to D2.

47. The conjugate of claim 42 wherein L2 comprises an anionic phosphonate moiety.

48. The conjugate of claim 47 wherein the anionic phosphonate moiety is a phosphonate monoester moiety, and the phosphorus atom of the phosphonate monoester moiety is located in L2 within a chain of linker atoms that connect D1 to D2.

49. The conjugate of claim 42 wherein L2 comprises a carboxylic acid moiety.

50. The conjugate of claim 49 wherein the carboxylic acid moiety is a carboxy benzene moiety.

51. The conjugate of claim 42 wherein L2 comprises up to 20 linker chain atoms.

52. The conjugate of claim 42 wherein D1 is a donor dye and D2 is an acceptor dye.

53. The conjugate of claim 42 wherein L1 comprises one of the following moieties:

—

CC≡

CH₂NH—

, —

C≡

CCH₂NHC(O)CH₂)₅NH—

, —

C═

CC(O)NH(CH₂)₅NH—

, —

C≡

CCH₂OCH₂CH₂NH—

, —

C≡

CCH₂OCH₂CH₂OCH₂CH₂NH—

, —

C≡

C—

CH₂OCH₂ CH₂—

NH—

, and —

C≡

C(p-C₄H₆)OCH₂CH₂NH—

.

57. The conjugate of claim 5 wherein the anionic phosphate moiety is a non-bridging phosphate moiety.

58. The conjugate of claim 57 wherein the non-bridging phosphate moiety has a net charge of −

1 or −

2.

59. The conjugate of claim 58 wherein the non-bridging phosphate moiety has a net charge of −

1.

60. The conjugate of claim 58 wherein the non-bridging phosphate moiety has a net charge of −

2.

61. The conjugate of claim 57 wherein the non-bridging phosphate moiety is a phosphate monoester or a phosphate diester.

62. The conjugate of claim 57 wherein the non-bridging phosphate moiety is a phosphate monoester.

63. The conjugate of claim 57 wherein the non-bridging phosphate moiety is a phosphate diester of the formula:

wherein R is a masking group selected from alkyl, alkenyl, alkynyl, aryl or alkaryl.

64. The conjugate of claim 7 wherein the anionic phosphonate moiety is a non-bridging phosphonate moiety.

65. The conjugate of claim 64, wherein the non-bridging phosphonate moiety has a net charge of −

1 or −

2.

66. The conjugate of claim 65 wherein the non-bridging phosphonate moiety has a net charge of −

1.

67. The conjugate of claim 65 wherein the non-bridging phosphonate moiety has a net charge of −

2.

68. The conjugate of claim 64 wherein the non-bridging phosphonate moiety is a phosphonic acid or a phosphonate monoester.

69. The conjugate of claim 64 wherein the non-bridging phosphonate moiety is a phosphonic acid.

70. The conjugate of claim 64 wherein the non-bridging phosphonate moiety is a phosphonate monoester of the formula:

wherein R is a masking group selected from alkyl, alkenyl, alkynyl, aryl or alkaryl.

71. The conjugate of claim 64 wherein the non-bridging phosphonate moiety is a phosphonate monoester of the formula:

wherein R is a masking group selected from alkyl, alkenyl, alkynyl, aryl or alkaryl.

72. The conjugate of claim 12 wherein D comprises at least one rhodamine dye.

73. The conjugate of claim 12 wherein D comprises at least one fluorescein dye.

74. The conjugate of claim 17 wherein the anionic phosphate moiety is a non-bridging phosphate moiety.

75. The conjugate of claim 74 wherein the non-bridging phosphate moiety has a net charge of −

1 or −

2.

76. The conjugate of claim 75 wherein the non-bridging phosphate moiety has a net charge of -1.

77. The conjugate of claim 75 wherein the non-bridging phosphate moiety has a net charge of −

2.

78. The conjugate of claim 74 wherein the non-bridging phosphate moiety is a phosphate monoester or a phosphate diester.

79. The conjugate of claim 74 wherein the non-bridging phosphate moiety is a phosphate monoester.

80. The conjugate of claim 74 wherein the non-bridging phosphate moiety is a phosphate diester of the formula:

wherein R is a masking group selected from alkyl, alkenyl, alkynyl, aryl or alkaryl.

81. The conjugate of claim 19 wherein the anionic phosphonate moiety is a non-bridging phosphonate moiety.

82. The conjugate of claim 81 wherein the non-bridging phosphonate moiety has a net charge of −

1 or −

2.

83. The conjugate of claim 82 wherein the non-bridging phosphonate moiety has a net charge of −

1.

84. The conjugate of claim 82 wherein the non-bridging phosphonate moiety has a net charge of −

2.

