Nucleic acid compositions with scissile linkage useful for detecting nucleic acid sequences

• US 4,876,187 A
• Filed: 12/05/1985
• Issued: 10/24/1989
• Est. Priority Date: 12/05/1985
• Status: Expired due to Term

• Current Assignee: ID Biomedical Corporation (GSK plc)
• Original Assignee: Meiogenics, Inc.
• Inventors: Crosby, William, Robertson, John G., Bender, Robert, Duck, Peter
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US06/805,279
• Time in Patent Office: 1,419 Days
• Field of Search: 435/6, 435/91, 436/501, 935/77, 935/78, 536/26, 536/27, 536/28
• US Class Current: 435/5
• 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 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• 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 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides

Abstract

A synthetic, non-naturally occurring molecule having the structure:

[NA.sub.1 --S--NA.sub.2 ].sub.n

wherein NA₁ and NA₂ are noncomplementary nucleic acid sequences;

wherein --S-- is a scissile linkage; and

wherein n is an integer from 1 to 4.

Variations of this molecule and methods for using the molecules for detecting nucleic acid sequences are provided.

Claims

1. A synthetic, non-naturally occurring composition comprising the structure:

space="preserve" listing-type="equation">--NA.sub.1 --S--NA.sub.2 ].sub.n

wherein NA₁ and NA₂ are different noncomplementary nucleic acid sequences;

wherein --S-- is a scissile linkage which is capable of being cleaved or disrupted without cleaving or disrupting the nucleic acid sequences of NA₁ or NA₂ or of a target nucleic acid sequence capable of hybridizing to the NA₁ and NA₂ sequences, or to the NA₁ and NA₂ sequences and the scissile linkage of said composition, wherein if the scissile linkage is a nucleic acid sequence it is RNA when both NA₁ and NA₂ are DNA sequences, or the scissile linkage is DNA when both NA₁ and NA₂ are RNA sequences; and

wherein n is an integer from 1 to 4.

2. The composition of claim 1, wherein the dashed lines represent chemical bonds.

3. The composition of claim 2, wherein the chemical bonds are covalent bonds.

4. The composition of claim 1, wherein the scissile linkage is selected from the group consisting of RNA sequences, DNA sequences, amino acid sequences, abasic nucleic acid sequences or carbohydrate polymers.

5. The composition of claim 1, wherein the scissile linkage is a nucleic acid sequence.

6. The composition of claim 1 immobilized on a solid support.

7. The composition of claim 1, wherein NA₁ and NA₂ are DNA sequences.

8. The composition of claim 1, wherein NA₁ and NA₂ are RNA sequences.

9. The composition of claim 1, wherein when the scissile linkage is other than a nucleic acid sequence NA₁ is either an RNA or DNA sequence and NA₂ is either an RNA or DNA sequence.

10. The molecule of claim 1, wherein NA₁ or NA₂ comprises a sequence which is not a naturally occurring nucleic acid sequence.

11. The composition of claim 1, wherein NA₁ or NA₂ is a naturally occurring nucleic acid sequence.

12. The composition of claim 1, wherein in each -NA₁ --S--NA₂ ] a marker is attached to one of NA₁ or NA₂.

13. The composition of claim 12 immobilized on a solid support.

14. The composition of claim 12, wherein the marker is a radioisotope, a radiolabelled molecule, a fluorescent molecule, biotin, an enzyme, or a ligand.

15. The composition of claim 1, wherein NA₁ and NA₂ each comprise between 8 nucleotides and 10,000 nucleotides.

16. The composition of claim 1, wherein n is greater than 1 and each [NA₁ --S--NA₂ ] unit is the same.

17. The composition of claim 1, wherein n is greater than 1 and each [NA₁ --S--NA₂ ] unit is different.

46. A method of detecting in a sample the presence of a target nucleic acid sequence of interest comprising:

(a) contacting the sample with a composition of claim 12 which comprises a nucleic acid sequence substantially complementary to the target nucleic acid sequence of interest, under hybridizing conditions to form a hybridized complex;

(b) immobilizing said complex on a solid support;

(c) treating the immobilized complex so as to cleave the scissile linkage;

(d) separately recovering the immobilized complex; and

(e) detecting the presence of the marker on the immobilized complex and thereby the target nucleic acid sequence of interest.

49. A diagnostic method for detecting the presence of a foreign pathogen in a sample which comprises detecting a nucleic acid sequence characteristic of the pathogen using the method of any of claims 46-48.

