Detectably labeled dual conformation oligonucleotide probes, assays and kits

• US 5,925,517 A
• Filed: 05/12/1995
• Issued: 07/20/1999
• Est. Priority Date: 11/12/1993
• Status: Expired due to Term

• Current Assignee: The Public Health Research Institute of the City of New York, Inc.
• Original Assignee: The Public Health Research Institute of the City of New York, Inc.
• Inventors: Kramer, Fred R., Tyagi, Sanjay, Lizardi, Paul M.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/439,819
• Time in Patent Office: 1,530 Days
• Field of Search: 536/24.3, 435/6
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

Unimolecular and bimolecular hybridization probes for the detection of nucleic acid target sequences comprise a target complement sequence, an affinity pair holding the probe in a closed conformation in the absence of target sequence, and either a label pair that interacts when the probe is in the closed conformation or, for certain unimolecular probes, a non-interactive label. Hybridization of the target and target complement sequences shifts the probe to an open conformation. The shift is detectable due to reduced interaction of the label pair or by detecting a signal from a non-interactive label. Certain unimolecular probes can discriminate between target and non-target sequences differing by as little as one nucleotide. Also, universal stems and kits useful for constructing said probes. Also, assays utilizing said probes and kits for performing such assays.

Claims

1. A signaling unitary hybridization probe useful in an assay having conditions that include a detection temperature for detecting at least one nucleic acid strand containing a preselected nucleic acid target sequence, said probe comprising:

a single-stranded target complement sequence having from 10 to about 140 nucleotides, having a 5'"'"' terminus and a 3'"'"' terminus, and being complementary to the target sequence;

flanking the target complement sequence, a pair of oligonucleotide arms consisting of a 5'"'"' arm sequence covalently linked to said 5'"'"' terminus and a 3'"'"' arm sequence covalently linked to said 3'"'"' terminus, said pair of oligonucleotide arms forming a stem duplex 3-25 nucleotides in length, said stem duplex having a melting temperature above said detection temperature under said assay conditions; and

at least one interacting label pair, each pair comprising a first label moiety conjugated to the 5'"'"' arm sequence and a second label moiety conjugated to the 3'"'"'arm sequence,

said probe having, under said assay conditions in the absence of said target sequence, a characteristic signal whose level is a function of the degree of interaction of said first and second label moieties, said signal having a first level at 10°

C. below said melting temperature, a second level at 10°

C. above said melting temperature and a third level at said detection temperature,

wherein under the assay conditions at the detection temperature and in the presence of an excess of said target sequence, hybridization of the target complement sequence to the target sequence alters the level of said characteristic signal from said third level toward the second level by an amount of at least ten percent of the difference between the first and second levels.

2. A probe according to claim 1 wherein said unitary probe is a unimolecular probe.

3. A probe according to claim 1 wherein said unitary probe is a bimolecular probe consisting of a first molecule containing approximately half of said target complement sequence including said 5'"'"' terminus, the 5'"'"' arm sequence and the first label moiety;

and a second molecule containing approximately half of said target complement sequence including said 3'"'"' terminus, the 3'"'"' arm sequence and the second label moiety.

4. A probe according to claim 1 wherein said at least one label pair is a FRET pair.

5. A probe according to claim 1 wherein said melting temperature is at least 5°

C. above said detection temperature.

6. A probe according to claim 5 wherein said at least one label pair is a FRET pair.

7. A unimolecular probe according to claim 6.

8. A unimolecular probe according to claim 5, wherein said first arm sequence has 3-15 nucleotides immediately adjacent to said 5'"'"' terminus that are complementary to nucleotides of said second arm sequence immediately adjacent to said 3'"'"' terminus.

9. A probe according to claim 6 wherein said first and second label moieties are covalently linked into said unitary probe.

10. A unimolecular probe according to claim 9.

11. A probe according to claim 6 wherein said melting temperature is at least 10°

C. above said detection temperature.

12. A unimolecular probe according to claim 11.

13. A probe according to claim 6 wherein said FRET pair is a fluorescer and a quencher.

14. A unimolecular probe according to claim 13.

15. A probe according to claim 14 wherein said FRET pair is EDANS and DABCYL.

16. A probe according to claim 1 wherein at least one of said 5'"'"' and 3'"'"' arm sequences is at least partially complementary to the nucleic acid strand containing the target sequence.

