CA2330570A1 - Nucleic acid enzyme for rna cleavage - Google Patents
Nucleic acid enzyme for rna cleavage Download PDFInfo
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- CA2330570A1 CA2330570A1 CA002330570A CA2330570A CA2330570A1 CA 2330570 A1 CA2330570 A1 CA 2330570A1 CA 002330570 A CA002330570 A CA 002330570A CA 2330570 A CA2330570 A CA 2330570A CA 2330570 A1 CA2330570 A1 CA 2330570A1
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- enzyme
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- nucleotide
- cleavage site
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
- C12N2310/111—Antisense spanning the whole gene, or a large part of it
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
- C12N2310/123—Hepatitis delta
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- Engineering & Computer Science (AREA)
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- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
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- Wood Science & Technology (AREA)
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- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A method is described for cleaving a nucleic acid substrate with a nucleic acid enzyme at a cleavage site comprising mixing the substrate with the enzyme, wherein the substrate includes a 7 nucleotide sequence with at least 6 nucleotides (3') to the cleavage site and at least I nucleotide (5') to the cleavage site and of formula:
5'-H'.dwnarw.GNNHNN-3' wherein each N is a nucleotide which may be the same or different, H is a nucleotide selected from the group consisting of A, U, C, and T, and j is the site of cleavage, and H' is a ribonucleotide selected from the group consisting of A, U, and C, wherein (i) the first nucleotide 3' to the cleavage site is capable of forming a wobble pair with the enzyme, (ii) the second, third, fifth, and sixth nucleotides 3' to the cleavage site are capable of forming conventional Watson-Crick base pairs with the enzyme, (iii) the fourth nucleotide 3' to the cleavage site is capable of forming a non-conventional Watson-Crick base pair with the enzyme, and (iv) the first nucleotide 5' to the cleavage site does not form a base pair with the enzyme; and the enzyme comprises a substrate binding portion which is capable of base pairing to the 6 nucleotides 3' to the cleavage site of the substrate and which binding portion comprises the sequence: 3'-UNNXNN-5' wherein each N is a nucleotide which may be the same or different, and X is a nucleotide selected from the group consisting of T, U, A, and G, whereby binding of the substrate to the enzyme effects cleavage of the substrate at the cleavage site.
5'-H'.dwnarw.GNNHNN-3' wherein each N is a nucleotide which may be the same or different, H is a nucleotide selected from the group consisting of A, U, C, and T, and j is the site of cleavage, and H' is a ribonucleotide selected from the group consisting of A, U, and C, wherein (i) the first nucleotide 3' to the cleavage site is capable of forming a wobble pair with the enzyme, (ii) the second, third, fifth, and sixth nucleotides 3' to the cleavage site are capable of forming conventional Watson-Crick base pairs with the enzyme, (iii) the fourth nucleotide 3' to the cleavage site is capable of forming a non-conventional Watson-Crick base pair with the enzyme, and (iv) the first nucleotide 5' to the cleavage site does not form a base pair with the enzyme; and the enzyme comprises a substrate binding portion which is capable of base pairing to the 6 nucleotides 3' to the cleavage site of the substrate and which binding portion comprises the sequence: 3'-UNNXNN-5' wherein each N is a nucleotide which may be the same or different, and X is a nucleotide selected from the group consisting of T, U, A, and G, whereby binding of the substrate to the enzyme effects cleavage of the substrate at the cleavage site.
Claims (30)
1. A method for cleaving a nucleic acid substrate with a nucleic acid enzyme at a cleavage site comprising mixing the substrate with the enzyme, wherein the substrate includes a 7 nucleotide sequence with at least 6 nucleotides 3' to the cleavage site and at least 1 nucleotide 5' to the cleavage site and of formula:
5' -H' ~GNNHNN-3' wherein each N is a nucleotide which may be the same or different, H is a nucleotide selected from the group consisting of A, U, C, and T, and ~ is the site of cleavage, and H' is a ribonucleotide selected from the group consisting of A, U, and C, wherein (i) the first nucleotide 3' to the cleavage site is capable of forming a wobble pair with the enzyme, (ii) the second, third, fifth, and sixth nucleotides 3' to the cleavage site are capable of forming conventional Watson-Crick base pairs with the enzyme, (iii) the fourth nucleotide 3' to the cleavage site is capable of forming a triplet with the enzyme comprising a non-conventional Watson-Crick base pair and a conventional Watson-Crick base pair, and (iv) the ribonucleotide directly 5' to the cleavage site does not form a base pair with the enzyme; and the enzyme comprises a substrate binding portion which is capable of base pairing to the 6 nucleotides 3' to the cleavage site of the substrate and which binding portion comprises the sequence:
3'-UNNXNN-5' wherein each N is a nucleotide which may be the same or different, and X is a nucleotide selected from the group consisting of T, U, A, and G, whereby binding of the substrate to the enzyme effects cleavage of the substrate at the cleavage site.