50. A method for quantitatively determining in a sample the amount of a target nucleic acid sequence of interest comprising:

(a) contacting the sample with a predetermined amount of a composition of claim 12, which comprises a nucleic acid sequence substantially complementary to the target nucleic acid sequence of interest, under hybridizing conditions to form a hybridized complex;

(b) immobilizing said complex on a solid support;

(c) treating the immobilized complex so as to cleave the scissile linkage;

(d) separately recovering the immobilized complex; and

(e) quantitatively determining the amount of marker present on the immobilized complex and thereby the amount of target nucleic acid sequence of interest.

53. A diagnostic method for measuring the amount of a foreign pathogen in a sample which comprises detecting a nucleic acid of the pathogen using the method of any of claims 50-52.

18. A synthetic, non-naturally occurring composition comprising the structure:

space="preserve" listing-type="equation">--NA.sub.1 --S--NA.sub.2 ].sub.n

wherein NA₁ and NA₂ are nucleic acid sequences;

wherein --S-- is a scissile linkage which is capable of being cleaved or disrupted without cleaving or disrupting the nucleic acid sequences of NA₁ or NA₂ or of a target nucleic acid sequence capable of hybridizing to the NA₁ and NA₂ sequences, or to the NA₁ and NA₂ sequences and the scissile linkage of said composition, wherein if the scissile linkage is a nucleic acid sequence it is RNA when both NA₁ and NA₂ are DNA sequences, or the scissile linkage is DNA when both NA₁ and NA₂ are RNA sequences; and

wherein n is an integer from 2 to 4.

19. The composition of claim 18, wherein a marker is attached to one of NA₁ or NA₂.

20. The composition of claim 18 immobilized on a solid support.

21. A composition comprising the structure:

space="preserve" listing-type="equation">S--L[NA.sub.1 --S--NA.sub.2 ].sub.n M

wherein NA₁ and NA₂ are nucleic acid sequences;

wherein --S-- is a scissile linkage which is capable of being cleaved or disrupted without cleaving or disrupting the nucleic acid sequences of NA₁ or NA₂ or of a target nucleic acid sequence capable of hybridizing to the NA₁ and NA₂ sequences, or to the NA₁ and NA₂ sequences and the scissile linkage of said composition, wherein if the scissile linkage is a nucleic acid sequence it is RNA when both NA₁ and NA₂ are DNA sequences, or the scissile linkage is DNA when both NA₁ and NA₂ are RNA sequences;

wherein n is an integer from 1 to 4;

wherein the solid lines represent chemical bonds;

wherein X is a solid support;

wherein L is a chemical entity which links NA₁ to the solid support; and

wherein M is a marker.

22. The composition of claim 21, wherein X is a silicaceous, cellulosic, or plastic material or controlled pore glass.

23. The composition of claim 21, wherein the chemical bonds are covalent bonds.

24. The composition of claim 21, wherein the chemical bonds are hydrogen or covalent bonds.

25. The composition of claim 21, wherein M is a radioisotope, a radiolabelled molecule, a fluorescent molecule, or a ligand.

26. The composition of claim 21, wherein the dashed lines represent chemical bonds.

27. The composition of claim 26, wherein the chemical bonds represented by the dashed lines are covalent bonds.

28. The composition of claim 21, wherein the scissile linkage is selected from the group consisting of RNA sequences, DNA sequences, amino acid sequences, abasic nucleotide sequences or carbohydrate polymers.

29. The composition of claim 21, wherein NA₁ and NA₂ each comprise between 8 nucleotides and 10,000 nucleotides.

30. The composition of claim 21, wherein n is greater than 1 and each [NA₁ --S--NA₂ ] unit is the same.

31. The composition of claim 21, wherein n is greater than 1 and each [NA₁ --S--NA₂ ] unit is different.

47. A method of detecting in a sample the presence of a target nucleic acid sequence of interest comprising:

(a) contacting the sample with a composition of claim 21 which comprises a nucleic acid sequence substantially complementary to the target nucleic acid sequence of interest, under hybridizing conditions to form a hybridized complex;

(b) treating said complex so as to cleave the scissile linkage;

(c) separately recovering the immobilized complex; and

(d) detecting the presence of the marker on the immobilized complex and thereby the target nucleic acid sequence of interest.

