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EP0360257A2
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EP
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RNA catalyst for cleaving specific RNA sequences
Assignee: Northern Illinois University
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Filing: 1989-09-20
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Granted: —
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Expires: 2009-09-20
Abstract
Abstract A synthetic RNA catalyst capable of cleaving an RNA substrate, the catalyst comprising a substrate binding portion and a "hairpin" portion. The invention also provides an engineered DNA molecule and a vector, each comprising a DNA sequence coding for an RNA catalyst according to the invention. The invention further comprises host cells transformed with the vectors of the invention which are capable of expressing the RNA substrate. Finally, the invention provides a method of cleaving an RNA substrate which comprises contacting the substrate with a synthetic RNA catalyst according to the invention.
Inventors
- 👤 Arnold E. Hampel
- 👤 Richard H. Tritz
- 👤 Margaret F. Hicks
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-05 15:08
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/113; C12N15/63; C12N15/79; C12N15/82; C12N15/8216; C12N15/8218; C12N2310/00; C12N2310/10; C12N2310/11; C12N2310/111; C12N2310/12; C12N2310/122
Claims Analysis
Total Claims: 47
Claim 1
Independent Claim
169 words
A synthetic RNA catalyst capable of cleaving an RNA substrate which contains the sequence: 5′-F₁-CS-F₂-3′, wherein CS is a cleavage sequence; and F₁ and F₂ each is a sequence of bases flanking the cleavage sequence; the catalyst comprising a substrate binding portion and a "hairpin" portion, the substrate binding portion of the catalyst having the sequence: 3′-F₄-L₁-F₃-5′, wherein, F₃ is a sequence of bases selected so that F₃ is substantially base paired with F₂ when the catalyst is bound to the substrate; F₄ is a sequence of bases selected so that F₄ is substantially base paired with F₁ when the catalyst is bound to the substrate; the sequences of F₃ and F₄ being selected so that each contains an adequate number of bases to achieve sufficient binding of the RNA substrate to the RNA catalyst so that cleavage of the substrate can take place; and L₁ is a sequence of bases selected so that L₁ does not base pair with CS when the catalyst is bound to the substrate.
Claim 2
Dependent Claim
169 words
A synthetic RNA catalyst capable of cleaving an RNA substrate which contains the sequence: 5′-F₁-CS-F₂-3′, wherein CS is a cleavage sequence; and F₁ and F₂ each is a sequence of bases flanking the cleavage sequence; the catalyst comprising a substrate binding portion and a "hairpin" portion, the substrate binding portion of the catalyst having the sequence: 3′-F₄-L₁-F₃-5′, wherein, F₃ is a sequence of bases selected so that F₃ is substantially base paired with F₂ when the catalyst is bound to the substrate; F₄ is a sequence of bases selected so that F₄ is substantially base paired with F₁ when the catalyst is bound to the substrate; the sequences of F₃ and F₄ being selected so that each contains an adequate number of bases to achieve sufficient binding of the RNA substrate to the RNA catalyst so that cleavage of the substrate can take place; and L₁ is a sequence of bases selected so that L₁ does not base pair with CS when the catalyst is bound to the substrate.
Claim 3
Dependent Claim
108 words
An RNA catalyst according to Claim 1, the "hairpin" portion of the catalyst having the sequence: wherein, P₁ and P₄ each is a sequence of bases, the sequences of P₁ and P₄ being selected so that P₁ and P₄ are substantially base paired; P₁ is covalently linked to F₄; S₁ and S₂ each is a sequence of bases, the sequences of S₁ and S₂ being selected so that S₁ and S₂ are substantially unpaired; P₂ and P₃ each is a sequence of bases, the sequences of P₂ and P₃ being selected so that P₂ and P₃ are substantially base paired; and L₂ is a sequence of unpaired bases.
Claim 4
Dependent Claim
41 words
An RNA catalyst according to Claim 1 or 2, which is capable of cleaving an RNA substrate in which CS has the sequence 5′-NGUC-3′, wherein N is any base and the substrate is cleaved by the catalyst between N and G.
Claim 5
Dependent Claim
13 words
An RNA catalyst according to Claim 3 wherein L₁ has the sequence 3′-AAGA-5′.
Claim 6
Dependent Claim
46 words
An RNA catalyst according to Claim 1 or 2 wherein F₃ is at least 3 bases in length and F₄ is from 3 to 5 bases in length, and the catalyst cleaves a substrate wherein F₁ and F₂ each is at least 3 bases in length.
