US5589332A
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Ribozyme amplified diagnostics
Assignee: Yale University
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Filing: 1994-05-09
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Granted: 1996-12-31
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Expires: 2014-05-09
Abstract
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.
Inventors
- 👤 Andy Shih
- 👤 Jeffrey M. Bockman
- 👤 Shaji T. George
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 06:54
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/113; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6823; C12N2310/00; C12N2310/10; C12N2310/12; C12N2310/121; C12N2310/30; C12N2310/35; C12N2310/351; C12N2310/3517
Claims Analysis
Total Claims: 27
Claim 1
Independent Claim
181 words
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.
Claim 2
Independent Claim
181 words
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.
Claim 3
Dependent Claim
39 words
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.
Claim 4
Dependent Claim
19 words
The method of claim 1 wherein the target nucleic acid molecule hydridizes with the labelled co-target nucleic acid molecule.
Claim 5
Dependent Claim
14 words
The method of claim 3 wherein the target nucleic acid molecule encodes a protein.
Claim 6
Dependent Claim
18 words
The method of claim 1 wherein the labelled cotarget nucleic acid molecule is immobilized on a solid support.
Claim 7
Dependent Claim
37 words
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.
Claim 8
Dependent Claim
21 words
The method of claim 1 wherein the ribozyme is conformationally active when the ribozyme hybridizes to the target nucleic acid molecule.
Claim 9
Dependent Claim
30 words
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.
Claim 10
Dependent Claim
36 words
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.
Claim 11
Dependent Claim
35 words
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.
Claim 12
Dependent Claim
34 words
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.
Claim 13
Dependent Claim
142 words
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.
Claim 14
Dependent Claim
18 words
The kit of claim 12 wherein the co-target nucleic acid molecule hybridizes to the target nucleic acid molecule.
Claim 15
Dependent Claim
13 words
The kit of claim 12 wherein the co-target nucleic acid molecule is labelled.
Claim 16
Dependent Claim
14 words
The kit of claim 12 wherein the target nucleic acid molecule encodes a protein.
Claim 17
Dependent Claim
36 words
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.
Claim 18
Dependent Claim
20 words
The kit of claim 12 wherein the ribozyme is conformationally active when hybridized the first portion of the first molecule.
Claim 19
Dependent Claim
41 words
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.
Claim 20
Dependent Claim
23 words
The kit of claim 12 wherein the enzymatic activity of the ribozyme is altered by binding of a regulating molecule to the ribozyme.
Claim 21
Dependent Claim
19 words
The kit of claim 12 wherein the ribozyme is conformationally active at a temperature between 37° and 60° .
Claim 22
Dependent Claim
18 words
The kit of claim 14 wherein the labelled co-target nucleic acid molecule is immobilized on a solid support.
Claim 23
Dependent Claim
36 words
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.
Claim 24
Dependent Claim
44 words
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.
Claim 25
Dependent Claim
45 words
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.
Claim 26
Independent Claim
85 words
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.
Claim 27
Independent Claim
85 words
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.
References Cited (Backward Citations)
US4775619A
Cited
WO1989005852A1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1999-12-31 | 2000-07-01 | 2000-12-31 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2003-12-31 | 2004-07-01 | 2004-12-31 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2007-12-31 | 2008-07-01 | 2008-12-31 | $7700.00 | Window Closed / Lapsed |