LABELLING STRATEGIES FOR THE SENSITIVE DETECTION OF ANALYTES

• US 20090215635A1
• Filed: 04/28/2006
• Published: 08/27/2009
• Est. Priority Date: 05/02/2005
• Status: Active Grant

• Current Assignee: Baseclick GmbH (BASF SE)
• Original Assignee: BASF SE
• Inventors: Gierlich, Johannes, Mofid, Mohammad Reza, Burley, Glenn A., Schwögler, Anja, Carell, Thomas
• Granted Patent: US 8,129,315 B2
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to methods and reagents for detecting analytes, e.g. nucleic acids. The new methods and reagents allow a simple and sensitive detection even in complex biological samples.

Claims

1. A method for detecting an analyte in a sample comprising the steps:

(i) providing a sample;

(ii) contacting the sample with a functionalized compound comprising at least one functional group selected from an aldehyde group, a protected aldehyde group, an aldehyde precursor group or a handle group for introducing an aldehyde group, a protected aldehyde group or an aldehyde precursor group, under conditions wherein said compound forms an association product with the analyte to be detected;

(iii) if necessary, reacting the handle group with a reaction partner, comprising an aldehyde group, a protected aldehyde group or an aldehyde precursor group, (iv) if necessary, converting protected aldehyde groups and aldehyde precursor groups to aldehyde groups,

(v) contacting said association product having aldehyde groups with a marker reagent under conditions wherein marker groups are formed around aldehyde groups in said association product, and 0 (vi) detecting said marker groups.

2. The method of claim 1, comprising a qualitative detection.

3. The method of claim 1, comprising a quantitative detection.

4. The method of claim 1 wherein the analyte is selected from nucleic acids and nucleoside-, nucleotide- or nucleic acid-binding molecules.

5. The method of claim 1, wherein the analyte to be detected is a nucleic acid selected from DNA and RNA.

6. The method of claim 1, wherein the sample is a biological sample.

7. The method of claim 6, wherein the sample is an agricultural or nutritional sample.

8. The method of claim 6, wherein the sample is a clinical sample.

9. The method of claim 1, wherein the detection is carried out directly without amplification.

10. The method of claim 1, wherein the detection is carried out in combination with an amplification step.

11. The method of claim 1, wherein a sequence-specific detection of a nucleic acid analyte is carried out.

12. The method of claim 11, wherein the forming of said association product comprises a sequence-specific hybridization reaction.

13. The method of claim 1, wherein the functionalized compound comprises

(i) an aliphatic or cycloaliphatic aldehyde group, a protected aliphatic or cycloaliphatic aldehyde group or an aliphatic or cycloaliphatic aldehyde precursor group, (ii) an aromatic or heteroaromatic aldehyde group or an aromatic or heteroaromatic aldehyde precursor group, and/or (iii) an alkyne aldehyde group, a protected alkyne aldehyde group or an alkyne aldehyde precursor group.

14. The method of claim 1, wherein the protected aldehyde groups are selected from acetal groups, hemiacetal groups, e.g. hemiacetal groups in a sugar or a sugar-related compound, thio and dithioacetal groups, imidazolone groups and benzothiazol groups.

15. The method of claim 1, wherein the aldehyde precursor groups are selected from carboxylic acids and carboxylic acid derivatives such as nitriles and primary alcohols.

16. The method of claim 1, wherein the handle groups are selected from azide and alkyne groups.

17. The method of claim 16, wherein said azide or alkyne groups are reacted by performing a Click reaction with a reaction partner comprising an aldehyde, protected aldehyde or aldehyde precursor group.

18. The method of claim 1, wherein the functional group is attached to a nucleobase of a nucleosidic or nucleotidic compound or an oligomer or polymer thereof.

19. The method of claim 18, wherein the nucleobase is selected from naturally occurring and non-naturally occurring purine and pyrimidine bases.

20. The method of claim 19, wherein the functional group is attached to position 5 and 6, preferably position 5, of a pyrimidine nucleobase or to positions 7 and 8, preferably position 7, of a purine nucleobase.

21. The method of claim 1, wherein the functional group is attached to the compound via a direct bond or spacer.

22. The method of claim 21 wherein said spacer is a flexible spacer or an at least partially rigid spacer.

23. The method of claim 22, wherein the spacer comprises at least one group selected from alkene groups, alkyne groups, cyclic groups, particularly aromatic and heteraaromatic groups, and combinations thereof.

