Methods, kits and compositions pertaining to linear beacons

• US 6,485,901 B1
• Filed: 10/26/1998
• Issued: 11/26/2002
• Est. Priority Date: 10/27/1997
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Boston Probes, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Fiandaca, Mark J., Coull, James M., Gildea, Brian D., Hyldig-Nielsen, Jens J.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/179,162
• Time in Patent Office: 1,492 Days
• Field of Search: 536/23.1, 536/24.3, 435/5, 435/6, 435/810
• US Class Current: 435/5
• CPC Class Codes: C07K 14/003 Peptide-nucleic acids (PNAs)
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention is directed to methods, kits and compositions pertaining to Linear Beacons. In the absence of a target sequence, Linear Beacons facilitate efficient energy transfer between the donor and acceptor moieties linked to opposite ends of the probe. Upon hybridization of the probe to a target sequence, there is a measurable change in at least one property of at least one donor or acceptor moiety of the probe which can be used to detect, identify or quantitate the target sequence in a sample.

Claims

1. A polymer comprising at least one linked energy donor moiety and at least one linked energy acceptor moiety wherein said donor and acceptor moieties are separated by at least a portion of a probing nucleobase sequence and wherein said polymer does not form a stem and loop hairpin and is further characterized in that the efficiency of transfer of energy between said donor and acceptor moieties, when the polymer is solvated in aqueous solution, is substantially independent of at least two variables selected from the group consisting of:

a) nucleobase sequence length separating the at least one energy donor moiety from the at least one energy acceptor moiety;

b) spectral overlap of the at least one linked energy donor moiety and the at least one linked energy acceptor moiety;

c) presence or absence of magnesium in the aqueous solution; and

the

d) ionic strength of the aqueous solution.

2. The polymer of claim 1, wherein the polymer is a PNA.

19. The polymer of claim 1, wherein the probing nucleobase sequence is in the range of between 5-30 subunits in length.

20. The polymer of claim 1, wherein the probing nucleobase sequence is in the range of between 8-18 subunits in length.

21. The polymer of claim 1, wherein the probing nucleobase sequence is in the range of between 11-17 subunits in length.

22. The polymer of claim 1, wherein subunits of the probing nucleobase sequence have the formula:

wherein,

each J is the same or different and is selected from the group consisting of;

H, R¹, OR¹, SR¹, NHR¹, NR¹₂, F, Cl, Br and I;

each K is the same or different and is selected from the group consisting of;

O, S, NH and NR¹;

each R¹ is the same or different and is an alkyl group having one to five carbon atoms which may optionally contain a heteroatom or a substituted or unsubstituted aryl group;

each A is selected from the group consisting of a single bond, a group of the formula;

—

(CJ₂)_s— and

a group of the formula;

—

(CJ₂)_sC(O)—

wherein, J is defined above and each s is an integer from one to five;

each t is 1 or 2;

each u is 1 or 2; and

each L is the same or different and is independently selected from the group consisting of J, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturally occurring nucleobase analogs, other non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties, biotin and fluorescein.

23. The polymer of claim 22, wherein each subunit consists of a naturally or a non-naturally occurring nucleobase attached to the aza nitrogen of a N-[2-(aminoethyl)]glycine backbone through a methylene carbonyl linkage.

24. The polymer of claim 1, wherein the efficiency of transfer of energy between the at least one linked donor moiety and the at least one linked acceptor moiety is substantially independent of at least three of the selected variables.

25. The polymer of claim 1, wherein the efficiency of transfer of energy between the at least one linked donor moiety and the at lease one linked acceptor moiety is substantially independent of all four selected variables.

26. The polymer of claim 1, wherein the polymer is a PNA and the at least one energy acceptor moiety is linked to the C-terminus of the probing nucleobase sequence and the at least one energy donor moiety is linked to the N-terminus of the probing nucleobase sequence.

27. The polymer of claim 1, wherein the at least one energy donor moiety is a fluorophore selected from the group consisting of 5(6)-carboxyfluorescein, 5-(2′

-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), bodipy, rhodamine, Cy2, Cy3, Cy3.5, Cy5, Cy5.5 and texas red.

