Assay procedure using fluorogenic tracers

• US 20030099999A1
• Filed: 10/31/2002
• Published: 05/29/2003
• Est. Priority Date: 07/23/1993
• Status: Active Grant

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Wei, Ai-Ping, Herron, James N.
• Granted Patent: US 7,153,654 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Fluorescent energy transfer dyes capable of moving between a more stacked configuration to exhibit fluorescent quenching and a more spaced configuration to exhibit fluorescence can be conjugated to a peptide epitope or nucleic acid for use in the detection of an unknown antibody in bulk solution. The resulting labeled peptide reagent can be used in an immunoassay procedure by placing it in bulk solution along with the unknown antibody to be detected. When the antibody binds to the peptide epitope, the pair of dyes carried by the peptide epitope will have their configuration altered from a stacked to an unstacked configuration and will exhibit a fluorescent increase in response thereto.

Claims

1. An assay procedure for detecting a target nucleic acid, said assay procedure comprising:

providing a detector probe comprising a nucleic acid sequence labeled with two chromophores, one of the two chromophores attached proximate the 3′

end and the second of the two chromophores attached proximate the 5′

end of the nucleic acid sequence, wherein the detector probe is capable of moving between a stacked configuration that exhibits fluorescence quenching and a spaced configuration that exhibits fluorescence;

adding the detector probe to a sample potentially containing a target strand of nucleic acid, the configuration of the detector probe moving between the stacked configuration and the spaced configuration upon hybridization of the detector probe to the target strand of nucleic acid; and

detecting the presence or absence of the target strand of nucleic acid by measuring a change in fluorescence in the sample.

2. The assay procedure of claim 1, wherein the fluorescence quenching occurs as a result of physical interaction between the two chromophores.

3. The assay procedure of claim 1, wherein the fluorescence occurs by increasing the distance between the two chromophores upon hybridization of the detector probe to the target strand of nucleic acid.

4. The assay procedure of claim 1, wherein providing a detector probe comprising a nucleic acid sequence comprises providing a detector probe comprising a DNA sequence or an RNA sequence.

5. The assay procedure of claim 1, wherein providing a detector probe comprising a nucleic acid sequence comprises providing a detector probe comprising at least one nucleotide analogue.

6. The assay procedure of claim 1, wherein providing a detector probe comprising a nucleic acid sequence comprises providing a detector probe comprising an oligonucleotide.

7. The assay procedure of claim 1, wherein providing a detector probe comprising a nucleic acid sequence labeled with two chromophores comprises attaching the two chromophores to nucleotides positioned proximal to but not at the 3′

and 5′

ends of the nucleic acid sequence.

8. The assay procedure of claim 1, wherein providing a detector probe comprising a nucleic acid sequence labeled with two chromophores comprises attaching the two chromophores proximate the 3′

and 5′

ends of the nucleic acid sequence via a spacer structure.

9. The assay procedure of claim 1, wherein the stacked configuration occurs by dimerization of the two chromophores.

10. The assay procedure of claim 1, wherein the spaced configuration occurs when the target strand of nucleic acid hybridizes to the nucleic acid sequence of the detector probe.

11. The assay procedure of claim 1, wherein at least one of the chromophores is a fluorophore.

12. The assay procedure of claim 1, wherein the two chromophores are two planar, aromatic fluorophores capable of forming homodimers or heterodimers.

13. The assay procedure of claim 12, wherein the planar, aromatic fluorophores are selected from the group consisting of fluoresceins, rhodamines, cyanines, Texas Red, rhodamine B, and tetramethylrhodamine.

14. The assay procedure of claim 1, wherein providing a detector probe comprising a nucleic acid sequence labeled with two chromophores comprises providing the detector probe immobilized on a solid support.

15. A probe capable of hybridizing with a target nucleic acid strand, said probe comprising:

a nucleotide strand and a pair of chromophores attached proximate the 3′ and

5′

ends of the nucleotide strand, at least one of the pair of chromophores comprising a fluorophore, wherein the pair of chromophores interact with one another to quench fluorescence when the probe is in a stacked conformation, the pair of chromophores exhibit increased fluorescence when the probe is in a spaced conformation, and a distance between the pair of chromophores is changed upon hybridization of the nucleotide strand with the target nucleic acid strand.

16. The probe of claim 15, wherein the probe is in the stacked conformation when the probe is not hybridized to the target nucleic acid strand.

17. The probe of claim 15, wherein the probe is in the stacked conformation when the pair of chromophores dimerizes with each other.

