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 finction. 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 examining the function of a gene in a cell or organism comprising: 
 (a) introducing RNA of from about 21 to about 23 nucleotides that targets mRNA of the gene for degradation into the cell or organism, thereby producing a test cell or test organism;    (b) maintaining the test cell or test organism under conditions under which degradation of mRNA of the gene occurs, thereby producing a test cell or test organism in which mRNA of the gene is degraded; and    (c) observing the phenotype of the test cell or test organism produced in (b) and, optionally, comparing the phenotype observed to that of an appropriate control cell or control organism,    thereby providing information about the function of the gene.    
     
     
         2 . The method of  claim 1  wherein the RNA introduced in (a) is chemically synthesized.  
     
     
         3 . A method of examining the function of a gene in a cell or organism comprising 
 (d) combining double-stranded RNA that corresponds to a sequence of the gene with a soluble extract that mediates RNA interference, thereby producing a combination;    (e) maintaining the combination produced in (a) under conditions under which the double-stranded RNA is processed to RNA of about 21 to about 23 nucleotides, whereby RNA of about 21 to about 23 nucleotides is produced; 
 (c) isolating RNA of about 21 to about 23 nucleotides produced in (b);  
   (d) introducing the RNA isolated in (c) into the cell or organism, thereby producing a test cell or test organism;    (e) maintaining the test cell or test organism under conditions under which degradation of mRNA of the gene occurs, thereby producing a test cell or test organism in which mRNA of the gene is degraded; and    (f) observing the phenotype of the test cell or test organism produced in (e) and, optionally, comparing the phenotype observed to that of an appropriate control,    thereby providing information about the function of the gene.    
     
     
         4 . The method of  claim 1  or  3 , wherein the RNA of about 21 to about 23 nucleotides comprises a terminal 3′ hydroxyl group.  
     
     
         5 . The method of  claim 3 , wherein the soluble extract is derived from syncytial blastoderm Drosophila embryos.  
     
     
         6 . The method of  claim 3 , wherein the RNA is isolated using gel electrophoresis.  
     
     
         7 . The method of  claim 1 , wherein the RNA is introduced into the cell or organism by a recombinant DNA method.  
     
     
         8 . The method of  claim 7 , wherein the RNA is introduced into the cell or organism which encodes the RNA.  
     
     
         9 . The method of  claim 8 , wherein the RNA encoded by the DNA is processed to RNA segments of about 21 to about 23 nucleotides in length.  
     
     
         10 . The method of  claim 3 , wherein the RNA is isolated by chromatography.  
     
     
         11 . The method of  claim 3 , wherein the RNA is isolated by size-exclusion chromatography.  
     
     
         12 . The method of  claim 3 , wherein the RNA is isolated by glycerol gradient centrifugation.  
     
     
         13 . The method of  claim 3 , wherein the double-stranded RNA ranges in size from about 21 nucleotides to about 500 nucleotides.  
     
     
         14 . The method of  claim 13 , wherein double-stranded RNA is chemically synthesized.  
     
     
         15 . The method of  claim 13 , wherein double-stranded RNA is recombinantly produced.  
     
     
         16 . The method of  claim 1  or  3 , wherein the mRNA of the gene to be degraded is a mammalian cellular mRNA.  
     
     
         17 . The method of  claim 1  or  3 , wherein the mRNA of the gene to be degraded is a viral mRNA.  
     
     
         18 . The method of  claim 1 , wherein one or more nucleotides of the RNA introduced in (a) is a non-naturally occurring nucleotide.  
     
     
         19 . The method of  claim 1 , wherein one or more nucleotides of the RNA introduced in (a) is a deoxyribonucleotide.  
     
     
         20 . The method of  claim 1 , wherein one or more nucleotides of the RNA introduced in (a) is a non-standard nucleotide.  
     
     
         21 . The method of  claim 1  or  3 , wherein the gene to be degraded encodes a disease-associated protein.