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 assessing whether an agent acts on a gene product comprising: 
 (a) introducing RNA of from about 21 to about 23 nucleotides which targets the mRNA of the gene for degradation into a cell or organism;    (b) maintaining the cell or organism of (a) under conditions in which degradation of the mRNA occurs, 
 (c) introducing the agent into the cell or organism of (b); and  
   (d) determining whether the agent has an effect on the cell or organism,    wherein if the agent has no effect on the cell or organism then the agent acts on the gene product or on a biological pathway that involves the gene product.    
     
     
         2 . The method of  claim 1 , wherein the RNA of from about 21 to about 23 nucleotides is chemically synthesized.  
     
     
         3 . A method of assessing whether a gene product is a suitable target for drug discovery comprising: 
 (a) introducing RNA of from about 21 to about 23 nucleotides which targets the mRNA of the gene for degradation into a cell or organism;    (b) maintaining the cell or organism of (a) under conditions in which degradation of the mRNA occurs resulting in decreased expression of the gene; and    (c) determining the effect of the decreased expression of the gene on the cell or organism, wherein if decreased expression has an effect, then the gene product is a target for drug discovery.    
     
     
         4 . The method of claim  40 , wherein the RNA of from about 21 to about 23 nucleotides is synthetic RNA.  
     
     
         5 . The method of claim  38 , wherein the RNA is introduced into the cell or organism by a recombinant DNA method.  
     
     
         6 . The method of  claim 5 , wherein the RNA is introduced into the cell or organism as DNA which encodes the RNA.  
     
     
         7 . The method of  claim 6 , wherein the RNA encoded by the DNA is processed to RNA segments of about 21 to about 23 nucleotides in length.  
     
     
         8 . The method of  claim 3 , wherein the RNA is introduced into the cell or organism by a recombinant DNA method.  
     
     
         9 . The method of  claim 8 , wherein the RNA is introduced into the cell or organism as DNA which encodes the RNA.  
     
     
         10 . The method of  claim 9 , wherein the RNA is introduced into the cell or organism as DNA which encodes the RNA.  
     
     
         11 . The method of  claim 1  or  3 , wherein the RNA comprises a terminal 3′ hydroxyl group.  
     
     
         12 . The method of  claim 1  or  3 , wherein the mRNA of the gene to be degraded is a mammalian cellular mRNA.  
     
     
         13 . The method of  claim 1  or  3 , wherein the mRNA of the gene to be degraded is a viral mRNA.  
     
     
         14 . The method of  claim 1  or  3 , wherein one or more nucleotides of the RNA introduced in (a) is a non-naturally occurring nucleotide.  
     
     
         15 . The method of  claim 1  or  3 , wherein one or more nucleotides of the RNA introduced in (a) is a deoxyribonucleotide.  
     
     
         16 . The method of  claim 1  or  3 , wherein one or more nucleotides of the RNA introduced in (a) is a non-standard nucleotide.  
     
     
         17 . The method of  claim 1  or  3 , wherein the gene to be degraded encodes a disease-associated protein.