US4987071A
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RNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods
Assignee: University Patents Inc
•
Filing: 1986-12-03
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Granted: 1991-01-22
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Expires: 2006-12-03
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 ribosomal 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
2026-11-01 11:11
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: 77
Claim 1
Independent Claim
49 words
An enzymatic RNA molecule not naturally occurring in nature having an endonuclease activity independent of any protein, said endonuclease activity being 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 2
Dependent Claim
49 words
An enzymatic RNA molecule not naturally occurring in nature having an endonuclease activity independent of any protein, said endonuclease activity being 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 3
Dependent Claim
55 words
A purified enzymatic RNA molecule separated from other RNA molecules and oligonucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site comprising single-stranded RNA in said separate RNA molecule by a transesterification reaction.
Claim 4
Dependent Claim
55 words
A purified enzymatic RNA molecule separated from other RNA molecules and oligonucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site comprising single-stranded RNA in said separate RNA molecule by a transesterification reaction.
Claim 5
Dependent Claim
41 words
An enzymatic RNA molecule not naturally occurring in nature having an endonuclease activity independent of any protein, said endonuclease activity being specific for a nucleotide sequence defining a cleavage site consisting essentially of a single-stranded region of a separate RNA molecule.
Claim 6
Dependent Claim
41 words
An enzymatic RNA molecule not naturally occurring in nature having an endonuclease activity independent of any protein, said endonuclease activity being specific for a nucleotide sequence defining a cleavage site consisting essentially of a single-stranded region of a separate RNA molecule.
Claim 7
Dependent Claim
54 words
A purified enzymatic RNA molecule separated from other RNA molecules and oligonucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site consisting essentially of a single-stranded region in said separate RNA molecule.
Claim 8
Dependent Claim
54 words
A purified enzymatic RNA molecule separated from other RNA molecules and oligonucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site consisting essentially of a single-stranded region in said separate RNA molecule.
Claim 9
Dependent Claim
21 words
The enzymatic RNA molecule of claim 3 wherein said endonuclease activity causes cleavage at said cleavage site by a transesterification reaction.
Claim 10
Dependent Claim
19 words
The enzymatic RNA molecule of claim 4 wherein said cleavage of said cleavage site is by a transesterification reaction.
Claim 11
Dependent Claim
22 words
The enzymatic RNA molecule of any of claims 1-6 wherein said nucleotide sequence defining said cleavage site comprises four or more bases.
Claim 12
Dependent Claim
40 words
The enzymatic RNA molecule of any of claims 1-6 wherein said enzymatic RNA molecule comprises an active site region responsible for said cleavage substantially similar to the active site region of the ribosomal RNA intervening sequence naturally occurring in Tetrahymena.
Claim 13
Dependent Claim
22 words
The enzymatic RNA molecule of claim 7 wherein said nucleotide sequence defining said cleavage site is chosen from GUCU, CUGU, and CGCU.
Claim 14
Dependent Claim
21 words
The enzymatic RNA molecule of any of claims 1-6 having a pH optimum for said endonuclease activity between 7.5 and 8.0.
Claim 15
Dependent Claim
22 words
The enzymatic RNA molecule of claim 8, said endonuclease activity requiring the presence of magnesium ions, a denaturant or a guanine derivative.
Claim 16
Dependent Claim
19 words
The enzymatic RNA molecule of any of claims 1-6, said endonuclease activity requiring the presence of a divalent cation.
Claim 17
Dependent Claim
15 words
The enzymatic RNA molecule of claim 12, wherein said divalent cation is Mg 2+ .
Claim 18
Dependent Claim
29 words
The enzymatic RNA molecule of any of claims 1-6 wherein said enzymatic RNA molecule further comprises a second endonuclease activity, independent of any protein, for nucleic acid comprising deoxynucleotides.
Claim 19
Dependent Claim
19 words
The enzymatic RNA molecule of any of claims 1-6 wherein said enzymatic RNA molecule further comprises a nucleotidyltransferase activity.
Claim 20
Dependent Claim
19 words
The enzymatic RNA molecule of any of claims 1-6 wherein said enzymatic RNA molecule further comprises a dephosphorylase activity.
Claim 21
Dependent Claim
19 words
The enzymatic RNA molecule of claim 15 wherein said nucleotidyltransferase activity causes polymerization of oligocytidylic acid to polycytidylic acid.