17. A unimolecular probe according to claim 16.

18. A probe according to claim 1 wherein each of said 5'"'"' and 3'"'"' arm sequences contains a pair of complementary sequences capable of forming a hairpin having a stem of at least three nucleotides.

19. A unimolecular probe according to claim 18.

20. A probe according to claim 1 wherein the probe sequences are selected from the group consisting of DNA, RNA, and mixtures of DNA and RNA.

21. A probe according to claim 1 wherein said first and second label moieties are covalently linked into said unitary probe.

22. A probe according to claim 21 wherein said first label moiety is linked through an alkyl spacer to said 5'"'"' arm sequence and said second label moiety is linked through an alkyl spacer to said 3'"'"' arm sequence.

23. A unimolecular probe according to claim 22.

24. A probe according to claim 1 tethered to a solid surface.

25. A unimolecular probe according to claim 24.

26. A probe according to claim 1 wherein said melting temperature is at least 5°

C. above said detection temperature, wherein said 5'"'"' and 3'"'"' arm sequences are covalently linked directly to said target complement sequence, wherein said first and second label moieties are covalently linked to said 5'"'"' and 3'"'"' arm sequences, and said at least one label pair is a fluorescer and a quencher.

27. A unimolecular probe according to claim 26.

28. A probe according to claim 26 tethered to a solid support.

29. A unimolecular probe according to claim 28.

30. A probe according to claim 26 wherein said target complement sequence has from 20 to 60 nucleotides.

31. A unimolecular probe according to claim 30.

32. A probe according to claim 31 tethered to a solid support.

46. An assay for detecting in a sample the presence of at least one nucleic acid strand containing a preselected target sequence, said assay having conditions including a detection temperature, comprising the steps of:

contacting said sample with a unitary probe according to claim 1, and

ascertaining any change in the level of said characteristic signal at said detection temperature.

47. An assay according to claim 46 wherein said step of ascertaining comprises measuring.

48. A real-time assay according to claim 47.

49. A real-time assay according to claim 46.

50. An assay according to claim 47, wherein said step of ascertaining comprises measuring as a function of time.

51. An assay according to claim 47 further comprising a step of adding to a target-less control the probe according to claim 1 measuring any change in said level for said control, wherein said step of ascertaining includes calculating a difference between the change in level for said control and the change in level for said sample.

52. An assay according to claim 46 further comprising a step of adding to a target-less control the probe according to claim 1.

53. An assay according to claim 46 wherein said probe is a bimolecular probe, and the assay is performed entirely below the melting temperature of said probe.

54. An assay according to claim 46 wherein the melting temperature of said probe is at least 5°

C. above said detection temperature, wherein said 5'"'"' and 3'"'"' arm sequences are covalently linked directly to said target complement sequence, wherein said first and second label moieties are covalently linked to said 5'"'"' and 3'"'"' arm sequences, and said at least one label pair is a fluorescer and a quencher.

55. A real-time assay according to claim 54.

56. An assay according to claim 54 wherein said probe is a bimolecular probe, and the assay is performed entirely below the melting temperature of said probe.

57. An assay according to claim 46 wherein the step of ascertaining is quantitative.

58. An assay according to claim 46 wherein said probe is a unimolecular probe.

59. An assay according to claim 58 wherein the step of ascertaining is quantitative.

60. An assay according to claim 58 further comprising a step of adding to a target-less control said unimolecular probe.

61. An assay according to claim 58 wherein the melting temperature of said probe is at least 5°

C. above the detection temperature, wherein said 5'"'"' and 3'"'"' arm sequences are covalently linked directly to said target complement sequence, wherein said first and second label moieties are covalently linked to said 5'"'"' and 3'"'"' arm sequences, and said at least one label pair is a fluorescer and a quencher.

62. An assay according to claim 58 wherein said nucleic acid target sequence is a product of an amplification reaction, and wherein said step of adding said probe to a sample precedes completion of said amplification reaction.

63. An assay according to claim 62 further comprising a step of adding said probe to a negative control, which step precedes completion of said amplification reaction on said negative control.

64. A real-time assay according to claim 63.

65. An assay according to claim 64 wherein said step of ascertaining includes calculating a difference between the change in level for said control and the change in level for said sample.

