Nucleic acid binding oligonucleotides

• US 20080269152A1
• Filed: 09/26/2007
• Published: 10/30/2008
• Est. Priority Date: 10/31/2003
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: Harvard University, Licensing & Industry Sponsored Research
• Inventors: Verdine, Gregory L., Santos, Webster
• Application Number:
  US11/904,612
• Publication Number:
  US 20080269152A1
• Time in Patent Office: Days
• US Class Current: 514/44
• CPC Class Codes: A61P 31/12 Antivirals
• 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 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present application pertains to products and methods related to the ability of short nucleotide oligomers to bind the tertiary or globular structure of nucleic acids. This application discloses libraries of short oligomers and methods for using these libraries.

Claims

1. A library of nucleic acid oligomers or nucleotide analog oligomers, wherein:

each oligomer is a 15mer or shorter; and

each oligomer is immobilized on a support.

2. The library of claim 1, wherein the library comprises from 50-99%, inclusive, of all permutations of four nucleotides in an oligomer of a particular length.

3. The library of claim 1, wherein the library provides at least one instance of each permutation of four nucleotides in an oligomer of a particular length.

4. The library of any one of claims 1-3, wherein the oligomer comprises a nucleotide analog in place of at least one of the four nucleotides.

9. The library or array of any one of claims 1-8, wherein the nucleic acid oligomer comprises RNA.

10. The library or array of any one of claims 1-8, wherein the nucleic acid oligomer consists of RNA.

11. The library or array of any one of claims 1-8, wherein the nucleic acid oligomer comprises DNA.

12. The library or array of any one of claims 1-8, wherein the nucleic acid oligomer consists of DNA.

13. The library or array of any one of claims 1-8, wherein at least one address comprises a nucleotide analog.

14. The library or array of any one of claims 1-8, wherein the oligomer consists of a nucleotide analog at each position of the oligomer.

15. The library or array of claim 13 or 14, wherein the nucleotide analog is one of the following:

a phosphodiester, a peptide nucleic acid, a phosphoramidate, a phosphorodiamidate, a phosphorothioate, a methylphosphonate, a morpholino, a tenofovir disoproxil fumarate, a hydroxyurea, tricyclo (tc)-DNA, a 2′

-deoxy-2′

-fluoro-D-arabinonucleic acid (FANA), a 2′

deoxy-2′

fluoro, a 2′

deoxy-2′

amine, a 2′

-O-alkyl analog, a 2′

(R or S)-deoxy-2′

C-alkyl, or a 2′

-C-alkyl.

16. The library or array of claim 13 or 14, wherein the nucleotide analog comprises one of the following:

Abacavir, ddI, 3TC, d4T, ddC, AZT, Emtricitabine, 2-amino-5-(2′

-deoxy-beta-d-ribofuranosyl)pyridine-5′

-triphosphate (d*CTP), or 5-(2′

-deoxy-beta-d-ribofuranosyl)-3-methyl-2-pyridone-5′

-triphosphate (d*TTP).

21. The library or array of any one of claims 1-20, wherein the oligomer is bound or attached to the support or substrate by a spacer.

22. The array of any one of claims 1-20, wherein the oligomer is bound or attached directly to the support or substrate, without a spacer.

23. The array of claim 21, wherein the oligomer is bound or attached to the support or substrate with a nucleic acid spacer.

24. The array of claim 21, wherein the oligomer bound or attached to the support or substrate with a non-nucleic acid spacer.

25. The library or array of any one of claims 1-20, wherein the oligomer is conjugated to a protein carrier molecule.

26. The array of any one of claims 6-26, wherein the array further comprises a target nucleic acid molecule comprising a tertiary or globular structure bound to one or more selected addresses of the array.

27. The array of claim 26, wherein the target nucleic acid molecule comprises a label.

28. The array of claim 27, wherein the label is a fluorescent label.

29. The array of claim 27, wherein the target nucleic acid molecule is radiolabeled.

30. The array of claim 26, wherein the target nucleic acid molecule comprises a moiety suitable for secondary labeling of the target nucleic acid molecule.

31. The array of claim 26, wherein the target nucleic acid comprises an RNA molecule comprising a tertiary or globular structure.

32. The array of claim 31, wherein the RNA molecule is labeled.

33. The array of claim 32, wherein the RNA molecule is conjugated to a reporter molecule comprising a fluorescent label.

34. The array of claim 32, wherein the RNA molecule is radiolabeled.

35. The array of claim 31, wherein the RNA molecule is conjugated to a moiety that enables secondary labeling of the target nucleic acid molecule.

