Method for determining the presence of an RNA analyte in a sample using a modified oligonucleotide probe

• US 7,070,925 B1
• Filed: 05/05/2000
• Issued: 07/04/2006
• Est. Priority Date: 07/16/1996
• Status: Expired due to Term

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Brentano, Steven T., Becker, Michael M., Majlessi, Mehrdad
• Primary Examiner(s): TUNG, JOYCE
• Application Number:
  US09/565,427
• Time in Patent Office: 2,251 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 536/24.31, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/6.1
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

The present invention concerns oligonucleotides containing one or more modified nucleotides which increase the binding affinity of the oligonucleotides to target nucleic acids having a complementary nucleotide base sequence. These modified oligonucleotides hybridize to the target sequence at a faster rate than unmodified oligonucleotides having an identical nucleotide base sequence. Such modified oligonucleotides include oligonucleotides containing at least one 2′-O-methylribofuranosyl moiety joined to a nitrogenous base. Oligonucleotides can be modified in accordance with the present invention to preferentially bind RNA targets. The present invention also concerns methods of using these modified oligonucleotides and kits containing the same.

Claims

1. A diagnostic method for the specific detection of an RNA analyte in a sample, said method comprising:

a) processing a sample to allow for or enhance discrimination between an RNA analyte and non-analyte nucleic acids present in said sample;

b) contacting said sample with an oligonucleotide probe having a detectable label under conditions such that a first base region of said probe stably and specifically hybridizes to a second base region of said RNA analyte to form a probe;

analyte hybrid containing at least one ribonucleotide modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety, wherein said probe does not stably hybridize under said conditions to non-analyte nucleic acids present in said sample, and wherein said probe is free in solution when contacted with said sample; and

c) determining whether said hybrid is present in said sample as an indication of the presence or absence of said RNA analyte in said sample.

2. The method of claim 1, wherein said first base region contained within said hybrid includes a cluster of at least 4 ribonucleotides modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety.

3. The method of claim 1, wherein said first base region contained within said hybrid includes at least one nucleotide which is not a ribonucleotide modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety.

4. The method of claim 1, wherein each nucleotide of said probe is a ribonucleotide modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety.

5. The method of claim 1, wherein said probe includes a conjugate molecule.

6. The method of claim 2, wherein said probe includes a conjugate molecule joined to said probe at a site located within the cluster of said first base region.

7. The method of claim 1, wherein said probe is between 10 and 100 bases in length.

8. The method of claim 1, wherein said probe is between 10 and 16 bases in length.

9. The method of claim 1, wherein said label is a chemiluminescent or a fluorescent agent.

10. The method of claim 1, wherein said RNA analyte consists of ribosomal RNA.

11. The method of claim 1, wherein said sample contains DNA.

12. The method of claim 1, wherein said second base sequence is contained in a product of an amplification procedure.

13. The method of claim 12, wherein said amplification procedure is a transcription-based method of amplification.

14. The method of claim 1 further comprising the step of quantifying said target organism or virus determined to be present in said sample.

15. The method of claim 12 further comprising the step of quantifying said RNA analyte determined to be present in said sample.

16. The method of claim 1, wherein said RNA analyte is directly or indirectly immobilized by a solid support in said processing step.

17. The method of claim 1 further comprising the step of providing to said sample a nuclease inhibitor other than a polynucleotide modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety of a ribonucleotide.

18. The method of claim 1, wherein said sample is obtained from a clinical source, an environmental source or a food source.

19. The method of claim 1, wherein said determining step provides an indication of the presence or absence of a particular species of microorganisms in said sample.

20. The method of claim 1, wherein said determining step provides an indication of the presence or absence of a virus in said sample.

21. The method of claim 20, wherein said sample contains chromosomal DNA.

22. The method of claim 1, wherein said sample is obtained from a human.

23. The method of claim 1, wherein said RNA analyte consists of mRNA.

24. The method of claim 1, wherein said RNA analyte consists of tRNA.

25. The method of claim 1 further comprising contacting said sample with one or more helper probes.

26. The method of claim 1, wherein the rate of hybridization of said probe to said second base region is greater than the rate of hybridization of said probe containing an unmodified form of said first base region having the same base sequence and length to said second base region under identical nucleic acid conditions.