18. The probe of claim 15, wherein the probe is in the spaced conformation when the probe is hybridized to the target nucleic acid strand.

19. The probe of claim 15, wherein the fluorophore is selected from the group consisting of a fluorescein, a rhodamine, a cyanine, Texas Red, rhodamine B, and tetramethylrhodamine.

20. The probe of claim 15, wherein the pair of chromophores are planar, aromatic fluorophores capable of forming homodimers or heterodimers.

21. The probe of claim 15, wherein the distance between the pair of chromophores is increased upon the hybridization of the nucleotide strand with the target nucleic acid strand.

22. The probe of claim 15, wherein the pair of chromophores is attached proximal to but not at the 3′

and 5′

ends of the nucleic acid sequence.

23. The probe of claim 15, wherein the pair of chromophores is attached proximate the 3′

and 5′

ends of the nucleotide strand via a spacer structure.

24. The probe of claim 15, wherein the nucleotide strand comprises DNA or RNA.

25. The probe of claim 15, wherein the nucleotide strand comprises at least one nucleotide analogue.

26. The probe of claim 15, wherein the nucleotide strand comprises an oligonucleotide.

27. The probe of claim 15, wherein the probe is immobilized on a solid support.

28. The probe of claim 27, wherein the solid support is a waveguide.

29. An assay procedure for detecting and quantifying a target nucleic acid, comprising:

providing a detector probe comprising a nucleic acid sequence labeled with a pair of chromophores attached proximate the 3′ and

5′

ends of the nucleic acid sequence, at least one of the pair of chromophores comprising a fluorophore, wherein the detector probe is capable of moving between a first configuration in which the chromophores physically interact to exhibit fluorescence quenching and a second configuration in which the distance between the chromophores is increased to exhibit fluorescence;

adding the detector probe to a sample potentially containing a target strand of nucleic acid, the configuration of the detector probe shifting between the first configuration and the second configuration upon hybridization of the detector probe to the target strand of nucleic acid;

detecting the presence or absence of the target strand of nucleic acid by measuring changes in fluorescence in the sample; and

quantifying the presence of the target strand of nucleic acid by measuring the changed fluorescence in the sample.

30. A probe comprising:

a nucleotide strand and a pair of chromophores attached to the nucleotide strand, at least one of the pair of chromophores comprising a fluorophore, wherein the pair of chromophores interact with one another to quench fluorescence when the probe is in a stacked conformation and the pair of chromophores exhibit increased fluorescence when the probe is in a spaced conformation.

31. The probe of claim 30, wherein the pair of chromophores is attached to the nucleotide strand proximate the 3′

and 5′

ends.

32. The probe of claim 30, wherein the pair of chromophores is attached to the nucleotide strand at modified nucleotides positioned in the middle of the probe.

33. An assay procedure for detecting a target nucleic acid, said assay procedure comprising:

providing a detector probe comprising a nucleic acid sequence labeled with two chromophores, one of the two chromophores attached proximate the 3′

end and the second of the two chromophores attached proximate the 5′

end of the nucleic acid sequence, wherein the detector probe is capable of moving between a first configuration that exhibits fluorescence quenching and a second configuration that exhibits fluorescence;

adding the detector probe to a sample potentially containing a target strand of nucleic acid, the configuration of the detector probe moving between the first configuration and the second configuration upon hybridization of the detector probe to the target strand of nucleic acid; and

detecting the presence or absence of the target strand of nucleic acid by measuring a change in fluorescence in the sample.

34. A probe capable of hybridizing with a target nucleic acid strand, said probe comprising:

a nucleotide strand and a pair of chromophores, each chromophore attached proximate a 3′ and

5′

end of the nucleotide strand, at least one of the pair of chromophores comprising a fluorophore, wherein the pair of chromophores interact with one another to quench fluorescence when the probe is in a first conformation, the pair of chromophores exhibit increased fluorescence when the probe is in a second conformation, and the distance between the pair of chromophores is changed upon hybridization of the nucleotide strand with the target nucleic acid strand.

35. A probe comprising:

a nucleic acid sequence and a pair of chromophores attached proximate the 3′ and

5′

ends of the nucleic acid sequence, at least one of the pair of chromophores comprising a fluorophore, wherein the pair of chromophores interact with one another to quench fluorescence when the probe is in a first conformation and the pair of chromophores exhibit increased fluorescence when the probe is in a second conformation.

36. The probe of claim 35, wherein the pair of chromophores is closely spaced in the first conformation and are spaced a greater distance apart in the second conformation.