66. An assay according to claim 62 wherein said step of ascertaining includes measuring repeatedly over time during said amplification reaction.

67. An assay according to claim 66 wherein the melting temperature of said probe is at least 5°

C. above the detection temperature, wherein said 5'"'"' and 3'"'"' arm sequences are covalently linked directly to said target complement sequence, wherein said first and second label moieties are covalently linked to said 5'"'"' and 3'"'"' arm sequences, and said at least one label pair is a fluorescer and a quencher.

68. An assay according to claim 62 wherein said amplification reaction is amplification of an RNA reporter by an RNA polymerase.

69. An assay according to claim 62 wherein said amplification reaction is a PCR reaction.

70. An assay according to claim 62 wherein said amplification reaction is an SDA reaction.

71. An assay according to claim 62 wherein said label pair is a FRET pair.

72. An assay according to claim 58 wherein said probe is tethered to a solid surface.

73. An assay according to claim 72 including at least one additional tethered probe according to this invention having a different target complement sequence and linked to the same support surface at a predetermined location.

79. A kit comprising a probe according to claim 1 and instructions for performing an assay for detecting at least one nucleic acid strand containing a preselected target sequence, said instructions including;

a) assay conditions, including a detection temperature,

b) a step of adding said probe, and

c) a step of ascertaining any change in the probe'"'"'s characteristic signal at said detection temperature.

80. An amplification kit according to claim 79 wherein the probe is unimolecular.

81. A kit according to claim 80 further including one or more reagents selected from the group consisting of salts, buffers, nuclease inhibitors, restriction enzymes and denaturants.

82. A kit according to claim 79 wherein said instructions include an amplification step and said kit includes one or more components selected from the group consisting of primers, nucleotides, polymerases and polymerase templates.

83. A kit according to claim 79 including one or more components suitable for performing a vital stain assay, said components being selected from the group consisting of primers, nucleotides, polymerases and polymerase templates.

84. A kit according to claim 79 including one or more components suitable for performing an in situ assay, said components being selected from the group consisting of permeabilizing agents, liposome precursors and counterstains.

85. A kit according to claim 79 wherein said probe is a tethered probe.

86. A kit according to claim 85 wherein said probe is unimolecular.

87. A kit according to claim 86 including at least one additional tethered probe according to this invention having a different target complement sequence and linked to the same support surface at a predetermined location.

88. A kit according to claim 86 including a positive control probe according to this invention.

107. A probe according to claim 10 wherein said target complement sequence has 15-35 nucleotides and said first arm sequence has 5-8 nucleotides immediately adjacent to said 5'"'"' terminus that are complementary to nucleotides of said second arm sequence immediately adjacent to said 3'"'"' terminus.

33. A unitary hybridization probe useful in an assay for the detection of at least one nucleic acid target containing a preselected target sequence, said probe being capable of assuming a closed conformation and an open conformation and comprising:

a) a target complement sequence of from 10 to about 140 nucleotides complementary to said preselected nucleic acid target sequence, having a 5'"'"' terminus and a 3'"'"' terminus,

b) an affinity pair comprising a first affinity moiety covalently linked to said 5'"'"' terminus and a second affinity moiety covalently linked to said 3'"'"' terminus, said affinity pair interacting sufficiently to hold said probe in the closed conformation in said assay in the absence of said nucleic acid target, and

c) a label pair comprising a first label moiety conjugated to said first affinity moiety and a second label moiety conjugated to said second affinity moiety, said label moieties interacting to affect a measurable characteristic of at least one of them when said probe is in the closed conformation,

wherein hybridization of said target complement sequence to said target sequence in said assay causes said probe to assume its open conformation, in which said label moieties do not so interact.

34. A probe according to claim 33 wherein said unitary probe is a unimolecular probe.

35. A probe according to claim 33 wherein said unitary probe is a bimolecular probe consisting of a first molecule containing approximately half of said target complement sequence including said 5'"'"' terminus, the first affinity moiety and the first label moiety;

and a second molecule containing approximately half of said target complement sequence including said 3'"'"' terminus, the second affinity moiety and the second label moiety.

36. A probe according to claim 33 wherein said first label moiety is conjugated to said first affinity moiety and said second label moiety is conjugated to said second affinity moiety.

