Patent US5116742A successfully refreshed from Google Patents.
US5116742A
Expired / Lapsed
US
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RNA ribozyme restriction endoribonucleases and methods
Assignee: University Patents Inc
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Filing: 1989-03-24
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Granted: 1992-05-26
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Expires: 2009-03-24
Abstract
Abstract New RNA endoribonuclease ribozymes are found with new conditions to prevent mismatch cleavage and able to cleave RNA after 6 different sets of ribonucleotide 4 base sequences.
Inventors
- 👤 Thomas R. Cech
- 👤 Felicia L. Murphy
- 👤 Arthur J. Zaug
- 👤 Cheryl Grosshans
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-08 08:03
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/113; C12N15/10; C12N2310/00; C12N2310/10; C12N2310/11; C12N2310/111; C12N2310/12; C12N2310/124; C12N2310/1241
Claims Analysis
Total Claims: 27
Claim 1
Independent Claim
128 words
A method for preventing non-complementary cleavage by an enzymatic RNA molecule of a separate RNA molecule, comprising the steps of: providing an enzymatic RNA molecule having an RNA substrate-binding site comprising a first nucleotide sequence and an endonuclease activity independent of any protein in vitro, said endonuclease activity being active at or at a location proximal to a second nucleotide sequence in the separate RNA molecule complementary to said first nucleotide sequence, and contacting said enzymatic RNA molecule and the separate RNA molecule in the presence of a denaturing agent to allow said endonuclease activity to cause specific cleavage of said separate RNA molecule at or proximal to said second nucleotide sequence but not at any other nucleotide sequence which is not complementary to said first nucleotide sequence.
Claim 2
Independent Claim
128 words
A method for preventing non-complementary cleavage by an enzymatic RNA molecule of a separate RNA molecule, comprising the steps of: providing an enzymatic RNA molecule having an RNA substrate-binding site comprising a first nucleotide sequence and an endonuclease activity independent of any protein in vitro, said endonuclease activity being active at or at a location proximal to a second nucleotide sequence in the separate RNA molecule complementary to said first nucleotide sequence, and contacting said enzymatic RNA molecule and the separate RNA molecule in the presence of a denaturing agent to allow said endonuclease activity to cause specific cleavage of said separate RNA molecule at or proximal to said second nucleotide sequence but not at any other nucleotide sequence which is not complementary to said first nucleotide sequence.
Claim 3
Dependent Claim
15 words
The method of claim 1, wherein said denaturing agent is chosen from urea and formamide.
Claim 4
Dependent Claim
19 words
The method of claim 2, wherein said denaturing agent is provided at a concentration between 1.5 and 3.0 Molar.
Claim 5
Dependent Claim
18 words
The method of claim 1, wherein said first and second nucleotide sequences comprise between 4 and 6 nucleotides.
Claim 6
Independent Claim
136 words
A method for cleaving a cleavage site in a separate RNA molecule, comprising the steps of: providing an enzymatic RNA molecule comprising an RNA substrate-binding site formed from at least four nucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein in vitro, said endonuclease activity being active at or at a site proximal to a nucleotide sequence complementary to said at least four nucleotides at the RNA substrate-binding site of said enzymatic molecule, providing the separate RNA molecule, wherein said cleavage site in the separate RNA molecule is complementary to said at least four nucleotides at at least two of said four nucleotides; and contacting said enzymatic RNA molecule and the separate RNA molecule to allow said endonuclease activity to cleave said separate RNA molecule at or proximal to said cleavage site.
Claim 7
Independent Claim
136 words
A method for cleaving a cleavage site in a separate RNA molecule, comprising the steps of: providing an enzymatic RNA molecule comprising an RNA substrate-binding site formed from at least four nucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein in vitro, said endonuclease activity being active at or at a site proximal to a nucleotide sequence complementary to said at least four nucleotides at the RNA substrate-binding site of said enzymatic molecule, providing the separate RNA molecule, wherein said cleavage site in the separate RNA molecule is complementary to said at least four nucleotides at at least two of said four nucleotides; and contacting said enzymatic RNA molecule and the separate RNA molecule to allow said endonuclease activity to cleave said separate RNA molecule at or proximal to said cleavage site.
Claim 8
Dependent Claim
27 words
The method of claim 5, wherein said separate RNA molecule and said enzymatic RNA molecule are contacted together in the presence of less than 1 Molar denaturant.
Claim 9
Dependent Claim
30 words
The method of claim 5 or 6, wherein said separate RNA molecule and said enzymatic RNA molecule are contacted together in the presence of less than 5 millimolar magnesium ion.
Claim 10
Independent Claim
136 words
A method for cleaving a cleavage site in a separate RNA molecule, comprising the steps of: providing an enzymatic RNA molecule comprising an RNA substrate-binding site formed from at least five nucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein in vitro, said endonuclease activity being active at or at a site proximal to a nucleotide sequence complementary to said at least five nucleotides at the RNA substrate-binding site of said enzymatic molecule, providing the separate RNA molecule, wherein said cleavage site in the separate RNA molecule is complementary to said at least five nucleotides at at least two of said five nucleotides; and contacting said enzymatic RNA molecule and the separate RNA molecule to allow said endonuclease activity to cleave said separate RNA molecule at or proximal to said cleavage site.
