Non-nucleotide linking reagents for nucleotide probes

• US 5,696,251 A
• Filed: 06/07/1995
• Issued: 12/09/1997
• Est. Priority Date: 09/21/1987
• Status: Expired due to Term

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Bhatt, Ram S., Reynolds, Mark A., Arnold, Lyle J. Jr.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US08/485,629
• Time in Patent Office: 916 Days
• Field of Search: 525/155, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/25.32, 536/25.33, 536/25.34, 536/26.1, 536/26.11, 536/26.13, 536/26.14, 536/26.2, 536/26.21 , 536/26.22, 536/26.23, 536/26.26, 536/26.3, 536/26.5, 536/26.6, 536/26.7, 536/26.8, 536/26.9, 558/70, 558/199, 558/202, 562/8, 562/10, 562/23
• US Class Current: 536/23.1
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• 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

A versatile reagent with a non-nucleotide monomeric unit having a ligand, and first and second coupling groups which are linked to the non-nucleotide monomeric unit. The ligand can be either a chemical moiety, such as a label or intercalator, or a linking arm which can be linked to such a moiety. Such reagent permits preparation of versatile nucleotide/non-nucleotide polymers, having any desired sequence of nucleotide and non-nucleotide monomeric units, each of the latter of which bear a desired ligand. These polymers can for example, be used as probes which can exhibit enhanced sensitivity and/or which are capable of detecting a genus of nucleotides each species of which has a common target nucleotide sequence of interest bridged by different sequences not of interest.

Claims

1. A substituted oligonucleotide consisting of a chain of from 2 to 100 covalently coupled nucleotides and one or more non-nucleotide linker units, wherein each said non-nucleotide linker unit has the formula:

space="preserve" listing-type="equation">X.sup.1 --R.sup.2 (X.sup.3 --M).sub.n --Z.sup.1

where,

X¹ is O, S, NH or NNH,

R² is a chain of atoms having a length of 2-20 atoms, consisting essentially of a hydrocarbon chain optionally substituted by one or more heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur, said R² being stable to DNA synthesis deprotection conditions and joined to X^l and Z¹ and each said X³ --M,

each X³ is independently NH, O, S, NNH or a chain having a length of 2-25 atoms ending in NH, O, S, or NNH, wherein said X³ chain consists essentially of a hydrocarbon chain optionally substituted by one or more heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur, said X³ being stable to DNA synthesis deprotection conditions,

each M is independently H, a label, a solid-phase support, or a protecting group,

n is a positive integer,

Z¹ is a phosphorus-containing group, or may be O if it is at the 3'"'"'-end of said oligonucleotide where it is attached to H, and

wherein each X¹ and Z¹ is independently covalently bonded to a moiety selected from the group consisting of a protecting group, H, a chain of one or more said nucleotides, and another said linker unit.

2. The oligonucleotide of claim 1 wherein R² is a chain having a length of 2 to 10 atoms.

3. The oligonucleotide of claim 1 wherein R² is acyclic.

4. The oligonucleotide of claim 2 wherein R² is an acyclic hydrocarbon chain.

5. The oligonucleotide of claim 4 wherein said acyclic hydrocarbon chain has a length of 2 to 3 carbon atoms.

6. The oligonucleotide of any one of claim 1-5 wherein each X³ is linked to R² by a carbon and to M by a nitrogen.

7. The oligonucleotide of any one of claims 1-5 wherein n=1.

8. The oligonucleotide of any one of claims 1-5 wherein X¹ is O.

9. The oligonucleotide of any one of claims 1-5 wherein M is H.

10. The oligonucleotide of any one of claims 1-5 wherein M is a label.

11. The oligonucleotide of claim 10 wherein said label is selected from the group consisting of fluorescein, biotin, and acridinium ester derivatives.

24. The oligonucleotide of claim 6 wherein n=1.

25. The oligonucleotide of claim 6 wherein X¹ is O.

26. The oligonucleotide of claim 7 wherein X¹ is O.

27. The oligonucleotide of claim 6 wherein M is H.

28. The oligonucleotide of claim 7 wherein M is H.

29. The oligonucleotide of claim 8 wherein M is H.

30. The oligonucleotide of claim 6 wherein M is a label.

31. The oligonucleotide of claim 7 wherein M is a label.

32. The oligonucleotide of claim 8 wherein M is a label.

12. A substituted oligonucleotide consisting of a chain of from 2 to 100 covalently coupled nucleotides and one or more non-nucleotide linker units, wherein each said non-nucleotide linker unit has the formula:

##STR3## where, X¹ is O, S, NH or NNH,

X⁵ is H, or (CH₂)_k --NH--(CO--(CH₂)_q NH)_f --M,

each M is independently H, fluorenylmethoxycarbonyl, trifluoroacetyl, a label, or a solid-phase support,

i is 0, 1, 2 or 3, j is 0, 1, 2 or 3, and i+j is at least 1,

each k, q and r independently is an integer such that k+1+(2+q)r is an integer between 1 and 25 inclusive,

Z¹ is a phosphorus-containing group wherein said phosphorus-containing group is coupled to the OH group of a nucleotide or to X^x of another said linker unit, or may be 0 if it is at the 3'"'"'-end of said oligonucleotide where it is attached to H, and

wherein each X¹ and Z¹ is independently covalently bonded to a moiety selected from the group consisting of a protecting group, H, a chain of one or more said nucleotides, and another said linker unit.

13. The oligonucleotide of claim 12 wherein M is H.

14. The oligonucleotide of claim 12 wherein M is a solid-phase support.

15. The oligonucleotide of claim 12 wherein M is a label.

16. The oligonucleotide of claim 15 wherein said label is selected from the group consisting of fluorescein, biotin, and acridinium ester derivatives.

17. The oligonucleotide of any one of claims 12-16 wherein X⁵ is H.

18. The oligonucleotide of any one of claims 12-16 whereto i+j is 1 or 2.

19. The oligonucleotide of any one of claims 12-16 wherein each k is independently an integer between 1 and 3, inclusively.

20. The oligonucleotide of any one of claims 12-16 wherein each q is an integer between 1 and 6, inclusively.

21. The oligonucleotide of any one of claims 12-16 wherein r is 2.

22. The oligonucleotide of any one of claims 12-16 wherein r is 1.

23. The oligonucleotide of any one of claims 12-16 wherein r is O.

33. The oligonucleotide of claim 17 wherein i+j is 1 or 2.

34. The oligonucleotide of claim 17 wherein each k is independently an integer between 1 and 3, inclusively.

35. The oligonucleotide of claim 18 wherein each k is independently an integer between 1 and 3, inclusively.

36. The oligonucleotide of claim 17 wherein each q is an integer between 1 and 6, inclusively.

37. The oligonucleotide of claim 18 wherein each q is an integer between 1 and 6, inclusively.

38. The oligonucleotide of claim 19 wherein each q is an integer between 1 and 6, inclusively.

39. The oligonucleotide of claim 17 wherein r is 2.

40. The oligonucleotide of claim 18 wherein r is 2.

41. The oligonucleotide of claim 19 wherein r is 2.

42. The oligonucleotide of claim 20 wherein r is 2.

43. The oligonucleotide of claim 17 whereto r is 1.

44. The oligonucleotide of claim 18 wherein r is 1.

45. The oligonucleotide of claim 20 wherein r is 1.

46. The oligonucleotide of claim 17 wherein r is 0.

47. The oligonucleotide of claim 18 wherein r is 0.

48. The oligonucleotide of claim 19 wherein r is 0.

49. The oligonucleotide of claim 19 wherein r is 1.