RNA sequence-specific mediators of RNA interference

Abstract

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 .- 16 . (canceled) 
     
     
         17 . A method of mediating RNA interference of mRNA of a gene in a cell or organism comprising:
 (a) introducing RNA of from about 21 to about 23 nucleotides which targets the mRNA of the gene for degradation into the cell or organism;   (b) maintaining the cell or organism produced in (a) under conditions under which degradation of the mRNA occurs, thereby mediating RNA interference of the MRNA of the gene in the cell or organism.   
     
     
         18 . The method of  claim 17  wherein the RNA of (a) is a chemically synthesized RNA or an analog of naturally occurring RNA. 
     
     
         19 . The method of  claim 17 , wherein the gene encodes a cellular mRNA or a viral mRNA. 
     
     
         20 . A method of mediating RNA interference of mRNA of a gene in a cell or organism in which RNA interference occurs, comprising:
 (a) combining double-stranded RNA that corresponds to a sequence of the gene with a soluble extract that mediates RNA interference, thereby producing a combination;   (b) maintaining the combination producing in (a) under conditions under which the double-stranded RNA is processed to RNA of from about 21 to about 23 nucleotides, thereby producing RNA of from about 21 to about 23 nucleotides;   (c) isolating RNA of from about 21 to about 23 nucleotides produced in (b);   (d) introducing RNA isolated in (c) into the cell or organism; and   (e) maintaining the cell or organism produced in (d) under conditions under which degradation of mRNA of the gene occurs, thereby mediating RNA interference of the mRNA of the gene in the cell or organism.   
     
     
         21 . The method of  claim 20 , wherein the soluble extract is derived from syncytial blastoderm Drosophila embryos. 
     
     
         22 . The method of  claim 20 , wherein the RNA is isolated using gel electrophoresis. 
     
     
         23 . A method of mediated RNA interference of mRNA of a gene in a cell or organism in which RNA interference occurs, comprising: (a) introducing into the cell or organism RNA of from about 21 to about 23 nucleotides that mediates RNA interference of mRNA of the gene, thereby producing a cell or organism that contains the RNA and (b) maintaining the cell or organism that contains the RNA under conditions under which RNA interference occurs, thereby mediating RNA interference of mRNA of the gene in the cell or organism. 
     
     
         24 . The method of  claim 23 , wherein the RNA of from about 21 to about 23 nucleotides is chemically synthesized RNA or an analog of RNA that mediates RNA interference. 
     
     
         25 . The method of  claim 23 , wherein the gene encodes a cellular mRNA or a viral mRNA. 
     
     
         26 .- 35 . (canceled) 
     
     
         36 . A method of treating a disease or condition associated with the presence of a protein in an individual comprising administering to the individual RNA of from about 21 to about 23 nucleotides that targets the mRNA of the protein for degradation. 
     
     
         37 . The method of  claim 36  wherein RNA of from about 21 to about 23 nucleotides is chemically synthesized or an analog of RNA that mediates RNA interference. 
     
     
         38 .- 50 . (canceled)