28. The polymer of claim 1, wherein the at least one energy acceptor moiety is 4-((-4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl).

29. The polymer of claim 1, wherein the polymer is immobilized to a support.

30. The polymer of claim 1, wherein the polymer is one component polymer of an array.

3. A method for the detection, identification or quantitation of a target sequence in a sample, said method comprising:

a) contacting the sample with a polymer comprising at least one linked energy donor moiety and at least one linked energy acceptor moiety wherein said donor and acceptor moieties are separated by at least a portion of a probing nucleobase sequence and wherein said polymer does not form a stem and loop hairpin and is further characterized in that the efficiency of transfer of energy between said donor and acceptor moieties, when the polymer is solvated in aqueous solution is, substantially independent of at least two variables selected from the group consisting of;

i) nucleobase sequence length separating the at least one energy donor moiety from the at least one energy acceptor moiety;

ii) spectral overlap of the at least one linked energy donor moiety and the at least one energy acceptor moiety;

iii) presence or absence of magnesium in the aqueous solution; and

the

iv) ionic strength of the aqueous solution; and

b) detecting, identifying or quantitating the hybridization of the polymer to the target sequence, under suitable hybridization conditions, wherein the presence, absence or amount of target sequence present in the sample is correlated with a change in detectable signal associated with at least one donor or acceptor moiety of the polymer.

4. The method of claim 3, wherein the nucleobase sequence of the polymer is a probing nucleobase sequence.

5. The method of claim 3, wherein the polymer is a PNA.

39. The method of claim 3, wherein the efficiency of transfer of energy between the at least one donor moiety and the at least one acceptor moiety of the polymer is substantially independent of at least three of the selected variables.

40. The method of claim 3, wherein the efficiency of transfer of energy between the at least one donor moiety and the at least one acceptor moiety of the polymer is substantially independent of all four selected variables.

41. The method of claim 3, wherein the target sequence is detected, identified or quantitated in a closed tube (homogeneous) assay.

42. The method of claim 41, wherein the target sequence that is detected, identified or quantitated is synthesized or amplified in a reaction occurring in the closed tube (homogeneous) assay.

43. The method of claim 42, wherein the target sequence is synthesized or amplified using a process selected from the group consisting of:

Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), Transcription-Mediated Amplification (TMA), Rolling Circle Amplification (RCA) and Q-beta replicase.

44. The method of claim 43, wherein the synthesized or amplified target sequence is measured in real-time.

45. The method of claim 43, wherein the PCR reaction is an asymmetric PCR reaction.

6. An array comprising two or more support bound polymers wherein at least one polymer of the array comprises at least one linked energy donor moiety and at least one linked energy acceptor moiety wherein said donor and acceptor moieties are separated by at least a portion of a probing nucleobase sequence and wherein said polymer does not form a stem and loop hairpin and is further characterized in that the efficiency of transfer of energy between said donor and acceptor moieties, when the polymer is solvated in aqueous solution, is substantially independent of at least two variables selected from the group consisting of:

i) nucleobase sequence length separating the at least one energy donor moiety from the at least one energy acceptor moiety;

ii) spectral overlap of the at least one linked energy donor moiety and the at least one linked energy acceptor moiety;

iii) presence or absence of magnesium in the aqueous solution; and

the

iv) ionic strength of the aqueous solution; and

wherein said polymer is suitable for detecting, identifying or quantitating a target sequence present in a sample.

7. The array of claim 6, wherein the array is suitable for regeneration by treatment with one or more regeneration catalysts selected from the group consisting of heat, nuclease enzyme and chemical denaturant.

8. A kit suitable for performing an assay which detects the presence, absence or amount of target sequence in a sample, wherein said kit comprises:

a) at least one polymer having at least one linked energy donor moiety and at least one linked energy acceptor moiety, wherein said donor and acceptor moieties are separated by at least a portion of a probing nucleobase sequence and wherein said polymer does not form a stem and loop hairpin and is further characterized in that the efficiency of transfer of energy between said donor and acceptor moieties, when the polymer is solvated in aqueous solution, is substantially independent of at least two variables selected from the group consisting of;

i) nucleobase sequence length separating the at least one energy donor moiety from the at least one energy acceptor moiety;

ii) spectral overlap of the at least one linked energy donor moiety and the at least one linked energy acceptor moiety;

iii) presence or absence of magnesium in the aqueous solution; and

the

iv) ionic strength of the aqueous solution; and

b) other reagents or compositions necessary to perform the assay.

