Releasable nonvolatile mass label molecules

• US 6,635,452 B1
• Filed: 12/10/1997
• Issued: 10/21/2003
• Est. Priority Date: 12/10/1996
• Status: Expired due to Fees

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Shaler, Thomas A., Monforte, Joseph A., Pollart, Daniel J., Becker, Christopher H.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US08/988,024
• Time in Patent Office: 2,141 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3, 536/24.31, 536/24.33, 536/25.3, 536/25.32, 436/173
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2333/922 Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
• CPC Class Codes: G01N 2333/95 Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
• CPC Class Codes: G01N 2458/15 Non-radioactive isotope labels, e.g. for detection by mass spectrometry
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: Y10S 977/775 Nanosized powder or flake, e.g. nanosized catalyst
• CPC Class Codes: Y10S 977/958 Of biomolecule property

Abstract

Using nonvolatile, releasable, mass-labels, the present invention provides for the synthesis and use of mass-labeled compounds to specifically interact with biomolecular targets. Following binding of the mass-labeled compounds to the target molecule, the unique mass-label can be analyzed using mass spectrometry to identify and characterize the target molecule. In one embodiment of the invention, a mass-labeled oligonucleotide probe is used to identify a specific gene sequence. A myriad of mass-labeled compounds may be produced for use in a wide variety of interactions such as oligonucleotide-oligonucleotide hybridization, polynucleotide-polynucleotide interactions, enzyme-substrate or substrate analog/intermediate interactions, polypeptide-nucleic acid interactions, protein-ligand interactions, receptor-ligand interactions, polypeptide-metal interactions, nucleic acid-metal interactions or antigen-antibody interactions. Also contemplated are combinatorial processes for creating large libraries of compounds permitting rapid screening for a wide variety of targets.

Claims

1. A method for detecting a target molecule, comprising:

(a) obtaining a target molecule;

(b) amplifying the target molecule to produce an amplified target molecule;

(c) obtaining a probe comprising a polynucleotide reactive group, a release group and a mass label;

(d) hybridizing the amplified target molecule to the probe to produce a probe;

amplified target molecule complex;

(e) releasing the mass label from the probe;

amplified target molecule complex to obtain a released mass label; and

(f) determining the mass of the released mass label by mass spectrometry, thereby detecting the target molecule.

2. The method of claim 1, wherein the amplified target molecule comprises a functional group capable of being immobilized on a solid support.

3. The method of claim 2, wherein the functional group comprises a biotin or digoxigenin.

4. The method of claim 2, wherein the functional group is attached to an oligonucleotide primer incorporated into the amplified target molecule during the amplification step.

5. The method of claim 2, wherein the functional group is attached to a nucleotide incorporated into the amplified target molecule during the amplification step.

6. The method of claim 2, wherein the amplified target molecule is immobilized onto the solid support and any probe not part of a probe:

amplified target molecule complex is removed by washing.

7. The method of claim 1, wherein the mass label is released by an enzyme.

8. The method of claim 7, wherein the enzyme comprises a nuclease.

9. The method of claim 8, wherein the nuclease comprises a restriction endonuclease.

10. The method of claim 8, wherein the nuclease comprises an exonuclease.

14. The method of claim 10, wherein the exonuclease is specific for single-stranded DNA.

15. The method of claim 1, wherein the release group comprises a chemically cleavable linkage.

16. The method of claim 15, wherein the chemically cleavable linkage comprises a modified base, a modified sugar, a disulfide bond, a chemically cleavable group incorporated into the phosphate backbone, or a chemically cleavable linker.

17. The method of claim 16, wherein the chemically cleavable linkage further comprises a moiety cleavable by acid, base, oxidation, reduction, heat, light, metal ion catalyzed, displacement, or elimination chemistry.

18. The method of claim 17, wherein the chemically cleavable linkage comprises a disulfide bond.

19. The method of claim 1, wherein the polynucleotide reactive group further comprises a nucleotide or oligonucleotide added after hybridization of the probe to the amplified target molecule.

20. The method of claim 19, wherein the added nucleotide or oligonucleotide further comprises a functional group capable of being immobilized on a solid support.

21. The method of claim 20, wherein the nucleotide is added by a polymerase.

22. The method of claim 20, wherein the oligonucleotide is added by a ligase.

23. The method of claim 20, further comprising:

(a) immobilizing the polynucleotide reactive group onto the solid support after addition of the nucleotide or oligonucleotide; and

(b) removing any probes having unbound reactive groups prior to releasing the mass label of any probe having a bound reactive group.

