Patent US5093246A successfully refreshed from Google Patents.
US5093246A
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Rna ribozyme polymerases, dephosphorylases, restriction endoribo-nucleases and methods
Assignee: University Patents Inc
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Filing: 1990-08-03
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Granted: 1992-03-03
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Expires: 2010-08-03
Abstract
Abstract RNA enzymes or ribozymes can act as endoribonucleases, catalyzing the cleavage of RNA molecules with a sequence specificity of cleavage greater than that of known ribonucleases and approaching that of the DNA restriction endonucleases, thus serving as RNA sequence specific endoribonucleases. An example is a shortened form of the self-splicing ribonsomal RNA intervening sequence of Tetrahymena (L-19 IVS RNA). Site-specific mutagenesis of the enzyme active site of the L-19 IVS RNA alters the substrate sequence specificity in a predictable manner, allowing a set of sequence-specific endoribonucleases to be synthesized. Varying conditions allow the ribozyme to act as a polymerase (nucleotidyltransferase), a dephosphorylase (acid phosphatase or phosphotransferase) or a sequence-specific endoribonuclease.
Inventors
- 👤 Thomas R. Cech
- 👤 Arthur J. Zaug
- 👤 Michael D. Been
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 09:18
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: 43
Claim 1
Independent Claim
52 words
Method for dephosphorylation of an RNA substrate comprising the steps of: providing an enzymatic RNA molecule having a dephosphorylation activity independent of any protein, and contacting said enzymatic RNA molecule with said RNA substrate under conditions suitable for said enzymatic RNA molecule to remove the 3' terminal phosphate of said RNA substrate.
Claim 2
Dependent Claim
52 words
Method for dephosphorylation of an RNA substrate comprising the steps of: providing an enzymatic RNA molecule having a dephosphorylation activity independent of any protein, and contacting said enzymatic RNA molecule with said RNA substrate under conditions suitable for said enzymatic RNA molecule to remove the 3' terminal phosphate of said RNA substrate.
Claim 3
Independent Claim
88 words
A method for locating a nucleotide sequence in an RNA molecule having a first length, comprising the steps of: providing an enzymatic RNA molecule having an endonuclease activity independent of any protein, contacting said enzymatic RNA molecule with said RNA molecule wherein said enzymatic RNA molecule will cleave said RNA molecule at any said nucleotide sequence present in said RNA molecule, and determining the length of resulting RNA molecules resulting from said contacting step as an indication of the location of the nucleotide sequence in said RNA molecule.
Claim 4
Independent Claim
88 words
A method for locating a nucleotide sequence in an RNA molecule having a first length, comprising the steps of: providing an enzymatic RNA molecule having an endonuclease activity independent of any protein, contacting said enzymatic RNA molecule with said RNA molecule wherein said enzymatic RNA molecule will cleave said RNA molecule at any said nucleotide sequence present in said RNA molecule, and determining the length of resulting RNA molecules resulting from said contacting step as an indication of the location of the nucleotide sequence in said RNA molecule.
Claim 5
Independent Claim
121 words
A method for locating a plurality of nucleotide sequences in an RNA molecule having a first length, comprising the steps of: providing a plurality of different enzymatic RNA molecules having an endonuclease activity independent of any protein, each said different enzymatic RNA molecule cleaving said RNA molecule at a different said nucleotide sequence of said plurality of nucleotide sequences; contacting said plurality of different enzymatic RNA molecules with said RNA molecule wherein each said different enzymatic RNA molecule will cleave said RNA molecule at any said different nucleotide sequence present in said RNA molecule, and determining the length of resulting RNA molecules resulting from said contacting step as an indication of the location of the nucleotide sequence in said RNA molecule.
Claim 6
Independent Claim
121 words
A method for locating a plurality of nucleotide sequences in an RNA molecule having a first length, comprising the steps of: providing a plurality of different enzymatic RNA molecules having an endonuclease activity independent of any protein, each said different enzymatic RNA molecule cleaving said RNA molecule at a different said nucleotide sequence of said plurality of nucleotide sequences; contacting said plurality of different enzymatic RNA molecules with said RNA molecule wherein each said different enzymatic RNA molecule will cleave said RNA molecule at any said different nucleotide sequence present in said RNA molecule, and determining the length of resulting RNA molecules resulting from said contacting step as an indication of the location of the nucleotide sequence in said RNA molecule.