24. The method of claim 1, wherein said functionalized compound is selected from:

(i) a functionalized nucleic acid or nucleic acid analogue building block and/or (ii) a functionalized nucleic acid or nucleic acid analogue.

25. The method of claim 24, wherein the forming of said association product comprises incorporating said functionalized building block into a nucleic acid or nucleic acid analogue molecule.

26. The method of claim 25, wherein said functionalized nucleotide or nucleotide analogue is enzymatically incorporated into a nucleic acid molecule.

27. The method of claim 25, wherein the forming of said association product comprises binding said functionalized nucleic acid or nucleic acid analogue to the analyte to be detected.

28. The method of claim 27, wherein the binding comprises hybridization to a nucleic acid analyte.

29. The method of claim 24, comprising a primer extension reaction optionally in combination with a nucleic acid amplification reaction.

30. The method of claim 29 comprising:

providing at least one primer molecule which hybridizes under the assay conditions with the nucleic acid analyte to be detected or the complement thereof and extending said primer, wherein at least one functionalized nucleotide or nucleotide analogue is incorporated into the extension product.

31. The method of claim 29 comprising:

providing at least one functionalized primer molecule which hybridizes under the assay conditions with the nucleic acid analyte to be detected or the complement thereof, extending said primer molecule, wherein an extended primer molecule is formed.

32. The method of claim 28 comprising:

providing a functionalized hybridization probe which hybridizes under the assay conditions with the nucleic acid analyte to be detected or the complement thereof, wherein a hybridization product is formed.

33. The method of claim 26 comprising:

providing a functionalized hybridization probe which hybridizes under the assay conditions with the nucleic acid analyte to be detected or the complement thereof, and subjecting the hybridization product to a treatment wherein a hybridization product containing at least one mismatch is dissolved, wherein the presence of an undissolved hybridization product is indicative for the presence and/or amount of a nucleic acid which has a fully complementary sequence to the hybridization probe.

34. The method of claim 33, wherein the treatment comprises a mismatch digestion treatment.

35. The method of claim 34, wherein the mismatch digestion treatment comprises contacting the hybridization product with a mismatch-glycosylase.

36. The method of claim 33, wherein the treatment comprises a differential hybridization treatment.

37. The method of claim 1, wherein said marker reagent comprises fluorescent or redox active groups.

38. The method of claim 1, wherein said marker reagent comprises metal groups.

39. The method of claim 1, wherein said groups are selected from metal atoms and/or ions such as Ag, Au₁ Bi, Cu, Pd, Pt which can be selectively deposited around aldehyde groups in said association product.

40. The method of claim 38, wherein said marker reagent comprises an Ag-ammonium complex.

41. The method of claim 1, wherein the detection is carried out by optical means.

42. The method of claim 1, wherein the detection is carried out by electrical means.

43. The method of claim 1 comprising a parallel detection of a plurality of nucleic acids.

44. The method of claim 1, wherein the detection is carried out on a solid surface, e.g. on a microarray chip.

45. A reagent kit for detecting an analyte in a sample, comprising:

(a) a functionalized compound comprising a functional group selected from an aldehyde group, a protected aldehyde group or an aldehyde precursor group, or a handle group for introducing an aldehyde group, a protected aldehyde group or an aldehyde precursor group,

(b) optionally a reaction partner for the handle group comprising an aldehyde group, a protected aldehyde group or an aldehyde precursor group,

(c) optionally an aldehyde-forming reagent capable of converting protected aldehyde groups and aldehyde precursor groups to aldehyde groups, and

(d) a marker reagent.

46. (canceled)

58. The compound of claim 46 which is a building block for the synthesis of peptide nucleic acids, morpholino nucleic acids or locked nucleic acids.

47. A compound of the formula (I):

A-S—

N

wherein A is a functional group selected from an aldehyde group, a protected aldehyde group or an aldehyde precursor group, S is a spacer or a bond, and

N is a nucleic acid or nucleic acid analogue building block such as a nucleosidic or nucleotidic compound.

48. The compound of claim 47, wherein the protected aldehyde groups are selected from acetal groups, hemiacetal groups, thio- and dithioacetal groups, imidazolone groups and benzothiazole groups.

