Ribozyme amplified diagnostics

• US 5,589,332 A
• Filed: 05/09/1994
• Issued: 12/31/1996
• Est. Priority Date: 12/04/1992
• Status: Expired due to Term

• Current Assignee: Ribozyme Pharmaceuticals, Inc. (Alnylam Pharmaceuticals Incorporated), Yale University
• Original Assignee: Innovir Laboratories, Inc. (Baxter International Inc.)
• Inventors: Bockman, Jeffrey M., George, Shaji T., Shih, Andy
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Whisenant, Ethan
• Application Number:
  US08/240,081
• Time in Patent Office: 967 Days
• Field of Search: 435/6, 435/91.2, 536/24.5, 536/23.1
• US Class Current: 435/5
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/121 Hammerhead
• CPC Class Codes: C12N 2310/3517 Marker; Tag
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2521/337 Ribozyme

Abstract

A system is described for the use of a ribozyme as a diagnostic tool for detecting the presence of a nucleic acid, protein, or other molecule, in which the formation of an active ribozyme and cleavage of an assayable marker is dependent on the presence or absence of the specific target molecule. The essential component is a ribozyme specifically but reversibly binding a selected target in combination with a labelled co-target, preferably immobilized on a support structure. When both the target and co-target are bound, the ribozyme cleaves the label from the co-target, which is then quantifiable. Since the ribozyme is reversibly bound by target and co-target, it can reassociate with additional co-target, cleaving more label, thereby amplifying the reaction signal. In one embodiment, the target is a nucleic acid hybridizing to complementary sequences that form part of the ribozyme; in a second embodiment, the target is a protein or other macromolecule which is bound by interactions with a portion of the ribozyme molecule in another embodiment, a thermostable ribozyme is used, so that improperly bound ribozyme is destabilized and inactive at elevated temperatures. A method for isolating regularable ribozymes is also disclosed. The regulatable ribozymes are useful in the method for detecting the presence of a specific macromolecule, or can be used in in vitro or in vivo methods for inactivation or activation of the cleavage of target RNA molecules.

Claims

1. A method for detecting a target nucleic acid molecule in a solution, comprising:

providing in the solution under conditions wherein two complementary nucleotide molecules will hybridize, a ribozyme molecule, a labelled co-target nucleic acid molecule and the target nucleic acid molecule, wherein the co-target and the target molecules have different sequences and wherein the ribozyme molecule comprises two regions complementary to portions of the co-target and target nucleic acid molecules, wherein the first portion is present on the labelled co-target nucleic acid molecule which contains a cleavage site for the ribozyme and the second portion is present on the target nucleic acid molecule, wherein the complementary regions include at least the minimum number of complementary nucleotides to obtain hybridization between the ribozyme molecule and the co-target and target nucleic acid molecules,

allowing the ribozyme molecule to react with the labelled co-target nucleic acid molecule and the target nucleic acid molecule, and

detecting the presence of free label when the target nucleic acid molecule is present in solution as compared with when the target nucleic acid molecule is not present in solution.

2. The method of claim 1 wherein the ribozyme is derived from the group consisting of Tetrahymena ribozymes, RNAase P, ribozymes derived from newt satellite ribozyme, hammerhead ribozymes derived from plant viroids, and axehead ribozymes derived from hepatitis delta virus.

3. The method of claim 1 wherein the target nucleic acid molecule hydridizes with the labelled co-target nucleic acid molecule.

4. The method of claim 3 wherein the target nucleic acid molecule encodes a protein.

5. The method of claim 1 wherein the labelled cotarget nucleic acid molecule is immobilized on a solid support.

6. The method of claim 1 wherein the labelled co-target nucleic acid molecule is labelled with a label selected from the group consisting of dyes, enzymes reactive with a chromogenic substrate, fluorescent labels, chemiluminescent labels, and radioactive labels.

