Method for the in vitro synthesis of short double stranded rnas

• US 20040259097A1
• Filed: 05/04/2004
• Published: 12/23/2004
• Est. Priority Date: 11/05/2001
• Status: Active Grant

• Current Assignee: Janssen Pharmaceutica NV (Johnson & Johnson)
• Original Assignee: Janssen Pharmaceutica NV (Johnson & Johnson)
• Inventors: Harris, Adam N, De Backer, Marianne Denise
• Granted Patent: US 7,659,391 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence

Abstract

The present invention relates to the field of synthesis of short double-stranded RNAs. An in vitro transcription method using bacteriophage polymerases and target sequence-specific single-stranded DNA oligonucleotides as templates is disclosed. The present invention finds particularly advantageous use in the synthesis of short interfering RNAs (siRNAs) that have been shown to function as key intermediates in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates and vertebrate systems.

Claims

1. A method for the synthesis of target-specific short double stranded RNAs comprising the steps of:

a) combining a target-specific sense oligonucleotide template and a chain extending enzyme in a reaction mixture such that the template extended, sense oligoribonucleotide, product is formed;

b) combining a target specific antisense oligonucleotide template and a chain extending enzyme in a reaction mixture such that the template extended, antisense oligoribonucleotide, product is formed; and

c) hybridizing the sense oligoribonucleotide product obtained in step a) with the complementary antisense oligoribonucleotide product obtained in step b).

2. A method according to claim 1 wherein the chain extending enzyme is an RNA polymerase.

3. A method according to claim 2 wherein the RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase of and SP6 polymerase.

4. A method according to claim 1 wherein the oligonucleotide templates of step a) and b) comprise an RNA polymerase promoter sequence consisting of dsDNA.

5. A method according to claim 4 wherein the RNA polymerase promoter sequence is recognized by an RNA polymerase selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase.

6. A method according to claim 4 wherein the RNA polymerase promoter sequence is recognized by T7 RNA polymerase.

7. (Currently Amended) A method according to any one of claims 4 to 6 claim 4 wherein the oligonucleotide templates of step a) and b) are further characterized by being partially double stranded DNA oligo templates comprising a double stranded RNA polymerase promoter sequence which is extended at the 5′

end of the template strand with the target specific template sequence.

8. A method according to claim 4 wherein the RNA polymerase promoter sequence consists of the truncated T7 RNA polymerase promoter sequence as shown in FIG. 1.

9. A method according to claim 1 wherein the target-specific short double stranded RNA is less than 30 nucleotides long.

10. A method according to claim 1 wherein the target-specific short double stranded RNA is characterized by comprising at the 5′

-end nucleotides transcribed from the promoter sequence and at the 3′

-end nucleotides complementary to the nucleotides transcribed from the promoter sequence.

25. Isolated RNA obtainable by the method of claim 1.

26. Isolated RNA according to claim 25 wherein said RNA consists of target-specific short double stranded RNA of less than 50 nucleotides, characterized by comprising at the 5′

end nucleotides transcribed from an RNA polymerase promoter sequence and at the 3′

end nucleotides complementary to the nucleotides transcribed from said RNA polymerase promoter sequence.

27. Isolated RNA according to claim 25 wherein the number of nucleotides transcribed from the promoter sequence and the number of nucleotides complementary to the nucleotides transcribed from the promoter sequence consist of 2, 3 or 4 nucleotides.

28. Isolated RNA according to claim 25 wherein the sense sequence of said RNA molecule can be represented by 5′

-xx(n_12-30)yy-3′

wherein x refers to the nucleotides transcribed from the promoter sequence, y refers to the nucleotides complementary to the nucleotides transcribed from the promoter sequence, and n refers to any oligonucleotide of 12 to 30 nucleotides.

29. Isolated RNA according to claim 25 wherein the sense sequence of said RNA molecule can be represented by 5′

-gg(n_15-30)cc-3′

wherein g refers to the nucleotide guanosine transcribed from the truncated T7 RNA polymerase promoter sequence (as shown in FIG.

     1), c refers to the nucleotide cytosine complementary to the nucleotides transcribed form the truncated T7 RNA polymerase promoter (as shown in FIG.

     1) sequence, and n_15-30 refers to any oligonucleotide of 15 to 30 nucleotides.

30. Isolated RNA according to claim 25 wherein the target-specific short double stranded RNA is extended at the 3 ′

ends with 2 or 3 additional nucleotides.

32. A target-specific antisense oligonucleotide template for use in a method according to claim 1.

11. A method for the synthesis of small interfering RNAs (siRNAs) comprising the steps of;

a) combining a sense siRNA template with a chain extending enzyme in a reaction mixture such that the template extended sense oligoribonucleotide product is formed;

b) combining an antisense siRNA template with a chain extending enzyme in a reaction mixture such that the template extended antisense oligoribonucleotide product is formed; and

c) hybridizing the sense oligoribonucleotide product obtained in step a) with the antisense oligoribonucleotide product obtained in step b);

whereby the siRNA templates of step a) and b) comprise a double stranded RNA polymerase promoter sequence extended at the 5′

-end of the template strand with the target-specific template sequence and 2 or 3 additional nucleotides.

