Non-aggregating non-quenching oligomer comprising nucelotide analogs, method of synthesis and use thereof

• US 20040116689A1
• Filed: 11/04/2003
• Published: 06/17/2004
• Est. Priority Date: 11/23/1999
• Status: Active Grant

• Current Assignee: ELITechGroup Inc. (ELITech Group SAS)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Vermeulen, Nicolaas M.J., Gall, Alexander A., Dempcy, Robert O., Kutyavin, Igor V.
• Granted Patent: US 6,972,328 B2
• Time in Patent Office: Days
• US Class Current: 536/27.1
• CPC Class Codes: A61K 31/519 ortho- or peri-condensed with heterocyclic rings
• CPC Class Codes: C07D 487/04 Ortho-condensed systems
• 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: C07K 14/003 Peptide-nucleic acids (PNAs)
• CPC Class Codes: C07K 5/06026 the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
• CPC Class Codes: C07K 5/06139 with the first amino acid being heterocyclic
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid

Abstract

The invention provides compositions and methods for improved hybridization analysis utilizing DNA, RNA, PNA and chimeric oligomers in which one or more purine bases are substituted by a pyrazolo[5,4-d]pyrimidine or by a 7-deazapurine purine analogue. Reduced self-aggregation and reduced fluorescence quenching are obtained when the oligomers are used in various methods involving hybridization. Methods of synthesis, as well as novel synthetic precursors, are also provided.

Claims

1. A composition comprising

13. An oligomer according to claim 1 wherein the oligomer is a PNA/DNA chimera.

2. A composition comprising

3. The composition of claim 2 wherein:

R₁ is —

OH;

R₂ is —

NHCOCH(CH₃)₂;

R₃ is —

H;

R₄ is MMTr; and

R₅ is —

H.

4. The composition of claim 2 wherein:

R₁ is —

OH;

R₂ is —

N═

CH—

N(CH₃)₂;

R₃ is —

H;

R₄ is MMTr; and

R₅ is —

H.

5. The composition of claim 2 wherein:

R₁ is —

NHAn;

R₂ is —

H;

R₃ is —

H;

R₄ is MMTr; and

R₅ is —

H.

6. The composition of claim. 2 wherein:

R₁ is —

N═

CH—

N(CH₃)₂;

R₂ is —

H;

R₃ is —

H;

R₄ is MMTr; and

R₅ is —

H.

7. The composition of claim 2 wherein:

R₁ is —

OH;

R₂ is —

H;

R₃ is —

H;

R₄ is MMTr; and

R₅ is —

H.

8. A method for synthesizing an oligomer, wherein the oligomer comprises one or more peptide nucleic acid residues, wherein the method comprises stepwise cycles of monomer addition to a growing oligomer chain that is optionally attached to a solid support, wherein the growing oligomer chain contains a blocked growing end, and wherein one or more of the monomer addition cycles comprises the following steps:

a) treatment of the growing chain under conditions that de-block the growing end;

b) treatment of the composition of claim 2 with an activating agent, if R₅ is —

H;

c) condensation of the products of steps (a) and (b);

d) optionally repeating the cycle of steps (a)-(c); and

e) at the completion of synthesis of the oligomer, exposure of the completed oligomer to conditions that remove all blocking groups and, if necessary, cleave the completed oligomer from the support.

9. The method of claim 8, wherein the oligomer is a peptide nucleic acid.

10. The method of claim 8, wherein the oligomer is a PNA/DNA chimera.

11. An oligomer synthesized according to the method of claim 8.

12. An oligomer according to claim 11, wherein the oligomer is a peptide nucleic acid.

75. An oligomer microarray, comprising one or more oligomers according to claim 11.

77. A method for detecting a target sequence in a polynucleotide by hybridization, wherein the improvement comprises:

use of the oligomer of claim 11 as a probe.

81. The method of any of claims 77-80 wherein the target sequence is distinguished from a related sequence having a single-nucleotide mismatch with respect to the target sequence.

14. An oligomer comprising one or more peptide nucleic acid (PNA) residues, wherein one or more of said PNA residues comprises a base having the formula

15. An oligomer according to claim 14, wherein the oligomer comprises one or more DNA nucleotides.

16. An oligomer according to claim 14, wherein the oligomer is a peptide nucleic acid.

17. An oligomer according to claim 14, wherein the oligomer is a PNA/DNA chimera.

18. An oligomer according to claim 14, further comprising a minor groove binder.

19. An oligomer according to claim 14, further comprising a fluorophore.

20. An oligomer according to claim 19, further comprising a fluorescence quencher.

21. An oligomer according to claim 20, further comprising a minor groove binder.

22. An oligomer according to claim 17, further comprising a fluorophore.

23. An oligomer according to claim 22, further comprising a fluorescence quencher.

24. An oligomer according to claim 23, further comprising a minor groove binder.

25. An oligomer according to claim 17, further comprising a minor groove binder.

26. An oligomer according to claim 25, further comprising a fluorophore.

76. An oligomer microarray, comprising one or more oligomers according to claim 14.

78. A method for detecting a target sequence in a polynucleotide by hybridization, wherein the improvement comprises:

use of the oligomer of claim 14 as a probe.