Claim 7
Dependent Claim
47 words
An RNA catalyst according to Claim 5 wherein F₃ is from 6 to 12 bases in length and F₄ is 4 bases in length, and the catalyst cleaves a substrate wherein F₁ is 4 bases in length and F₂ is from 6 to 12 bases in length.
Claim 8
Dependent Claim
20 words
An RNA catalyst according to Claim 2 wherein P₁ and P₄ each is from 3 to 6 bases in length.
Claim 9
Dependent Claim
19 words
An RNA catalyst according to Claim 7 wherein P₁ has the sequence 5′-ACCAG-3′ and P₄ has the sequence 5′-CUGGUA-3′.
Claim 10
Dependent Claim
20 words
An RNA catalyst according to Claim 2 wherein S₁ and S₂ each is from 4 to 7 bases in length.
Claim 11
Dependent Claim
19 words
An RNA catalyst according to Claim 9 wherein S₁ has the sequence 5′-AGAA-3′ and S₂ has the sequence 5′-AUAUUAC-3′.
Claim 12
Dependent Claim
20 words
An RNA catalyst according to Claim 2 wherein P₂ and P₃ each is from 3 to 6 bases in length.
Claim 13
Dependent Claim
19 words
An RNA catalyst according to Claim 11 wherein P₂ has the sequence 5′-ACACAC-3′ and P₃ has the sequence 5′-GUGGU-3′.
Claim 14
Dependent Claim
16 words
An RNA catalyst according to Claim 2 wherein L₂ is at least 3 bases in length.
Claim 15
Dependent Claim
13 words
An RNA catalyst according to Claim 13 wherein L₂ has the sequence 5′-GUU-3′.
Claim 16
Dependent Claim
13 words
An RNA catalyst according to Claim 2 wherein 5′-S₁-P₂-L₂ has the sequence 5′AGAAACACACGUU-3′.
Claim 17
Dependent Claim
39 words
An RNA catalyst according to Claim 1 or 2 which is capable of cleaving an RNA substrate selected from the group consisting of messenger RNA, transfer RNA, ribosomal RNA, viral RNA, nuclear RNA, organellar RNA and other cellular RNA.
Claim 18
Dependent Claim
27 words
The catalyst of Claim 16 which is capable of cleaving an RNA substrate selected from the group consisting of HIV-1 virus RNA and tobacco mosaic virus RNA.
Claim 19
Dependent Claim
19 words
An RNA catalyst according to Claim 16 which is capable of cleaving messenger RNA coding for chloramphenicol acetyl transferase.
Claim 20
Dependent Claim
60 words
An RNA catalyst according to Claim 2 containing the sequence: wherein, F₁, F₂, F₃, F₄, L₁, L₂, S₁, S₂, P₁, P₂, P₃ and P₄ are as defined in Claims 1 and 2; and L₃ is a sequence of unpaired bases that covalently links the catalyst portion of the molecule with the substrate portion to produce a synthetic autocatalytic RNA catalyst.
Claim 21
Dependent Claim
30 words
An RNA catalyst according to Claim 19 wherein CS has the sequence 5′-NGUC-3′, wherein N is any base, and the substrate is cleaved by the catalyst between N and G.
Claim 22
Dependent Claim
13 words
An RNA catalyst according to Claim 20 wherein L₁ has the sequence 3′-AAGA-5′.
Claim 23
Dependent Claim
13 words
An RNA catalyst according to Claim 21 wherein 5′-P₁-S₁-P₂-L₂-P₃-S₂-P₄-3′ has the sequence 5′-ACCAGAGAAACACACGUUGUGGUAUAUUACCUGGUA-3′.
Claim 24
Dependent Claim
13 words
An RNA catalyst according to Claim 22 wherein L₃ has the sequence 3′-CCUCC-5′.
Claim 25
Dependent Claim
156 words
A synthetic RNA catalyst which is capable of cleaving an RNA substrate containing the sequence: 5′-F₁-CS-F₂-3′, the catalyst containing the sequence: 5′-F₃-L₁-F₄-ACCAGAGAAACACACGUUGUGGUAUAUUACCUGGUA-3′, and active variants thereof, wherein, CS is a cleavage sequence; F₁ and F₂ each is a sequence of bases flanking the cleavage sequence; F₃ is a sequence of bases selected so that F₃ is substantially base paired with F₂ when the catalyst is bound to the substrate; F₄ is a sequence of bases selected so that F₄ is substantially base paired with F₁ when the catalyst is bound to the substrate; the sequences of F₃ and F₄ being selected so that each contains an adequate number of bases to achieve sufficient binding of the RNA substrate to the RNA catalyst so that cleavage of the substrate can take place; and L₁ is a sequence of bases selected so that L₁ does not base pair with CS when the catalyst is bound to the substrate.
