SIRNA AND METHODS OF MANUFACTURE
Double-stranded RNA of about 19 to about 25 nucleotides in length capable of regulating gene expression by RNA interference is provided. Such double-stranded RNA are particularly useful for treating disease or conditions associated with a target mRNA or gene. Methods of manufacture and methods of use of the double-stranded RNA are also provided.
1 . A method of preparing a double-stranded RNA molecule comprising
(a) synthesizing a first set of two or more RNA sub-strands, wherein the combined length of the first set of sub-strands is from about 19 to about 25 nucleotides,
(b) synthesizing a second set of two or more RNA sub-strands, wherein the combined length of the second set of sub-strands is from about 19 to about 25 nucleotides;
(c) combining the synthesized first and second set of RNA sub-strands under conditions, wherein a double-stranded RNA molecule is formed
2 . The method according to claim 1 , wherein the RNA strands are chemically synthesized.
3 . The method according to claim 1 , wherein the RNA strands are enzymatically synthesized.
4 . The method of claim 1 , wherein the double-stranded RNA molecule comprises a single double stranded region and single stranded regions of about 1 to about 5 nucleotides at the 3′ ends of at least one of the strands of the double-stranded RNA molecule.
5 . The method of claim 1 , wherein both strands of the double-stranded RNA molecule each have a 3′-overhang from about 1 to about 5 nucleotides.
6 . The method of claim 1 , wherein both strands of the double-stranded RNA molecule each have a 3′-overhang of about 2 nucleotides.
7 . The method of claim 1 , wherein each strand has a length from 20-22 nucleotides.
8 . The method of claim 1 , wherein the double-stranded RNA molecule comprises at least one sugar-modified nucleotide, wherein the 2′-OH group of said sugar-modified nucleotide is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, N(R) 2 or CN, wherein R is C 1 -C 6 alkyl, alkenyl or alkynyl and halo is F, C, Br or I.
9 . The method of claim 1 , wherein the double stranded RNA molecule comprises at least one backbone-modified nucleotide containing a phosphorothioate group.
10 .- 18 . (canceled)
19 . A method of preparing a double-stranded RNA molecule comprising
(a) synthesizing a first set of two or more RNA sub-strands, wherein the combined length of the first set of sub-strands is 25 or fewer nucleotides,
(b) synthesizing a second set of two or more RNA sub-strands, wherein the combined length of the second set of sub-strands is 25 or fewer nucleotides;
(c) combining the synthesized first and second set of RNA sub-strands under conditions, wherein a double-stranded RNA molecule is formed.
20 . The method of claim 19 , wherein the double-stranded RNA molecule comprises a single double stranded region and single stranded regions of about 1 to about 5 nucleotides at the 3′ ends of at least one of the strands of the double-stranded RNA molecule.
21 . The method of claim 20 , wherein each strand has a length from 20-22 nucleotides.
22 .- 24 . (canceled)