27. A method for detecting a target sequence in a polynucleotide wherein the improvement comprises:

use of a probe, complementary to the target sequence, wherein one or more residues in the probe comprises a base analogue, wherein the base analogue is selected from the group consisting of pyrazolopyrimidines and deazapurines,

such that the base analogues retain the base-pairing specificity of the bases for which they are substituted and reduce the tendency of the probe to self-associate.

28. The method of claim 27 wherein the probe comprises DNA.

29. The method of claim 27 wherein the probe comprises peptide nucleic acid (PNA).

30. The method of claim 27 wherein the probe comprises a PNA/DNA chimera.

31. The method of claim 27, wherein the base analogue has the structure

32. The method of claim 31 wherein one or more guanine bases in the probe are substituted by PPG.

33. The method of claim 31 wherein one or more adenine bases in the probe are substituted by PPA.

34. The method of claim 27, wherein the probe further comprises a minor groove binder.

35. The method of claim 27, wherein the probe further comprises a fluorophore.

36. The method of claim 35, wherein the probe further comprises a fluorescence quencher.

37. The method of claim 35, wherein the probe further comprises a minor groove binder.

38. The method of claim 36, wherein the probe further comprises a minor groove binder.

39. The method of claim 29, wherein the probe further comprises a minor groove binder.

40. The method of claim 29, wherein the probe further comprises a fluorophore.

41. The method of claim 40, wherein the probe further comprises a fluorescence quencher.

42. The method of claim 40, wherein the probe further comprises a minor groove binder.

43. The method of claim 41, wherein the probe further comprises a minor groove binder.

44. A method for the detection of a target sequence in a polynucleotide using a probe comprising a polymeric portion and a fluorescent portion, wherein the polymeric portion comprises a plurality of monomers;

the improvement comprising;

one or more of the monomers in the probe comprises a base analogue, wherein the base analogue is selected from the group consisting of pyrazolopyrimidines and deazapurines;

such that the base analogues retain the base-pairing specificity of the bases for which they are substituted and reduce the tendency of the polymeric portion of the probe to quench emission from the fluorescent portion.

45. The method of claim 44 wherein the polymeric portion of the probe comprises DNA.

46. The method of claim 44 wherein the polymeric portion of the probe comprises peptide nucleic acid (PNA).

47. The method of claim 44 wherein the polymeric portion of the probe comprises a PNA/DNA chimera.

48. The method of claim 44, wherein the base analogue has the structure

49. The method of claim 48 wherein one or more guanine bases in the polymeric portion of the probe are substituted by PPG.

50. The method of claim 49 wherein three or more consecutive guanine bases in the polymeric portion of the probe are substituted by PPG.

51. The method of claim 49, wherein a PPG residue is adjacent to the fluorescent portion of the probe.

52. The method of claim 44, wherein the probe further comprises a quenching portion.

53. The method of claim 51, wherein the probe further comprises a quenching portion.

54. The method of claim 44, wherein the probe further comprises a minor groove binder.

55. The method of claim 53, wherein the probe further comprises a minor groove binder.

57. The composition of claim 55, wherein the base analogue has the structure

64. The composition of claim 57, wherein X is ═

N—

.

65. The composition of claim 64, wherein the base analogue is selected from the group consisting of PPA, PPG and PPI.

66. The composition of claim 65 wherein three or more consecutive guanine bases in the polymer are substituted by PPG.

67. The composition of claim 57, wherein X is ═

CH—

.

68. The composition of claim 67, wherein the base analogue is selected from the group consisting of 7-deazadenine and 7-deazaguanine.

80. A method for detecting a target sequence in a polynucleotide by hybridization, wherein the improvement comprises:

use of the composition of claim 57 as a probe.

56. A composition comprising:

(a) a polymer comprising a plurality of monomer units; and

(b) a fluorophore;

wherein one or more of the monomer units comprises a base analogue, wherein the base analogue is selected from the group consisting of pyrazolopyrimidines and pyrrolopyrimidines, such that the base analogues retain the base-pairing specificity of the bases for which they are substituted and lead to reduced quenching of the fluorophore and/or reduced self-association of the polymer.

58. The composition of claim 56, further comprising a fluorescence quencher.

59. The composition of claim 56 wherein the polymer comprises DNA.

60. The composition of claim 56 wherein the polymer comprises peptide nucleic acid (PNA).

61. The composition of claim 56 wherein the polymer comprises a PNA/DNA chimera.

62. The composition of claim 61, wherein the base analogue is present in the DNA portion of the polymer.

63. The composition of claim 61, wherein the base analogue is present in the PNA portion of the polymer.

69. The composition of claim 58, wherein the fluorophore emits between 400 and 800 nm.

70. The composition of claim 69, wherein the quencher absorbs between 400 and 800 nm.

71. The composition of claim 56, further comprising a minor groove binder.

72. A method for detecting a target sequence in an amplification reaction (hydrolyzable probe assay) wherein the improvement comprises:

the use of the composition of claim 56 as a probe.

73. A method for detecting a target sequence in an amplification reaction (hydrolyzable probe assay) wherein the improvement comprises:

the use of the composition of claim 58 as a probe.

74. A method for detecting a target sequence in an amplification reaction (hydrolyzable probe assay) wherein the improvement comprises:

the use of the composition of claim 59 as a probe.

79. A method for detecting a target sequence in a polynucleotide by hybridization, wherein the improvement comprises:

use of the composition of claim 56 as a probe.