METHODS AND COMPOSITIONS FOR THE SPECIFIC INHIBITION OF GENE EXPRESSION BY DOUBLE-STRANDED RNA
The invention is directed to compositions and methods for selectively reducing the expression of a gene product from a desired target gene in a cell, as well as for treating diseases caused by the expression of the gene. More particularly, the invention is directed to compositions that contain double stranded RNA (“dsRNA”), and methods for preparing them, that are capable of reducing the expression of target genes in eukaryotic cells. The dsRNA has a first oligonucleotide sequence that is between 25 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions. In addition, a region of one of the sequences of the dsRNA having a sequence length of at least 19 nucleotides is sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to trigger the destruction of the target RNA by the RNAi machinery.
1 . A method for preparing an isolated double stranded nucleic acid capable of reducing the expression of a target gene comprising selecting a target sequence of an RNA of a target gene, wherein the target sequence comprises at least 19 nucleotides, synthesizing a second oligonucleotide strand having a nucleotide sequence that is complementary to the selected target sequence and synthesizing a first oligonucleotide strand that is capable of annealing to the second oligonucleotide strand under biological conditions,
wherein the first oligonucleotide strand is 25-30 nucleotides in length and contains 1-3 DNA bases on the 3′ end of the first oligonucleotide strand;
wherein the second oligonucleotide strand that is 25-30 nucleotides in length comprising an overhang domain and a primary domain wherein the overhang domain is comprised of modified RNA monomers and the primary domain is capable of hybridizing to the first oligonucleotide strand and is comprised of a combination of unmodified and modified monomers,
thereby preparing the double stranded nucleic acid.