37. The probe of claim 35, wherein the probe is immobilized on a solid support.

38. A system for detecting a target nucleic acid comprising:

a probe that hybridizes with the target nucleic acid, the probe comprising a nucleic acid sequence and two chromophores attached proximate the 3′ and

5′

ends of the nucleic acid sequence,

at least one of the two chromophores comprising a fluorophore, wherein the two chromophores interact with one another to quench fluorescence when the probe is in a stacked conformation, the pair of chromophores exhibit increased fluorescence when the probe is in a spaced conformation, and a distance between the pair of chromophores is changed upon hybridization of the nucleotide strand with the target nucleic acid strand.

39. A detector probe comprising:

an amino acid sequence labeled with two chromophores, each chromophore attached proximate to an end of the amino acid sequence, wherein the detector probe is of a sufficient length to fold and move between a stacked configuration that exhibits fluorescence quenching and a spaced configuration that exhibits fluorescence.

40. The detector probe of claim 39, wherein one of the two chromophores is attached to the N-terminal residue and the second of the two chromophores is attached to the carboxy-terminus of the amino acid sequence.

41. The detector probe of claim 39, wherein at least one of the two chromophores is attached proximate an end of the peptide epitope via a spacer structure.

42. The detector probe of claim 39, wherein one of the two chromophores is attached to the N-terminal residue via a spacer structure and the second of the two chromophores is attached to the carboxy-terminus of the amino acid sequence via a spacer structure.

43. The detector probe of claim 39, wherein at least one of the two chromophores is a fluorophore.

44. The detector probe of claim 39, wherein the two chromophores are fluorophores.

45. The detector probe of claim 44, wherein one of the fluorophores is a fluorescent energy transfer donor and the second of the fluorophores is a fluorescent energy transfer acceptor.

46. The detector probe of claim 44, wherein the fluorophores are selected from the group consisting of fluoresceins, rhodamines, cyanines, Texas Red, rhodamine B, and tetramethylrhodamine.

47. The probe of claim 44, wherein the pair of chromophores are planar, aromatic fluorophores capable of forming homodimers or heterodimers

48. The detector probe of claim 39, wherein the amino acid sequence is a length of about 6 to 13 amino acids.

49. The detector probe of claim 39, wherein the two chromophores interact with one another to quench fluorescence when the detector probe is in the stacked configuration, the two chromophores exhibit increased fluorescence when the detector probe is in the spaced configuration, and a distance between the two chromophores is changed upon recognition of the amino acid sequence by a protein.

50. A detector probe comprising:

a peptide epitope labeled with two chromophores, each chromophore attached proximate an end of the peptide epitope, wherein the detector probe is of a sufficient length to fold and unfold and move between a stacked configuration that exhibits fluorescence quenching and a spaced configuration that exhibits fluorescence.

51. The detector probe of claim 50, wherein the peptide epitope is a flexible peptide.

52. The detector probe of claim 50, wherein the peptide epitope is an amino acid sequence of about 6 to 13 amino acids.

53. The detector probe of claim 50, wherein the peptide epitope is an antigen or antigen-mimicking peptide.

54. The detector probe of claim 50, wherein one of the two chromophores is attached to an N-terminal residue and the second of the two chromophores is attached to a carboxy-terminus of the peptide epitope.

55. The detector probe of claim 50, wherein at least one of the two chromophores is attached proximate an end of the peptide epitope via a spacer structure.

56. The detector probe of claim 50, wherein one of the two chromophores is attached to the N-terminal residue via a spacer structure and the second of the two chromophores is attached to the carboxy-terminus of the peptide epitope via a spacer structure.

57. The detector probe of claim 50, wherein the two chromophores are planar, aromatic fluorophores capable of forming homodimers or heterodimers.

58. The detector probe of claim 50, wherein at least one of the two chromophores is a fluorophore.

59. The detector probe of claim 58, wherein one of the fluorophores is a fluorescent energy transfer donor and the second of the fluorophores is a fluorescent energy transfer acceptor.

60. The detector probe of claim 50, wherein the two chromophores are fluorophores.

61. The detector probe of claim 60, wherein the fluorophores are fluorescent energy transfer dyes or a fluorescent energy transfer pair.

62. The detector probe of claim 60, wherein the fluorophores are selected from the group consisting of fluoresceins, rhodamines, cyanines, Texas Red, rhodamine B, and tetramethylrhodamine.

63. The detector probe of claim 50, wherein the two chromophores interact with one another to quench fluorescence when the detector probe is in the stacked configuration, the two chromophores exhibit increased fluorescence when the detector probe is in the spaced configuration, and the distance between the two chromophores is changed upon recognition of the peptide epitope by the protein.

