Arrays of modified nucleic acid probes and methods of use

• US 6,156,501 A
• Filed: 04/03/1996
• Issued: 12/05/2000
• Est. Priority Date: 10/26/1993
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: McGall, Glenn Hugh, Tan, Jennifer Dee, Chee, Mark S., Cronin, Maureen T., Miyada, Charles Garrett
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/630,427
• Time in Patent Office: 1,707 Days
• Field of Search: 422/50, 422/68.1, 435/6, 435/810, 435/283.1, 435/287.1, 435/287.2, 435/288.3, 435/288.7, 435/289.1, 435/299.1, 435/305.1, 436/501, 536/23.1, 536/24.1, 536/22.1, 536/24.3-24.33, 935/77, 935/78
• US Class Current: 506/9
• 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

Abstract

Oligonucleotide analogue arrays attached to solid substrates and methods related to the use thereof are provided. The oligonucleotide analogues hybridize to nucleic acids with either higher or lower specificity than corresponding unmodified oligonucleotides. Target nucleic acids which comprise nucleotide analogues are bound to oligonucleotide and oligonucleotide analogue arrays.

Claims

1. A composition for analyzing interactions between oligonucleotide targets and oligonucleotide probes comprising an array of a plurality of oligonucleotide analogue probes having different sequences, wherein said oligonucleotide analogue probes are coupled to a solid substrate at known locations and wherein said plurality of oligonucleotide analogue probes are selected to bind to complementary oligonucleotide targets with a similar hybridization stability across the array.

2. The composition of claim 1, wherein at least one of said oligonucleotide analogue probes is selected to maintain hybridization specificity or mismatch discrimination with said complementary oligonucleotide targets.

3. The composition of claim 1, wherein at least one of said oligonucleotide analogue probes has increased the thermal stability between said oligonucleotide analogue probe and said complementary oligonucleotide target as compared to an oligonucleotide probe that is the perfect complement to the complementary oligonucleotide target with which said oligonucleotide analogue probe anneals.

4. The composition of claim 1, wherein at least one of said oligonucleotide analogue probes has decreased the thermal stability between said oligonucleotide analogue probe and said complementary oligonucleotide target as compared to an oligonucleotide probe that is the perfect complement to the complementary oligonucleotide target with which said oligonucleotide analogue probe anneals.

5. The composition of claim 2, wherein at least one of said oligonucleotide analogue probes has increased the thermal stability between said oligonucleotide analogue probe and said complementary oligonucleotide target as compared to an oligonucleotide probe that is the perfect complement to the complementary oligonucleotide target with which said oligonucleotide analogue probe anneals.

6. The composition of claim 2, wherein at least one of said oligonucleotide analogue probes has decreased the thermal stability between said oligonucleotide analogue probe and said complementary oligonucleotide target as compared to an oligonucleotide probe that is the perfect complement to the complementary oligonucleotide target with which said oligonucleotide analogue probe anneals.

7. The composition of claims 1-5 or 6, wherein said solid substrate is selected from the group consisting of silica, polymeric materials, glass, beads, chips, and slides.

8. The composition of claims 1-5 or 6, wherein said composition comprises an array of oligonucleotide analogue probes 5 to 20 nucleotides in length.

9. The composition of claims 1-5 or 6, wherein said array of oligonucleotide analogue probes comprises a nucleoside analogue with the formula ##STR3## wherein:

the nucleoside analogue is not a naturally occurring DNA or RNA nucleoside;

R¹ is selected from the group consisting of hydrogen, methyl, hydroxyl, alkoxy, alkythio, halogen, cyano, and azido;

R² is selected from the group consisting of hydrogen, methyl, hydroxyl, alkoxy, alkythio, halogen, cyano, and azido;

Y is a heterocyclic moiety;

and wherein said nucleoside analogue is incorporated into the oligonucleotide analogue by attachment to a 3'"'"' hydroxyl of the nucleoside analogue, to a 5'"'"' hydroxyl of the nucleoside analogue, or both the 3'"'"' nucleoside and the 5'"'"' hydroxyl of the nucleoside analogue.