49. The compound of claim 47, wherein the aldehyde precursor groups are selected from carboxylic acids and carboxylic acid derivatives such as nitriles and primary alcohols.

50. The compound of claim 47, wherein the functional group is attached to a nucleobase.

51. The compound of claim 50, wherein the nucleobase is selected from naturally occurring and non-naturally purine and pyrimidine bases.

52. The compound of claim 50, wherein the nucleobase is selected from adenine, 7-deazaadenine, guanine, 7-deazaguanine, cytosine, thymidine, uracil, inosine and xanthine.

53. The compound of claim 51, wherein the functional group is attached to positions 5 and 6, preferably position 5, of a pyrimidine nucleobase, or to positions 7 and 8, preferably position 7 of a purine nucleobase.

54. The compound of claim 47, wherein said spacer is an at least partially rigid spacer.

55. The compound of claim 54, wherein the spacer comprises at least one group selected from alkene groups, alkyne groups, cyclic groups, particularly aromatic and heteroaromatic groups, and combinations thereof.

56. The compound of claim 47 which is a building block for the chemical or enzymatic synthesis of nucleic acids or nucleic acid analogues or a precursor thereof.

57. The compound of claim 47 which is a nucleoside triphosphate, particularly a ribose, 2′

-deoxyribose or 2′

,3′

-dideoxyribose nucleoside triphosphate or a nucleoside phosphoramidite, H—

phosphonate or phosphotriester.

59. A nucleic acid or a nucleic acid analogue molecule having incorporated therein at least one compound of formula (I) according to claim 47.

60. The molecule of claim 59 which is a nucleic acid selected from DNA and RNA₁ or a nucleic acid analogue selected from peptidic nucleic acids, morpholino nucleic acids or locked nucleic acids.

61. A method of synthesizing a nucleic acid or nucleic acid analogue molecule comprising incorporating at least one nucleotide or nucleotide analogue building block comprising a compound (I) of claim 47 into a nucleic acid or nucleic acid analogue molecule.

62. The method of claim 61 comprising a chemical synthesis and/or an enzymatic synthesis.

63. A metallization product of the nucleic acid or nucleic acid analogue molecule of claim 59.

64. An association product of the nucleic acid or nucleic acid analogue molecule of claim 59.

65. The product of claim 64 which is a hybridization product with a complementary nucleic acid.

77. The compound of claim 76 having the features as defined in any one of claims 50 to 58.

66. A method for detecting a nucleic acid in a sample comprising the steps:

(i) providing a sample;

(ii) contacting the sample with a functionalized compound comprising at least one functional group which is a first reaction partner for a Click reaction under conditions wherein said compound forms an association product with the nucleic acid to be detected or the complement thereof, (iii) contacting the association product with a second reaction partner for a Click reaction under conditions wherein a Click reaction between the first and second reaction partner occurs, wherein the second reaction partner further comprises a marker group or a marker precursor group, (iv) if necessary, converting marker precursor groups to marker groups, and (v) detecting said marker groups.

67. The method of claim 66 comprising a qualitative detection.

68. The method of claim 66, wherein the functional group is selected from azide groups or alkyne groups.

69. The method of claim 66, wherein the Click reaction comprises a (3+2) cycloaddition.

70. The method of claim 69, wherein a 5-membered heterocycle is formed by the Click reaction.

71. The method of claim 66, wherein the second reaction partner comprises a marker group selected from optically detectable marker groups, particularly fluorescence marker groups or from redox active marker groups.

72. The method of claim 66, wherein the second reaction partner comprises a marker precursor group selected from aldehyde groups and protected aldehyde groups and aldehyde precursor groups.

73. The method of claim 72, wherein converting the marker precursor groups to marker groups comprises forming metal depositions around aldehyde groups.

74. A reagent kit for detecting an analyte in a sample, comprising:

(a) a functionalized compound comprising at least one functional 5 group which is a first reaction partner for a Click reaction,

(b) a second reaction partner for a Click reaction, wherein the second reaction partner further comprises a marker group or a marker precursor group, and

(c) optionally a marker forming reagent capable of converting marker o precursor groups to marker groups.

