WO1995010609B1 - Asymmetric hammerhead ribozymes and nucleotide sequences for their construction - Google Patents
Asymmetric hammerhead ribozymes and nucleotide sequences for their constructionInfo
- Publication number
- WO1995010609B1 WO1995010609B1 PCT/EP1994/003391 EP9403391W WO9510609B1 WO 1995010609 B1 WO1995010609 B1 WO 1995010609B1 EP 9403391 W EP9403391 W EP 9403391W WO 9510609 B1 WO9510609 B1 WO 9510609B1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hammerhead ribozyme
- anyone
- sequence
- helix
- asymmetric
- Prior art date
Links
- 229920001941 Hammerhead ribozyme Polymers 0.000 title claims abstract 30
- 229920001850 Nucleic acid sequence Polymers 0.000 title claims abstract 26
- 238000010276 construction Methods 0.000 title abstract 2
- 229920000160 (ribonucleotides)n+m Polymers 0.000 claims abstract 15
- 230000003197 catalytic Effects 0.000 claims abstract 5
- 108090000994 Catalytic RNA Proteins 0.000 claims abstract 4
- 229920002033 ribozyme Polymers 0.000 claims abstract 4
- 239000000203 mixture Substances 0.000 claims abstract 3
- 238000004519 manufacturing process Methods 0.000 claims abstract 2
- 108090001102 hammerhead ribozyme Proteins 0.000 claims 26
- 239000002773 nucleotide Substances 0.000 claims 19
- 125000003729 nucleotide group Chemical group 0.000 claims 19
- 108020004707 nucleic acids Proteins 0.000 claims 7
- 150000007523 nucleic acids Chemical class 0.000 claims 7
- 229920003013 deoxyribonucleic acid Polymers 0.000 claims 5
- 108091007521 restriction endonucleases Proteins 0.000 claims 3
- 230000000875 corresponding Effects 0.000 claims 2
- 238000000034 method Methods 0.000 claims 2
- 238000002360 preparation method Methods 0.000 claims 2
- 239000000969 carrier Substances 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 230000000295 complement Effects 0.000 claims 1
- 239000003085 diluting agent Substances 0.000 claims 1
- 201000010099 disease Diseases 0.000 claims 1
- 230000000694 effects Effects 0.000 claims 1
- 230000002458 infectious Effects 0.000 claims 1
- 238000003786 synthesis reaction Methods 0.000 claims 1
- 230000035897 transcription Effects 0.000 claims 1
- 230000024881 catalytic activity Effects 0.000 abstract 1
Abstract
Nucleotide sequences are described which can be used for target RNA-specific construction of asymmetric hammerhead ribozymes having high catalytic activity. Further, methods for the production of these asymmetric ribozymes and DNA sequences encoding them as well as compositions containing such ribozymes or DNA sequences are described.
Claims
1. A nucleotide sequence comprising the following general formula
3 ' - [N] n- [N] Lo- [N] 10- [N] 9_3- [N] -5 '
wherein N represents any nucleotide and may be the same or different, [N] represents no nucleotide, nucleotide 2.1 or nucleotides 2.x till 2.1 capable of forming Helix I in a hammerhead ribozyme/target-complex, [N] 9.3 represents nucleotides 3 to 9 of a hammerhead ribozyme, [N] 10 represents nucleotides 10.1 till 10.n and [N] 1:L represents no nucleotide, nucleotide 11.m or comprises at least a part of nucleotides 11.m till 11.1, x is an integer ranging from 2 to 8, m is an integer, n is an integer and represents the length of Helix II, wherein, if [N] .1 comprises at least a part of nucleotides 11.m till 11.1, n and m may be the same or different, Lo is an integer and [N] Lo represents any sequence capable of forming Loop 2 of a hammerhead ribozyme, wherein said nucleotide sequence does not contain the Helix Ill-forming region of a hammerhead ribozyme/target-complex.
2. The nucleotide sequence according to claim l, wherein the region 3 ' - [N] n- [N] L0- [N] IO-5 ' contains at least one recognition sequence for one or more restriction enzymes and/or a recognition sequence for a restriction enzyme that overlaps with the sequence following 3 ' to [N] n .
3. The nucleotide sequence according to claim 1 or 2, wherein n ranges from 3 to 50.
4. The nucleotide sequence according to anyone of claims 1 to 3, wherein x is 3 and/or n is 4 and/or m ranges from 2 to 4.
