Process for preparing double-stranded RNA, and its applications

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

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435 911, 935 77, 935 78, C12P 1934, C12Q 168, C12N 1500

Patent

active

057957150

DESCRIPTION:

BRIEF SUMMARY
This application is a 371 filing of PCT/FR92/01209 filed 18 Dec. 1992.


FIELD OF THE INVENTION

The present invention relates to a process for the synthesis of double-stranded RNA, and to its applications.


DESCRIPTION OF THE BACKGROUND

Double-stranded ribonucleic acids are naturally rare and they are indeed present only in certain microorganisms such as yeasts or viruses. Up until recently, double-stranded RNA was only considered as a molecule of essentially theoretical interest, and whose possible applications were relevant only in basic research.
However, it has been demonstrated that double-stranded RNAs can, transiently, be involved in phenomena of regulation of expression, as well al., Methods in Enzymology, 1981, 78, 291 and WU-LI, J. Biol, Chem., 1990, 265, 5470)! and that they had anti-proliferative properties, which makes it possible also to envisage therapeutic applications (AUBEL et al., Proc. Natl. Acad. Sci., USA 1991, 88, 906).
It is therefore important to be able practically to synthesize double-stranded RNA (dsRNA) of determined sequence and length.
However, the production of double-stranded RNA is considered difficult. The few processes described up until now can be divided into three categories.
1) Extraction of double-stranded RNA from biological material
The extraction of double strands of RNA from viruses has been described, for example by BOCCARDO G. et al. (Double stranded RNA viruses, 1983, Bishop Eds. Elsevier, New York) and more recently by DULIEU et al. (J. Virol. Methods, 1989, 24, 77-84).
The presence of dsRNA in certain yeasts has been demonstrated by FRIED et al. (Proc. Natl. Acad. Sci. USA, 1978, 75, 4225) and by AL-HAKEEM et al. (Anal. Biochem., 1987, 163, 433-439).
While the extraction of dsRNA from yeasts is advantageous from the quantitative point of view, the sequences and lengths of the dsRNA are of course determined by the microorganism itself. It is a good route for production of dsRNA as raw material, but does not at all permit the synthesis of a predetermined sequence.
2) Hybridization of 2 single-stranded RNAs
The synthesis of dsRNA by hybridization of two complementary single-stranded RNAs has been described by SADHER et al., (Biochem. Int., 1987, 14, 1015). Each RNA chain is then synthesized by in vitro transcription of a recombinant plasmid containing, downstream of a promoter sequence of a DNA-dependent RNA polymerase, the DNA sequence to be transcribed. The single-stranded RNAs are then purified and quantified and then combined to form, by hybridization, a double strand of RNA.
More recently, BHATTACHARYYA (Nature, 1990, 343, 484) uses the RNA synthesis described by MILLIGAN (Nucleic Acids Res., 1987, 21, 8783) consisting in the transcription of a synthetic DNA template to RNA. The single-stranded RNAs are then combined and hybridized with each other.
These techniques are relatively long and difficult to implement because they necessitate the preparation of two recombinant or synthetic template DNAs and the purification of the two RNA strands produced prior to the hybridization.
3) Production of homopolymeric double-stranded RNA
J. YANO et al. (French Patent Application 2 617 403) describe a process for preparing double-stranded RNA of defined length, but of repetitive or homopolymeric sequence. In no case is this technique applicable to the synthesis of RNA of more complex sequence.
However, the inventors have now developed a process permitting the synthesis of double-stranded RNA from a DNA template of given sequence. They have indeed observed that if the simultaneous transcription of the two complementary strands of a DNA sequence is carried out under determined conditions and in the same reaction compartment, the two transcripts formed hybridize immediately between themselves, thus giving rise to a double-stranded RNA.


SUMMARY OF THE INVENTION

The subject of the present invention is a process for preparing double-stranded RNA, which process is characterized in that the simultaneous transcription is carried out of the two complementary strands of a

REFERENCES:
patent: 4766072 (1988-08-01), Jendrisak et al.
Sadhu et al., Biochemistry International 14(6):1015-1022 (1987).

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