10. The composition of claims 1-5 or 6, wherein said array of

oligonucleotide analogue probes comprises a nucleoside analogue with the formula ##STR4## wherein:

the nucleoside analogue is not a naturally occurring DNA or RNA nucleoside;

R¹ is selected from the group consisting of hydrogen, hydroxyl, methyl, methoxy, ethoxy, propoxy, allyloxy, propargyloxy, Fluorine, Chlorine, and Bromine;

R² is selected from the group consisting of hydrogen, hydroxyl, methyl, methoxy, ethoxy, propoxy, allyloxy, propargyloxy, Fluorine, Chlorine, and Bromine; and

Y is a base selected from the group consisting of purines, purine analogues pyrimidines, pyrimidine analogues, 3-nitropyrrole and 5-nitroindole;

and wherein said nucleoside analogue is incorporated into the oligonucleotide analogue by attachment to a 3'"'"' hydroxyl of the nucleoside analogue, to a 5'"'"' hydroxyl of the nucleoside analogue, or both the 3'"'"' nucleoside and the 5'"'"' hydroxyl of the nucleoside analogue.

11. The composition of claims 1-5 or 6, wherein each probe of said plurality of oligonucleotide analogue probes has at least one oligonucleotide analogue, and wherein at least one of said oligonucleotide analogues comprises a peptide nucleic acid.

12. The composition of claims 1-5 or 6, wherein at least one of said plurality of oligonucleotide analogue probes said array of oligonucleotide analogue probes is resistant to RNAase A.

13. The composition of claims 1-5 or 6, wherein said solid substrate is attached to over 1000 different oligonucleotide analogue probes.

14. The composition of claims 1-5 or 6, wherein each probe of said plurality of oligonucleotide analogue probes has at least one oligonucleotide analogue, and wherein at least one of said oligonucleotide analogues comprises 2'"'"'-O-methyl nucleotides.

15. The composition of claims 1-5 or 6, wherein said array of oligonucleotide analogue probes and said solid substrate comprises a plurality of different oligonucleotide analogue probes, each oligonucleotide analogue probes having the formula:

space="preserve" listing-type="equation">Y--L.sup.1 --X.sup.1 --L.sup.2 --X.sup.2

wherein,

Y is a solid substrate;

X¹ and X² are complementary oligonucteotides containing at least one nucleotide analogue;

L¹ is a spacer;

L² is a linking group having sufficient length such that X¹ and X² form a double-stranded oligonucleotide.

16. The composition of claim 15, wherein said composition comprises a library of unimolecular double-stranded oligonucleotide analogue probes.

17. The composition of claims 1-5 or 6, wherein said array of oligonucleotide analogue probes comprises a conformationally restricted array of oligonucleotide analogue probes with the formula:

space="preserve" listing-type="equation">--X.sup.11 --Z--X.sup.12

wherein X¹¹ and X¹² are complementary oligonucleotides or oligonucleotide analogues and Z is a presented moiety.

18. The composition of claims 1-5 or 6, wherein each probe of said plurality of oligonucleotide analogue probes has at least one oligonucleotide analogue, and wherein at least one of said oligonucleotide analogues comprises a nucleotide with a base selected from the group of bases consisting of 5-propynyluracil, 5-propynylcytosine, 2-aminoadenine, 7-deazaguanine, 2-aminopurine, 8-aza-7-deazaguanine, 1H-purine, and hypoxanthine.

19. The composition of claims 1-5 or 6, wherein said plurality of oligonucleotide analogue probes are coupled to said solid substrate by light-directed chemical coupling.

20. The composition of claim 19, wherein said solid substrate is derivitized with a silane reagent prior to synthesis of said plurality of oligonucleotide analogue probes.

21. The composition of claims 1-5 or 6, wherein said plurality of oligonucleotide analogue probes are coupled to said solid substrate by flowing oligonucleotide analogue reagents over known locations of the solid substrate.

22. The composition of claim 21, wherein said solid substrate is derivitized with a silane reagent prior to synthesis of said plurality of oligonucleotide analogue probes.

23. The composition of claims 1-5 or 6, wherein at least one of plurality of said oligonucleotide analogue probes forms a first duplex with a target oligonucleotide sequence, wherein said oligonucleotide analogue probe has a corresponding oligonucleotide sequence that forms a second duplex with said target oligonucleotide sequence, wherein said second duplex is rich in A-T or G-C nucleotide pairs, and wherein said oligonucleotide analogue probe has at least one nucleotide analogue in place of an A, T, G, or C nucleotide of said corresponding oligonucleotide sequence at a position within said oligonucleotide analogue probe such that said first duplex has an increased hybridization stability than said second duplex.

