Multisignal labeling reagents, and processes and uses therefor

• US 20040198971A1
• Filed: 04/03/2003
• Published: 10/07/2004
• Est. Priority Date: 04/03/2003
• Status: Active Grant

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Stavrianopoulos, Jannis G., Donegan, James J., Rabbani, Elazar
• Granted Patent: US 7,514,551 B2
• Time in Patent Office: Days
• US Class Current: 536/24.3
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

The present invention provides multisignal labeling reagents and these are useful in a number of biochemical applications, including the manufacture of biomolecular probes and their use in detecting or amplifying analyte-specific moieties.

Claims

1. A composition of matter comprising an oligomer or polymer comprising

a) two or more labeled moieties wherein the label or labels are chemically linked to said oligomer or polymer;

b) one or more reactive groups; and

c) one or more charged groups wherein said charged groups

(i) are covalently linked to said oligomer or polymer;

or

(ii) comprise part of the backbone of said oligomer or polymer;

or

(iii) any combination of the foregoing.

2. The composition of claim 1, wherein said oligomer or polymer is homopolymeric, heteropolymeric or chimeric.

3. The composition of claims 1 or 2, wherein said oligomer or polymer comprises one or more monomeric subunits selected from the group consisting of amino acids, nucleotides, carbohydrates, sugars, aromatic compounds, derivatized organic compounds that form oligomeric or polymeric moieties and analogs of the foregoing.

4. The composition of claim 3, wherein one or more of said monomeric subunits has been modified.

5. The composition of claims 3 or 4, wherein said oligomer or polymer comprises a nucleic acid, abasic nucleic acid, peptide nucleic acid, polypeptide, protein, oligosaccharide, polysaccharide, organic polymer or any combination thereof.

6. The composition of claim 1 wherein said labels are selected from the group consisting of fluorescent compounds, phosphorescent compounds, chemiluminescent compounds, chelating compounds, electron dense compounds, magnetic compounds, intercalating compounds and energy transfer compounds or any combination thereof.

7. The composition of claim 1, wherein said reactive group is selected from the group consisting of isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, aziridine, sulfonyl halogen substituted diazine, maleimide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyldithio)-proprionamide, glyoxal, aldehyde, carbon-carbon double bonds, mercury salts, and any group capable of reacting with carbon-carbon double bonds, amines, hydroxyl groups, sulfhydryl groups and halogens.

8. The composition of claim 1, wherein said reactive group is attached to the oligomer or polymer through a linker.

9. The composition of claim 8, wherein said linker comprises one or more carbon, nitrogen, oxygen, phosphorus or sulfur atoms in any combination.

10. The composition of claim 9, wherein said linker comprises one or more peptide bonds, alkyl chains, alkene groups, alkyl groups, aryl groups, conjugated systems, sugars or derivatives thereof.

11. The composition of claim 1, wherein said charged group is selected from the group consisting of phosphate, carboxylic, sulfone, amine and hydroxy groups.

12. The composition of claim 1, wherein said charged group has a negative charge.

13. The composition of claim 1, wherein said oligomer or polymer is linear or branched.

14. The composition of claim 1, wherein said oligomer or polymer comprises a complex formed from more than one oligomer or polymer.

15. The composition of claim 14, wherein said complex comprises two or more nucleic acid strands and said complex is formed through base-pairing or triple strand structures.

16. The composition of claim 15, wherein at least one of said nucleic acid strands comprises one or more nucleotide analogs or modified nucleotides.

69. A process for labeling a target molecule that comprises attaching or binding the compositions of claims 1, 17, 39 or 55 to said target molecule.

70. The process of claim 69, wherein said target molecule is selected from the group consisting of peptides, proteins, antibodies, enzymes, enzyme substrates, ligands, hormones, receptors, antigens, haptens, lectins, avidin, streptavidin, toxins, carbohydrates, oligosaccharides, polysaccharides, ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, analogs of deoxynucleotides, ribonucleotides and dideoxynucleotides, modified deoxynucleotides, modified ribonucleotides, modified dideoxynucleotides oligonucleotides and polynucleotides.