75. (canceled)

76. A compound of the formula (II):

C—

S—

N

wherein C is a functional group which is a first reaction partner 0 for a Click reaction,

S is a spacer or a bond, and

N is a nucleic acid or nucleic acid analogue building block such as a nucleosidic or nucleotidic compound.

78. The compound of claim 76, wherein the functional group is attached to the nucleobase via a spacer having a chain length of up to o 10 atoms.

79. The compound of claim 78, wherein the chain length is of up to 3 atoms.

80. A nucleic acid or a nucleic acid analogue molecule having incorporated therein at least one compound of formula (II) according to claim 76.

81. The nucleic acid or a nucleic acid analogue molecule of claim 80 which is a nucleic acid selected from DNA and RNA, or a nucleic acid analogue molecule selected from peptidic nucleic acids, morpholino nucleic acids or locked nucleic acids.

82. A method of synthesizing a nucleic acid or nucleic acid analogue molecule comprising incorporating a nucleotide or nucleotide analogue building block comprising a compound (II) of claim 76 into a nucleic acid or nucleic acid analogue molecule.

83. The method of claim 82 comprising a chemical synthesis and/or an enzymatic synthesis.

84. The method of claim 82 further comprising contacting the nucleic acid or nucleic acid analogue molecule with a second reaction partner of compound (II) and performing a Click reaction between the first and second reaction partners.

85. An association product of the nucleic acid or nucleic acid analogue molecule of claim 80 with an analyte.

86. The product of claim 85 which is a hybridization product with a complementary nucleic acid.

87. A method for detecting an analyte in a sample comprising the steps:

(i) providing a sample

(ii) contacting the sample with a functionalized compound comprising at least one functional group selected from a photosensitizer group or a handle group for introducing a photosensitizer group under conditions wherein said compound forms an association product with the analyte to be detected;

(iii) if necessary, reacting the handle group with a reaction partner comprising a photosensitizer group;

(iv) irradiating said association product in contact with a photosensitive medium under conditions wherein marker groups are formed in said photosensitive medium in the presence of photosensitizer groups in said association product, and (v) detecting said marker groups.

88. The method of claim 87 comprising a qualitative detection.

89. The method of claim 87 wherein the photosensitizer groups are selected from fluorescent dye groups.

90. The method of claim 89 wherein the photosensitizer groups are selected from cyanine-based indoline groups and quinoline groups.

91. The method of claim 87 wherein the photosensitive medium comprises metal atoms or ions capable of forming metal nuclei.

92. The method of claim 91 wherein the metal is Ag.

93. The method of claim 87 wherein the photosensitive medium is a light sensitive paper such as photographic paper or a light sensitive emulsion or gel on a supportive material.

94. The method of claim 87 wherein the irradiating step (iv) is carried out with long wave visible light and/or with infrared light.

95. A reagent kit for detecting an analyte in a sample comprising:

(a) a functionalized compound comprising at least one functional group selected from a photosensitizer group or a handle group for introducing a photosensitizer group;

(b) optionally a reaction partner for the handle group comprising a photosensitizer group, and (c) a photosensitive medium which forms marker groups upon irradiation in the presence of photosensitizer groups.

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117. A method for detecting an analyte in a sample comprising the steps:

(i) providing a sample,

(ii) providing a reporter molecule comprising a photosensitizer group or a handle group for introducing a photosensitizer group and a quencher group wherein the photosensitizer group is quenched in the absence of the analyte to be detected,

(iii) contacting the sample with the reporter molecule under conditions wherein the quenching of the photosensitizer group is at least partially reduced or terminated in the presence of the analyte, (iv) if necessary, reacting the handle group with a reaction partner comprising a photosensitizer group

(v) irradiating said reporter molecule in contact with a photosensitive medium under conditions wherein marker groups are formed in said photosensitive medium in the presence of unquenched photosensitizer groups in said reporter molecule and (vi) detecting said marker groups.

118. A reagent kit for detecting an analyte in a sample comprising:

(a) a reporter molecule comprising a photosensitizer group or a handle group for introducing a photosensitizer group and a quencher group wherein the photosensitizer group is quenched in the absence of the analyte to be detected,

(b) optionally a reaction parker for the handle group comprising a photosensitizer group and

(c) a photosensitive medium which forms marker groups upon irradiation of unquenched photosensitizer groups.