24. The composition of claim 23, wherein said oligonucleotide analogue probe contains fewer bases than said corresponding oligonucleotide sequence.

25. The composition of claims 1-5 or 6, wherein said oligonucleotide analogue probe forms a first duplex with a target oligonucleotide sequence, wherein said oligonucleotide analogue probe has a corresponding oligonucleotide sequence that forms a second duplex with said target polynucleotide sequence, and wherein said oligonucleotide analogue probe is shorter than said corresponding polynucleotide sequence.

26. A composition for analyzing the interaction between an oligonucleotide target and an oligonucleotide probe comprising an array of a plurality of oligonucleotide probes having different sequences hybridized to complementary oligonucleotide analogue targets, wherein said oligonucleotide analogue targets bind to complementary oligonucleotide probes with a similar hybridization stability across the array.

27. The composition of claim 26, wherein at least one of said oligonucleotide analogue target is selected to maintain hybridization specificity or mismatch discrimination with said complementary oligonucleotide probes.

28. The composition of claim 26, wherein at least one of said oligonucleotide analogue targets has increased the thermal stability between said oligonucleotide analogue target and said complementary oligonucleotide probe as compared to an oligonucleotide target that is the perfect complement to the complementary oligonucleotide probe with which said oligonucleotide analogue target anneals.

29. The composition of claim 26, wherein at least one of said oligonucleotide analogue targets has decreased the thermal stability between said oligonucleotide analogue target and said complementary oligonucleotide probe as compared to an oligonucleotide target that is the perfect complement to the complementary oligonucleotide probe with which said oligonucleotide analogue target anneals.

30. The composition of claim 27, wherein at least one of said oligonucleotide analogue targets has increased the thermal stability between said oligonucleotide analogue target and said complementary oligonucleotide probe as compared to an oligonucleotide target that is the perfect complement to the complementary oligonucleotide probe with which said oligonucleotide analogue target anneals.

31. The composition of claim 27, wherein at least one of said oligonucleotide analogue targets has decreased the thermal stability between said oligonucleotide analogue target and said complementary oligonucleotide probe as compared to an oligonucleotide target that is the perfect complement to the complementary oligonucleotide probe with which said oligonucleotide analogue target anneals.

32. The composition of claims 26-30 or 31, wherein the oligonucleotide analogue target is a PCR amplicon.

33. The composition of claims 26-30 or 31, wherein at least one of said plurality of oligonucleotide probes comprise at least one oligonucleotide analogue.

34. The composition of claims 26-30 or 31, wherein at least one target oligonucleotide analogue acid is an RNA nucleic acid.

35. A method analyzing interactions between an oligonucleotide target and an oligonucleotide probe, comprising the steps of:

(a). synthesizing an oligonucleotide analogue array comprising a plurality of oligonucleotide analogue probes having different sequences, wherein said oligonucleotide analogue probes are coupled to a solid substrate at known locations, said solid substrate having a surface;

(b). exposing said oligonucleotide analogue probe array to a plurality of oligonucleotide targets under hybridization conditions such that said plurality of oligonucleotide analogue probes bind to complementary oligonucleotide targets with a similar hybridization stability across the array; and

(c). determining whether an oligonucleotide analogue probe of said oligonucleotide analogue probe array binds to at least one of said target nucleic acids.

36. The method in accordance of claim 35, wherein at least one of said oligonucleotide analogue probes is selected to maintain hybridization specificity or mismatch discrimination with said complementary oligonucleotide targets.

37. The method in accordance of claim 35, wherein at least one of said oligonucleotide analogue probes has increased the thermal stability between said oligonucleotide analogue probe and said complementary oligonucleotide target as compared to an oligonucleotide probe that is the perfect complement to the complementary oligonucleotide target with which said oligonucleotide analogue probe anneals.

38. The method in accordance of claim 35, wherein at least one of said oligonucleotide analogue probes has decreased the thermal stability between said oligonucleotide analogue probe and said complementary oligonucleotide target as compared to an oligonucleotide probe that is the perfect complement to the complementary oligonucleotide target with which said oligonucleotide analogue probe anneals.