5' -H' ~GNNHNN-3' wherein each N is a nucleotide which may be the same or different, H is a nucleotide selected from the group consisting of A, U, C, and T, and ~ is the site of cleavage, and H' is a ribonucleotide selected from the group consisting of A, U, and C, wherein (i) the first nucleotide 3' to the cleavage site is capable of forming a wobble pair with the enzyme, (ii) the second, third, fifth, and sixth nucleotides 3' to the cleavage site are capable of forming conventional Watson-Crick base pairs with the enzyme, (iii) the fourth nucleotide 3' to the cleavage site is capable of forming a triplet with the enzyme comprising a non-conventional Watson-Crick base pair and a conventional Watson-Crick base pair, and (iv) the ribonucleotide directly 5' to the cleavage site does not form a base pair with the enzyme; and the enzyme comprises a substrate binding portion which is capable of base pairing to the 6 nucleotides 3' to the cleavage site of the substrate and which binding portion comprises the sequence:
3'-UNNXNN-5' wherein each N is a nucleotide which may be the same or different, and X is a nucleotide selected from the group consisting of T, U, A, and G, whereby binding of the substrate to the enzyme effects cleavage of the substrate at the cleavage site.
2. The method of claim 1, wherein in the substrate, H' and a nucleotide immediately 5' to it are not both pyrimidine nucleotides.
3. The method of claim 1 or 2 wherein the substrate comprises the sequence 5'-H'~GNNHNNN.
4. The method of claim 3 wherein the substrate comprises the sequence 5'-NNRH'~GNNHNNN-3', wherein R is G or A.
5. The method of claim 4, wherein the substrate comprises the sequence 5'-RRRH'~GNNHNNN-3'.
6. The method of claim 5, wherein the substrate comprises the sequence selected from the group consisting of 5'-GGGC~GNNUNNN-3', 5'-GGGC~GNNCNNN-3', 5'-GGGU~GNNUNNN-3', 5'-GGGU~GNNCNNNN-3', and 5'-AAAC~GNNUNNN-3'.
7. The method of claim 4, wherein the substrate comprises the sequence 5'-YHRH'~GNNHNNN-3', wherein Y is C, U, or T.
8. The method of any one of claims 1 to 7, wherein the substrate binding protein of the enzyme comprises the sequence 3'-UNNXNNN-5'.
9. The method of claim 8, wherein the substrate binding portion of the enzyme comprises the sequences 3'-UNNANNN-5', 3'-UNNGNNN-5', or 3'-UNNCNNN-5'.
10. The method of any one of claims 1 to 9, wherein the enzyme comprises three or more distinct double-stranded regions, and two or more distinct single-stranded regions, wherein the substrate binding portion is comprised by one of the single-stranded regions.
11. The method of any one of claims 1 to 10, wherein the enzyme is derived from hepatitis delta virus.
12. The method of any one of claim 1 to 11 wherein the substrate is composed of ribonucleotides.
13. The method of any one of claim 1 to 11, wherein the substrate is composed of a mixture of ribonucleotides and deoxyribonucleotides.
14. The method of any one of claims 1 to 13 wherein the enzyme is composed of ribonucleotides.
15. The method of any one of claims 1 to 13 wherein the enzyme is composed of a mixture of ribonucleotides and deoxyribonucleotides.