Claim 7
Independent Claim
87 words
A method for detecting the presence of a nucleotide sequence in an RNA molecule, comprising the steps of: providing an enzymatic RNA molecule having an endonuclease activity independent of any protein, contacting said enzymatic RNA molecule with said RNA molecule wherein said enzymatic RNA molecule will cleave said RNA molecule of any said nucleotide sequence present in said RNA molecule to produce two or more fragments of said RNA molecule, and detecting the presence of said fragments as an indication of the presence of said nucleotide sequence.
Claim 8
Independent Claim
87 words
A method for detecting the presence of a nucleotide sequence in an RNA molecule, comprising the steps of: providing an enzymatic RNA molecule having an endonuclease activity independent of any protein, contacting said enzymatic RNA molecule with said RNA molecule wherein said enzymatic RNA molecule will cleave said RNA molecule of any said nucleotide sequence present in said RNA molecule to produce two or more fragments of said RNA molecule, and detecting the presence of said fragments as an indication of the presence of said nucleotide sequence.
Claim 9
Dependent Claim
31 words
Method of claim 2, 3 or 4 wherein a radiolabelled guanosine derivative is provided in said contacting step to cause radiolabelling of the 5' end of any cleaved said RNA molecule.
Claim 10
Dependent Claim
101 words
Method for producing an enzymatic RNA molecule having an endonuclease activity independent of any protein, comprising the steps of: (a) transcribing recombinant DNA encoding a precursor RNA molecule to said enzymatic RNA molecule with an RNA polymerase in vitro to produce said precursor RNA; (b) incubating said precursor RNA under conditions suitable for promoting self-splicing and cyclization of said precursor RNA to produce cyclized RNA; (c) contacting said cyclized RNA with MgCl 2 at pH 9.0 to promote conversion of said cyclized RNA to said enzymatic RNA molecule; and (d) purifying said enzymatic RNA molecule by polyacrylamide gel electrophoresis and chromatography.
Claim 11
Dependent Claim
101 words
Method for producing an enzymatic RNA molecule having an endonuclease activity independent of any protein, comprising the steps of: (a) transcribing recombinant DNA encoding a precursor RNA molecule to said enzymatic RNA molecule with an RNA polymerase in vitro to produce said precursor RNA; (b) incubating said precursor RNA under conditions suitable for promoting self-splicing and cyclization of said precursor RNA to produce cyclized RNA; (c) contacting said cyclized RNA with MgCl 2 at pH 9.0 to promote conversion of said cyclized RNA to said enzymatic RNA molecule; and (d) purifying said enzymatic RNA molecule by polyacrylamide gel electrophoresis and chromatography.
Claim 12
Dependent Claim
13 words
Method of claim 6 wherein said recombinant DNA is cloned in E. coli.
Claim 13
Dependent Claim
18 words
Method of claim 6 wherein said recombinant DNA comprises an intron of a Tetrahymena gene encoding ribosomal RNA.
Claim 14
Dependent Claim
20 words
Method of claim 6 wherein the expression of said DNA is regulated by the SP6 or T7 RNA polymerase promoter.
Claim 15
Independent Claim
50 words
A method for producing an enzymatic RNA molecule having an endonuclease activity of any protein, comprising the steps of: providing DNA encoding RNA consisting essentially of said enzymatic RNA molecule under the transcriptional control of a promoter, and transcribing said DNA from said promoter to produce said enzymatic RNA molecule.
Claim 16
Independent Claim
50 words
A method for producing an enzymatic RNA molecule having an endonuclease activity of any protein, comprising the steps of: providing DNA encoding RNA consisting essentially of said enzymatic RNA molecule under the transcriptional control of a promoter, and transcribing said DNA from said promoter to produce said enzymatic RNA molecule.
Claim 17
Dependent Claim
50 words
A vector for production of an enzymatic RNA molecule having an endonuclease activity independent of any protein, comprising: DNA encoding said enzymatic RNA molecule, and a promoter positioned relative to said DNA to cause transcription of said DNA to form an RNA molecule consisting essentially of said enzymatic RNA molecule.
Claim 18
Dependent Claim
50 words
A vector for production of an enzymatic RNA molecule having an endonuclease activity independent of any protein, comprising: DNA encoding said enzymatic RNA molecule, and a promoter positioned relative to said DNA to cause transcription of said DNA to form an RNA molecule consisting essentially of said enzymatic RNA molecule.
Claim 19
Dependent Claim
39 words
The vector of claim 11 wherein said endonuclease activity is specific for a nucleotide sequence defining a cleavage site comprising single stranded RNA in a separate RNA molecule and causing cleavage at said cleavage site by a transesterification reaction.
Claim 20
Dependent Claim
31 words
The vector of claim 11 wherein said endonuclease activity is specific for a nucleotide sequence defining a cleavage site consisting essentially of a single stranded region of a separate RNA molecule.