85. The conjugate of claim 81 wherein the non-bridging phosphonate moiety is a phosphonic acid or a phosphonate monoester.

86. The conjugate of claim 81 wherein the non-bridging phosphonate moiety is a phosphonic acid.

87. The conjugate of claim 81 wherein the non-bridging phosphonate moiety is a phosphonate monoester of the formula:

wherein R is a masking group selected from alkyl, alkenyl, alkynyl, aryl or alkaryl.

88. The conjugate of claim 81 wherein the non-bridging phosphonate moiety is a phosphonate monoester of the formula:

wherein R is a masking group selected from alkyl, alkenyl, alkynyl, aryl or alkaryl.

89. The conjugate of claim 34 wherein the anionic phosphate moiety is a non-bridging phosphate moiety.

90. The conjugate of claim 89 wherein the non-bridging phosphate moiety has a net charge of −

1 or −

2.

91. The conjugate of claim 90 wherein the non-bridging phosphate moiety has a net charge of −

1.

92. The conjugate of claim 90 wherein the non-bridging phosphate moiety has a net charge of −

2.

93. The conjugate of claim 89 wherein the non-bridging phosphate moiety is a phosphate monoester or a phosphate diester.

94. The conjugate of claim 89 wherein the non-bridging phosphate moiety is a phosphate monoester.

95. The conjugate of claim 89 wherein the non-bridging phosphate moiety is a phosphate diester of the formula:

wherein R is a masking group selected from alkyl, alkenyl, alkynyl, aryl or alkaryl.

96. The conjugate of claim 36 wherein the anionic phosphonate moiety is a non-bridging phosphonate moiety.

97. The conjugate of claim 96 wherein the non-bridging phosphonate moiety has a net charge of −

1 or −

2.

98. The conjugate of claim 97 wherein the non-bridging phosphonate moiety has a net charge of −

1.

99. The conjugate of claim 97 wherein the non-bridging phosphonate moiety has a net charge of −

2.

100. The conjugate of claim 96 wherein the non-bridging phosphonate moiety is a phosphonic acid or a phosphonate monoester.

101. The conjugate of claim 96 wherein the non-bridging phosphonate moiety is a phosphonic acid.

102. The conjugate of claim 96 wherein the non-bridging phosphonate moiety is a phosphonate monoester of the formula:

wherein R is a masking group selected from alkyl, alkenyl, alkynyl, aryl or alkaryl.

103. The conjugate of claim 96 wherein the non-bridging phosphonate moiety is a phosphonate monoester of the formula:

wherein R is a masking group selected from alkyl, alkenyl, alkynyl, aryl or alkaryl.

104. The conjugate of claim 18 wherein L2 comprises a sulfonic acid moiety.

105. The conjugate of claim 18 wherein L2 comprises a sulfonated benzene moiety.

106. The conjugate of claim 20 wherein L2 is not an anionic linker.

54. A method of sequencing a target polynucleotide sequence, the method comprising

(a) forming four classes of polynucleotides which are complementary to a target polynucleotide sequence, by template-dependent primer extension, wherein the polynucleotides in each class terminate with a different terminator subunit type that contains a distinct conjugate comprising a dye labeled nucleobase of the form:

(1) B-L-D, wherein B is a nucleobase, L is an anionic linker, and D comprises at least one fluorescent dye that comprises a xanthene, a rhodamine or a fluorescein, or

(2) B-L1-D1-L2-D2, wherein B is a nucleobase, L1 and L2 are linkers such that at least one of L1 and L2 is an anionic linker, and D1 and D2 are members of a fluorescent donor/acceptor pair, such that one of D1 and D2 is a donor dye capable of absorbing light at a first wavelength and emitting energy in response thereto, and the other of D1 and D2 is an acceptor dye capable of absorbing energy emitted by the donor dye and fluorescing at a second wavelength in response thereto, and at least one of D1 and D2 comprises a xanthene, a rhodamine or a fluorescein,

wherein L or at least one of L1 and L2 comprises at least one anionic phosphate or anionic phosphonate to identify the polynucleotides in that class, and

(b) separating the polynucleotides of the four classes on the basis of size to obtain a mobility pattern, and determining the sequence of the target polynucleotide sequence from the mobility pattern.

55. The method of claim 54 wherein the terminator subunits are nonextendable.

56. The method of claim 54 wherein the terminator subunits contain a 3′

-hydroxyl group.

107. The method of claim 54 wherein the dye-labeled nucleobase is of the form B-L-D.

108. The method of claim 107 wherein L comprises a sulfonic acid moiety.

109. The method of claim 107 wherein L comprises a sulfonated benzene moiety.

110. The method of claim 107 wherein L comprises an anionic phosphate moiety.

111. The method of claim 110 wherein the anionic phosphate moiety is a phosphate diester moiety, and the phosphorus atom of the phosphate diester moiety is located in L within a chain of linker atoms that connect B to D.