16. A nucleic acid enzyme capable of recognizing and cleaving a nucleic acid substrate at a cleavage site comprising a substrate binding portion which is capable of base pairing to the 6 nucleotides 3' to the cleavage site of the substrate and which binding portion comprises the sequence:
3'-UNNXNN-5' wherein each N is a nucleotide which may be the same or different, and X is a nucleotide selected from the group consisting of T, U, A, G, and the substrate includes a 7 nucleotide sequence with at least 6 nucleotides 3' to the cleavage site and at least 1 nucleotide 5' to the cleavage site of formula:
5'-H'~GNNHNN-3' wherein each N is a nucleotide which may be the same or different, H is a nucleotide selected from the group consisting of A, U, C, and T, ~ is the site of cleavage, and H' is a ribonucleotide selected from the group consisting of A, U, and C, wherein (i) the first nucleotide 3' to the cleavage site is capable of forming a wobble pair with the enzyme, (ii) the second, third, fifth, sixth, and seventh nucleotides 3' to the cleavage site are capable of forming conventional Watson-Crick base pairs with the enzyme, (iii) the fourth nucleotide 3' to the cleavage site is capable of forming a triplet with the enzyme comprising a non-conventional Watson-Crick base pair and a conventional Watson-Crick base pair, and (iv) the ribonucleotide directly 5' to the cleavage site does not form a base pair with the enzyme; and
3'-UNNXNN-5' wherein each N is a nucleotide which may be the same or different, and X is a nucleotide selected from the group consisting of T, U, A, G, and the substrate includes a 7 nucleotide sequence with at least 6 nucleotides 3' to the cleavage site and at least 1 nucleotide 5' to the cleavage site of formula:
5'-H'~GNNHNN-3' wherein each N is a nucleotide which may be the same or different, H is a nucleotide selected from the group consisting of A, U, C, and T, ~ is the site of cleavage, and H' is a ribonucleotide selected from the group consisting of A, U, and C, wherein (i) the first nucleotide 3' to the cleavage site is capable of forming a wobble pair with the enzyme, (ii) the second, third, fifth, sixth, and seventh nucleotides 3' to the cleavage site are capable of forming conventional Watson-Crick base pairs with the enzyme, (iii) the fourth nucleotide 3' to the cleavage site is capable of forming a triplet with the enzyme comprising a non-conventional Watson-Crick base pair and a conventional Watson-Crick base pair, and (iv) the ribonucleotide directly 5' to the cleavage site does not form a base pair with the enzyme; and
17. The nucleic acid enzyme of claim 16, wherein in the substrate, H' and a nucleotide immediately 5' to it are not both pyrimidine nucleotides.
18. The nucleic acid enzyme of claim 16 or 17 wherein the substrate comprises the sequence 5'-H'~GNNHNNN-3'.
19. The method of claim 18 wherein the substrate comprises the sequence 5'-NNRH'~GNNHNNN-3', wherein R is G or A.
20. The nucleic acid enzyme of claim 19, wherein the substrate comprises the sequence 5'-RRRH'~GNNHNNN-3'.
21. The nucleic acid enzyme of claim 20, wherein the substrate comprises the sequence selected from the group consisting of 5'-GGGC~GNNUNNN-3', 5'-GGGC~GNNCNNN-3', 5'-GGGU~GNNUNNN-3', 5'-GGGU~GNNCNNNN-3', and 5'-AAAC~GNNUNNN-3'.
22. The nucleic acid enzyme of claim 19 wherein the substrate comprises the sequence 5'-YHRH'~GNNHNNN-3', wherein Y
is C, U, or T.
is C, U, or T.
23. The nucleic acid enzyme of any one of claims 16 to 22, wherein the substrate binding protein of the enzyme comprises the sequence 3'-UNNXNNN-5'.
24. The nucleic acid enzyme of claim 23, wherein the substrate binding portion of the enzyme comprises the sequences 3'-UNNANNN-5', 3'-UNNGNNN-5', or 3'-UNNCNNN-5'.
25. The nucleic acid enzyme of any one of claims 16 to 24 wherein the enzyme comprises three or more distinct double-stranded regions, and two or more distinct single-stranded regions, wherein the substrate binding portion is comprised by one of the single-stranded regions.
26. The nucleic acid enzyme of any one of claims 16 to 25, wherein the enzyme is derived from hepatitis delta virus.
27. The nucleic acid enzyme of any one of claim 16 to 26, wherein the substrate is composed of ribonucleotides.
28. The nucleic acid enzyme of any one of claim 16 to 26, wherein the substrate is composed of a mixture of ribonucleotides and deoxyribonucleotides.
29. The nucleic acid enzyme of any one of claims 16 to 28, wherein the enzyme is composed of ribonucleotides.
30. The nucleic acid enzyme of any one of claims 16 to 28 wherein the enzyme is composed of a mixture of ribonucleotides and deoxyribonucleotides.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002330570A CA2330570C (en) | 1998-04-29 | 1999-04-29 | Nucleic acid enzyme for rna cleavage |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA2,230,203 | 1998-04-29 | ||
| CA002230203A CA2230203A1 (en) | 1998-04-29 | 1998-04-29 | Delta ribozyme for rna cleavage |
| CA002330570A CA2330570C (en) | 1998-04-29 | 1999-04-29 | Nucleic acid enzyme for rna cleavage |
| PCT/CA1999/000391 WO1999055856A2 (en) | 1998-04-29 | 1999-04-29 | Nucleic acid enzyme for rna cleavage |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2330570A1 true CA2330570A1 (en) | 1999-11-04 |
| CA2330570C CA2330570C (en) | 2010-01-12 |
Family
ID=25680061
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002330570A Expired - Fee Related CA2330570C (en) | 1998-04-29 | 1999-04-29 | Nucleic acid enzyme for rna cleavage |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2330570C (en) |
-
1999
- 1999-04-29 CA CA002330570A patent/CA2330570C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CA2330570C (en) | 2010-01-12 |
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| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |