RNA-interference by single-stranded RNA molecules
The present invention relates to sequence and structural features of single-stranded (ss)RNA molecules required to mediate target-specific nucleic acid modifications by RNA-interference (RNAi), such as target mRNA degradation and/or DNA methylation.
1. Purified human RISC, wherein said RISC is a ribonucleoprotein complex containing a single stranded RNA component, and wherein said RNA component comprises at least one modified nucleotide analogue which is selected from sugar backbone and nucleobase-modified ribonucleotides and combinations thereof, wherein said RNA component is a single-stranded RNA molecule wherein the single-stranded RNA molecule has a length from 19-29 nucleotides, and wherein said RISC has a molecular weight of less than about 160 kDa.
2. The RISC of claim 1 comprising at least one member of the Argonaute family of proteins.
3. The RISC of claim 2 wherein said at least one member of the Argonaute family of proteins is elF2C1 and/or elFC2.
4. The RISC of claim 3 , further comprising at least one of elFC3, elFC4, HILI or HIWI.
5. The RISC of claim 1 , wherein at least the 14-20 5′ most nucleotides are substantially complementary to a target transcript.
6. The RISC of claim 1 , wherein said RNA molecule has a free 5′ hydroxyl moiety or a moiety selected from phosphate groups or analogues thereof.
7. The RISC of claim 6 , wherein said RNA molecule has a 5′-moiety selected from the group consisting of 5′-monophosphate ((HO) 2 (O)P—O-5′), 5′-diphosphate ((HO) 2 (O)P—O—P(HO)(O)—O-5′), 5′-triphosphate ((HO) 2 (O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O -5′), 5′-monothiophosphate (phosphorothioate; (HO) 2 (S)P—O-5′), 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO) 2 (O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates, 5′-phosphoramidates ((HO) 2 (O)P—NH-5′, (HO)(NH 2 )(O)P—O-5′), 5′-alkylphosphonates, and 5′-alkyletherphosphonates.
8. The RISC of claim 1 , wherein said RNA molecule is completely complementary to said target transcript or completely complementary to said target transcript with the exception of nucleotides that extend beyond position 20 counted from the 5′ terminus.
9. The RISC of claim 1 , wherein said covalent coupling is via the 3′-terminus of the RNA molecule.
10. A host cell or non-human host organism capable of overexpressing the RISC according to claim 1 .
11. A method of enhancing RNAi in a cell or an organism comprising causing said cell or organism to overexpress at least one component of RISC according to claim 1 .
12. A composition comprising the RISC according to claim 1 in combination with a pharmaceutical carrier.
13. The RISC according to claim 7 , wherein said sulfur replaced monophosphate, diphosphate or triphosphate is 5′-alpha-thiotriphosphate or 5′-gamma-thiotriphosphate, said 5′-alkylphosphonate is RP(OH)(O)—O-5′- or (OH)2(O)P-5′-CH 2 —), and said 5′-alkyletherphosphonate is RP(OH)(O)—O-5′-.
14. The RISC according to claim 1 , wherein the 3′ end of said RNA component is stabilized against degradation.
15. The RISC according to claim 14 , wherein the 3′ end of said RNA component is stabilized against degradation by incorporating non-nucleotide chemical derivatives selected from the group consisting of aminolinkers, thiol linkers, carboxyl linkers, and non-nucleotidic spacers.
16. The RISC according to claim 1 , wherein the 3′ end of said RNA component is modified with biotin or fluoresceine.