Methods and Compositions Relating to Gene Silencing

Abstract

A method for obtaining a mixture of heterogenous short double-stranded RNA molecules suitable for use in gene silencing (hsiRNA) by subjecting large double-stranded RNA to enzymatic cleavage under specified conditions. The resulting mixture consistently includes enhanced representation of fragments having a size of 21-22 nucleotides absent any fractionation step. The fragments contain sequences that collectively span the entire length of the large double-stranded RNA from which they are derived. Double-stranded RNA with sequences that individually represent segments of a target mRNA may be analyzed using the methods described herein to identify the most active subset of hsiRNA fragments or individual siRNA fragments for achieving gene silencing for any gene or transcribed sequences. A method is additionally provided for preparing and cloning DNA encoding selected siRNA, hsiRNA mixtures or hairpin sequences to provide a continuous supply of a gene silencing reagent derived from any long double-stranded RNA.

1 . A method comprising steps of:
 (a) combining in a mixture:
 (i) a large double-stranded RNA substrate at least 40 nucleotides in length; 
 (ii) a prokaryotic RNase III present at a ratio of enzyme to substrate (w/w) which is greater than or equal to about 0.25:1; and 
 (iii) a divalent transition metal cation at a concentration of at least 5 mM; and 
   (b) incubating the mixture under conditions and for a time sufficient for the RNA substrate to be cleaved such that more than about 50% of the RNA in the mixture is within the size range of about 15-25 nucleotides as determined by ethidium bromide acrylamide gel electrophoresis.   
     
     
         2 . A method according to  claim 1 , wherein the more than about 50% of the RNA further comprises a plurality of overlapping fragments. 
     
     
         3 . A method according to  claim 1 , wherein the transition metal cation is manganese. 
     
     
         4 . A method according to  claim 1 , wherein the mixture can be incubated for a time period of 60-180 seconds. 
     
     
         5 . A method according to  claim 1 , wherein the mixture is suitable for use in gene silencing absent an additional purification step. 
     
     
         6 . A method comprising steps of:
 (a) combining in a mixture:
 (i) a large double-stranded RNA substrate at least 40 nucleotides in length; 
 (ii) a prokaryotic RNase III present at a ratio of enzyme to double-stranded substrate (w/w) which is greater than or equal to about 0.4:1; and 
 (iii) a divalent transition metal cation; and 
   (b) incubating the mixture under conditions and for a time sufficient for the RNA substrate to be cleaved such that more than about 50% of the RNA in the mixture is within the size range of about 15-30 nucleotides as determined by ethidium bromide acrylamide gel electrophoresis.   
     
     
         7 . A reaction mixture, comprising a prokaryotic RNase III, a buffer containing manganese ions and a set of double-stranded RNA fragments, comprising a plurality of overlapping double strand RNA fragments of a size in the range of about 15-30 nucleotides, the fragments in the set collectively representing a substantial portion of a sequence of one or more large double-stranded RNAs of a size of at least 40 nucleotides from which the fragments are derived by in vitro cleavage with the RNase III, one strand of each of the large double-stranded RNAs having a sequence complementary to part or all of a target RNA in a eukaryotic cell such that the mixture is capable of gene silencing when introduced into the eukaryotic cell.