Claim 22
Dependent Claim
25 words
The enzymatic RNA molecule of claim 16 wherein said dephosphorylase activity causes removal of a 3'-terminal phosphate of a nucleic acid comprising a 3' ribonucleotide.
Claim 23
Dependent Claim
18 words
The enzymatic RNA molecule of claim 18 wherein said ephosphorylase activity is specific to a specific nucleotide sequence.
Claim 24
Dependent Claim
43 words
The enzymatic RNA molecule of any of claims 1-6 comprising the ribonucleotide base sequence of L-19 IVS RNA said enzymatic RAN molecule having a ribonucleotide base sequence at its active site responsible for said cleavage different from that of L-19 isolated from Tetgrahymena.
Claim 25
Dependent Claim
16 words
The enzymatic RNA molecule of claim 2 comprising the ribonucleotide base sequence of L-19 IVS RNA.
Claim 26
Independent Claim
103 words
A method for specifically cleaving a separate RNA molecule at a cleavage site comprising single-stranded RNA, comprising the steps of: providing an enzymatic RNA molecule not naturally occurring in nature having an endonuclease activity independent of any protein, said endonuclease activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded RNA in said separate RNA molecule, and causing cleavage at said cleavage site by a transesterification reaction, and contacting said enzymatic RNA molecule with said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule.
Claim 27
Independent Claim
103 words
A method for specifically cleaving a separate RNA molecule at a cleavage site comprising single-stranded RNA, comprising the steps of: providing an enzymatic RNA molecule not naturally occurring in nature having an endonuclease activity independent of any protein, said endonuclease activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded RNA in said separate RNA molecule, and causing cleavage at said cleavage site by a transesterification reaction, and contacting said enzymatic RNA molecule with said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule.
Claim 28
Independent Claim
101 words
A method for specifically cleaving a separate RNA molecule at a cleavage site comprising single-stranded RNA, comprising the steps of: providing a purified enzymatic RNA molecule separated from other RNA molecules and oligonucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of said separate RNA molecule at a specific nucleotide sequence, defining said cleavage site, by a transesterification reaction, and contacting said enzymatic RNA molecule with said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule.
Claim 29
Independent Claim
101 words
A method for specifically cleaving a separate RNA molecule at a cleavage site comprising single-stranded RNA, comprising the steps of: providing a purified enzymatic RNA molecule separated from other RNA molecules and oligonucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of said separate RNA molecule at a specific nucleotide sequence, defining said cleavage site, by a transesterification reaction, and contacting said enzymatic RNA molecule with said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule.
Claim 30
Independent Claim
93 words
A method for specifically cleaving a separate RNA molecule at a cleavage site comprising single-stranded RNA, comprising the steps of: providing an enzymatic RNA molecule not naturally occurring in nature and having an endonuclease activity independent of any protein, said endonuclease activity being specific for a said cleavage site consisting essentially of a single-stranded region of said separate RNA molecule, and contacting said enzymatic RNA molecule with said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule.
Claim 31
Independent Claim
93 words
A method for specifically cleaving a separate RNA molecule at a cleavage site comprising single-stranded RNA, comprising the steps of: providing an enzymatic RNA molecule not naturally occurring in nature and having an endonuclease activity independent of any protein, said endonuclease activity being specific for a said cleavage site consisting essentially of a single-stranded region of said separate RNA molecule, and contacting said enzymatic RNA molecule with said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule.
Claim 32
Independent Claim
104 words
A method for specifically cleaving a separate RNA molecule comprising a cleavage site comprising single-stranded RNA, at the steps of: providing a purified enzymatic RNA molecule separated from other RNA molecules and oligonucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of said separate RNA molecule at a said cleavage site consisting essentially of a single-stranded region in said separate RNA molecule, and contacting said enzymatic RNA molecule with said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule.
Claim 33
Independent Claim
104 words
A method for specifically cleaving a separate RNA molecule comprising a cleavage site comprising single-stranded RNA, at the steps of: providing a purified enzymatic RNA molecule separated from other RNA molecules and oligonucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of said separate RNA molecule at a said cleavage site consisting essentially of a single-stranded region in said separate RNA molecule, and contacting said enzymatic RNA molecule with said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule.
Claim 34
Dependent Claim
21 words
The method of claim 24 or 25 wherein said endonuclease activity causes cleavage at said cleavage site by a transesterification reaction.
Claim 35
Independent Claim
24 words
The method of any of claims 22, 23, 24 and 25 wherein said nucleotide sequence defining said cleavage site comprises four or more bases.