24. The method of claim 1, wherein the reactive and release groups are the same.

25. The method of claim 1, wherein the release group is contained within the reactive group.

26. The method of claim 1, wherein the probe comprises at least two mass labels having different masses.

27. The method of claim 1, wherein the target molecule is contacted with a plurality of probes.

28. The method of claim 27, wherein each reactive group is associated with a unique mass label.

29. The method of claim 27, wherein each reactive group is associated with a unique set of mass labels.

30. The method of claim 29, wherein the released mass label comprises a unique set of mass labels.

31. The method of claim 30, wherein each member of the set of mass labels is attached to a different probe.

32. The method of claim 1, wherein the amplified target molecule comprises a double-stranded molecule, each strand having a 3′

end and a 5′

end, said double-stranded molecule containing a mismatch and the 3′

ends are not capable of being digested by an exonuclease.

33. The method of claim 32, further comprising

cleaving at least one strand of the double-stranded molecule at the mismatch;

and selectively releasing the mass label by digesting the cleaved strand with a 3′

to 5′

exonuclease.

34. The method of claim 33, wherein the mismatch is cleaved by an enzyme.

35. The method of claim 34, wherein the enzyme comprises mutHLS, T4 endonuclease VII, mutY DNA glycosylase, thymine mismatch DNA glycosylase, or endonuclease V.

36. The method of claim 33, wherein the mismatch is cleaved by a chemical.

37. The method of claim 36, wherein the chemical comprises OsO₄, HONH₂, or KMnO₄.

38. The method of claim 33, wherein the 3′

to 5′

exonuclease comprises exonuclease III.

11. The method of 10, wherein the exonuclease is specific for double-stranded DNA.

12. The method of claim 11, wherein the exonuclease is selected from the group consisting of exonuclease III, T4 endonuclease VII, lambda exonuclease, and DNA polymerase.

13. The method of claim 11, wherein the release of the mass label occurs upon nuclease digestion of the probe:

amplified target molecule complex.

39. A method for detecting a target molecule, said method comprising the steps of:

(a) obtaining a probe comprising a polynucleotide reactive group, a release group and a nonvolatile mass label;

(b) obtaining a target molecule;

(c) contacting the target molecule with the probe to produce a probe;

target molecule complex;

(d) selectively releasing the mass label from the probe;

target molecule complex; and

(e) determining the mass of the mass label by mass spectrometry, thereby detecting the target molecule.

40. The method of claim 39, wherein the mass label is selectively released by an enzyme.

41. The method of claim 40, wherein the enzyme comprises a nuclease.

42. The method of claim 41, wherein the enzyme comprises a restriction endonuclease.

43. The method of claim 41, wherein the enzyme comprises an exonuclease.

46. the method of claim 39, wherein the release group is located within a reactive group and the polynucleotide reactive group is an oligonucleotide.

47. The method of claim 46, wherein the selective release of the mass label is inhibited by the presence of a double-stranded oligonucleotide at said release group.

48. The method of claim 47, wherein contacting the probe with the target molecule results in the release group being present in a single-stranded region.

49. The method of claim 48, wherein the release group comprises a chemically cleavable release group.

50. The method of claim 49, wherein the chemically cleavable release group comprises 3′

-(S)-phosphorothioate, 5′

-(S)-phosphorothioate, 3′

-(N)phosphoroamidate, 5′

-(N)-phosphoroamidate, or ribose.

51. The method of claim 48, wherein the release group is cleavable by a single-strand-specific nuclease.

52. The method of claim 39, wherein the polynucleotide reactive group comprises a polynucleotide or an oligonucleotide.

53. The method of claim 52, wherein the polynucleotide reactive group further comprises a nucleotide or an oligonucleotide added after hybridization to the target molecule.

54. The method of claim 53, wherein the nucleotide is added by a polymerase.

55. The method of claim 53, wherein the oligonucleotide is added by a ligase.

56. The method of claim 53, wherein the nucleotide or oligonucleotide further comprises a functional group capable of being immobilized on a solid support.

57. The method of claim 56, wherein the functional group comprises a biotin or digoxigenin.

58. The method of claim 56 further comprising:

(a) immobilizing the polynucleotide reactive group onto the solid support after the addition of the nucleotide or oligonucleotide; and

(b) removing any probes having unbound reactive groups prior to releasing their mass labels.

44. The method of 43, wherein the exonuclease is specific for double-stranded DNA.

45. The method of claim 44, wherein the selective release of the mass label by the exonuclease occurs upon hybridization of the probe to the target molecule and nuclease digestion of the probe:

target molecule complex.