73. A composition comprising a target molecule that has been labeled by the process of claim 69 or 71.

74. The composition of claim 73, wherein said target molecule is selected from the group consisting of peptides, proteins, antibodies, enzymes, enzyme substrates, ligands, hormones, receptors, antigens, haptens, lectins, avidin, streptavidin, toxins, carbohydrates, oligosaccharides, polysaccharides, ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, analogs of deoxynucleotides, ribonucleotides and dideoxynucleotides, modified deoxynucleotides, modified ribonucleotides, modified dideoxynucleotides oligonucleotides and polynucleotides.

76. The composition of claim 73 wherein said Target is selected from the group consisting of peptides, proteins, antibodies, enzymes, enzyme substrates, ligands, hormones, receptors, antigens, haptens, lectins, avidin, streptavidin, toxins, carbohydrates, oligosaccharides, polysaccharides, ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, analogs of deoxynucleotides, ribonucleotides and dideoxynucleotides, modified deoxynucleotides, modified ribonucleotides, modified dideoxynucleotides oligonucleotides and polynucleotides.

79. A process for detecting or quantifying an analyte comprising the steps of:

i) providing the composition of claim 74, 76 or 78, wherein said target is an analyte specific moiety;

ii) contacting said composition with a specimen suspected of containing said analyte; and

iii) measuring the amount of said composition bound to analytes in said specimen to detect or quantify said analyte.

80. The process of claim 79, wherein in said providing step (i), when said composition of claim 74, 76 or 78, comprises an oligonucleotide or polynucleotide that comprises labeled nucleotides or labeled nucleotide analogs, a second composition is provided comprising an unlabeled oligonucleotide or polynucleotide, said second composition being added prior to, during or after said contacting step (ii).

81. The process of claim 80, wherein said unlabeled oligonucleotide or polynucleotide comprises sequences that are identical to all or a portion of said labeled oligonucleotide or polynucleotide.

17. A composition of matter comprising a nucleic acid strand or a complex of two or more nucleic acid strands wherein said strand or complex comprises one or more reactive groups and two or more labeled nucleotides or labeled nucleotide analogs.

18. The composition of claim 17, wherein said nucleic acid strand or complex comprise one or more unlabeled nucleotide analogs.

19. The composition of claims 17 or 18, wherein said nucleotide analogs are selected from the group comprising abasic nucleotides, spacer groups or peptide nucleic acid subunits.

20. The composition of claim 17, wherein said label is chosen from the group comprising fluorescent compounds, phosphorescent compounds, chemiluminescent compounds, chelating compounds, electron dense compounds, magnetic compounds, intercalating compounds and energy transfer compounds or any combination thereof.

21. The composition of claim 17, wherein said reactive group is selected from the group consisting of isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, aziridine, sulfonyl halogen substituted diazine, maleimide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal, aldehyde, carbon-carbon double bonds, mercury salts, and any group capable of reacting with carbon-carbon double bonds, amines, hydroxyl groups, sulfhydryl groups and halogens.

22. The composition of claim 17, wherein said reactive group is attached to said strand or complex through a linker.

23. The composition of claim 22, wherein said linker is attached to a nucleotide or nucleotide analog comprising said strand or complex and said attachment is through a base, sugar, phosphate or an analog thereof.

24. The composition of claim 22, wherein said linker comprises one or more carbon, nitrogen, oxygen, phosphorus or sulfur atoms in any combination.

25. The composition of claim 22, wherein said linker comprises one or more peptide bonds, alkyl chains, alkene groups, alkyl groups, aryl groups, conjugated systems, sugars or derivatives thereof.

26. The composition of claim 17, wherein said nucleic acid strands are linear or branched.

27. The composition of claim 20, wherein said complex is formed through base-pairing or triple strand formation.

28. A composition of matter comprising a nucleic acid strand or a complex of two or more nucleic acid strands wherein said strand or complex comprises (i) two or more labeled nucleotides or labeled nucleotide analogs, and (ii) one or more binding partners different from said labels.

29. The composition of claim 28, wherein said strand or complex comprises one or more unlabeled nucleotide analogs.

30. The composition of claims 28 or 29, wherein said nucleotide analogs are selected from the group consisting of abasic nucleotides, spacer groups or peptide nucleic acid subunits.