12. A method according to claim 11 wherein the chain-extending enzyme is an RNA polymerase.

13. A method according to claim 12 wherein the RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase.

14. A method according to claim 12 wherein the RNA polymerase consists of T7 RNA polymerase.

15. A method according to claim 11 wherein the RNA polymerase promoter sequence is recognized by an RNA polymerase selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase.

16. A method according to claim 15 wherein the RNA polymerase promoter sequence is recognized by T7 RNA polymerase.

17. A method according to claim 16 wherein the RNA polymerase promoter sequence consists of the truncated T7 RNA polymerase promoter sequence as shown in FIG. 1.

18. A method according to claim 11 wherein the target-specific template sequence is further characterized by comprising at the 5′

end two guanosine (g) nucleotides and at the 3′

end two cytosine (c) nucleotides.

31. A target-specific sense oligonucleotide template for use in a method according claim 18.

33. The target-specific oligonucleotide template of claim 31 wherein said oligonucleotide templates comprise an RNA polymerase promoter sequence consisting of dsRNA which is extended at the 5′

end of the template strand with the target-specific template sequence.

34. The target-specific oligonucleotide templates of claim 31 wherein the RNA polymerase promoter sequence consists of the truncated T7 RNA polymerase promoter sequence as shown in FIG. 1.

35. The target-specific oligonucleotide templates of claim 31 wherein the target-specific short double stranded RNA is less than 30 nucleotides long.

36. The target-specific oligonucleotide templates of claim 31 wherein the target-specific short double stranded RNA is characterized by comprising at the 5′

-end nucleotides transcribed from the promoter sequence and at the 3′

-end nucleotides complementary to the nucleotides transcribed from the promoter sequence.

19. A method to inhibit expression of a target gene in a cell comprising introduction of RNA into a cell wherein said RNA comprises target-specific short double stranded RNA of less than 50 nucleotides characterized by comprising at the 5′

end nucleotides transcribed from an RNA polymerase promotor sequence and at the 3′

end nucleotides complementary to the nucleotides transcribed from said RNA polymerase promotor sequence.

20. A method according to claim 19 wherein the number of nucleotides transcribed from the promoter sequence and the number of nucleotides complementary to the nucleotides transcribed from the promoter sequence consist of 2, 3 or 4 nucleotides.

21. A method according to claim 19 wherein the target-specific short double stranded RNA is extended at the 3′

ends with 2 or 3 additional nucleotides.

22. A method according to claim 19 wherein the RNA polymerase promoter sequence is being recognized by an RNA polymerase selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase.

23. A method according to claim 19 wherein the RNA polymerase promoter sequence consists of the truncated T7 RNA polymerase promoter sequence as shown in FIG. 1.

24. A method according to claim 19 wherein the target gene is a cellular gene, an endogenous gene, a transgene, or a gene from a pathogen.

37. A kit for the synthesis of short double stranded target-specific RNAs the kit comprising at least one of the following components;

a) instructions to design target-specific sense and antisense oligonucleotide templates;

b) a chain extending enzyme;

c) transcriptionbuffers;

d) the nucleoside triphosphates (NTPs) for the four required ribonucleotide bases;

e) purification means to obtain the sense and antisense oligoribonucleotide products.

38. A kit according to claim 37 wherein the instructions to design the target-specific sense and antisense oligonucleotide templates consists of the method comprising the following steps;

1) look for a target-specific sequence located within the coding sequence of the target gene and having the following sequence 5′

-xx(n_12-30)yy-3′

. Wherein, x refers to the nucleotides transcribed from the promoter, y refers to the nucleotides complementary to the nucleotides transcribed form the promoter sequence, and n_12-30 refers to any oligonucleotide of 12 to 30 nucleotides

2) design a sense oligonucleotide template comprising the double stranded RNA polymerase promoter sequence according to the invention extended at the 5′

end of the template strand with the complement oligonucleotide sequence of the target-specific sequence located in step

     1), optionally extended with two additional nucleotides.

3) design an antisense oligonucleotide template comprising the double stranded RNA polymerase promoter sequence according to the invention extended at the 5′

end of the template strand with the reverse oligonucleotide sequence of the target-specific sequence located in step

     1), optionally extended with two additional nucleotides.

39. A kit according to claim 37 wherein the chain extending enzyme consists of an RNA polymerase, preferably an RNA polymerase selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase.

40. A kit according to claim 37 wherein the purification means consist of size exclusion chromatography columns.