37. A unimolecular probe according to claim 36.

38. A probe according to claim 33 wherein said affinity pair comprises complementary oligonucleotide arm sequences 3 to 25 nucleotides in length.

39. A probe according to claim 38 wherein said first label moiety is covalently linked to said first affinity moiety and said second label moiety is covalently linked to said second affinity moiety.

40. A unimolecular probe according to claim 39.

41. A probe according to claim 36 wherein said label pair comprises a FRET pair.

42. A unimolecular probe according to claim 41.

43. A probe according to claim 33 wherein said affinity pair comprises an antibody and an antigen.

44. A probe according to claim 33 tethered to a solid surface.

45. A unimolecular probe according to claim 44.

74. An assay for detecting at least one nucleic acid target containing a preselected target sequence comprising the steps of:

adding to a sample a unitary probe according to claim 36, and

ascertaining any change in the level of said measurable characteristic.

75. An assay according to claim 74 wherein said step of ascertaining comprises measuring.

76. An assay according to claim 75 wherein said measuring comprises visually comparing to a standard.

77. An assay according to claim 74 wherein said nucleic acid target is a product of an amplification reaction, and wherein said probe is unimolecular.

78. An assay according to claim 77 wherein said step of adding said probe precedes completion of said amplification reaction.

89. A labeled, single-stranded probe for detecting the presence or absence in a sample of a preselected nucleic acid target sequence in an assay having conditions including a detection temperature, said probe comprising:

(a) an oligonucleotide sequence comprising

(i) a target complement sequence perfectly complementary to the target sequence, said target complement sequence having 7 to 25 nucleotides, a 5'"'"' terminus, and a 3'"'"' terminus,

(ii) a first arm sequence covalently linked to said 5'"'"' terminus and a second arm sequence covalently linked to said 3'"'"' terminus, said first arm sequence having 3 to 8 nucleotides immediately adjacent to said 5'"'"' terminus that are complementary to nucleotides of said second arm sequence immediately adjacent said 3'"'"' terminus, said arms forming a stem duplex having a melting temperature at least 5°

C. above said detection temperature under said assay conditions, said oligonucleotide sequence, when oppositely terminally labeled with a fluorescer/quencher pair producing under said conditions upon addition of excess model target, an increase in fluorescence that is at least 10 percent as great as the increase in fluorescence caused by heating it from 10°

C. below to 10°

C. above said melting temperature; and

(b) at least one detectable label,

wherein said probe produces a detectable signal above background in said assay of a sample containing an excess of said target sequence that is at least five times higher above background than in said assay of a sample containing an excess of a sequence differing from said target sequence by a single internally located nucleotide.

90. The probe according to claim 89 wherein said label is a non-interactive label selected from the group consisting of radioisotopes, enzymes, fluorophores and luminescent moieties.

91. A probe according to claim 89 wherein said at least one label is at least one interactive label pair, one member of which is conjugated to said first arm sequence and the other member of which is conjugated to said second arm sequence, said members interacting when said arms form said stem duplex, and wherein said probe produces a detectable signal above background in said assay of a sample containing an excess of said target sequence that is at least ten times greater above background than in said assay of a sample containing an excess of a sequence differing from said target sequence by a single internally located nucleotide.

92. The probe according to claim 91 wherein said interactive label pair is a fluorescer and a quencher.

93. The probe according to claim 92 wherein said label pair is DABCYL and EDANS.

94. An assay for detecting the presence or absence in a sample of a preselected nucleic acid target sequence, said assay having conditions including a detection temperature, comprising the steps of

a) incubating said sample with a probe that is a probe according to claim 91 under said assay conditions, and

b) detecting whether or not adding said sample resulted in an increase in said signal at said detection temperature.

95. An assay according to claim 94 wherein said interactive label pair is a fluorescer and a quencher.

96. An assay according to claim 95 wherein said label pair is DABCYL and EDANS.

97. An assay for detecting the presence or absence in a sample of nucleic acid strands containing a preselected nucleic acid target sequence, said assay having conditions including a detection temperature, comprising the steps of

a) incubating said sample with at least one capture probe complementary to said strands but not to said preselected target sequence,

b) binding said at least one capture probe to a solid surface,

c) incubating said sample with a probe that is a probe according to claim 90 under said assay conditions,

d) after completion of steps a, b and c, washing said solid surface, and

e) detecting for said signal at said detection temperature.