Claim 26
Dependent Claim
156 words
A synthetic RNA catalyst which is capable of cleaving an RNA substrate containing the sequence: 5′-F₁-CS-F₂-3′, the catalyst containing the sequence: 5′-F₃-L₁-F₄-ACCAGAGAAACACACGUUGUGGUAUAUUACCUGGUA-3′, and active variants thereof, wherein, CS is a cleavage sequence; F₁ and F₂ each is a sequence of bases flanking the cleavage sequence; F₃ is a sequence of bases selected so that F₃ is substantially base paired with F₂ when the catalyst is bound to the substrate; F₄ is a sequence of bases selected so that F₄ is substantially base paired with F₁ when the catalyst is bound to the substrate; the sequences of F₃ and F₄ being selected so that each contains an adequate number of bases to achieve sufficient binding of the RNA substrate to the RNA catalyst so that cleavage of the substrate can take place; and L₁ is a sequence of bases selected so that L₁ does not base pair with CS when the catalyst is bound to the substrate.
Claim 27
Dependent Claim
44 words
An RNA catalyst according to Claim 24 wherein F₃ is at least 3 bases in length and F₄ is from 3 to 5 bases in length, and the catalyst cleaves a substrate wherein F₁ and F₂ each is at least 3 bases in length.
Claim 28
Dependent Claim
47 words
An RNA catalyst according to Claim 25 wherein F₃ is from 6 to 12 bases in length and F₄ is 4 bases in length, and the catalyst cleaves a substrate wherein F₁ is 4 bases in length and F₂ is from 6 to 12 bases in length.
Claim 29
Dependent Claim
39 words
An RNA catalyst according to Claim 24 which is capable of cleaving an RNA substrate in which CS has the sequence 5′-NGUC-3′, wherein N is any base and the substrate is cleaved by the catalyst between N and G.
Claim 30
Dependent Claim
13 words
AN RNA catalyst according to Claim 27 wherein L₁ has the sequence 3′-AAGA-5′.
Claim 31
Dependent Claim
37 words
An RNA catalyst according to Claim 24 which is capable of cleaving an RNA substrate selected from the group consisting of messenger RNA, transfer RNA, ribosomal RNA, viral RNA, nuclear RNA, organellar RNA and other cellular RNA.
Claim 32
Dependent Claim
29 words
An RNA catalyst according to Claim 29 which is capable of cleaving an RNA substrate selected from the group consisting of HIV-1 virus RNA and tobacco mosaic virus RNA.
Claim 33
Dependent Claim
19 words
An RNA catalyst according to Claim 29 which is capable of cleaving messenger RNA coding for chloramphenicol acetyl transferase.
Claim 34
Dependent Claim
17 words
An engineered DNA molecule coding for an RNA catalyst according to Claim 1, 2, 19 or 24.
Claim 35
Dependent Claim
29 words
A vector comprising a DNA sequence coding for an RNA catalyst according to Claim 1, 2, 19 or 24, the DNA sequence being operatively linked to expression control sequences.
Claim 36
Dependent Claim
13 words
The vector of Claim 33 which is capable of self-replication in a host.
Claim 37
Dependent Claim
42 words
The vector of Claim 33 wherein the RNA catalyst encoded by the vector is capable of cleaving an RNA substrate selected from the group consisting of messenger RNA, transfer RNA, ribosomal RNA, viral RNA, nuclear RNA, organellar RNA and other cellular RNA.
Claim 38
Dependent Claim
34 words
The vector of Claim 35 wherein the RNA catalyst encoded by the vector is capable of cleaving an RNA substrate selected from the group consisting of HIV-1 virus RNA and tobacco mosaic virus RNA.
Claim 39
Dependent Claim
24 words
The vector of Claim 35 wherein the RNA catalyst encoded by the vector is capable of cleaving messenger RNA coding for chloramphenicol acetyl transferase.
Claim 40
Dependent Claim
20 words
A host cell transformed with a vector according to Claim 33 and which is capable of expressing the RNA substrate.
Claim 41
Independent Claim
175 words
A method of cleaving an RNA substrate which contains the sequence: 5′F₁-CS-F₂-3′, wherein CS is a cleavage sequence; and F₁ and F₂ each is a sequence of bases flanking the cleavage sequence; the method comprising contacting the substrate with a synthetic RNA catalyst comprising a substrate binding portion and a "hairpin" portion, the substrate binding portion of the catalyst having the sequence: 3′F₄-L₁-F₃-5′, wherein, F₃ is a sequence of bases selected so that F₃ is substantially base paired with F₂ when the catalyst is bound to the substrate; F₄ is a sequence of bases selected so that F₄ is substantially base paired with F₁ when the catalyst is bound to the substrate; the sequences of F₃ and F₄ being selected so that each contains an adequate number of bases to achieve sufficient binding of the RNA substrate to the RNA catalyst so that cleavage of the substrate can take place; and L₁ is a sequence of bases selected so that L₁ does not base pair with CS when the catalyst is bound to the substrate.