Claim 21
Dependent Claim
20 words
The vector of claim 11 or 13 wherein the nucleotide sequence defining said cleavage site comprises four or more bases.
Claim 22
Dependent Claim
19 words
The vector of claim 13 wherein said endonuclease activity causes cleavage at said cleavage site by a transesterification reaction.
Claim 23
Dependent Claim
36 words
The vector of claim 11 wherein said enzymatic RNA molecule comprises an active site region responsible for said cleavage substantially identical to the active site region of the ribosomal RNA intervening sequence naturally occurring in Tetrahymena.
Claim 24
Dependent Claim
20 words
The vector of claim 16 wherein said nucleotide sequence defining said cleavage site is chosen from GUCU, CUGU, and CGCU.
Claim 25
Dependent Claim
22 words
The vector of claim 11 wherein said enzymatic RNA molecule has a pH optimum for said endonuclease activity between 7.5 and 8.
Claim 26
Dependent Claim
21 words
The vector of claim 11 wherein said endonuclease activity requires the presence of magnesium ions, a denaturant, or a guanine derivative.
Claim 27
Dependent Claim
18 words
The vector of claim 11 wherein said endonuclease activity is enhanced by the presence of a divalent cation.
Claim 28
Dependent Claim
11 words
The vector of claim 20 wherein said divalent cation is magnesium.
Claim 29
Dependent Claim
40 words
The vector of claim 11 wherein said enzymatic RNA molecule further comprises a second endonuclease activity, independent of any protein, for nucleic acid comprising deoxyribonucleotides wherein said endonuclease activity and said second endonuclease activity may be the same or different.
Claim 30
Dependent Claim
16 words
The vector of claim 11 wherein said enzymatic RNA molecule further has a nucleotidyl transferase activity.
Claim 31
Dependent Claim
18 words
The vector of claim 23 wherein said nucleotidyl transferase activity causes polymerization of oligocytidylic acid to polycytidylic acid.
Claim 32
Dependent Claim
15 words
The vector of claim 11 wherein said enzymatic RNA molecule further has a dephosphorylase activity.
Claim 33
Dependent Claim
24 words
The vector of claim 25 wherein said dephosphorylase activity causes removal of a 3' terminal phosphate of a nucleic acid comprising a 3' ribonucleotide.
Claim 34
Dependent Claim
16 words
The vector of claim 26 wherein said dephosphorylase activity is specific to a specific nucleotide sequence.
Claim 35
Dependent Claim
42 words
The vector of claim 11 wherein said enzymatic RNA molecule comprises the ribonucleotide base sequence of L-19 RNA, said enzymatic RNA molecule having a ribonucleotide base sequence at the active site responsible for said cleavage different from that of L-19 from Tetrahymena.
Claim 36
Dependent Claim
18 words
The vector of claim 11 wherein said enzymatic RNA molecule comprises the ribonucleotide base sequence of L-19 RNA.
Claim 37
Dependent Claim
14 words
The vector of claim 29 wherein said L-19 comprises bases 19-331 of Tetrahymena L-IVS.
Claim 38
Dependent Claim
57 words
The vector of claim 11 wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site comprising single stranded RNA in a separate RNA molecule, said enzymatic RNA molecule not causing specific cleavage of said separate RNA molecule immediately adjacent in the direction 3' to the nucleotide sequence CUCU.
Claim 39
Dependent Claim
10 words
The vector of claim 11, said vector being a plasmid.
Claim 40
Dependent Claim
22 words
The vector of claim 11 wherein said promoter is selected from the group consisting of the bacteriophage SP6 and bacteriophage T7 promoters.
Claim 41
Dependent Claim
16 words
The vector of claim 11, wherein said enzymatic RNA molecule comprises bases 21-331 of Tetrahymena L-IVS.
Claim 42
Dependent Claim
21 words
A plasmid selected from the group consisting of pSPTT1A3, pT7-TT1A3, pBGST7, pBG/-2G:23C, pBG/23C, pBG/-3G:24C, pBG/24C, pBG/-4G:25C, pBG/25C, and pBG/23A 4 .
Claim 43
Dependent Claim
21 words
A plasmid selected from the group consisting of pSPTT1A3, pT7-TT1A3, pBGST7, pBG/-2G:23C, pBG/23C, pBG/-3G:24C, pBG/24C, pBG/-4G:25C, pBG/25C, and pBG/23A 4 .
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1995-03-03 | 1995-09-02 | 1996-03-03 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1999-03-03 | 1999-09-02 | 2000-03-03 | $3760.00 | Window Closed / Lapsed |
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