31. The composition of claim 28, wherein said label is selected from the group consisting of fluorescent compounds, phosphorescent compounds, chemiluminescent compounds, chelating compounds, electron dense compounds, magnetic compounds, intercalating compounds and energy transfer compounds, or any combination thereof.

32. The composition of claim 28, wherein said binding partners are selected from the group consisting of ligand/receptor, hormone/receptor, biotin/avidin, biotin/strepavidin and antigen/antibody pairs.

33. The composition of claim 28, wherein said binding partner is attached to said strand or said complex through a linker.

34. The composition of claim 33, wherein said linker is attached to a nucleotide or nucleotide analog comprising said strand or complex and said attachment is through a base, sugar, phosphate or an analog thereof.

35. The composition of claim 33, wherein said linker comprises one or more carbon, nitrogen, oxygen, phosphorus or sulfur atoms, or a combination thereof.

36. The composition of claim 33, wherein said linker comprises one or more peptide bonds, alkyl chains, alkene groups, alkyl groups, aryl groups, conjugated systems, sugars or derivatives thereof.

37. The composition of claim 28, wherein said nucleic acid strands are linear or branched.

38. The composition of claim 28, wherein said complex is formed through base-pairing or triple strand formation.

82. A process for detecting or quantifying an analyte in a specimen comprising the steps of

(i) providing

(a) a specimen where an analyte is sought to be detected or quantified;

(b) the composition of claim 28 wherein said composition comprises a first non-hybridizing binding partner and

(c) an analyte specific moiety that comprises a second non-hybridizing binding partner wherein said first binding partner and said second binding partner comprise a binding pair;

(ii) binding said analyte specific moiety (c) to any analytes that may be present in the specimen (a);

(iii) binding said composition (b) to any bound analyte specific moieties from step (ii) through interactions between said first binding partners and said second binding partners; and

(iv) measuring signal generation and thereby detecting the presence or quantity of said analyte.

83. The process of claim 82, wherein said first and second binding partners are paired members selected from the group consisting of biotin/avidin, biotin/streptavidin, antigen/antibody, hormone/hormone receptor, lectin/sugar, enzyme/enzyme substrate, enzyme/substrate analog, enzyme/enzyme inhibitor, co-factor/co-factor enzyme binding site, and chelator/chelate.

84. The process of claim 82, wherein said analyte specific moiety comprises an antibody or an antigen.

85. The process of claim 82, wherein said process further comprises providing (d) one or more unlabeled nucleic acids.

86. The process of claim 85, wherein said unlabeled nucleic acids comprises some or all of the sequences of the nucleic acid portion in said composition of claim 28.

87. A process for detecting or quantifying an analyte in a specimen comprising the steps of

(i) providing

(a) a specimen where an analyte is sought to be detected or quantified;

(b) the composition of claim 28 wherein said composition comprises a first non-hybridizing binding partner;

(c) a linking moiety comprising a second non-hybridizing binding partner and a third non-hybridizing binding partner;

(d) an analyte specific moiety that comprises a fourth non-hybridizing binding partner;

wherein said first binding partner and said second binding partner comprise a first binding pair, and said third binding partner and said fourth binding partner comprise a second binding pair;

(ii) performing or carrying out at least one binding reaction comprising;

(a) binding said analyte specific moiety (d) to any analytes that may be present in the specimen (a);

(b) binding said third binding partner to said fourth binding partner;

(c) binding said first binding partner to said second binding partner, to form a complex that comprises the analyte, the analyte specific moiety, the linking moiety and the composition of claim 28;

and

(iii) detecting or quantifying the amount of signal generation in said complex.

88. The process of claim 87, wherein said performing or carrying out step (ii), the binding reactions are performed or carried out sequentially or concomitantly.

89. The process of claim 88, wherein said sequential binding reactions comprise step (ii)(a) followed by step (ii)(b) and then step (ii)(c).

90. The process of claim 88, wherein said sequential binding reactions comprise step (ii)(b) followed by (ii)(c) and then step (ii)(a).

91. The process of claim 88, wherein said sequential binding reactions comprise step (ii)(a) followed by stepm (ii)(c) and then step (ii)(b).