39. The method in accordance of claim 36, wherein at least one of said oligonucleotide analogue probes has increased the thermal stability between said oligonucleotide analogue probe and said complementary oligonucleotide target as compared to an oligonucleotide probe that is the perfect complement to the complementary oligonucleotide target with which said oligonucleotide analogue probe anneals.

40. The method in accordance of claim 36, wherein at least one of said oligonucleotide analogue probes has decreased the thermal stability between said oligonucleotide analogue probe and said complementary oligonucleotide target as compared to an oligonucleotide probe that is the perfect complement to the complementary oligonucleotide target with which said oligonucleotide analogue probe anneals.

41. The method of claims 35-39 or 40, wherein said oligonucleotide target is selected from the group comprising genomic DNA, cDNA, unspliced RNA, mRNA, and rRNA.

42. The method of claims 35-39 or 40, wherein said target nucleic acid is amplified prior to said hybridization step.

43. The method of claims 35-39 or 40, wherein said plurality of oligonucleotide analogue probes is synthesized on said solid support by light-directed synthesis.

44. The method of claims 35-39 or 40, wherein said plurality of said oligonucleotide analogue probes is synthesized on said solid support by causing oligonucleotide analogue synthetic reagents to flow over known locations of said solid support.

45. The method of claims 35-39 or 40, wherein said step (a). comprises the steps of:

i). forming a plurality of channels adjacent to the surface of said substrate;

ii). placing selected reagents in said channels to synthesize oligonucleotide analogue probes at known locations; and

iii). repeating steps i). and ii). thereby forming an array of oligonucleotide analogue probes having different sequences at known locations on said substrate.

46. The method of claims 35-39 or 40, wherein said solid substrate is selected from the group consisting of beads, slides, and chips.

47. The method of claims 35-39 or 40, wherein said solid substrate is comprised of materials selected from the group consisting of silica, polymers and glass.

48. The method of claims 35-39 or 40, wherein the oligonucleotide analogue probes of said array are synthesized using photoremovable protecting groups.

49. The method of claims 35-39 or 40, further comprising selectively incorporating MeNPoc onto the 3'"'"' or 5'"'"' hydroxyl of at least one nucleoside analogue and selectively incorporating said nucleoside analogue into at least one of said oligonucleotide analogue probes.

50. The method of claims 35-39 or 40, wherein at least one of said oligonucleotide analogue probes is synthesized from phosphoramidite nucleoside reagents.

51. A method of detecting an oligonucleotide target, comprising enzymatically copying an oligonucleotide target using at least one nucleotide analogue, thereby producing multiple oligonucleotide analogue targets, selecting said oligonucleotide analogue targets such that said oligonucleotide analogue targets bind to the complementary oligonucleotide probes coupled to a solid surface at known locations of an array with a similar hybridization stability across the array, hybridizing the oligonucleotide analogue targets to complementary oligonucleotide probes, and detecting whether at least one of said oligonuclotide analogue targets binds to said complementary oligonucleotide acid probe.

52. The method of claim 51, wherein at least one of said oligonucleotide analogue targets is selected to maintain hybridization specificity or mismatch discrimination with said complementary oligonucleotide probes.

53. The method of claim 51, wherein at least one of said oligonucleotide analogue targets has increased the thermal stability between said oligonucleotide analogue target and said complementary oligonucleotide probe as compared to an oligonucleotide target that is the perfect complement to the complementary oligonucleotide probe with which said oligonucleotide analogue target anneals.

54. The method of claim 51, wherein at least one of said oligonucleotide analogue targets has decreased the thermal stability between said oligonucleotide analogue target and said complementary oligonucleotide probe as compared to an oligonucleotide target that is the perfect complement to the complementary oligonucleotide probe with which said oligonucleotide analogue target anneals.

55. The method of claim 52, wherein at least one of said oligonucleotide analogue targets has increased the thermal stability between said oligonucleotide analogue target and said complementary oligonucleotide probe as compared to an oligonucleotide target that is the perfect complement to the complementary oligonucleotide probe with which said oligonucleotide analogue target anneals.

56. The method of claim 52, wherein at least one of said oligonucleotide analogue targets has decreased the thermal stability between said oligonucleotide analogue target and said complementary oligonucleotide probe as compared to an oligonucleotide target that is the perfect complement to the complementary oligonucleotide probe with which said oligonucleotide analogue target anneals.