7. The method of claim 1 wherein the ribozyme is conformationally active when the ribozyme hybridizes to the target nucleic acid molecule.

8. The method of claim 1 wherein the ribozyme is conformationally active when the ribozyme is hybridized to both the target nucleic acid molecule and the labelled co-target nucleic acid molecule.

9. The method of claim 1 wherein the ribozyme is stable to elevated temperatures in excess of 37°

C. and the target nucleic acid molecule hybridizes to the ribozyme at a temperature in excess of 37°

C.

10. The method of claim 1 wherein when the target nucleic acid molecule hybridizes to the ribozyme molecule there is less label detected than when the target nucleic acid is not hybridized to the ribozyme molecule.

11. The method of claim 1 wherein when the target nucleic acid molecule hybridizes to the ribozyme there is more label detected than when the target nucleic acid is not hybridized to the ribozyme molecule.

12. A kit for use in the method of claim 1 comprising

a ribozyme molecule,

a co-target nucleic acid molecule which contains a cleavage site for the ribozyme, and

a target nucleic acid molecule,

wherein the co-target and target molecules have different sequences, and

wherein the ribozyme comprises two regions complementary to portions of the co-target and target nucleic acid molecules, wherein the first portion is present on the co-target nucleic acid molecule and is complementary to a first region of the ribozyme and the second portion is present on the target nucleic acid molecule and is complementary to a second region of the ribozyme, wherein the complementary regions of the ribozyme include at least the minimum number of complementary nucleotides to obtain hybridization between the ribozyme molecule and the first portion on the co-target and the second portion on the target molecule.

13. The kit of claim 12 wherein the co-target nucleic acid molecule hybridizes to the target nucleic acid molecule.

14. The kit of claim 12 wherein the co-target nucleic acid molecule is labelled.

15. The kit of claim 12 wherein the target nucleic acid molecule encodes a protein.

16. The kit of claim 12 wherein the ribozyme is derived from the group consisting of Tetrahymena ribozymes, RNAase P, newt satellite ribozyme, hammerhead ribozymes derived from plant viroids, and axehead ribozymes derived from hepatitis delta virus.

17. The kit of claim 12 wherein the ribozyme is conformationally active when hybridized the first portion of the first molecule.

18. The kit of claim 12 wherein the ribozyme is conformationally active when the first complementary region is hybridized to the first portion on the co-target molecule and the second complementary region is hybridized to the second portion on the target molecule.

19. The kit of claim 12 wherein the enzymatic activity of the ribozyme is altered by binding of a regulating molecule to the ribozyme.

20. The kit of claim 12 wherein the ribozyme is conformationally active at a temperature between 37°

and 60°

.

21. The kit of claim 14 wherein the labelled co-target nucleic acid molecule is immobilized on a solid support.

22. The kit of claim 14 wherein the labelled co-target nucleic acid is labelled with a label selected from the group consisting of dyes, enzymes reactive with a chromogenic substrate, fluorescent labels, chemiluminescent labels, and radioactive labels.

23. The kit of claim 14 wherein when the target nucleic acid is hybridized to either the ribozyme or the co-target molecule there is less label detected than when the target nucleic acid molecule is not hybridized to either the ribozyme or the co-target molecule.

24. The kit of claim 14 wherein when the target nucleic acid molecule is hybridized to either the ribozyme or the co-target molecule there is more label detected than when the target nucleic acid molecule is not hybridized to either the ribozyme or the co-target molecule.

25. A method for detecting a targeted nucleic acid molecule in a solution, comprising:

adding to the solution a trans-acting ribozyme labelled with a label selected from the group consisting of dyes, enzymes reactive with a chromogenic substrate, fluorescent labels, chemiluminescent labels, and radioactive labels, and having two regions of complementarity to the targeted nucleic acid molecule, wherein the label is cleaved from the ribozyme when the ribozyme hybridizes to the targeted molecule; and

detecting the targeted molecule by detecting the label cleaved from the ribozyme.