Claim 36
Independent Claim
40 words
The method of any of claims 22, 23, 24 and 25 wherein said enzymatic RNA molecule comprises an active site region responsible for said cleavage substantially similar to the active site region of an RNA molecule naturally occurring in Tetrahymena.
Claim 37
Independent Claim
26 words
The method of any of claims 22, 23, 24, and 25 wherein said nucleotide sequence defining said cleavage site is chosen from GUCU, CUGU, and CGCU.
Claim 38
Independent Claim
25 words
The method of any of claims 22, 23, 24, and 25 which said contacting is in a medium having a pH between 7.5 and 8.0.
Claim 39
Independent Claim
27 words
The method of any of claims 22, 23, 24, and 25 which said contacting is in a medium having magnesium ions, a denaturant or a guanine derivative.
Claim 40
Independent Claim
22 words
The method of any of claims 22, 23, 24, and 25 which said contacting is in a medium having a divalent cation.
Claim 41
Independent Claim
19 words
The method of any of claims 22, 23, 24, and 25 wherein said divalent cation is Mg 2+ .
Claim 42
Independent Claim
31 words
The method of any of claims 22, 23, 24, and 25 wherein said enzymatic RNA molecule further comprises a second endonuclease activity, independent of any protein, for nucleic acid comprising deoxynucleotides.
Claim 43
Independent Claim
21 words
The method of any of claims 22, 23, 24, and 25 wherein said enzymatic RNA molecule further comprises a nucleotidyltransferase activity.
Claim 44
Independent Claim
21 words
The method of any of claims 22, 23, 24, and 25 wherein said enzymatic RNA molecule further comprises a dephosphorylase activity.
Claim 45
Independent Claim
23 words
The method of any of claims 22, 23, 24, and 25 wherein said nucleotidyltransferase activity causes polymerization of oligocytidylic acid to polycytidylic acid.
Claim 46
Independent Claim
29 words
The method of any of claims 22, 23, 24, and 25 wherein said dephosphorylase activity causes removal of a 3'-terminal phosphate of a nucleic acid comprising a 3' ribonucleotide.
Claim 47
Independent Claim
22 words
The method of any of claims 22, 23, 24, and 25 wherein said dephosphorylase activity is specific to a specific nucleotide sequence.
Claim 48
Independent Claim
46 words
The method of any of claims 22, 23, 24, and 25 wherein said enzymatic RNA molecule comprises the ribonucleotide base sequence of L-19 IVS RNA having a ribonucleotide base sequence at its active site responsible for said cleavage different from that of L-19 isolated from Tetrahymena.
Claim 49
Independent Claim
25 words
The method of any of claims 22, 23, 24, and 25 wherein said enzymatic RNA molecule comprises the ribonucleotide base sequence of L-19 IVS RNA.
Claim 50
Independent Claim
30 words
The method of any of claims 22, 23, 24, and 25 wherein said active site comprises those ribonucleotides at positions 22 through 25 in an IVS RNA isolated from Tegrahymena.
Claim 51
Dependent Claim
66 words
A purified enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site consisting essentially of single-stranded RNA in said 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 52
Dependent Claim
66 words
A purified enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site consisting essentially of single-stranded RNA in said 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 53
Dependent Claim
16 words
The purified RNA molecule of claim 43 wherein said activity causes cleavage by a transesterification reaction.
Claim 54
Independent Claim
95 words
A method for specifically cleaving a separate RNA molecule at a cleavage site comprising single-stranded RNA, comprising the steps of: providing an enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site consisting essentially of single-stranded RNA in a separate RNA molecule, and contacting said enzymatic RNA molecule and said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule.
Claim 55
Independent Claim
95 words
A method for specifically cleaving a separate RNA molecule at a cleavage site comprising single-stranded RNA, comprising the steps of: providing an enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site consisting essentially of single-stranded RNA in a separate RNA molecule, and contacting said enzymatic RNA molecule and said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule.
Claim 56
Dependent Claim
29 words
The method of claim 45 wherein said enzymatic RNA molecule is chosen from one not causing specific cleavage of said separate RNA molecule immediately after the nucleotide sequence CUCU.
Claim 57
Dependent Claim
19 words
The method of claim 45 or 46, wherein said enzymatic RNA molecule is purified prior to said providing step.
Claim 58
Dependent Claim
47 words
A purified enzymatic RNA molecule, separated from other RNA molecules and oligonucleotides, having endonuclease activity independent of any protein, said activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded RNA in a separate RNA molecule, said enzymatic RNA molecule being synthesized from DNA.