Claim 11
Independent Claim
136 words
A method for cleaving a cleavage site in a separate RNA molecule, comprising the steps of: providing an enzymatic RNA molecule comprising an RNA substrate-binding site formed from at least five nucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein in vitro, said endonuclease activity being active at or at a site proximal to a nucleotide sequence complementary to said at least five nucleotides at the RNA substrate-binding site of said enzymatic molecule, providing the separate RNA molecule, wherein said cleavage site in the separate RNA molecule is complementary to said at least five nucleotides at at least two of said five nucleotides; and contacting said enzymatic RNA molecule and the separate RNA molecule to allow said endonuclease activity to cleave said separate RNA molecule at or proximal to said cleavage site.
Claim 12
Independent Claim
136 words
A method for cleaving a cleavage site in a separate RNA molecule, comprising the steps of: providing an enzymatic RNA molecule comprising an RNA substrate-binding site formed from at least six nucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein in vitro, said endonuclease activity being active at or at a site proximal to a nucleotide sequence complementary to said at least six nucleotides at the RNA substrate-binding site of said enzymatic molecule, providing the separate RNA molecule, wherein said cleavage site in the separate RNA molecule is complementary to said at least six nucleotides at at least two of said six nucleotides; and contacting said enzymatic RNA molecule and the separate RNA molecule to allow said endonuclease activity to cleave said separate RNA molecule at or proximal to said cleavage site.
Claim 13
Independent Claim
136 words
A method for cleaving a cleavage site in a separate RNA molecule, comprising the steps of: providing an enzymatic RNA molecule comprising an RNA substrate-binding site formed from at least six nucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein in vitro, said endonuclease activity being active at or at a site proximal to a nucleotide sequence complementary to said at least six nucleotides at the RNA substrate-binding site of said enzymatic molecule, providing the separate RNA molecule, wherein said cleavage site in the separate RNA molecule is complementary to said at least six nucleotides at at least two of said six nucleotides; and contacting said enzymatic RNA molecule and the separate RNA molecule to allow said endonuclease activity to cleave said separate RNA molecule at or proximal to said cleavage site.
Claim 14
Dependent Claim
27 words
The method of claim 5, 8, or 9, wherein said separate RNA molecule is complementary at at least three of said nucleotides of said RNA substrate-binding site.
Claim 15
Dependent Claim
27 words
The method of claim 5, 8, or 9, wherein said separate RNA molecule is complementary at at least four of said nucleotides of said RNA substrate-binding site.
Claim 16
Dependent Claim
26 words
The method of claim 8 or 9, wherein said separate RNA molecule is complementary at at least five of said nucleotides of said RNA substrate-binding site.
Claim 17
Dependent Claim
24 words
The method of claim 1, wherein said separate RNA molecule is complementary at at least six of said nucleotides of said RNA substrate-binding site.
Claim 18
Dependent Claim
25 words
The method of claim 5, 8 or 9 wherein said cleavage site of said separate RNA molecule is completely complementary to said RNA substrate-binding site.
Claim 19
Dependent Claim
24 words
The method of claim 5, 8, or 9 wherein said complementary nucleotides are chosen from the base pair combinations A:T, A:U, G:C, and G:U.
Claim 20
Dependent Claim
21 words
The method of claim 11 wherein said complementary nucleotides are chosen form the base pair combinations A:T, A:U, G:C, and G:U.
Claim 21
Dependent Claim
21 words
The method of claim 12 wherein said complementary nucleotides are chosen from the base pair combinations A:T, A:U, G:C, and G:U.
Claim 22
Dependent Claim
21 words
The method of claim 13 wherein said complementary nucleotides are chosen from the base pair combinations A:T, A:U, G:C, and G:U.
Claim 23
Dependent Claim
39 words
A DNA vector comprising DNA encoding L-21 ScaI enzymatic RNA, said DNA being constructed and arranged to cause production of the L-21 ScaI enzymatic RNA molecule when contacted with ScaI restriction endonuclease and an RNA polymerase under transcriptional conditions.
Claim 24
Dependent Claim
39 words
A DNA vector comprising DNA encoding L-21 ScaI enzymatic RNA, said DNA being constructed and arranged to cause production of the L-21 ScaI enzymatic RNA molecule when contacted with ScaI restriction endonuclease and an RNA polymerase under transcriptional conditions.
Claim 25
Dependent Claim
29 words
The vector of claim 19, wherein said DNA comprises a T7 promoter arranged to interact with said RNA polymerase to cause transcription of said L-21 ScaI enzymatic RNA molecule.
Claim 26
Dependent Claim
11 words
The vector of claim 20, wherein said DNA is PT7 L-21.
Claim 27
Dependent Claim
19 words
The vector of claim 21, wherein said PT7 L-21 is that DNA present as a deposit designated ATCC 40291.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1995-05-26 | 1995-11-25 | 1996-05-26 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1999-05-26 | 1999-11-25 | 2000-05-26 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2003-05-26 | 2003-11-25 | 2004-05-26 | $7700.00 | Window Closed / Lapsed |