27. The method of claim 26, wherein the unmodified form of said first base region consists of deoxynucleotides.

28. The method of claim 27, wherein said probe hybridizes to said second base region at least about 1.6-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

29. The method of claim 27, wherein said probe hybridizes to said second base region at least about 2.0-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

30. The method of claim 27, wherein said probe hybridizes to said second base region at least about 2.3-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

31. The method of claim 27, wherein said probe hybridizes to said second base region at least about 2.6-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

32. The method of claim 27, herein said probe hybridizes to said second base region at least about 2.9-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

33. The method of claim 27, wherein said probe hybridizes to said second base region at least about 3.1-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

34. The method of claim 1, wherein said contacting step is not followed by the addition of a nuclease to said sample.

35. The method of claim 1, wherein said first base region targets a double-stranded region contained within said second base region.

36. The method of claim 1 further comprising providing to said sample a detergent.

37. The method of claim 36, wherein said first base region targets a double-stranded region contained within said second base region.

107. The method of claim 1, wherein said probe:

analyte hybrid forms in solution, provided said RNA analyte is present in said sample.

38. A diagnostic method for the specific detection of an RNA analyte in a sample, said method comprising:

a) contacting a sample with an oligonucletide probe having a detectable label in the presence of a detergent under conditions such that a first base region of said probe stably and specifically hybridizes to a second base region of an RNA analyte to form a probe;

analyte hybrid containing at least one ribonucleotide modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety, wherein said probe does not stably hybridize under said conditions to non-analyte nucleic acids present in said sample; and

b) determining whether said hybrid is present in said sample as an indication of the presence or absence of said RNA analyte in said sample.

39. The method of claim 38, wherein said first base region contained within said hybrid includes a cluster of at least 4 ribonucleotides modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety.

40. The method of claim 38, wherein said first base region contained within said hybrid includes at least one nucleotide which is not a ribonucleolide modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety.

41. The method of claim 38, wherein each nucleotide of said probe is a ribonucleotide modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety.

42. The method of claim 38, wherein said probe includes a conjugate molecule.

43. The method of claim 39, wherein said probe includes a conjugate molecule joined to said probe at a site located within the cluster of said first base region.

44. The method of claim 38, wherein said probe is between 10 and 100 bases in length.

45. The method of claim 38, wherein said probe is between 10 and 16 bases in length.

46. The method of claim 38, wherein said label is a chemiluminescent or a fluorescent agent.

47. The method of claim 38, wherein said RNA analyte consists of ribosomal RNA.

48. The method of claim 38, wherein said sample contains DNA.

49. The method of claim 38, wherein said second base sequence is contained in a product of an amplification procedure.

50. The method of claim 49, wherein said amplification procedure is a transcription-based method of amplification.

51. The method of claim 38 further comprising the step of quantifying said RNA analyte determined to be present in said sample.

52. The method of claim 49 further comprising the step of quantifying said RNA analyte determined to be present in said sample.

53. The method of claim 38, wherein at least one of said probe and said RNA analyte is directly or indirectly immobilized by a solid support.

54. The method of claim 38 further comprising the step of providing to said sample a nuclease inhibitor other than a polynucleotide modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety of a ribonucleotide.

55. The method of claim 38, wherein said sample is obtained from a clinical source, an environmental source or a food source.

56. The method of claim 38, wherein said determining step provides an indication of the presence or absence of a particular species of microorganisms in said sample.

57. The method of claim 38, wherein said determining step provides an indication of the presence or absence of a virus in said sample.

58. The method of claim 57, wherein said sample contains chromosomal DNA.

59. The method of claim 38, wherein said sample is obtained from a human.

60. The method of claim 38, wherein said RNA analyte consists of mRNA.

61. The method of claim 38, wherein said RNA analyte consists of tRNA.

62. The method of claim 38 further comprising contacting said sample with one or more helper probes.

63. The method of claim 38, wherein the rate of hybridization of said probe to said second base region is greater than the rate of hybridization of said probe containing an unmodified form of said first base region having the same base sequence and length to said second base region under identical nucleic acid conditions.

64. The method of claim 63, wherein the unmodified form of said first base region consists of deoxynucleotides.

65. The method of claim 64, wherein said probe hybridizes to said second base region at least about 1.6-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

66. The method of claim 64, wherein said probe hybridizes to said second base region at least about 2.0-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

67. The method of claim 64, wherein said probe hybridizes to said second base region at least about 2.3-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

68. The method of claim 64, wherein said probe hybridizes to said second base region at least about 2.6-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

69. The method of claim 64, wherein said probe hybridizes to said second base region at least about 2.9-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

70. The method of claim 64, wherein said probe hybridizes to said second base region at least about 3.1-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

71. The method of claim 38, wherein said contacting step is not followed by the addition of a nuclease to said sample.

72. The method of claim 38, wherein said probe is free in solution during said contacting step.

108. The method of claim 72, wherein said probe:

analyte hybrid forms in solution, provided said RNA analyte is present in said sample.

