RNA INTERFERENCE MEDIATING SMALL RNA MOLECULES
Double-stranded RNA (dsRNA) induces sequence-specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 19-23 nt short RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3′ ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the produced siRNP complex.
1 . Isolated double-stranded RNA molecule, wherein each RNA strand has a length from 19-25 nucleotides, wherein said RNA molecule is capable of target-specific nucleic acid modifications.
2 . The RNA molecule of claim 1 wherein at least one strand has a 3′-overhang from 1-5 nucleotides.
3 . The RNA molecule of claim 1 capable of target-specific RNA interference and/or DNA methylation.
4 . The RNA molecule of claim 1 , wherein each strand has a length from 19-23, particularly from 20-22 nucleotides.
5 . The RNA molecule of claim 2 , wherein the 3′-overhang is from 1-3 nucleotides.
6 . The RNA molecule of claim 2 , wherein the 3′-overhang is stabilized against degradation.
7 . The RNA molecule of claim 1 , which contains at least one modified nucleotide analogue.
8 . The RNA molecule of claim 7 , wherein the modified nucleotide analogue is selected from sugar- or backbone-modified ribonucleotides.
9 . The RNA molecule according to claim 7 , wherein the nucleotide analogue is a sugar-modified ribonucleotide, wherein the 2′—OH group 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, Cl, Br or I.
10 . The RNA molecule of claim 7 , wherein the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphothioate group.
11 . (canceled)
12 . The RNA molecule of claim 11 , wherein the identity is at least 70 percent.
13 . A method of preparing a double-stranded RNA molecule of claim 1 comprising the steps: (a) synthesizing two RNA strands each having a length from 19-25 nucleotides, wherein said RNA strands are capable of forming a double-stranded RNA molecule, (b) combining the synthesized RNA strands under conditions, wherein a double-stranded RNA molecule is formed, which is capable of target-specific nucleic acid modifications.
14 .- 15 . (canceled)
16 . A method of mediating target-specific nucleic acid modifications in a cell or an organism comprising the steps: (a) contacting said cell or organism with the double-stranded RNA molecule of claim 1 under conditions wherein target-specific nucleic acid modifications can occur, and (b) mediating a target-specific nucleic acid modification effected by the double-stranded RNA towards a target nucleic acid having a sequence portion substantially corresponding to the double-stranded RNA.
17 . The method of claim 16 , wherein the nucleic acid modification is RNA interference and/or DNA methylation.
18 . The method of claim 16 wherein said contacting comprises introducing said double-stranded RNA molecule into a target cell in which the target-specific nucleic acid modification can occur.
19 .- 26 . (canceled)
27 . Pharmaceutical composition containing as an active agent at least one double-stranded RNA molecule of claim 1 and a pharmaceutical carrier.
28 .- 29 . (canceled)
30 . A eukaryotic cell or a eukaryotic non-human organism exhibiting a target gene-specific knockout phenotype wherein said cell or organism is transfected with at least one double-stranded RNA molecule capable of inhibiting the expression of an endogenous target gene or with a DNA encoding at least one double-stranded RNA molecule capable of inhibiting the expression of at least one endogenous target gene.
31 . The cell or organism of claim 30 which is a mammalian cell.
32 . The cell or organism of claim 31 which is a human cell.
33 .- 47 . (canceled)