92. The process of claim 88, wherein said sequential binding reactions comprise step (ii)(c) followed by step (ii)(b) and then step (ii)(a).

93. The process of claim 87, wherein said first and second binding partners and said third and fourth binding partners are paired members selected from the group consisting of biotin/avidin, biotin/streptavidin, antigen/antibody, hormone/hormone receptor, lectin/sugar, enzyme/enzyme substrate, enzyme/substrate analog, enzyme/enzyme inhibitor, co-factor/co-factor enzyme binding site, and chelator/chelate.

94. The process of claim 93, wherein said providing step (i), the first binding pair and the second binding pair comprise the same pair of binding partners.

95. The process of claim 93, wherein said providing step (i), the first binding pair and the second binding pair comprise different pairs of binding partners.

96. The process of claim 87, wherein said analyte specific moiety comprises an antibody or an antigen.

97. The process of claim 87, wherein said process further comprises providing (e) one or more unlabeled nucleic acids.

98. The process of claim 97, wherein said unlabeled nucleic acids comprises some or all of the sequences of the nucleic acid portion in said composition of claim 28.

39. A composition of matter comprising a compound having the structure:

40. The composition of claim 39, wherein said non-inherent charged group Q is selected from the group consisting of phosphate, carboxylic, sulfone, amine and hydroxy groups.

41. The composition of claim 39, wherein said Q has a negative charge.

42. The composition of claim 39, wherein said label D is selected from the group consisting of fluorescent compounds, phosphorescent compounds, chemiluminescent compounds, chelating compounds, electron dense compounds, magnetic compounds, intercalating compounds and energy transfer compounds or any combination thereof.

43. The composition of claim 39, wherein said reactive group R is selected from the group consisting of isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, aziridine, sulfonyl halogen substituted diazine, maleimide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl-dithio)-proprionamide, glyoxal, aldehyde, carbon-carbon double bonds, mercury salts, and any group capable of reacting with carbon-carbon double bonds, amines, hydroxyl groups, sulfhydryl groups and halogens.

44. The composition of claim 39, wherein said reactive group R is attached to said oligomer or polymer P through a linker.

45. The composition of claim 44, wherein said linker comprises one or more carbon, nitrogen, oxygen, phosphorus or sulfur atoms, or a combination thereof.

46. The composition of claim 44, wherein said linker comprises one or more peptide bonds, alkyl chains, alkene groups, alkyl groups, aryl groups, conjugated systems, sugars or derivatives thereof.

47. The composition of claim 39, wherein said oligomer or polymer P comprises one or more monomeric subunits selected from the groups consisting of amino acids, nucleotides, carbohydrates, sugars, aromatic compounds, derivatized organic compounds that form oligomeric or polymeric moieties and analogs of the foregoing.

48. The composition of claim 47, wherein one or more of said monomeric subunits has been modified 49. The composition of claim 39, wherein said oligomer or polymer P is homopolymeric, hetropolymeric or chimeric.

50. The composition of claim 39 wherein said oligomer or polymer P comprises a nucleic acid, abasic nucleic acid, peptide nucleic acid polypeptide, protein, oligosaccharide, polysaccharide, organic polymer or a combination thereof.

51. The composition of claim 40, wherein said oligomer or polymer P is linear or branched.

52. The composition of claim 40, wherein said oligomer or polymer P comprises a complex formed from two or more oligomers or polymers.

53. The composition of claim 52, wherein said complex comprises two or more nucleic acid strands and said complex is formed through base-pairing or triple strand structures.

54. The composition of claim 53, wherein at least one of said nucleic acid strands comprises one or more nucleotide analogs or modified nucleotides.

55. A composition of matter comprising a compound having the structure:

56. The composition of claim 55, wherein said label D is selected from the group consisting of fluorescent compounds, phosphorescent compounds, chemiluminescent compounds, chelating compounds, electron dense compounds, magnetic compounds, intercalating compounds and energy transfer compounds or a combination thereof.

