METHODS AND COMPOSITIONS FOR USE IN PREPARRING siRNAs

Abstract

Methods and compositions for producing siRNAs, e.g., in the form of a d-siRNA composition, from dsRNAs are provided. In the subject methods, a dsRNA is contacted with a composition that includes an activity that cleaves dsRNA into siRNAs, where the composition efficiently cleaves dsRNA into siRNAs. siRNAs produced by the subject methods find use in a variety of applications, particularly in applications where the specific reduction or silencing of a gene is desired. Also provided are kits for use in practicing the subject invention.

1 . A method for producing siRNA from an initial dsRNA, said method comprising:
 contacting said dsRNA with a composition enriched for a protein having an activity that cleaves a dsRNA substrate into fragments having siRNA activity to produce said siRNA, wherein said composition efficiently produces siRNAs from dsRNA.   
     
     
         2 . The method according to  claim 1 , wherein said method is in vitro. 
     
     
         3 . The method according to  claim 1 , wherein said contacting occurs in the absence of ATP. 
     
     
         4 . The method according to  claim 1 , wherein said protein is a recombinantly produced protein. 
     
     
         5 . The method according to  claim 1 , wherein said protein is full-length Dicer or an active fragment thereof. 
     
     
         6 . The method according to  claim 1 , wherein said composition converts at least 80% of said dsRNA to siRNA. 
     
     
         7 . The method according to  claim 1 , wherein siRNAs produced by said method range in length from about 20 to 23 residues. 
     
     
         8 . The method according to  claim 7 , wherein siRNAs produced by said method are 20-21 residues in length. 
     
     
         9 . The method according to  claim 1 , wherein said method produces a d-siRNA preparation of siRNAs from said dsRNA. 
     
     
         10 . A method of at least reducing the expression of a gene in a target cell, said method comprising:
 producing an siRNA preparation for said gene using the method of  claim 1 ; and   introducing into said cell an effective amount of said siRNA preparation to at least reduce expression of said gene.   
     
     
         11 . The method according to  claim 10 , wherein said siRNA preparation is a d-siRNA preparation according to  claim 9 . 
     
     
         12 . The method according to  claim 10 , wherein said method is an in vitro method. 
     
     
         13 . The method according to  claim 10 , wherein said method is an in vivo method. 
     
     
         14 . The method according to  claim 10 , wherein said method is a method of silencing expression of said gene. 
     
     
         15 . The method according to  claim 10 , wherein said method is a loss of function assay. 
     
     
         16 . A kit for use in preparing siRNA from dsRNA, said kit comprising:
 a composition enriched for a protein having an activity that cleaves a dsRNA substrate into fragments having siRNA activity to produce said siRNA, wherein said composition efficiently produces siRNAs from dsRNA.   
     
     
         17 . A kit according to  claim 16 , wherein said composition is ATP free. 
     
     
         18 . The kit according to  claim 16 , wherein said protein is a recombinantly produced protein. 
     
     
         19 . The kit according to  claim 16 , wherein said protein is full length dicer or an active fragment thereof. 
     
     
         20 . The kit according to  claim 16 , wherein said kit further comprises instructions for practicing a method according to  claim 1 . 
     
     
         21 . A d-siRNA preparation producing according to the method  claim 1 .