RNA sequence-specific mediators of RNA interference
The present invention relates to a Drosophila in vitro system which was used to demonstrate that dsRNA is processed to RNA segments 21-23 nucleotides (nt) in length. Furthermore, when these 21-23 nt fragments are purified and added back to Drosophila extracts, they mediate RNA interference in the absence of long dsRNA. Thus, these 21-23 nt fragments are the sequence-specific mediators of RNA degradation. A molecular signal, which may be their specific length, must be present in these 21-23 nt fragments to recruit cellular factors involved in RNAi. This present invention encompasses these 21-23 nt fragments and their use for specifically inactivating gene function. The use of these fragments (or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of specific mRNAs for degradation in mammalian cells, where the use of long dsRNAs to elicit RNAi is usually not practical, presumably because of the deleterious effects of the interferon response. This specific targeting of a particular gene function is useful in functional genomic and therapeutic applications.
1 . A method of producing knockdown cells, comprising introducing into cells in which a gene is to be knocked down RNA of about 21 to about 23 nucleotides that targets the mRNA corresponding to the gene and maintaining the resulting cells under conditions under which RNAi occurs, resulting in degradation of the mRNA of the gene, thereby producing knockdown cells.
2 . The method of claim 1 , wherein the gene encodes a cellular mRNA
3 . The method of claim 1 , wherein the RNA of from about 21 to about 23 nucleotides is recombinantly produced.
4 . The method of claim 3 , wherein the RNA comprises a terminal 3′ hydroxyl group.
5 . The method of claim 1 , wherein the RNA of from about 21 to about 23 nucleotides is chemically synthesized.
6 . The method of claim 5 , wherein the RNA comprises a terminal 3′ hydroxyl group.
7 . The method of claim 5 , wherein one or more nucleotides of the isolated RNA is a non-naturally occurring nucleotide.
8 . The method of claim 5 , wherein one or more nucleotides of the isolated RNA is a deoxyribonucleotide.
9 . The method of claim 5 , wherein one or more nucleotides of the isolated RNA is a non-standard nucleotide.
10 . A knockdown cell generated by the method of claim 1 .
11 . The knockdown cell of claim 10 , wherein the cell or organism mimics a disease.
12 . A method of producing a knockdown organism, comprising introducing into the organism in which a gene is to be knocked down RNA of about 21 to about 23 nucleotides that targets the mRNA corresponding to the gene and maintaining the resulting organism under conditions under which RNAi occurs, resulting in degradation of the mRNA of the gene, thereby producing the knockdown organism.
13 . The method of claim 12 , wherein the gene encodes a cellular mRNA
14 . The method of claim 12 , wherein the RNA of from about 21 to about 23 nucleotides is recombinantly produced.
15 . The method of claim 14 , wherein the RNA comprises a terminal 3′ hydroxyl group.
16 . The method of claim 12 , wherein the RNA of from about 21 to about 23 nucleotides is chemically synthesized.
17 . The method of claim 16 , wherein the RNA comprises a terminal 3′ hydroxyl group.
18 . The method of claim 16 , wherein one or more nucleotides of the isolated RNA is a non-naturally occurring nucleotide
19 . The method of claim 16 , wherein one or more nucleotides of the isolated RNA is a deoxyribonucleotide.
20 . The method of claim 16 , wherein one or more nucleotides of the isolated RNA is a non-standard nucleotide.
21 . A knockdown organism generated by the method of claim 12 .
22 . The knockdown organism of claim 21 , wherein the cell or organism mimics a disease.