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 . A method of producing RNA of from about 21 to about 23 nucleotides in length comprising: 
 (a) combining double-stranded RNA with a soluble extract that mediates RNA interference, thereby producing a combination; and    (b) maintaining the combination of a) under conditions in which the double-stranded RNA is processed to RNA of from about 21 to about 23 nucleotides in length.    
     
     
         2 . The method of  claim 1 , wherein the soluble extract is derived from syncytial blastoderm Drosophila embryos.  
     
     
         3 . The method of  claim 1  further comprising isolating the RNA of from about 21 to about 23 nucleotides from the combination.  
     
     
         4 . A method of producing an RNA of from about 21 to about 23 nucleotides in length that mediates RNA interference of mRNA of a gene to be degraded, comprising: 
 (a) combining double-stranded RNA (dsRNA) that corresponds to a sequence of the gene to be degraded with a soluble extract that mediates RNA interference, thereby producing a combination; and    (b) maintaining the combination of (a) under conditions under which the dsRNA is processed to RNA of from about 21 to about 23 nucleotides that mediates RNA interference of the mRNA of the gene to be degraded, thereby producing RNA of from about 21 to about 23 nucleotides that mediates RNA interference of the mRNA.    
     
     
         5 . The method of  claim 4 , wherein the soluble extract is derived from syncytial blastoderm Drosophila embryos.  
     
     
         6 . The method of  claim 4 , further comprising isolating RNA of from about 21 to about 23 nucleotides from the combination.  
     
     
         7 . The method of  claim 4 , wherein the mRNA of the gene to be degraded is a mammalian cellular mRNA.  
     
     
         8 . The method of  claim 4 , wherein the RNA comprises a terminal 3′ hydroxyl group.  
     
     
         9 . The method of  claim 4 , wherein one or more nucleotides of the isolated RNA are a non-naturally occurring nucleotide  
     
     
         10 . The method of  claim 4 , wherein one or more nucleotides of the isolated RNA are a deoxyribonucleotide.  
     
     
         11 . The method of  claim 4 , wherein one or more nucleotides of the isolated RNA are a non-standard nucleotide.  
     
     
         12 . The method of  claim 6 , wherein the RNA is isolated by gel electrophoresis.  
     
     
         13 . The method of  claim 6 , wherein the RNA is isolated by chromatography.  
     
     
         14 . The method of  claim 6 , wherein the RNA is isolated by size-exclusion chromatography.  
     
     
         15 . The method of  claim 6 , wherein the RNA is isolated by glycerol gradient centrifugation.  
     
     
         16 . The method of  claim 6 , wherein the dsRNA ranges in size from about 21 nucleotides to about 500 nucleotides.  
     
     
         17 . A method of producing a double-stranded RNA of from about 21 to about 23 nucleotides in length that mediates RNA interference of mRNA of a gene to be degraded, comprising: 
 (a) synthesizing complementary RNA strands, wherein the sequence of one strand has sufficient homology to the mRNA of the gene to be degraded, and    (b) combining the strands of (a) under conditions under which the double-stranded RNA is produced, thereby producing the double-stranded RNA of from about 21 to about 23 nucleotides that mediates RNA interference of the mRNA of the gene to be degraded    
     
     
         18 . The method according to  claim 17 , wherein the RNA strands are chemically synthesized.  
     
     
         19 . The method according to  claim 17 , wherein the RNA strands are recombinantly produced.  
     
     
         20 . The method of  claim 17 , wherein the mRNA of the gene to be degraded is a mammalian cellular mRNA.  
     
     
         21 . The method of  claim 17 , wherein the RNA comprises a terminal 3′ hydroxyl group.  
     
     
         22 . The method of  claim 17 , wherein one or more nucleotides of the isolated RNA are a non-naturally occurring nucleotide  
     
     
         23 . The method of  claim 17 , wherein one or more nucleotides of the isolated RNA are a deoxyribonucleotide.  
     
     
         24 . The method of  claim 17 , wherein one or more nucleotides of the isolated RNA are a non-standard nucleotide.