64. The detector probe of claim 50, wherein the detector probe is in the stacked configuration when the two chromophores dimerize with each other.

65. A detector probe comprising:

an amino acid sequence labeled with two chromophores, each chromophore attached proximate to an end of the amino acid sequence, wherein the detector probe comprises a first configuration that exhibits fluorescence quenching and a second configuration that exhibits fluorescence.

66. A detector probe comprising:

an amino acid sequence labeled with two chromophores, each chromophore attached proximate to an end of the amino acid sequence, wherein the two chromophores dimerize to form a configuration that exhibits fluorescence quenching and wherein a distance between the two chromophores increases to form a configuration that exhibits fluorescence.

67. A detector probe comprising:

an amino acid sequence labeled with two chromophores, each chromophore attached to an end of the amino acid sequence, wherein the detector probe is capable of changing conformations from a first conformation that exhibits fluorescence quenching to a second conformation that exhibits fluorescence.

68. A method for detecting an analyte in a sample, comprising:

providing a detector probe comprising an amino acid sequence labeled with two chromophores, each chromophore attached to an end of the amino acid sequence, wherein the detector probe is capable of changing conformations from a first conformation that exhibits fluorescence quenching to a second conformation that exhibits fluorescence;

adding the sample potentially containing the analyte to the detector probe, the conformation of the detector probe changing from the first conformation to the second conformation upon recognition between the detector probe and the analyte; and

detecting the presence or absence of analyte by measuring a change in fluorescence in the sample.

69. A system for detecting an analyte comprising:

a probe comprising an amino acid sequence and two chromophores attached proximate the 3′ and

5′

ends of the nucleic acid sequence, at least one of the two chromophores comprising a fluorophore, wherein the two chromophores interact with one another to quench fluorescence when the probe is in a stacked conformation, the pair of chromophores exhibit increased fluorescence when the probe is in a spaced conformation, and a distance between the pair of chromophores is changed upon recognition between the detector probe and the analyte.

70. A probe capable of recognizing an analyte, said probe comprising:

a linker comprising a first end and a second end; and

two chromophores, one of the chromophore attached proximate the first end of the linker and the second of the chromophores attached proximate the second end of the linker, at least one of the two chromophores comprising a fluorophore, wherein the two chromophores interact with one another to quench fluorescence when the probe is in a first conformation, the two chromophores exhibit increased fluorescence when the probe is in a second conformation, and the distance between the two chromophores is changed upon recognition of the analyte by the linker.

71. The probe of claim 70, wherein the linker is a peptide sequence, a DNA sequence, an RNA sequence, or an organic molecule.

72. The probe of claim 70, wherein the linker is a sufficient length to fold and move between the first conformation that exhibits fluorescence quenching and the second conformation that exhibits fluorescence.

73. The probe of claim 70, wherein the linker is immobilized on a solid support.

74. The probe of claim 70, wherein the solid support is a waveguide.

75. The probe of claim 70, wherein the analyte is selected from the group consisting of an antigen, an amino acid sequence, an organic molecule, an epitope, an epitope mimic, and a nucleotide sequence.

76. A system for detecting an analyte, comprising:

a probe comprising a linker and two chromophores attached proximate each end of the linker, at least one of the two chromophores comprising a fluorophore, wherein the two chromophores interact with one another to quench fluorescence when the probe is in a first conformation, the pair of chromophores exhibit increased fluorescence when the probe is in a second conformation, and a distance between the pair of chromophores is changed upon recognition between the and the analyte.

77. The system of claim 76, wherein the linker is a peptide sequence, a DNA sequence, an RNA sequence, or an organic molecule.

78. The system of claim 76, wherein the linker is a sufficient length to fold and move between the first conformation that exhibits fluorescence quenching and the second conformation that exhibits fluorescence.

79. The system of claim 76, wherein the analyte is selected from the group consisting of an antigen, an amino acid sequence, an organic molecule, an epitope, an epitope mimic, and a nucleotide sequence.

80. A system for detecting multiple analytes, comprising:

a plurality of probes, each probe comprising a linker and two chromophores attached proximate each end of the linker, the plurality of probes having different linkers and chromophores, at least one of the two chromophores comprising a fluorophore, wherein the two chromophores interact with one another to quench fluorescence when the probe is in a first conformation, the pair of chromophores exhibit increased fluorescence when the probe is in a second conformation, and a distance between the pair of chromophores is changed upon recognition between the and the analyte.