Compositions and methods related to two-arm nucleic acid probes

• US 20030215864A1
• Filed: 04/23/2003
• Published: 11/20/2003
• Est. Priority Date: 04/23/2002
• Status: Abandoned Application

• Current Assignee: U.S. Genomics, Inc (Toxic Reports LLC)
• Original Assignee: U.S. Genomics, Inc (Toxic Reports LLC)
• Inventors: Goncalves, Nuno, Gilmanshin, Rudolf
• Application Number:
  US10/421,644
• Publication Number:
  US 20030215864A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof

Abstract

The invention provides compositions and methods of use relating to nucleic acid detection probes that comprise a Hoogsteen binding arm and a Watson-Crick binding arm that bind to adjacent but not identical target sites.

Claims

1. A composition comprising

a Hoogsteen binding arm that binds by Hoogsteen base pairing to a target nucleic acid molecule at a first target site, and

a Watson-Crick binding arm that binds by Watson-Crick base pairing to the target nucleic acid molecule at a second target site,

wherein the Hoogsteen binding arm and the Watson-Crick binding arm are conjugated to each other, and are comprised of nucleic acid or nucleic acid mimic elements.

2. The composition of claim 1, wherein the Hoogsteen binding arm is selected from the group consisting of a DNA, an RNA, a PNA, and an LNA.

3. The composition of claim 1, wherein the Watson-Crick binding arm is selected from the group consisting of a DNA, an RNA, a PNA, and an LNA.

4. The composition of claim 1, wherein the target nucleic acid molecule is a DNA or an RNA.

5. The composition of claim 1, wherein the Hoogsteen binding arm has at least one backbone modification.

6. The composition of claim 1, wherein the Watson-Crick binding arm has at least one backbone modification.

7. The composition of claim 5 or 6, wherein the at least one backbone modification is selected from the group consisting of a peptide modification, and a phosphorothioate modification.

8. The composition of claim 1, wherein the Hoogsteen binding arm and the Watson-Crick binding arm are conjugated to each other covalently.

9. The composition of claim 1, wherein the Hoogsteen binding arm and the Watson-Crick binding arm are conjugated to each other using a linker molecule.

10. The composition of claim 9, wherein the linker molecule is selected from the group consisting of 8-amino-3,6-dioxaoctanoic acid (O-linker), E-linker, and X-linker.

11. The composition of claim 9, wherein the linker molecule comprises a cleavable bond.

12. The composition of claim 9, wherein the linker molecule has a length of less than 100 Angstroms.

13. The composition of claim 1, wherein the Hoogsteen binding arm has a nucleotide sequence that is a homopurine nucleotide sequence or homopyrimidine nucleotide sequence.

14. The composition of claim 1, wherein the Watson-Crick binding arm has a nucleotide sequence that is random.

15. The composition of claim 1, wherein the Hoogsteen binding arm is 5-12 nucleotides in length.

16. The composition of claim 1, wherein the Watson-Crick binding arm is 5-12 nucleotides in length.

17. The composition of claim 1, wherein the Hoogsteen binding arm and the Watson-Crick binding arm have different lengths.

18. The composition of claim 1;

wherein the first target site and the second target site are spaced apart from each other by a distance selected from the group consisting of 1 base pair, 2 base pairs, 5 base pairs, 7 base pairs, 10 base pairs, 20 base pairs, and 25 base pairs.

19. The composition of claim 1, wherein the Hoogsteen binding arm and the Watson-Crick binding arm, when both are bound to their respective target sites, are spaced apart from each other by a distance selected from the group consisting of 1 base pair, 2 base pairs, 5 base pairs, 7 base pairs, 10 base pairs, 20 base pairs, and 25 base pairs.

20. The composition of claim 1, wherein the Hoogsteen binding arm is conjugated to an agent.

21. The composition of claim 1 or 20, wherein the Watson-Crick binding arm is conjugated to an agent.

22. The composition of claim 20 or 21, wherein the agent is a detectable label.

23. The composition of claim 22, wherein the detectable label is selected from the group consisting of an electron spin resonance molecule (e.g., nitroxyl radicals), a fluorescent molecule, a chemiluminescent molecule, a radioisotope, an enzyme substrate, a biotin molecule, an avidin molecule, an electrical charge transferring molecule, a semiconductor nanocrystal, a semiconductor nanoparticle, a colloid gold nanocrystal, a ligand, a microbead, a magnetic bead, a paramagnetic particle, a quantum dot, a chromogenic substrate, an affinity molecule, a protein, a peptide, a nucleic acid, a carbohydrate, an antigen, a hapten, an antibody, an antibody fragment, and a lipid.

24. The composition of claim 22, wherein the detectable label is detected using a detection system selected from the group consisting of a charge coupled device detection system, an electron spin resonance detection system, a fluorescent detection system, an electrical detection system, a photographic film detection system, a chemiluminescent detection system, an enzyme detection system, an atomic force microscopy (AFM) detection system, a scanning tunneling microscopy (STM) detection system, an optical detection system, a nuclear magnetic resonance (NMR) detection system, a near field detection system, and a total internal reflection (TIR) detection system.

25. The composition of claim 20 or 21, wherein the agent is a cytotoxic agent.

26. The composition of claim 1, wherein the target nucleic acid molecule is a genomic DNA molecule or a mitochondrial DNA molecule.

28. A method for labeling a target nucleic acid molecule comprising

a) contacting the target nucleic acid molecule with a composition of claim 1 or 27, and

b) allowing the composition to bind specifically to the target nucleic acid molecule.