Claim 59
Dependent Claim
47 words
A purified enzymatic RNA molecule, separated from other RNA molecules and oligonucleotides, having endonuclease activity independent of any protein, said activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded RNA in a separate RNA molecule, said enzymatic RNA molecule being synthesized from DNA.
Claim 60
Dependent Claim
29 words
A purified enzymatic RNA molecule of claim 48, wherein said recombinant DNA encodes a naturally-occurring RNA sequence in which the active site is different from the naturally-occurring active site.
Claim 61
Dependent Claim
29 words
The purified enzymatic RNA molecule of claim 48 or 49 wherein said cleavage site comprises four nucleotides and is different from the cleavage site in the naturally-occurring RNA sequence.
Claim 62
Dependent Claim
57 words
An enzymatic RNA molecule having an endonuclease activity independent of any protein, said activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded RNA in a separate RNA molecule, said enzymatic RNA molecule being chemically synthesized to correspond to naturally occurring RNA wherein the naturally-occurring cleavage site of said naturally-occurring RNA has been deleted.
Claim 63
Dependent Claim
57 words
An enzymatic RNA molecule having an endonuclease activity independent of any protein, said activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded RNA in a separate RNA molecule, said enzymatic RNA molecule being chemically synthesized to correspond to naturally occurring RNA wherein the naturally-occurring cleavage site of said naturally-occurring RNA has been deleted.
Claim 64
Dependent Claim
16 words
The enzymatic RNA molecule of claim 51 wherein said cleavage is caused by a transesterification reaction.
Claim 65
Independent Claim
82 words
A method for producing an enzymatic RNA molecule having an endonuclease activity independent of any protein, said activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded RNA in a separate first RNA molecule, comprising the steps of: identifying a second RNA molecule having intramolecular cleaving activity, determining the cleavage site of said second RNA molecule, said site comprising at least four bases, and synthesizing an enzymatic RNA molecule corresponding to said second RNA molecule lacking said cleavage site.
Claim 66
Independent Claim
82 words
A method for producing an enzymatic RNA molecule having an endonuclease activity independent of any protein, said activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded RNA in a separate first RNA molecule, comprising the steps of: identifying a second RNA molecule having intramolecular cleaving activity, determining the cleavage site of said second RNA molecule, said site comprising at least four bases, and synthesizing an enzymatic RNA molecule corresponding to said second RNA molecule lacking said cleavage site.
Claim 67
Dependent Claim
30 words
The method of claim 53 wherein said determining step comprises determining the nucleotide immediately adjacent in the 3' or 5' direction to said cleavage site of said second RNA molecule.
Claim 68
Dependent Claim
27 words
The method of claim 53 or 54 further comprising determining the sequence in said second RNA molecule that base-pairs with the nucleotide sequence comprising said cleavage site.
Claim 69
Dependent Claim
20 words
The method of claim 55 further comprising synthesizing an enzymatic RNA molecule lacking said nucleotide sequence comprising said cleavage site.
Claim 70
Dependent Claim
68 words
An enzymatic RNA molecule, isolated from its natural environment, having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site comprising single-stranded RNA in said 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 71
Dependent Claim
68 words
An enzymatic RNA molecule, isolated from its natural environment, having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site comprising single-stranded RNA in said 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 72
Dependent Claim
16 words
The enzymatic RNA molecule of claim 57 wherein said activity causes cleavage by a transesterification reaction.
Claim 73
Dependent Claim
30 words
The enzymatic RNA molecule of claim 1 or 3 wherein said cleavage site is immune to cleavage by any naturally occurring RNA molecule having endonuclease activity independent of any protein.
Claim 74
Dependent Claim
28 words
The method of claim 22 or 24 wherein said cleavage site is immune to cleavage by any naturally occurring RNA molecule having endonuclease activity independent of any protein.
Claim 75
Dependent Claim
14 words
The method of claim 45 wherein said activity causes cleavage by a transesterification reaction.
Claim 76
Dependent Claim
25 words
The purified enzymatic RNA molecule of claim 48, wherein the active site of said recombinant DNA is different from that of L-19 isolated from Tetrahymena.
Claim 77
Dependent Claim
14 words
The purified enzymatic RNA molecule of claim 48, wherein said DNa is recombinant DNA.
References Cited (Backward Citations)
No citation records indexed.
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