57. The method of claims 51-55 or 56, wherein the oligonucleotide probe array comprises at least one oligonucleotide analogue probe which is complementary to at least one of said oligonucleotide analogue targets.

58. A method of making an array of oligonucleotide probes, comprising providing a plurality of oligonucleotide analogue probes having at least one oligonucleotide analogue, said oligonucleotide analogue probes having different sequences at known locations on an array, selecting the oligonucleotide analogue probes to hybridize with complementary oligonucleotide target sequences under hybridization conditions such that said oligonucleotide analogue probes bind to complementary oligonucleotide targets with a similar hybridization stability, across the array.

59. The method of claim 58, wherein at least one of said oligonucleotide analogue probes is selected to maintain hybridization specificity or mismatch discrimination with said complementary oligonucleotide targets.

60. The method of claim 58, wherein at least one of said oligonucleotide analogue probes has increased the thermal stability between said oligonucleotide analogue probe and said complementary oligonucleotide target as compared to an oligonucleotide probe that is the perfect complement to the complementary oligonucleotide target with which said oligonucleotide analogue probe anneals.

61. The method of claim 58, wherein at least one of said oligonucleotide analogue probes has decreased the thermal stability between said oligonucleotide analogue probe and said complementary oligonucleotide target as compared to an oligonucleotide probe that is the perfect complement to the complementary oligonucleotide target with which said oligonucleotide analogue probe anneals.

62. The method of claim 59, wherein at least one of said oligonucleotide analogue probes has increased the thermal stability between said oligonucleotide analogue probe and said complementary oligonucleotide target as compared to an oligonucleotide probe that is the perfect complement to the complementary oligonucleotide target with which said oligonucleotide analogue probe anneals.

63. The method of claim 59, wherein at least one of said oligonucleotide analogue probes has decreased the thermal stability between said oligonucleotide analogue probe and said complementary oligonucleotide target as compared to an oligonucleotide probe that is the perfect complement to the complementary oligonucleotide target with which said oligonucleotide analogue probe anneals.

64. The method in accordance with claims 58-62, or 63, further comprising incorporating at least one oligonucleotide analogue into at least one of the oligonucleotide analogue probes of the array to reduce or prevent the formation of secondary structure in the oligonucleotide of the array.

65. The method in accordance with claims 58-62, or 63, further comprising incorporating at least one oligonucleotide analogue into at least one of the oligonucleotide target to reduce or prevent the formation of secondary structure in the target polynucleotide sequence.

66. The method in accordance with claims 58-62, or 63, further comprising incorporating at least one oligonucleotide analogue into at least one of the oligonucleotide analogue probes of the array to create secondary structure in the oligonucleotide of the array.

67. The method in accordance with claims 58-62, or 63, further comprising incorporating a base selected from the group consisting of 5-propynyluracil, 5-propynylcytosine, 2-aminoadenine, 7-deazaguanine, 2-aminopurine, 8-aza-7-deazaguanine, 1H-purine, and hypoxanthine into the oligonucleotide analogue probes of the array.

68. The method of claim 67 further comprising selecting said at least one oligonucleotide analogue such that the oligonucleotide analogue probe is a homopolymer.

69. The method in accordance with claims 58-62, or 63, further comprising selecting said at least one oligonucleotide analogue from the group consisting essentially of oligonucleotide analogues comprising 2'"'"'-O-methyl nucleotides and oligonucleotides comprising a base selected from the group of bases consisting of 5-propynyluracil, 5-propynylcytosine, 7-deazaguanine, 2-aminoadenine, 8-aza-7-deazaguanine, 1H-purine, and hypoxanthine.

70. The method in accordance with claims 58-62 or 63, further comprising selecting said at least one oligonucleotide analogue such that oligonucleotide analogue probes comprises at least one peptide nucleic acid.

71. The method in accordance with claims 58-62, or 63, further comprising selecting said at least one oligonucleotide analogue to increase image brightness when the oligonucleotide target and the oligonucleotide analogue probe hybridize in the presence of a fluorescent indicator, in comparison to a oligonucleotide probe without oligonucleotide analogs.

72. The method in accordance with claims 58-62, or 63, further comprising providing said plurality of oligonucleotide analogue probes in an array with at least 1000 other oligonucleotide analogue probes.