9. The kit of claim 8, wherein one of more of the polymers of the kit have a probing nucleobase sequence that is in the range of between 11-17 subunits in length.

10. The kit of claim 8, wherein two or more polymers are labeled with independently detectable moieties.

11. The kit of claim 10, wherein the independently detectable moieties are used to independently detect, identify or quantitate at least two different target sequences which may be present in the same sample.

12. The kit of claim 8, wherein the kit is used in an in-situ hybridization assay.

13. The kit of claim 8, wherein the kit is used to detect organisms in food, beverages, water, pharmaceutical products, personal care products, dairy products or environmental samples.

14. The kit of claim 8, wherein the kit is used to test raw materials, products or processes.

15. The kit of claim 8, wherein the kit is used to examine clinical samples selected from the group consisting of:

clinical specimens, equipment, fixtures and products used to treat humans or animals.

16. The kit of claim 8, wherein the kit is used to detect a target sequence which is specific for a genetically based disease or is specific for a predisposition to a genetically based disease.

17. The kit of claim 16, wherein the kit is used to detect a target sequence associated with a disease selected from the group consisting of β

-Thalassemia, sickle cell anemia, Factor-V Leiden, cystic fibrosis and cancer related targets such as p53, p10, BRC-1 and BRC-2.

18. The kit of claim 8, wherein the kit is used to detect a target sequence in a forensic technique selected from the group consisting of:

prenatal screening, paternity testing, identity confirmation and crime investigation.

31. A polymer comprising;

a) a probing nucleobase sequence for probing a target sequence to which the probing nucleobase sequence is complementary or substantially complementary;

b) at least one energy donor moiety that is linked to the probing nucleobase sequence; and

c) at least one energy acceptor moiety that is linked to the probing nucleobase sequence wherein the at least one donor moiety is separated from the at least one acceptor moiety by at least a portion of the probing nucleobase sequence.

32. The polymer of claim 31, wherein the at least one energy donor moiety is linked to one of a first or second end of the probing nucleobase sequence and the at least one energy acceptor moiety is linked to the other one of the first or the second end of the probing nucleobase sequence.

33. The polymer of claim 32, wherein the polymer is a PNA and the at least one energy acceptor moiety is linked to the C-terminus of the probing nucleobase sequence and the at least one energy donor moiety is linked to the N-terminus of the probing nucleobase sequence.

34. The polymer of claim 31, wherein subunits of the probing nucleobase sequence have the formula:

wherein,

each J is the same or different and is selected from the group consisting of;

H, R¹, OR¹, SR¹, NHR¹, NR¹₂, F, Cl, Br and I;

each K is the same or different and is selected from the group consisting of;

O, S, NH and NR¹;

each R¹ is the same or different and is an alkyl group having one to five carbon atoms which may optionally contain a heteroatom or a substituted or unsubstituted aryl group;

each A is selected from the group consisting of a single bond, a group of the formula;

—

(CJ₂)_s— and

a group of the formula;

—

(CJ₂)_sC(O)—

wherein, J is defined above and each s is an integer from one to five;

each t is 1 or 2;

each u is 1 or 2; and

each L is the same or different and is independently selected from the group consisting of J, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturally occurring nucleobase analogs, other non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties, biotin and fluorescein.

35. The polymer of claim 34, wherein the subunit consists of a naturally or a non-naturally occurring nucleobase attached to the aza nitrogen of a N-[2-(aminoethyl)]glycine backbone through a methylene carbonyl linkage.

36. The polymer of claim 31, wherein the probing nucleobase sequence is in the range of between 5-30 subunits in length.

37. The polymer of claim 31, wherein the probing nucleobase sequence is the range of between 8-18 subunits in length.

38. The polymer of claim 31, wherein the probing nucleobase sequence is the range of between 11-17 subunits in length.

81. The polymer of claim 31, wherein the at least one energy donor moiety is a fluorophore selected from the group consisting of 5(6)-carboxyfluorescein, 5-(2′

-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), bodipy, rhodamine, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, and texas red.