73. A diagnostic method for the specific detection of an RNA analyte in a sample, said method comprising:

a) contacting a sample with an oligonucleotide probe having a detectable label under conditions such that a first base region of said probe stably and specifically hybridizes to a second base region present in an RNA analyte to form a probe;

analyte hybrid containing at least one ribonucleotide modified to include a 240 -O-methyl substitution to the ribofuranosyl moiety, said first base region targeting a double-stranded region contained within said second base region, wherein said probe does not stably hybridize under said conditions to non-analyte nucleic acids present in said sample, and wherein said probe is free in solution when contacted with said sample; and

b) determining whether said hybrid is present in said sample as an indication of the presence or absence of said RNA analyte in said sample.

74. The method of claim 73, wherein said first base region contained within said hybrid includes a cluster of at least 4 ribonucleotides modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety.

75. The method of claim 73, wherein said first base region contained within said hybrid includes at least one nucleotide which is not a ribonucleotide modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety.

76. The method of claim 73, wherein each nucleotide of said probe is a ribonucleotide modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety.

77. The method of claim 73, wherein said probe includes a conjugate molecule.

78. The method of claim 74, wherein said probe includes a conjugate molecule joined to said probe at a site located within the cluster of said first base region.

79. The method of claim 73, wherein said probe is between 10 and 100 bases in length.

80. The method of claim 73, wherein said probe is between 10 and 16 bases in length.

81. The method of claim 73, wherein said label is a chemiluminescent or a fluorescent agent.

82. The method of claim 73, wherein said RNA analyte consists of ribosomal RNA.

83. The method of claim 73, wherein said sample contains DNA.

84. The method of claim 73, wherein said second base sequence is contained in a product of an amplification procedure.

85. The method of claim 84, wherein said amplification procedure is a transcription-based method of amplification.

86. The method of claim 73 further comprising the step of quantifying said RNA analyte determined to be present in said sample.

87. The method of claim 84 further comprising the step of quantifying said RNA analyte determined to be present in said sample.

88. The method of claim 73, wherein at least one of said probe and said RNA analyte is directly or indirectly immobilized by a solid support.

89. The method of claim 73 further comprising the step of providing to said sample a nuclease inhibitor other than a polynucleotide modified to include a 2′

-O-methyl substitution to the ribofuranosyl moiety of a ribonucleotide.

90. The method of claim 73, wherein said sample is obtained from a clinical source, an environmental source or a food source.

91. The method of claim 73, wherein said determining step provides an indication of the presence or absence of a particular species of microorganisms in said sample.

92. The method of claim 73, wherein said determining step provides an indication of the presence or absence of a virus in said sample.

93. The method of claim 92, wherein said sample contains chromosomal DNA.

94. The method of claim 73, wherein said sample is obtained from a human.

95. The method of claim 73, wherein said RNA analyte consists of mRNA.

96. The method of claim 73, wherein said RNA analyte consists of tRNA.

97. The method of claim 73 further comprising contacting said sample with one or more helper probes.

98. The method of claim 73, wherein the rate of hybridization of said probe to said second base region is greater than the rate of hybridization of said probe containing an unmodified form of said first base region having the same base sequence and length to said second base region under identical nucleic acid conditions.

99. The method of claim 98, wherein the unmodified form of said first base region consists of deoxynucleotides.

100. The method of claim 99, wherein said probe hybridizes to said second base region at least about 1.6-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

101. The method of claim 99, wherein said probe hybridizes to said second base region at least about 2.0-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

102. The method of claim 99, wherein said probe hybridizes to said second base region at least about 2.3-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

103. The method of claim 99, wherein said probe hybridizes to said second base region at least about 2.6-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

104. The method of claim 99, wherein said probe hybridizes to said second base region at least about 2.9-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

105. The method of claim 99, wherein said probe hybridizes to said second base region at least about 3.1-fold faster than said probe containing the unmodified form of said first base region does under said conditions.

106. The method of claim 73, wherein said contacting step is not followed by the addition of a nuclease to said sample.

109. The method of claim 73, wherein said probe:

analyte hybrid forms in solution, provided said RNA analyte is present in said sample.