Fluorescent nucleobase conjugates having anionic linkers
First Claim
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.
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Accused Products
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.
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Citations
152 Claims
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1. A conjugate comprising a dye labeled nucleobase of the form:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106)
(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.
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26. The conjugate of claim 25, wherein D1 is a donor dye and D2 is an acceptor dye.
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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.
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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.
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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.
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30. The conjugate of claim 25 wherein L2 comprises up to 20 linker chain atoms.
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31. The conjugate of claim 24 wherein D1 is a donor dye and D2 is an acceptor dye.
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32. The conjugate of claim 14 wherein L2 comprises a sulfonic acid moiety.
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33. The conjugate of claim 14 wherein L2 comprises a sulfonated benzene moiety.
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34. The conjugate of claim 14 wherein L2 comprises an anionic phosphate moiety.
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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.
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36. The conjugate of claim 14 wherein L2 comprises an anionic phosphonate moiety.
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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.
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38. The conjugate of claim 14 wherein L2 comprises a carboxylic acid moiety.
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39. The conjugate of claim 38 wherein the carboxylic acid moiety is a carboxy benzene moiety.
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40. The conjugate of claim 14 wherein L2 comprises up to 20 linker chain atoms.
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41. The conjugate of claim 14 wherein D1 is a donor dye and D2 is an acceptor dye.
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42. The conjugate of claim 14 wherein L2 is an anionic linker and L1 is not an anionic linker.
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43. The conjugate of claim 42 wherein L2 comprises a sulfonic acid moiety.
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44. The conjugate of claim 42 wherein L2 comprises a sulfonated benzene moiety.
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45. The conjugate of claim 42 wherein L2 comprises an anionic phosphate moiety.
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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.
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47. The conjugate of claim 42 wherein L2 comprises an anionic phosphonate moiety.
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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.
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49. The conjugate of claim 42 wherein L2 comprises a carboxylic acid moiety.
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50. The conjugate of claim 49 wherein the carboxylic acid moiety is a carboxy benzene moiety.
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51. The conjugate of claim 42 wherein L2 comprises up to 20 linker chain atoms.
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52. The conjugate of claim 42 wherein D1 is a donor dye and D2 is an acceptor dye.
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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—
.
- —
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57. The conjugate of claim 5 wherein the anionic phosphate moiety is a non-bridging phosphate moiety.
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58. The conjugate of claim 57 wherein the non-bridging phosphate moiety has a net charge of −
- 1 or −
2.
- 1 or −
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59. The conjugate of claim 58 wherein the non-bridging phosphate moiety has a net charge of −
- 1.
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60. The conjugate of claim 58 wherein the non-bridging phosphate moiety has a net charge of −
- 2.
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61. The conjugate of claim 57 wherein the non-bridging phosphate moiety is a phosphate monoester or a phosphate diester.
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62. The conjugate of claim 57 wherein the non-bridging phosphate moiety is a phosphate monoester.
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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.
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64. The conjugate of claim 7 wherein the anionic phosphonate moiety is a non-bridging phosphonate moiety.
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65. The conjugate of claim 64, wherein the non-bridging phosphonate moiety has a net charge of −
- 1 or −
2.
- 1 or −
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66. The conjugate of claim 65 wherein the non-bridging phosphonate moiety has a net charge of −
- 1.
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67. The conjugate of claim 65 wherein the non-bridging phosphonate moiety has a net charge of −
- 2.
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68. The conjugate of claim 64 wherein the non-bridging phosphonate moiety is a phosphonic acid or a phosphonate monoester.
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69. The conjugate of claim 64 wherein the non-bridging phosphonate moiety is a phosphonic acid.
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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.
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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.
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72. The conjugate of claim 12 wherein D comprises at least one rhodamine dye.
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73. The conjugate of claim 12 wherein D comprises at least one fluorescein dye.
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74. The conjugate of claim 17 wherein the anionic phosphate moiety is a non-bridging phosphate moiety.
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75. The conjugate of claim 74 wherein the non-bridging phosphate moiety has a net charge of −
- 1 or −
2.
- 1 or −
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76. The conjugate of claim 75 wherein the non-bridging phosphate moiety has a net charge of -1.
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77. The conjugate of claim 75 wherein the non-bridging phosphate moiety has a net charge of −
- 2.
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78. The conjugate of claim 74 wherein the non-bridging phosphate moiety is a phosphate monoester or a phosphate diester.
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79. The conjugate of claim 74 wherein the non-bridging phosphate moiety is a phosphate monoester.
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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.
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81. The conjugate of claim 19 wherein the anionic phosphonate moiety is a non-bridging phosphonate moiety.
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82. The conjugate of claim 81 wherein the non-bridging phosphonate moiety has a net charge of −
- 1 or −
2.
- 1 or −
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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.
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85. The conjugate of claim 81 wherein the non-bridging phosphonate moiety is a phosphonic acid or a phosphonate monoester.
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86. The conjugate of claim 81 wherein the non-bridging phosphonate moiety is a phosphonic acid.
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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.
-
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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.
-
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89. The conjugate of claim 34 wherein the anionic phosphate moiety is a non-bridging phosphate moiety.
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90. The conjugate of claim 89 wherein the non-bridging phosphate moiety has a net charge of −
- 1 or −
2.
- 1 or −
-
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.
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94. The conjugate of claim 89 wherein the non-bridging phosphate moiety is a phosphate monoester.
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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.
-
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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.
- 1 or −
-
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.
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100. The conjugate of claim 96 wherein the non-bridging phosphonate moiety is a phosphonic acid or a phosphonate monoester.
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101. The conjugate of claim 96 wherein the non-bridging phosphonate moiety is a phosphonic acid.
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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.
-
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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.
-
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104. The conjugate of claim 18 wherein L2 comprises a sulfonic acid moiety.
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105. The conjugate of claim 18 wherein L2 comprises a sulfonated benzene moiety.
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106. The conjugate of claim 20 wherein L2 is not an anionic linker.
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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. - View Dependent Claims (55, 56, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152)
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117. The method of claim wherein 107 comprises at least one fluorescein or rhodamine.
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130. The method of claim wherein L2 comprises up to 20 linker chain atoms.
Specification