98. A kit comprising

a) instructions for performing an assay, and

b) a probe that is a probe according to claim 89 under conditions of said assay.

99. A kit according to claim 98 wherein said instructions are for performing an amplification assay and wherein said kit includes one or more components selected from the group consisting of primers, nucleotides, polymerases and polymerase templates.

100. An assay according to claim 94 wherein said assay includes an amplification reaction and said nucleic acid target sequence is a product of said amplification reaction.

101. An assay according to claim 100 wherein the amplification is a PCR reaction.

102. An assay according to claim 101 wherein said probe is incubated with said sample during amplification.

103. An assay according to claim 100 wherein said probe is incubated with said sample during amplification.

104. An assay according to claim 103 wherein said interactive label pair is a fluorescer and a quencher.

105. A kit according to claim 98 wherein said probe contains an interactive label pair, one member of which is conjugated to said first arm sequence and the other member of which is conjugated to said second arm sequence, said members interacting when said arms form a stem duplex.

106. A kit according to claim 105 wherein said interactive label pair is a fluorescer and a quencher.

108. The probe according to claim 91 wherein said at least one label pair is a FRET pair.

109. The probe according to claim 91 wherein said one member is covalently linked to said first arm sequence and said other member is covalently linked to said second arm sequence.

110. The probe according to claim 109 wherein said one member is covalently linked to said first arm sequence through an alkyl spacer and said other member is covalently linked to said second arm sequence through an alkyl spacer.

111. The probe according to claim 91 wherein said melting temperature is at least 10°

C. above said detection temperature.

114. A probe according to claim 92 wherein said probe produces a detectable signal above background in said assay of a sample containing an excess of said target sequence that is at least twenty times higher above background than in said assay of a sample containing an excess of a sequence differing from said target sequence by a single internally located nucleotide.

115. An assay according to claim 95 wherein said probe produces a detectable signal above background in said assay of a sample containing an excess of said target sequence that is at least twenty times higher above background than in said assay of a sample containing an excess of a sequence differing from said target sequence by a single internally located nucleotide.

116. An assay according to claim 115 wherein at least one additional probe according to claim 91 complementary to a second preselected nucleic acid target sequence is added in step a).

117. An assay according to claim 103 wherein said probe produces a detectable signal above background in said assay of a sample containing an excess of said target sequence that is at least twenty times higher above background than in said assay of a sample containing an excess of a sequence differing from said target sequence by a single internally located nucleotide.

118. An assay according to claim 94 wherein the melting temperature of the probe'"'"'s stem duplex is at least 10°

C. above the detection temperature.

119. A kit according to claim 105 wherein said probe produces a detectable signal above background in said assay of a sample containing an excess of said target sequence that is at least twenty times higher above background than in said assay of a sample containing an excess of a sequence differing from said target sequence by a single internally located nucleotide.

112. A signaling unimolecular hybridization probe useful in an assay having conditions that include a detection temperature for detecting at least one nucleic acid strand containing a preselected nucleic acid target sequence, said probe comprising:

a single-stranded target complement sequence having from 10 to 60 nucleotides, have a 5'"'"' terminus and a 3'"'"', terminus, and being complementary to the target sequence;

flanking the target complement sequence, a pair of oligonucleotide arms consisting of a 5'"'"' arm sequence covalently linked to said 5'"'"' terminus and a 3'"'"' arm sequence covalently linked to said 3'"'"' terminus, said pair of oligonucleotide arms forming a stem duplex 3-25 nucleotides in length, said stem duplex having a melting temperature at least 5°

C. above said detection temperature under said assay conditions; and

at least interacting label pair covalently linked to said target complement sequence,

said probe having, under said assay conditions in the absence of said target sequence, a characteristic fluorescence signal whose level is a function of the degree of interaction of said label pair, said signal having a first level at 10°

C. below said melting temperature, a second level at 10°

C. above said melting temperature and a third level at said detection temperature,

wherein under the assay conditions at the detection temperature and in the presence of an excess of said target sequence, hybridization of the target complement sequence to the target sequence alters the level of said characteristic signal from said third level toward said second level by an amount of at least ten percent of the difference between the first and second levels.

113. The probe according to claim 112 wherein said label pair comprises a fluorescer and a quencher.