Claim 42
Independent Claim
175 words
A method of cleaving an RNA substrate which contains the sequence: 5′F₁-CS-F₂-3′, wherein CS is a cleavage sequence; and F₁ and F₂ each is a sequence of bases flanking the cleavage sequence; the method comprising contacting the substrate with a synthetic RNA catalyst comprising a substrate binding portion and a "hairpin" portion, the substrate binding portion of the catalyst having the sequence: 3′F₄-L₁-F₃-5′, wherein, F₃ is a sequence of bases selected so that F₃ is substantially base paired with F₂ when the catalyst is bound to the substrate; F₄ is a sequence of bases selected so that F₄ is substantially base paired with F₁ when the catalyst is bound to the substrate; the sequences of F₃ and F₄ being selected so that each contains an adequate number of bases to achieve sufficient binding of the RNA substrate to the RNA catalyst so that cleavage of the substrate can take place; and L₁ is a sequence of bases selected so that L₁ does not base pair with CS when the catalyst is bound to the substrate.
Claim 43
Dependent Claim
107 words
The method of Claim 39 wherein the "hairpin" portion of the catalyst has the sequence: wherein, P₁ and P₄ each is a sequence of bases, the sequences of P₁ and P₄ being selected so that P₁ and P₄ are substantially base paired; P₁ is covalently linked to F₄; S₁ and S₂ each is a sequence of bases, the sequences of S₁ and S₂ being selected so that S₁ and S₂ are substantially unpaired; P₂ and P₃ each is a sequence of bases, the sequences of P₂ and P₃ being selected so that P₂ and P₃ are substantially base paired; and L₂ is a sequence of unpaired bases.
Claim 44
Independent Claim
158 words
A method of cleaving an RNA substrate containing the sequence: 5′-F₁-CS-F₂-3′, comprising contacting the substrate with a synthetic RNA catalyst containing the sequence: 5′-F₃-L₁-F₄-ACCAGAGAAACACACGUUGUGGUAUAUUACCUGGUA-3′, and active variants thereof, wherein, CS is a cleavage sequence; F₁ and F₂ each is a sequence of bases flanking the cleavage sequence; F₃ is a sequence of bases selected so that F₃ is substantially base paired with F₂ when the catalyst is bound to the substrate; F₄ is a sequence of bases selected so that F₄ is substantially base paired with F₁ when the catalyst is bound to the substrate; the sequences of F₃ and F₄ being selected so that each contains an adequate number of bases to achieve sufficient binding of the RNA substrate to the RNA catalyst so that cleavage of the substrate can take place; and L₁ is a sequence of bases selected so that L₁ does not base pair with CS when the catalyst is bound to the substrate.
Claim 45
Independent Claim
158 words
A method of cleaving an RNA substrate containing the sequence: 5′-F₁-CS-F₂-3′, comprising contacting the substrate with a synthetic RNA catalyst containing the sequence: 5′-F₃-L₁-F₄-ACCAGAGAAACACACGUUGUGGUAUAUUACCUGGUA-3′, and active variants thereof, wherein, CS is a cleavage sequence; F₁ and F₂ each is a sequence of bases flanking the cleavage sequence; F₃ is a sequence of bases selected so that F₃ is substantially base paired with F₂ when the catalyst is bound to the substrate; F₄ is a sequence of bases selected so that F₄ is substantially base paired with F₁ when the catalyst is bound to the substrate; the sequences of F₃ and F₄ being selected so that each contains an adequate number of bases to achieve sufficient binding of the RNA substrate to the RNA catalyst so that cleavage of the substrate can take place; and L₁ is a sequence of bases selected so that L₁ does not base pair with CS when the catalyst is bound to the substrate.
Claim 46
Dependent Claim
15 words
The method of Claim 39, 40 or 41 wherein the cleavage occurs under physiological conditions.
Claim 47
Dependent Claim
41 words
The method of Claim 42 wherein the cleavage occurs in vivo in a host cell which has been transformed with a vector comprising a DNA sequence coding for the RNA catalyst, the DNA sequence being operatively linked to expression control sequences.
References Cited (Backward Citations)
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