82. The polymer of claim 31, wherein the at least one energy acceptor moiety is 4-((-4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl).

83. The polymer of claim 31, wherein at least one spacer moiety separates one or both of the donor and acceptor moieties from the probing nucleobase sequence to which it is linked.

84. The polymer of claim 31, wherein the probing nucleobase sequence is perfectly complementary to the target sequence.

85. The polymer of claim 31, wherein the polymer is immobilized to a support.

86. The polymer of claim 31, wherein the polymer is one component polymer of an array.

46. A method for the detection, identification or quantitation of a target sequence in a sample, said method comprising:

a) contacting the sample with a polymer comprising;

i) a probing nucleobase sequence for probing the target sequence to which the probing nucleobase sequence is complementary or substantially complementary;

ii) at least one energy donor moiety that is linked to the probing nucleobase sequence; and

iii) at least one energy acceptor moiety that is linked to the probing nucleobase sequence wherein the at least one donor moiety is separated from the at least one acceptor moiety by at least a portion of the probing nucleobase sequence; and

b) detecting, identifying or quantitating the hybridization of the polymer to the target sequence, under suitable hybridization conditions, wherein the presence, absence or amount of target sequence present in the sample is correlated with a change in detectable signal associated with at least one donor or acceptor moiety of the polymer.

47. The method of claim 46, wherein the at least one energy donor moiety is linked to one of a first or a second end of the probing nucleobase sequence of the polymer and the at least one energy acceptor moiety is linked to the other one of the first or the second end of the probing nucleobase sequence of the polymer.

48. The method of claim 47, wherein the polymer is a PNA and the at least one energy acceptor moiety is linked to the C-terminus of the probing nucleobase sequence and the at least one energy donor moiety is linked to the N-terminus of the probing nucleobase sequence.

49. The method of claim 46, wherein the probing nucleobase sequence of the polymer is in the range of between 5-30 subunits in length.

50. The method of claim 46, wherein the probing nucleobase sequence of the polymer is in the range of between 8-18 subunits in length.

51. The method of claim 46, wherein the probing nucleobase sequence of the polymer is in the range of between 11-17 subunits in length.

52. The method of claim 46, wherein subunits of the probing nucleobase sequence of the polymer have the formula:

wherein,

each J is the same or different and is selected from the group consisting of;

H, R¹, OR¹, SR¹, NHR¹, NR¹₂, F, Cl, Br and I;

each K is the same or different and is selected from the group consisting of;

O, S, NH and NR¹;

each R¹ is the same or different and is an alkyl group having one to five carbon atoms which may optionally contain a heteroatom or a substituted or unsubstituted aryl group;

each A is selected from the group consisting of a single bond, a group of the formula;

—

(CJ₂)_s— and

a group of the formula;

—

(CJ₂)_sC(O)—

wherein, J is defined above and each s is an integer from one to five;

each t is 1 or 2;

each u is 1 or 2; and

each L is the same or different and is independently selected from the group consisting of J, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine, 2-aminoptirine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturally occurring nucleobase analogs, other non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties, biotin and fluorescein.

53. The method of claim 52, wherein each subunit consists of a naturally or a non-naturally occurring nucleobase attached to the aza nitrogen of a N-[2-(aminoethyl)]glycine backbone through a methylene carbonyl linkage.

54. The method of claim 46, wherein the polymer is a polyamide and the at least one energy acceptor moiety is linked to the C-terminus of the probing nucleobase sequence and the at least one energy donor moiety is linked to the N-terminus of the probing nucleobase sequence.

55. The method of claim 46, wherein the at least one energy donor moiety of the polymer is a fluorophore selected from the group consisting of 5(6)-carboxyfluorescein, 5-(2′

-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), bodipy, rhodamine, Cy2, Cy3, Cy3.5, Cy5, Cy5.5 and texas red.

56. The method of claim 46, wherein the at least one energy acceptor moiety of the polymer is 4-((-4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl).