51. A method for quantitatively determining in a sample the amount of a target nucleic acid sequence of interest comprising:

(a) contacting the sample with a predetermined amount of a composition of claim 21, which comprises a nucleic acid sequence substantially complementary to the target nucleic acid sequence of interest, under hybridizing conditions to form a hybridized complex;

(b) treating the immobilized complex so as to cleave the scissile linkage;

(c) separately recovering the immobilized complex; and

(d) quantitatively determining the amount of marker present on the immobilized complex and thereby the amount of target nucleic acid sequence of interest.

32. A composition comprising the structure:

space="preserve" listing-type="equation">Y--NA.sub.1 --S--NA.sub.2 ].sub.n Z

wherein NA₁ and NA₂ are nucleic acid sequences;

wherein --S-- is a scissile linkage which is capable of being cleaved or disrupted without cleaving or disrupting the nucleic acid sequences of NA₁ or NA₂ or of a target nucleic acid sequence capable of hybridizing to the NA₁ and NA₂ sequences, or to the NA₁ and NA₂ sequences and the scissile linkage of said composition, wherein if the scissile linkage is a nucleic acid sequence it is RNA when both NA₁ and NA₂ are DNA sequences, or the scissile linkage is DNA when both NA₁ and NA₂ are RNA sequences;

wherein the solid lines represent chemical bonds;

wherein n is an integer from 1 to 4;

wherein Y is absent or is a chemical entity which confers an identifying characteristic; and

wherein Z is absent or is a chemical entity which confers a different identifying characteristic.

33. The composition of claim 32, wherein the chemical bonds are covalent bonds.

34. The composition of claim 32, wherein the identifying characteristic of Y is hydrophobicity or hydrophilicity and the identifying characteristic of Z is hydrophilicity or hydrophobicity.

35. The composition of claim 32, wherein when one of Y or Z is absent, the other is hydrophobic.

36. The composition of claim 32, wherein the dashed lines represent chemical bonds.

37. The composition of claim 36, wherein the chemical bonds represented by dashed lines are covalent bonds.

38. The composition of claim 32, wherein the scissile linkage is selected from the group consisting of RNA sequences, DNA sequences, amino acid sequences, abasic nucleotide sequences or carbohydrate polymers.

39. The composition of claim 32, wherein one of Y or Z is a marker and the other is hydrophobic.

40. The composition of claim 32, wherein a marker is attached to one of NA₁ or NA₂.

41. The compositiion of claim 32, wherein NA₁ and NA₂ are DNA sequences.

42. The composition of claim 32, wherein NA₁ and NA₂ are RNA sequences.

43. The composition of claim 32, wherein when the scissile linkage is other than a nucleic acid sequence NA₁ is either an RNA or DNA sequence and NA₂ is either an RNA or DNA sequence.

44. The composition of claim 32, wherein NA₁ and NA₂ comprises a sequence which is not a naturally occurring nucleic acid sequence.

45. The composition of claim 32, wherein NA₁ or NA₂ is a naturally occurring nucleic acid sequence.

48. A method of detecting in a sample a target nucleic acid sequence of interest comprising:

(a) contacting under hybridizing conditions the sample with an aqueous solution comprising a composition of claim 40 which comprises a nucleic acid sequence complementary to the target nucleic acid sequence of interest to form a hybridized complex, wherein Y is a hydrophobic entity and Z is a hydrophilic entity;

(b) mixing the resulting solution with a nonpolar solvent to form a biphasic solution in which the complex is present at the phase interface of the biphasic solution, Y being oriented in one phase and Z being oriented in the other phase;

(c) treating the complex so as to cleave the scissile linkage;

(d) recovering the complex so treated; and

(e) detecting the presence of the marker and thereby detecting the target nucleic acid sequence of interest.

52. A method for quantitatively determining in a sample the amount of a target nucleic acid sequence of interest comprising:

(a) contacting under hybridizing conditions the sample with an aqueous solution comprising a composition of claim 40 which comprises a nulceic acid sequence complementary to the target nucleic acid sequence of interest to form a hybridized complex, wherein Y is a hydrophobic entity and Z is a hydrophilic entity;

(b) mixing the resulting solution with a nonpolar solvent to form a biphasic solution in which the complex is present at the phase interface of the biphasic solution, Y being oriented in one phase and Z being oriented in the other phase;

(c) treating the complex so as to cleave the scissile linkage;

(d) recovering the complex so treated; and

(e) quantitatively determining the amount of a marker and thereby the target nucleic acid sequence of interest.