59. A method for detecting a target molecule, said method comprising the steps of:

(a) amplifying a target nucleic acid to produce amplified nucleic acid products;

(b) obtaining one or more first molecules, said first molecules comprising a polynucleotide reactive group, a release group and a nonvolatile mass label;

(c) incorporating said first molecules into the amplified nucleic acid product during the amplification process to process to produce incorporated mass labeled molecules and unincorporated mass labeled molecules;

(d) releasing the mass labels incorporated into the amplified nucleic acid products to produce released mass labels; and

(e) determining the mass of the released mass labels by mass spectrometry, thereby detecting the target molecule.

60. The method of claim 59, wherein the molecules are oligonucleotide primers.

61. The method of claim 59, wherein the molecules are nucleoside triphosphates.

62. The method of claim 59, wherein the amplified nucleic acid products are produced using polymerase chain reaction (PCR), reverse transcriptase PCR (rtPCR), ligase chain reaction (LCR), Qbeta Replicase, strand displacement amplification (SDA), cyclic probe reaction (CPR), transcription-based amplification systems (TAS), nucleic acid sequence based amplification (NASBA);

or multiple rounds of RNA transcription or some combination thereof.

63. The method of claim 62, wherein the amplified nucleic acid products are produced using PCR or reverse transcriptase PCR.

64. The method of claim 59, wherein the mass label is released by an enzyme.

65. The method of claim 64, wherein the enzyme comprises a nuclease.

66. The method of claim 65, wherein the nuclease comprises a restriction endonuclease.

67. The method of claim 66, wherein the restriction endonuclease comprises a Type IIS restriction endonuclease.

68. The method of claim 66, wherein the restriction endonuclease comprises a Type II restriction endonuclease.

69. The method of claim 65, wherein the nuclease comprises an exonuclease.

70. The method of claim 69, wherein the exonuclease is specific for double-stranded DNA.

71. The method of claim 70, wherein the exonuclease is selected from a group consisting of exonuclease III, T4 endonuclease VII, lambda exonuclease, and DNA polymerase.

72. The method of claim 59, wherein the release group comprises a chemically cleavable linkage.

73. The method of claim 72, wherein the chemically cleavable linkage comprises a modified base, a modified sugar, a disulfide bond, a chemically cleavable group incorporated into the phosphate backbone , or a chemically cleavable linker.

74. The method of claim 73, wherein the chemically cleavable linkage further comprises a moiety cleavable by acid, base, oxidation, reduction, heat, light, metal ion catalyzed, displacement or elimination chemistry.

75. The method of claim 74, wherein the chemically cleavable group comprises a chemically cleavable group incorporated into the phosphate backbone.

76. The method of claim 75, wherein the chemically cleavable group comprises dialkoxysilane, 3′

-(S)-phosphorothioate, 5′

-(S)-phosphorothioate, 3′

-(N)-phosphoroamidate, or 5′

-(N)-phosphoroamidate.

77. The method of claim 74, wherein the chemically cleavable linkage comprises a modified sugar.

78. The method of claim 77, wherein the modified sugar comprises ribose.

79. The method of claim 74, wherein the chemically cleavable linkage comprises a disulfide bond.

80. The method of claim 59, wherein one or more second molecules are incorporated into the amplification nucleic acid products, said second molecules comprising a functional group capable of being immobilized on a solid support.

81. The method of claim 80, wherein the functional group comprises a biotin or digoxigenin.

82. The method of claim 80, wherein the functional group comprises a linker molecule capable of forming a covalent linkage to a solid support.

83. The method of claim 80, wherein the second molecules are oligonucleotide primers.

84. The method of claim 80, wherein the second molecules are nucleoside triphosphates.

85. The method of claim 80, further comprising the steps of binding the functional group of the amplified nucleic acid products to a solid support, and separating incorporated mass labeled molecules from unincorporated mass labeled molecules.

86. The method of claim 59, further comprising the step of separating the amplified nucleic acid products from unincorporated mass labeled molecules.

87. The method of claim 86, further comprising binding the amplified nucleic acid products to a solid support.

88. The method of claim 86, further comprising hybridizing the amplified nucleic acid products to a polynucleotide bound to a solid support.

89. The method of claim 88, wherein the bound polynucleotide is an oligonucleotide, a polyribonucleotide, a plasmid, an M13, a cosmid, a P1 clone, a BAC or a YAC.

90. The method of claim 88, further comprising hybridizing the amplified nucleic acid products to a plurality of polynucleotides immobilized onto the solid support at spaced locations.