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 having a molecular weight of from up to about 150-160 kDa.
2 . The RISC of claim 1 comprising at least one member of the Argonaute family of proteins.
3 . The RISC of claim 1 containing eIF2C1 and/or eIFC2 and optionally at least one of eIFC3, eIFC4, HILI and HIWI.
4 . The RISC of claim 1 , further containing an RNA component, particularly a single-stranded RNA molecule.
5 . The RISC of claim 4 , wherein the single-stranded RNA molecule has a length from 14-50 nucleotides wherein at least the 14-20 5′ most nucleotides are substantially complementary to a target transcript.
6 . The RISC of claim 4 , wherein said RNA molecule has a length from 15-29 nucleotides.
7 . The RISC of claim 4 , wherein said RNA molecule has a free 5′ hydroxyl moiety or a moiety selected from phosphate groups or analogues thereof.
8 . The RISC of claim 7 , wherein said RNA molecule has a 5′-moiety selected from 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-0-5′-(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 (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-).
9 . The RISC of claim 1 , wherein said RNA molecule is completely complementary to said target transcript, optionally with the exception of nucleotides that extend beyond position 20 (counted from the 5′ terminus).
10 . The RISC of claim 1 , wherein said RNA molecule comprises at least one modified nucleotide analogue, which is preferably selected from sugar-backbone- and nucleobase-modified ribonucleotides and combinations thereof.
11 . The RISC of claim 1 , wherein said RNA molecule is associated with biodegradable polymers or microparticles, preferably wherein said association comprises a covalent coupling, in particular a covalent coupling via the 3′-terminus of the RNA molecule.
12 . A host cell or non-human host organism capable of overexpressing RISC according to claim 1 .
13 . 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 .
14 . The RISC molecule according to claim 1 for use as a target for diagnosis and/or therapy.
15 . The RISC according to claim 1 for use as a diagnostic and/or therapeutic agent itself, as a molecular-biological reagent or as component in a screening procedure for identification and/or characterization of pharmaceutical agents.