5. A nucleic acid comprising the nucleotide sequence of anyone of claims l to 4 and a second nucleotide sequence containing at least the nucleotides 12 to 14 of a hammerhead ribozyme and at least part of the Helix III-forming region of a hammerhead ribozyme/targe -complex, starting with nucleotide 15.1 till 15.y, wherein y is an integer and the number of nucleotides within the Helix I-forming part, corresponding to nucleotides 2.1 till 2.x, is smaller than the number of nucleotides of the Helix III-forming region, corresponding to nucleotides 15.1 till 15.y.
6. The nucleic acid according to claim 5, wherein the nucleotide sequence and the second nucleotide sequence are linked via one of the recognition sequences contained in 3 ' - [N] n" [N]L0- [N] ιo-5' or via a recognition sequence for a restriction enzyme that overlaps with the sequence following 3- to [N]1X.
7. The nucleic acid according to claim 5 or 6, further comprising a third nucleotide sequence adjacent to the Helix III-forming region of the second nucleotide sequence, containing at least 3 nucleotides capable of forming an intramolecular Helix III of a hammerhead ribozyme, followed directly by nucleotide 17 of a hammerhead ribozyme and a nucleotide sequence which is complementary to [N] .
8. An asymmetric hammerhead ribozyme comprising the nucleic acid of anyone of claims 5 to 7 or the nucleotide sequence of anyone of claims l to 4.
9. The asymmetric hammerhead ribozyme according to claim 8, wherein x ranges from 2 to 8.
10. An asymmetric hammerhead ribozyme, wherein the Helix I-forming region is smaller than the Helix Ill-forming region.
11. The asymmetric hammerhead ribozyme according to anyone of claims 8 to 10, wherein at least one nucleotide is chemically modified.
12. The asymmetric hammerhead ribozyme according to anyone of claims 8 to 11, which is chemically synthesized.
13. An RNA construct comprising at least two asymmetric hammerhead ribozymes of anyone of claims 8 to 12.
14. The RNA construct according to claim 13, wherein the Helix I- and Helix III-forming regions of the asymmetric hammerhead ribozymes may be the same or different.
15. A DNA sequence which upon transcription corresponds to the asymmetric hammerhead ribozyme of anyone of claims 8 to 12 or the RNA construct of claim 13 or 14.
16. A vector comprising the nucleotide sequence of anyone of claims l to 4, the nucleic acid of anyone of claims 5 to 7, the asymmetric hammerhead ribozyme of anyone of claims 8 to 12, the RNA construct of claim 13 or 14 or the DNA sequence of claim 15.
17. A host organism containing the nucleotide sequence of anyone of claims l to 4, the nucleic acid of anyone of claims 5 to 7, the asymmetric hammerhead ribozyme of anyone of claims 8 to 12, the RNA construct of claim 13 or 14, the DNA sequence of claim 15 or the vector of claim 16.
18. A method for the production of the nucleotide sequence of anyone of claims l to 4, the nucleic acid of anyone of claims 5 to 7, the asymmetric hammerhead ribozyme of anyone of claims 8 to 12, the RNA construct of claim 13 or 14, the DNA sequence of claim 15 or the vector of claim 16, comprising cultivating the host organism of claim 17 under suitable conditions and isolating the desired product from the culture.
19. A process for the preparation of the asymmetric hammerhead ribozyme of anyone of claims 8 to 12, the RNA construct of claim 13 or 14, or the DNA encoding said asymmetric hammerhead ribozyme or said RNA construct, comprising
(i) selecting a cleavable motif in the target RNA,
(ii) selecting the appropriate vector and/or DNA cassette that provides a Helix I Box matching to the selected cleavable motif and a part of the catalytic domain of a hammerhead ribozyme as defined by the general formula 3 ■ - [N] X1- [N]Lo- [N] 10- [N] 9_3- [N] -5' and
(iii) attaching the second sequence that corresponds to the Helix III-forming part and the residual part of the catalytic domain of a hammerhead ribozyme.
20. A process for the preparation of the asymmetric hammerhead ribozyme of anyone of claims 8 to 12, the RNA construct of claim 13 or 14, or the DNA encoding said asymmetric hammerhead ribozyme or said RNA construct, comprising introducing the catalytic domain and the Helix I- forming sequence into DNA by a single PCR step, in which one DNA oUgonucleotide provides the sequence of at least part of the catalytic domain and the Helix I-forming region, or the step of the chemical synthesis of at least a part of the asymmetric ribozymes, or the generation of multiple ribozymes by adding a short sense sequence forming an intramolecular Helix III- and Helix I-forming region, including a cleavable motif.