112. The method of claim 107 wherein L comprises an anionic phosphonate moiety.

113. The method of claim 112 wherein the anionic phosphonate moiety is a phosphonate monoester moiety, and the phosphorus atom of the phosphonate monoester moiety is located in L within a chain of linker atoms that connect B to D.

114. The method of claim 107 wherein L comprises a carboxylic acid moiety.

115. The method of claim 114 wherein the carboxylic acid moiety is a carboxyl benzene moiety.

116. The method of claim 107 wherein L comprises 4 to 20 chain atoms.

118. The method of claim 54 comprises adenine, 7-deazaadenine, 7-deaza-8-azaadenine, cytosine, guanine, 7-deazaguanine, 7-deaza-8-azaguanine, thymine, uracil, or inosine.

119. The method of claim 54 wherein the labeled nucleobase is of the form B-L1-D1-L2-D2.

120. The method of claim 119 wherein L1 comprises a sulfonic acid moiety.

121. The method of claim 119 wherein L1 comprises a sulfonated benzene moiety.

122. The method of claim 119 wherein L1 comprises an anionic phosphate moiety.

123. The method of claim 122 wherein the anionic phosphate moiety is a phosphate diester moiety, and the phosphorus atom of the phosphate diester moiety is located in L1 within a chain of linker atoms that connect B to D1.

124. The method of claim 119 wherein L1 comprises an anionic phosphonate moiety.

125. The method of claim 124 wherein the anionic phosphonate moiety is a phosphonate monoester moiety, and the phosphorus atom of the phosphonate monoester moiety is located in L1 within a chain of linker atoms that connect B to D1.

126. The method of claim 119 wherein L1 comprises a carboxylic acid moiety.

127. The method of claim 126 wherein the carboxylic acid moiety is a carboxy benzene moiety.

128. The method of claim 119 wherein L1 comprises 4 to 20 linker chain atoms.

129. The method of claim 119 wherein L2 is not an anionic linker.

131. The method of claim 129 wherein D1 is a donor dye and D2 is an acceptor dye.

132. The method of claim 119 wherein L2 comprises a sulfonic acid moiety.

133. The method of claim 119 wherein L2 comprises a sulfonated benzene moiety.

134. The method of claim 119 wherein L2 comprises an anionic phosphate moiety.

135. The method of claim 134 wherein the anionic phosphate moiety is a phosphate diester moiety, and the phosphorus atom of the phosphate diester moiety is located in L2 within a chain of linker atoms that connect D1 to D2.

136. The method of claim 119 wherein L2 comprises an anionic phosphonate moiety.

137. The method of claim 136 wherein the anionic phosphonate moiety is a phosphonate monoester moiety, and the phosphorus atom of the phosphonate monoester moiety is located in L2 within a chain of linker atoms that connect D1 to D2.

138. The method of claim 119 wherein L2 comprises a carboxylic acid moiety.

139. The method of claim 138 wherein the carboxylic acid moiety is a carboxy benzene moiety.

140. The method of claim 119 wherein L2 comprises up to 20 linker chain atoms.

141. The method of claim 119 wherein D1 is a donor dye and D2 is an acceptor dye.

142. The method of claim 119 wherein L2 is an anionic linker and L1 is not an anionic linker.

143. The method of claim 142 wherein L2 comprises a sulfonic acid moiety.

144. The method of claim 142 wherein L2 comprises a sulfonated benzene moiety.

145. The method of claim 142 wherein L2 comprises an anionic phosphate moiety.

146. The method of claim 145 wherein the anionic phosphate moiety is a phosphate diester moiety, and the phosphorus atom of the phosphate diester moiety is located in L2 within a chain of linker atoms that connect D1 to D2.

147. The method of claim 142 wherein L2 comprises an anionic phosphonate moiety.

148. The method of claim 147 wherein the anionic phosphonate moiety is a phosphonate monoester moiety, and the phosphorus atom of the phosphonate monoester moiety is located in L2 within a chain of linker atoms that connect D1 to D2.

149. The method of claim 142 wherein L2 comprises a carboxylic acid moiety.

150. The method of claim 149 wherein the carboxylic acid moiety is a carboxy benzene moiety.

151. The method of claim 142 wherein L2 comprises up to 20 linker chain atoms.

152. The method of claim 142 wherein D1 is a donor dye and D2 is an acceptor dye.

117. The method of claim wherein 107 comprises at least one fluorescein or rhodamine.

130. The method of claim wherein L2 comprises up to 20 linker chain atoms.