36. The array of any one of claims 26-35, wherein the target RNA molecule comprises the sequence of at least a fragment of any one of the following:

a mRNA, a rRNA, a tRNA, a non-protein coding RNA, a small RNA, a miRNA, a siRNA, a ribozyme, a spliceosome, a telomerase, or a signal recognition particle.

37. The array of any one of claims 26-35, wherein the target RNA molecule comprises the sequence of at least a fragment of any one of the following:

an untranslated 5′

or 3′

region within 100 nucleotides of an ATG start codon, a ribosome pause site, a rare codon, or an internal ribosome entry site.

38. The array of claim 26, wherein the target nucleic acid molecule comprises a fragment of a viral nucleic acid sequence.

39. The array of claim 38, wherein the target nucleic acid molecule comprises the full length viral nucleic acid sequence.

40. The array of claim 38 or 39, wherein the viral nucleic acid sequence is from one of the following:

a RNA virus, a retrovirus, a dsRNA virus, a (+)sense RNA virus, a (−

) RNA virus, a viroid, a satellite RNA, or a prion encoding gene.

41. The array of claim 40, wherein the RNA virus consists of one of the following:

Hepatitis C virus, Human Immnuodeficiency virus, Herpes virus, Kaposi'"'"'s sarcoma-associated herpesvirus, Coronavirus, Bovine Coronavirus, Bovine viral diarrhea virus, GB virus-B, GB virus-C, Classic swine fever virus, foot-and-mouth disease virus, Friend murine leukemia virus, Moloney murine leukemia virus, Rous'"'"' sarcoma virus, Harvey sarcoma virus, Rhopalosiphum padi virus, Cricket paralysis virus, poliovirus, rhinovirus, encephalomyocarditis virus, and hepatitis A virus, or Plautia stali intestine virus (PSIV).

42. The array of claim 26, wherein the target nucleic acid molecule is at least a fragment of the RNA transcript of one of the following genes:

an oncogene, a tumor suppressor gene, a cell adhesion molecule gene, or a telomerase.

43. The array of claim 42, wherein the target RNA is at least a fragment of an mRNA encoding one of the following:

a translation initiation factor, e.g. eIF4G or DAP5;

a transription factor e.g., c-myc, NF-B repressing factor (NRF);

a growth factor e.g. Vascular endothelial growth factor (VEGF), Fibroblast growth factor 2 (FGF-2), Platelet-derived growth factor B (PDGF-B);

a homeotic gene e.g. Antennapedia;

a Survival Protein e.g. X-linked inhibitor of apoptosis (XIAP), Apaf-1;

or BiP.

44. The array of any one of claims 26 or 38-43, wherein the target nucleic acid molecule is produced recombinantly in a host cell.

45. The array of any one of claims 26 or 38-43, wherein the target nucleic acid molecule is produced in vitro.

46. The array of any one of claims 26 or 38-43, wherein the target nucleic acid is harvested from infected cells or from cells that carry a gene expressing the target nucleic acid endogenously.

47. The array of any one of claims 38-46, wherein the target nucleic acid molecule comprises a radiolabel, a reporter molecule comprising a fluorescent label, or a moiety suitable for secondary labeling.

48. An apparatus comprising:

the library or array of any one of claims 1-47; and

a detector suitable for detecting an interaction between a target nucleic acid molecule and an oligomer comprised by the library or array.

49. The apparatus of claim 48, wherein the detector is a scanner.

50. The apparatus of claim 48, wherein the detector comprises a microscope.

51. The apparatus of any one of claims 48-50, further comprising a digital storage device suitable for storing information generated by the scanner or microscope.

52. The apparatus of claim 51, wherein the detector is a fluorescence scanner.

53. The apparatus of any one of claims 48-52, wherein the detector generates information that allows a determination of the address or oligomer to which a target molecule has bound.

5. An array comprising:

a substrate comprising a plurality of addresses, wherein each address is associated with at least one nucleic acid oligomer, or one nucleotide analog oligomer, that is a 9mer or shorter.

6. The array of claim 5, wherein each address comprises at least one nucleic acid oligomer, or one nucleotide analog oligomer, that is a 5mer or shorter.

7. The array of claim 5 or 6, wherein the array provides an address for from 50-99%, inclusive, of all permutations of the nucleic acid oligomers.

8. The array of claim 5 or 6, wherein the array provides an address for all permutations of the nucleic acid or nucleotide analog oligomers for a particular oligomer length.

