Method for the in vitro synthesis of short double stranded RNAs

• US 7,659,391 B2
• Filed: 10/30/2002
• Issued: 02/09/2010
• Est. Priority Date: 11/05/2001
• Status: Expired due to Fees

• Current Assignee: Janssen Pharmaceutica NV (Johnson & Johnson)
• Original Assignee: Janssen Pharmaceutica NV (Johnson & Johnson)
• Inventors: De Backer, Marianne Denise, Harris, Adam N.
• Primary Examiner(s): Whiteman; Brian
• Application Number:
  US10/494,663
• Publication Number:
  US 20040259097A1
• Time in Patent Office: 2,659 Days
• Field of Search: 536/25.3
• US Class Current: 536/25.3
• 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 of less than 30 nucleotide long comprising the steps of:

a) combining a target-specific sense oligonucleotide template and a T7 RNA polymerase in a reaction mixture such that a template extended, sense oligoribonucleotide, product is formed;

b) combining a target-specific antisense oligonucleotide template and T7 RNA polymerase in a reaction mixture such that a 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), characterized in that;

the oligonucleotide templates of step a) and b) comprise an RNA polymerase promoter sequence consisting of the truncated T7 RNA polymerase promoter sequence as set forth in SEQ ID NO;

56, extended at the 5′

-end of the template strand with the target-specific template sequence, wherein said target-specific template sequence comprises at the 5′

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

-end two cytosine (c) nucleotides, wherein said two cytosine nucleotides being the first two nucleotides of said promoter sequence.

3. A method for the synthesis of small interfering RNAs (siRNAs) of 12-30 nucleotides comprising the steps of;

a) combining a sense siRNA template with T7 RNA polymerase in a reaction mixture such that a template extended sense oligoribonucleotide product is formed;

b) combining an antisense siRNA template with T7 RNA polymerase in a reaction mixture such that a 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 consisting of the truncated T7 RNA polymerase sequence as set forth in SEQ ID NO;

56, extended at the 5′

-end of the template strand with the target-specific template sequence as defined in claim 1, wherein the template further contains 2 or 3 additional nucleotides.

2. A method for the synthesis of target-specific short double stranded RNAs of less than 30 nucleotide long comprising the steps of:

a) combining a target-specific sense oligonucleotide template and a T7 RNA polymerase in a reaction mixture such that a template extended, sense oligoribonucleotide, product is formed;

b) combining a target-specific antisense oligonucleotide template and T7 RNA polymerase in a reaction mixture such that a 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), characterized in that;

wherein the oligonucleotide templates of step a) and b) are characterized by being partially double stranded DNA oligo templates comprising a double stranded RNA polymerase promoter sequence consisting of the truncated T7 RNA polymerase promoter sequence as set forth in SEQ ID NO;

56, extended at the 5′

-end of the template strand with the target-specific template sequence, wherein said target-specific template sequence comprises at the 5′

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

-end two cytosine (c) nucleotides, wherein said two cytosine nucleotides being the first two nucleotides of said promoter sequence.