21. A composition containing the asymmetric hammerhead ribozyme of anyone of claims 8 to 12, the RNA construct of claim 13 or 14, the DNA sequence of claim 15 encoding said asymmetric hammerhead ribozyme or said RNA construct or the vector of claim 16 containing said asymmetric hammerhead ribozyme, said RNA construct or said DNA sequence, optionally in association with a pharmaceutically, veterinarian or agriculturally acceptable carrier and/or diluent.
22. A method of treating a human being, an animal or a plant in need thereof against a disease caused by the undesired activity of a gene or an infectious agent, comprising administering a composition of claim 21.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP94930172A EP0785998B1 (en) | 1993-10-15 | 1994-10-14 | Asymmetric hammerhead ribozymes and nucleotide sequences for their construction |
| DE69412146T DE69412146T2 (en) | 1993-10-15 | 1994-10-14 | ASYMETRIC "HAMMERHEAD" RIBOZYMS AND NUCLEOTIDE SEQUENCES FOR THEIR STRUCTURE |
| AU79378/94A AU7937894A (en) | 1993-10-15 | 1994-10-14 | Asymmetric hammerhead ribozymes and nucleotide sequences for their construction |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEPCT/EP93/02853 | 1993-10-15 | ||
| PCT/EP1993/002853 WO1995010608A1 (en) | 1993-10-15 | 1993-10-15 | Asymmetric hammerhead ribozymes and nucleotide sequences for their construction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1995010609A1 WO1995010609A1 (en) | 1995-04-20 |
| WO1995010609B1 true WO1995010609B1 (en) | 1995-05-04 |
Family
ID=8165777
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP1993/002853 WO1995010608A1 (en) | 1993-10-15 | 1993-10-15 | Asymmetric hammerhead ribozymes and nucleotide sequences for their construction |
| PCT/EP1994/003391 WO1995010609A1 (en) | 1993-10-15 | 1994-10-14 | Asymmetric hammerhead ribozymes and nucleotide sequences for their construction |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP1993/002853 WO1995010608A1 (en) | 1993-10-15 | 1993-10-15 | Asymmetric hammerhead ribozymes and nucleotide sequences for their construction |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0785998B1 (en) |
| AU (2) | AU5334294A (en) |
| DE (1) | DE69412146T2 (en) |
| WO (2) | WO1995010608A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0902836A4 (en) * | 1996-04-02 | 2004-12-22 | Gene Shears Pty Ltd | Asymmetric hammerhead ribozymes |
| US20040005546A1 (en) | 2002-02-28 | 2004-01-08 | Oncolytics Biotech Inc. | Use of ribozymes in the detection of adventitious agents |
| CA2374388C (en) * | 2002-02-28 | 2005-07-12 | Matthew C. Coffey | The use of ribozymes in the detection of adventitious agents |
| WO2005117557A2 (en) * | 2004-06-01 | 2005-12-15 | San Diego State University Foundation | Expression system |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0640688A1 (en) * | 1987-12-15 | 1995-03-01 | Gene Shears Pty. Limited | Ribozymes |
| WO1991019789A1 (en) * | 1990-06-19 | 1991-12-26 | Commonwealth Scientific And Industrial Research Organisation | Endonucleases |
| CA2088154A1 (en) * | 1990-07-26 | 1992-01-27 | Martin Tabler | Portable ribozyme cassettes, dna sequences containing them, ribozyme encoded by these dna sequences, and compositions containing these ribozymes |
-
1993
- 1993-10-15 WO PCT/EP1993/002853 patent/WO1995010608A1/en active Application Filing
- 1993-10-15 AU AU53342/94A patent/AU5334294A/en not_active Abandoned
-
1994
- 1994-10-14 EP EP94930172A patent/EP0785998B1/en not_active Expired - Lifetime
- 1994-10-14 WO PCT/EP1994/003391 patent/WO1995010609A1/en active IP Right Grant
- 1994-10-14 AU AU79378/94A patent/AU7937894A/en not_active Abandoned
- 1994-10-14 DE DE69412146T patent/DE69412146T2/en not_active Expired - Fee Related
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