17. The array of any one of claims 5-8, wherein the array comprises more than one plurality of addresses and at least one plurality comprises oligomers comprising DNA and at least one other plurality comprises oligomers comprising RNA.

18. The array of any one of claims 5-8, wherein the array comprises more than one plurality of addresses and at least one plurality comprises oligomers comprising DNA and at least one other plurality comprises oligomers comprising a nucleotide analogue.

19. The array of any one of claims 5-8, wherein the array comprises more than one plurality of addresses and at least one plurality comprises oligomers comprising RNA and at least one other plurality comprises oligomers comprising a nucleotide analog.

20. The array of any one of claims 5-8, wherein the array comprises at least three plurality of addresses and one plurality comprises oligomers comprising DNA, another plurality comprises oligomers comprising RNA, and yet another plurality comprises oligomers comprising a nucleotide analog.

54. A method of making a substrate suitable for evaluating oligomers, said method comprising:

providing a substrate comprising a plurality of addresses;

attaching at least one nucleic acid oligomer at each address, wherein the oligomer is a 9mer or shorter; and

wherein the substrate provides an address for at least 10% of all permutations of four nucleotides at each position of an oligomer of a particular length.

55. The method of claim 54, wherein the oligomer is covalently attached to the substrate at each address.

56. The method of claim 54, wherein the oligomer is non-covalently attached to the substrate at each address.

57. The method of claim 54, wherein the oligomer is restricted to one address on the substrate.

58. The method of claim 54, wherein the oligomer is synthesized at an address on the substrate.

59. The method of any one of claims 54-58, wherein the substrate provides an address for each permutation of an oligomer of particular length comprising one of four nucleotide bases at each position.

60. The method of any one of claims 54-59, wherein a distinct address is provided for every permutation of the nucleic acid oligomer.

61. The method of any one of claims 54-60, wherein a spacer separates the oligomer from the substrate.

62. The method of any one of claims 54-60, wherein the attached or synthesized oligomer is directly attached to the substrate, without a spacer.

63. The method of any one of claims 54-62, wherein the oligomer comprises DNA.

64. The method of any one of claims 54-62, wherein the oligomer comprises RNA.

65. The method of any one of claims 54-62, wherein the oligomer comprises a nucleic acid analogue.

66. The method of any one of claims 54-62, wherein the oligomer comprises a mixture of any two or more of DNA, RNA, or a nucleic acid analogue.

67. A method of making a substrate suitable for evaluating oligomers, said method comprising:

providing a substrate comprising a plurality of addresses;

attaching at least one nucleic acid oligomer at each address, wherein the oligomer is a 5mer or shorter; and

wherein the substrate provides an address for at least 10% of all permutations of four nucleotide bases at each position of a nucleic acid oligomer of a particular length.

68. The method of claim 67, wherein the oligomer is covalently attached to the substrate at each address.

69. The method of claim 67, wherein the oligomer is non-covalently attached to the substrate at each address.

70. The method of claim 67, wherein the oligomer is restricted to one address on the substrate.

71. The method of claim 67, wherein the oligomer is synthesized at an address on the substrate.

72. The method of any one of claims 67-71, wherein the substrate provides an address for every permutation of an oligomer of a particular length comprising one of four possible nucleotides at each position of the oligomer.

73. The method of any one of claims 67-72, wherein a distinct address is provided for each permutation of the nucleic acid oligomer.

74. The method of claims 67-73, wherein a spacer separates the oligomer from the substrate.

75. The method of claims 67-73, wherein the attached or synthesized oligomer is directly attached to the substrate, without a spacer.

76. The method of any one of claims 67-75, wherein the oligomer comprises DNA.

77. The method of any one of claims 67-75, wherein the oligomer comprises RNA.

78. The method of any one of claims 67-75, wherein the oligomer comprises a nucleic acid analogue.

79. The method of any one of claims 67-75, wherein the oligomer comprises a mixture of any two or more of DNA, RNA, or a nucleic acid analogue.

80. A method of identifying at least one nucleic acid oligomer that non-canonically binds a target RNA molecule, said method comprising:

providing an array that comprises a substrate comprising a plurality of addresses, wherein each address comprises at least one nucleic acid oligomer, wherein the oligomer is from a 2mer to a 15mer, inclusive; and

contacting the array with a target molecule that comprises a folded RNA.

81. The method of claim 80, wherein the target molecule comprises a folded RNA-protein complex.

82. The method of claim 80 or 81, further comprising the step of detecting a binding interaction between the target RNA molecule and the array.

83. The method of claim 82, wherein the binding interaction is detected qualitatively.

84. The method of claim 82, wherein the binding interaction is detected quantitavely.

85. The method of any one of claims 82-84, further comprising (the step of) identifying one or more addresses of the plurality to which the target RNA molecule has bound.

86. The method of claim 85, further comprising (the step of) entering data representing the address to which the target RNA molecule has bound into a database.

87. The method of any one of claims 82-85, further comprising identifying the oligomer, or a group of oligomers comprising the oligomer, to which the target RNA molecule has bound.

88. The method of claim 87, further comprising entering data representing the oligomer, or a group of oligomers comprising the oligomer, to which the target RNA molecule has bound.

89. The method of any one of claims 82-87, wherein prior to detecting the binding interaction, the substrate is washed under non-denaturing conditions.

90. The method of any one of claims 81-89, wherein the contacting or washing is done in the presence of at least one non-target RNA molecule that is either unlabeled or differently labeled from the target RNA.

91. The method of claim 90, wherein the non-target RNA differs from the target RNA by fewer than 10 nucleotides.

92. The method of claim 90, wherein the non-target RNA comprises at least one RNA sequence that is expressed in an organism.

93. The method of claim 90, wherein the target RNA and the non-target RNA comprise the same RNA sequence, but only one of the RNA molecules is in an RNA-protein complex.

94. The method of any one of claims 85-93, wherein the data entered into a database represents the oligomer, or a group of oligomers comprising the oligomer, to which the target RNA molecule has bound.

95. The method of any one of claims 80-94, wherein the array comprises from 20-99%, inclusive, of all permutations of four nucleotide bases at each position of a nucleic acid oligomer of a particular length.

96. The method of any one of claims 80-94, wherein the array comprises every permutation of four nucleotide bases at each position of a nucleic acid oligomer of a particular length.

97. The method of any one of claims 80-96, wherein the target RNA comprises a reporter molecule comprising a fluorescent label.

98. The method of any one of claims 80-96, wherein the target RNA molecule is radiolabeled.

99. The method of any one of claims 80-96, wherein the target RNA molecule comprises a moiety suitable for secondary labeling.

100. The methods of any one of claims 80-96, wherein the target RNA molecule is modified to facilitate detection.

101. A method of identifying an oligomer that preferentially interacts with one of two RNA molecules, the method comprising:

performing the method of any one of claims 80-100 with a first RNA;

repeating the method performed with a second RNA or a second RNA in an RNA protein complex;

comparing the results; and

identifying an oligomer that preferentially interacts with either the first or the second RNA.

102. The method of any one of claims 87-100, further comprising formulating the oligomer identified as interacting with the target RNA in a pharmaceutical composition.

103. The method of claim 101, further comprising formulating the oligomer that preferentially interacts with one of the RNA molecules in a pharmaceutical composition.

104. The method of any one of claims 102-103, further comprising administering the pharmaceutical composition to a cell or organism.

105. A method of using an oligomer identified as interacting or preferentially interacting with a target RNA molecule, the method comprising:

performing any one of the methods of claims 87-101, wherein an oligomer is identified as interacting or preferentially interacting with a target RNA molecule;

contacting the identified oligomer to a cell; and

evaluating the cell.

106. A method of using an oligomer identified as interacting or preferentially interacting with a target RNA molecule, the method comprising:

performing any one of the methods of claim 87-101, wherein an oligomer is identified as interacting or preferentially interacting with a target RNA molecule; and

further evaluating the interaction between the identified oligomer and the target molecule.

107. The method of claim 106, wherein the interaction between the oligomer and the target is evaluated in solution.

108. The method of claim 106, wherein the evaluation of the interaction comprises any of the following:

a gel shift assay, a footprinting experiment, susceptibility of oligomer or the target molecule to affinity cleavage.

109. A method of identifying an oligomer useful as a candidate for the treatment of an RNA virus, comprising any of the methods of claim 87-101, wherein the target RNA comprises at least a fragment of a viral nucleic acid sequence.

110. The method of claim 109, wherein the target RNA comprises at least a fragment of the viral nucleic acid sequence from one of the following:

a RNA virus, a retrovirus, a dsRNA virus, a (+)sense RNA virus, a (−

)sense RNA virus, a viroid, a satellite RNA, a prion encoding gene.

111. The method of claim 109 or 110, wherein the RNA virus consists of one of the following:

Hepatitis C virus, Human Immnuodeficiency virus, Herpes virus, Kaposi'"'"'s sarcoma-associated herpesvirus, Coronavirus, Bovine Coronavirus, Bovine viral diarrhea virus, GB virus-B, GB virus-C, Classic swine fever virus, foot-and-mouth disease virus, Friend murine leukemia virus, Moloney murine leukemia virus, Rous'"'"' sarcoma virus, Harvey sarcoma virus, Rhopalosiphum padi virus, Cricket paralysis virus, poliovirus, rhinovirus, encephalomyocarditis virus, and hepatitis A virus, or Plautia stali intestine virus (PSIV).

112. A method of making a pharmaceutical composition for treating an RNA virus, the method comprising:

performing the method of any one of claims 109-111; and

formulating a pharmaceutical composition comprising the oligomer identified as useful in a treatment of a virus.

113. A method of identifying an oligomer useful in the regulation of gene expression comprising any one of the methods of claims 87-101, wherein the target RNA comprises at least a fragment of an mRNA molecule.

114. The method of claim 113, wherein the target RNA is at least a fragment of an mRNA encoding one of the following:

a translation initiation factor, e.g. eIF4G or DAP5;

a transription factor e.g., c-myc, NF-B repressing factor (NRF);

a growth factor e.g. Vascular endothelial growth factor (VEGF), Fibroblast growth factor 2 (FGF-2), Platelet-derived growth factor B (PDGF-B);

a homeotic gene e.g. Antennapedia;

a Survival Protein e.g. X-linked inhibitor of apoptosis (XIAP), Apaf-1;

or BiP.

115. A method of making a pharmaceutical composition for regulating gene expression, the method comprising:

performing the method of claim 113 or 114; and

formulating a pharmaceutical composition comprising the oligomer identified as useful in regulating gene expression.

119. The method of any one of claims 87-100, further comprising the step of synthesizing a variant of the oligomer identified as binding or preferentially binding a target molecule.

120. The method of claim 119, wherein synthesizing the variant oligomer comprises making new oligomers that differs from the identified oligomer in one of the following manners:

the variant has an altered the sugar backbone relative to the identified oligomer, at least one nucleotide in the identified oligomer is replaced with a nucleotide analog, at least one DNA nucleotide in the identified oligomer is replaced with an RNA nucleotide, at least one RNA nucleotide in the identified oligomer is replaced with a DNA nucleotide, or at least one nucleotide analog in the oligomer is replaced with a nucleotide.

121. The method of claim 119 or 120, further comprising formulating the variant oligomer in a pharmaceutical composition.

116. A method of identifying a candidate therapeutic target sequence within an RNA molecule, comprising:

providing an array comprising a substrate having a plurality of addresses, each address comprising at least one nucleic acid oligomer, and wherein the oligomer is from a 2mer to a 15mer, inclusive;

contacting the array with a candidate RNA molecule; and

detecting whether the candidate RNA molecule has bound the array;

thereby identifying whether or not the RNA molecule comprises a candidate therapeutic target for a non-canonically binding oligomer.

117. The method of claim 116, further comprising identifying the oligomer to which RNA has bound.

118. A method of designing a nucleic acid oligomer that binds non-canonically to a target RNA molecule, comprising:

providing an array comprising a substrate having a plurality of addresses, each address comprising at least one nucleic acid oligomer, and wherein the oligomer is from a 2mer to a 15mer, inclusive;

contacting the array with the target RNA molecule;

detecting whether the candidate RNA molecule binds the array;

identifying the address to which the RNA molecule binds;

correlating the address to the structural features of an oligomer comprised by the address; and

using the structural features to design an oligomer that binds non-canonically to the target RNA molecule.

122. A kit for identifying oligomers that bind non-canonically to target nucleic acid molecules comprising:

a container; and

within that container is packaged an array comprising a substrate having a plurality of addresses, each address comprising at least one nucleic acid oligomer, and wherein the oligomer is from a 2mer to a 15mer, inclusive.

123. The kit of claim 122, wherein the array is packaged with instructions for using the array in a method for identifying oligomers that bind non-canonically to target nucleic acid molecules, e.g. RNA.

124. The kit of either claim 122 or claim 123, wherein the array further provides an address for all permutations of the nucleic acid oligomer.

125. The kit of any one of claims 122-124, wherein the short nucleic acid oligomer comprises RNA, or DNA, or a modified oligonucleotide.

126. The kit of claim 125, wherein the array is packaged with a target RNA molecule that is either labeled or conjugated to a moiety that enables secondary labeling.

127. The kit of claims 122-125, further comprising non-denaturing reagents.