57. The composition of claim 55, wherein said reactive group R is selected from the group consisting of isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, aziridine, sulfonyl halogen substituted diazine, maleimide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyldithio)-proprionamide, glyoxal, aldehyde, carbon-carbon double bonds, mercury salts, and any group capable of reacting with carbon-carbon double bonds, amines, hydroxyl groups, sulfhydryl groups and halogens.

58. The composition of claim 55, wherein said reactive group R is attached to said oligomer or polymer P through a linker.

59. The composition of claim 58, wherein said linker comprises one or more carbon, nitrogen, oxygen, phosphorus or sulfur atoms, or a combination thereof.

60. The composition of claim 58, wherein said linker comprises one or more peptide bonds, alkyl chains, alkene groups, alkyl groups, aryl groups, conjugated systems, sugars or derivatives thereof.

61. The composition of claim 55, wherein said oligomer or polymer P comprises one or more monomeric subunits selected from the group consisting of amino acids, nucleotides, carbohydrates, sugars, aromatic compounds, derivatized organic compounds that form oligomeric or polymeric moieties and analogs of the foregoing.

62. The composition of claim 61, wherein one or more of said monomeric subunits has been modified

63. The composition of claim 55, wherein said oligomer or polymer P is homopolymeric, hetropolymeric or chimeric.

64. The composition of claim 55, wherein said oligomer or polymer P comprises a nucleic acid, abasic nucleic acid, peptide nucleic acid polypeptide, protein, oligosaccharide, polysaccharide, organic polymer or a combination thereof.

65. The composition of claim 55, wherein said oligomer or polymer P is linear or branched.

66. The composition of claim 55, wherein P comprises a complex formed from two or more oligomers or polymers.

67. The composition of claim 66, wherein said complex comprises two or more nucleic acid strands and said complex is formed through base-pairing or triple strand structures.

68. The composition of claim 67, wherein at least one of said nucleic acid strands comprises one or more nucleotide analogs or modified nucleotides.

71. A process for labeling a target molecule that comprises attaching or binding a composition of matter to said target molecule, said composition comprising a nucleic acid strand or a complex of two or more nucleic acid strands wherein said strand or complex comprises (i) two or more labeled nucleotides or labeled nucleotide analogs, and (ii) one or more binding partners.

72. The process of claim 71, wherein said target molecule is selected from the group consisting of peptides, proteins, antibodies, enzymes, enzyme substrates, ligands, hormones, receptors, antigens, haptens, lectins, avidin, streptavidin, toxins, carbohydrates, oligosaccharides, polysaccharides, ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, analogs of deoxynucleotides, ribonucleotides and dideoxynucleotides, modified deoxynucleotides, modified ribonucleotides, modified dideoxynucleotides oligonucleotides and polynucleotides

75. A composition prepared by a target labeling process comprising the steps of:

(a) providing;

(i) a target for labeling; and

(ii) a labeling reagent having the formula

wherein Q represents a non-inherent charged group, n represents an integer of 1 or greater, D represents a label, m represents an integer equal to or greater than 2, R represents at least one reactive group and P represents an oligomer or polymer;

(b) reacting said target (i) and said labeling reagent (ii) to form said composition, said composition having the formula

wherein L represents a linkage or linker between said oligomer or polymer and said target.

77. A composition prepared by a target labeling process comprising the steps of:

(a) providing;

(i) a target for labeling; and

(ii) a labeling reagent having the formula

wherein D represents a label, m represents an integer equal to or greater than 2;

R represents at least one reactive group, P represents a synthetic or chimeric oligomer or polymer, and wherein D or at least one of the monomeric units of P comprises one or more charged groups;

(b) reacting said target (i) and said labeling reagent (ii) to form said composition, said composition having the formula

wherein L represents a linkage or linker between said oligomer or polymer and said target.

78. The composition of claim 77, wherein said target molecule is selected from the group consisting of peptides, proteins, antibodies, enzymes, enzyme substrates, ligands, hormones, receptors, antigens, haptens, lectins, avidin, streptavidin, toxins, carbohydrates, oligosaccharides, polysaccharides, ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, analogs of deoxynucleotides, ribonucleotides and dideoxynucleotides, modified deoxynucleotides, modified ribonucleotides, modified dideoxynucleotides oligonucleotides and polynucleotides.