29. The method of claim 28, further comprising detecting binding of the composition to the target nucleic acid molecule.

30. The method of claim 28, wherein the Hoogsteen binding arm is selected from the group consisting of a DNA, an RNA, a PNA, and an LNA.

31. The method of claim 28, wherein the Watson-Crick binding arm is selected from the group consisting of a DNA, an RNA, a PNA, and an LNA.

32. The method of claim 28, wherein the Hoogsteen binding arm has at least one backbone modification.

33. The method of claim 28, wherein the Watson-Crick binding arm has at least one backbone modification.

34. The method of claim 32 or 33, wherein the at least one backbone modification is selected from the group consisting of a peptide modification and a phosphorothioate modification.

35. The method of claim 28, wherein the Hoogsteen binding arm and Hoogsteen binding arm are conjugated to each other covalently.

36. The method of claim 28, wherein the Hoogsteen binding arm and Hoogsteen binding arm are conjugated to each other using a linker molecule.

37. The method of claim 36, wherein the linker molecule is selected from the group consisting of 8-amino-3,6-dioxaoctanoic acid (O-linker), E-linker, and X-linker.

38. The method of claim 36, wherein the linker molecule comprises a hydrolyzable cleavable.

39. The method of claim 36, wherein the linker molecule has a length of less than 100 Angstroms.

40. The method of claim 28, wherein the Hoogsteen binding arm has a nucleotide sequence that is a homopurine nucleotide sequence or homopyrimidine nucleotide sequence.

41. The method of claim 28, wherein the Watson-Crick binding arm has a nucleotide sequence that is random.

42. The method of claim 28, wherein the Hoogsteen binding arm is 5-12 nucleotides in length.

43. The method of claim 28, wherein the Watson-Crick binding arm is 5-12 nucleotides in length.

44. The method of claim 28, wherein the Hoogsteen binding arm and the Watson-Crick binding arm have different lengths.

45. The method of claim 28, wherein the first target site and the second target site are spaced apart from each other by a distance selected from the group consisting of 1 base pair, 2 base pairs, 5 base pairs, 7 base pairs, 10 base pairs, 20 base pairs, and 25 base pairs.

46. The method of claim 28, wherein the Hoogsteen binding arm and the Watson-Crick binding arm, when both are bound to their respective target sites, are spaced apart from each other by a distance selected from the group consisting of 1 base pair, 2 base pairs, 5 base pairs, 7 base pairs, 10 base pairs, 20 base pairs, and 25 base pairs.

47. The method of claim 28, wherein the Hoogsteen binding arm is conjugated to an agent.

48. The method of claim 28 or 47, wherein the Watson-Crick binding arm is conjugated to an agent.

49. The method of claim 47 or 48, wherein the agent is a detectable label.

50. The method of claim 49, wherein the detectable label is selected from the group consisting of an electron spin resonance molecule (e.g., nitroxyl radicals), a fluorescent molecule, a chemiluminescent molecule, a radioisotope, an enzyme substrate, a biotin molecule, an avidin molecule, an electrical charge transferring molecule, a semiconductor nanocrystal, a semiconductor nanoparticle, a colloid gold nanocrystal, a ligand, a microbead, a magnetic bead, a paramagnetic particle, a quantum dot, a chromogenic substrate, an affinity molecule, a protein, a peptide, a nucleic acid, a carbohydrate, an antigen, a hapten, an antibody, an antibody fragment, and a lipid.

51. The method of claim 49, wherein the detectable label is detected using a detection system selected from the group consisting of a charge coupled device detection system, an electron spin resonance detection system, a fluorescent detection system, an electrical detection system, a photographic film detection system, a chemiluminescent detection system, an enzyme detection system, an atomic force microscopy (AFM) detection system, a scanning tunneling microscopy (STM) detection system, an optical detection system, a nuclear magnetic resonance (NMR) detection system, a near field detection system, and a total internal reflection (TIR) detection system.

52. The method of claim 47 or 48, wherein the agent is a cytotoxic agent.

53. The method of claim 48, wherein the agent is a nucleic acid cleaving agent.

54. The method of claim 28, wherein the target nucleic acid molecule is a DNA or an RNA molecule.

55. The method of claim 28, wherein the target nucleic acid molecule is a genomic DNA molecule or a mitochondrial DNA molecule.

56. The method of claim 29, further comprising determining a pattern of binding of the composition to the target nucleic acid molecule.

57. The method of claim 56, wherein the pattern of binding is determined using a linear polymer analysis system, FISH, or optical mapping.

58. The method of claim 56, wherein the pattern of binding is determined by detecting and measuring cleavage products from the target nucleic acid molecule.

59. The method of claim 56, wherein the pattern of binding is indicative of a loss of transcription.

60. The composition of claim 1, wherein the Hoogsteen binding arm comprises a PNA.

61. The composition of claim 1 or claim 60, wherein the Watson-Crick binding arm comprises a PNA.

62. The method of claim 28, wherein the Hoogsteen binding arm comprises a PNA.

63. The method of claim 28, wherein the Watson-Crick binding arm comprises a PNA.

27. A composition comprising

a Hoogsteen binding arm that binds by Hoogsteen base pairing to a target nucleic acid molecule at a first target site, and

a Watson-Crick binding arm that binds by Watson-Crick base pairing to the target nucleic acid molecule at a second target site

wherein the Hoogsteen binding arm and the Watson-Crick binding arm are conjugated to each other through a linker.