57. The method of claim 46, wherein the target sequence is detected, identified or quantitated in a closed tube (homogeneous) assay.

58. The method of claim 57, wherein the target sequence that is detected, identified or quantitated is synthesized or amplified in a reaction occurring in the closed tube (homogeneous) assay.

59. The method of claim 58, wherein the target sequence is synthesized or amplified using a process selected from the group consisting of:

Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), Transcription-Mediated Amplification (TMA), Rolling Circle Amplification (RCA) and Q-beta replicase.

60. The method of claim 58, wherein the synthesized or amplified target sequence is measured in real-time.

61. The method of claim 59, wherein the PCR reaction is an asymmetric PCR reaction.

62. The method of claim 46, wherein the target sequence is detected, identified or quantitated in a cell or tissue, whether living or not.

63. The method of claim 62, wherein the target sequence in the cell or tissue is detected, identified or quantitated or using in situ hybridization.

64. The method of claim 46, wherein the sample is contacted with said polymer and one or more blocking probes.

65. The method of claim 46, wherein detection, identification or quantitation of the target sequence determines the presence or amount of an organism or virus in the sample.

66. The method of claim 46, wherein detection, identification or quantitation of the target sequence determines the presence or amount of one or more species of an organism in the sample.

67. The method of claim 46, wherein detection, identification or quantitation of the target sequence determines the effect of antimicrobial agents on the growth of one or more microorganisms in the sample.

68. The method of claim 46, wherein detection, identification or quantitation of the target sequence determines the presence or amount of a taxonomic group of organisms in the sample.

69. The method of claim 46, wherein detection, identification or quantitation of the target sequence facilitates a diagnosis of a condition of medical interest.

70. The method of claim 46, wherein the target sequence is immobilized to a surface.

71. The method of claim 46, wherein the polymer is immobilized to a surface.

72. The method of claim 46, wherein the polymer is one component polymer of an array.

73. An array comprising two or more support bound polymers wherein at least one polymer of the array comprises:

a) a probing nucleobase sequence for probing a target sequence to which the probing nucleobase sequence is complementary or substantially complementary;

b) at least one energy donor moiety that is linked to the probing nucleobase sequence; and

c) at least one energy acceptor moiety that is linked to the probing nucleobase sequence wherein the at least one donor moiety is separated from the at least one acceptor moiety by at least a portion of the probing nucleobase sequence; and

wherein said polymer is suitable for detecting, identifying or quantitating a target sequence present in a sample.

74. The array of claim 73, wherein the at least one energy donor moiety of the at least one polymer is linked to one of a first or a second end of the probing nucleobase sequence and the at least one energy acceptor moiety of the at least one polymer is linked to the other one of the first or second end of the probing nucleobase sequence.

75. The array of claim 73, wherein the array is suitable for regeneration by treatment with one or more regeneration catalysts selected from the group consisting of heat, nuclease enzyme and chemical denaturant.

76. A kit suitable for performing an assay which detects the presence, absence or amount of target sequence in a sample, wherein said kit comprises:

a) at least one polymer having;

i) a probing nucleobase sequence for probing a target sequence to which the probing nucleobase sequence is complementary or substantially complementary;

ii) at least one energy donor moiety that is linked to the probing nucleobase sequence; and

iii) at least one energy acceptor moiety that is linked to the probing nucleobase sequence wherein the at least one donor moiety is separated from the at least one acceptor moiety by at least a portion of the probing nucleobase sequence; and

b) other reagents or compositions necessary to perform the assay.

77. The kit of claim 76, wherein the at least one energy donor moiety of the at least one polymer is linked to one of a first or a second end of the probing nucleobase sequence and the at least one energy acceptor moiety of the at least one polymer is linked to the other one of the first or second end of the probing nucleobase sequence.

78. The kit of claim 76, wherein one of more of the polymers of the kit have a probing nucleobase sequence that is 11-17 subunits in length.

79. The kit of claim 76, wherein two or more polymers of the kit are labeled with independently detectable moieties.

80. The kit of claim 79, wherein the independently detectable moieties are used to independently detect, identify or quantitate at least two different target sequences which may be present in the same sample.