COMPOSITIONS AND METHODS FOR INHIBITING GENE SILENCING BY RNA INTERFERENCE
The present invention provides compositions and methods for inhibiting gene silencing by the RNAi pathway. The RNAi inhibitors of the invention have a reverse complement (RC) region to the target molecule of interest (e.g., miRNA) in association with at least one flanking region coupled to either at the 3′ or 5′ end of the RC region. The flanking regions can be single-stranded or can have one or more regions of double stranded nucleic acid with or without a hairpin loop. The RNAi inhibitors described herein can inhibit endogenous targets, including but not limited to microRNAs, or piRNAs, or can be used to inhibit the effects of exogenously introduced molecules, such as synthetic siRNAs, siRNAs expressed from vector constructs (e.g., viral expression systems), or siRNAs generated by enzymatic methods. Inhibition is specific, potent, prolonged, and can be performed on a single target or multiple targets simultaneously.
1 . An RNAi inhibitor comprising:
a first oligonucleotide comprising:
a central region having 3′ and 5′ ends, and having a central sequence that is from about 6 to about 37 nucleotides and having at least about 80% complementarity with a target RNA sequence, wherein the target RNA sequence is capable of silencing a target gene; and
at least one flanking region coupled to the 3′ or 5′ end of the central region, said at least one flanking region having a flanking sequence that is from about 10 to about 40 nucleotides and is substantially devoid of having complementarity with the target RNA sequence.
2 . An RNAi inhibitor as in claim 1 , wherein the 3′ flanking region is coupled to the 3′ end of the central region and the 5′ flanking region is coupled to the 5′ end of the central region.
3 . An RNAi inhibitor as in claim 2 , wherein the first oligonucleotide is single-stranded and the central region is about 17 to about 32 nucleotides and the 3′ flanking region and 5′ flanking region are about 12 to about 20 nucleotides.
4 . An RNAi inhibitor as in claim 3 , wherein said 3′ flanking region and 5′ flanking region are comprised of not more than about 70% pyrimidine nucleotides.
5 . An RNAi inhibitor as in claim 4 , wherein at least one of the central region, 3′ flanking or 5′ flanking region comprises at least one nucleotide having a 2′ modification.
6 . An RNAi inhibitor as in claim 5 , wherein the 2′ modification is a 2′-O-alkyl, 2′ orthoester, or 2′ ACE.
7 . An RNAi inhibitor as in claim 6 , wherein at least about 30% of nucleotides in the RNAi inhibitor have the 2′ modification.
8 . An RNAi inhibitor as in claim 7 , wherein about 100% of nucleotides in the RNAi inhibitor have the 2′ modification.
9 . An RNAi inhibitor as in claim 1 , wherein at least one of the 3′ flanking region or 5′ flanking region includes a duplex region.
10 . An RNAi inhibitor as in claim 9 , wherein the duplex region includes at least 10 consecutive nucleotides.
11 . An RNAi inhibitor as in claim 9 , wherein the at least one of the 3′ flanking region or 5′ flanking region includes a hairpin structure having the duplex region.
12 . An RNAi inhibitor as in claim 11 , wherein the duplex region includes at least 6 consecutive nucleotides.
13 . An RNAi inhibitor as in claim 11 , wherein the hairpin structure has a loop of about 4-15 nucleotides.
14 . An RNAi inhibitor as in claim 11 , wherein both the 3′ and 5′ flanking regions include a hairpin structure having the duplex region.
15 . An RNAi inhibitor as in claim 14 , wherein the central region includes about 21-28 nucleotides and the 3′ and 5′ flanking regions include a hairpin structure having the duplex region of about 10 base pairs and the loop of about 4-10 nucleotides.
16 . An RNAi inhibitor as in claim 9 , wherein at least one of the central region or flanking region comprises at least one nucleotide having a 2′ modification.
17 . An RNAi inhibitor as in claim 16 , wherein the 2′ modification is a 2′-O-alkyl, 2′ orthoester, or 2′ ACE.
18 . An RNAi inhibitor as in claim 17 , wherein at least about 30% of nucleotides in the RNAi inhibitor have the 2′ modification.
19 . An RNAi inhibitor as in claim 18 , wherein at least about 30% of nucleotides in the central region have the 2′ modification.
20 . An RNAi inhibitor as in claim 18 , wherein about 100% of nucleotides in the central region have the 2′ modification.
21 . An RNAi inhibitor as in claim 16 , wherein the central region includes a higher percentage of nucleotides with the 2′ modification compared to the flanking region.
22 . An RNAi inhibitor as in claim 1 , wherein the target RNA sequence is selected from the group consisting of a region of a RISC-entering strand of a miRNA, a region of pre-miRNA, a mature region and regions bordering the mature region of pri-miRNA, a RISC-entering strand of siRNA, a RISC-entering strand of a short hairpin siRNA, and a RISC-entering strand of a piRNA.
23 . An RNAi inhibitor comprising:
a first oligonucleotide with a reverse complement region having 3′ and 5′ ends, and having a reverse complement sequence that is from about 17 to about 37 nucleotides and having at least about 80% complementarity with a target RNA sequence, wherein the target RNA sequence is capable of silencing a target gene; and
a second oligonucleotide annealed to and having at least about 80% complementarity with the first oligonucleotide, said second oligonucleotide having from about 17 to about 37 nucleotides.
24 . An RNAi inhibitor as in claim 23 , wherein at least about 30% of nucleotides in the first oligonucleotide have a 2′ modification.
25 . An RNAi inhibitor as in claim 24 , wherein about 100% of nucleotides in the first oligonucleotide have the 2′ modification.
26 . An RNAi inhibitor as in claim 23 , further comprising one or more bulges or mismatches between the first and second oligonucleotides.
27 . An RNAi inhibitor as in claim 23 , wherein less than about 30% of nucleotides in the second oligonucleotide have the 2′ modification.
28 . An RNAi inhibitor as in claim 23 , wherein the second oligonucleotide is substantially devoid of having the 2′ modification.
29 . An RNAi inhibitor as in claim 23 , wherein the 2′ modification is a 2′-O-alkyl, 2′ orthoester, or 2′ ACE.
30 . An RNAi inhibitor as in claim 23 , further comprising a conjugate coupled to at least one oligonucleotide of the RNAi inhibitor.
31 . An RNAi inhibitor as in claim 30 , wherein the conjugate is coupled to the at least one oligonucleotide via a linker.
32 . A method of inhibiting an RNAi pathway in a cell so as to inhibit a target gene from being silenced by a target RNA sequence, the method comprising:
providing a cell capable of expressing the target RNA sequence;
introducing an RNAi inhibitor into the cell, the RNAi inhibitor comprising:
a central region having 3′ and 5′ ends and a central sequence that is from about 6 to about 37 nucleotides and has at least about 80% complementarity with the target RNA sequence; and
at least one flanking region coupled to the 3′ or 5′ end of the central region, said at least one flanking region having a flanking sequence that is from about 10 to about 40 nucleotides and is substantially devoid of having complementarity with the target RNA sequence;
maintaining the cell under conditions in which the silencing of the target gene by the target miRNA is inhibited.
33 . A method of inhibiting an RNAi pathway in a cell so as to inhibit a target gene from being silenced by a target RNA sequence, the method comprising:
providing a cell capable of expressing the target RNA sequence;
introducing an RNAi inhibitor into the cell, the RNAi inhibitor comprising:
a first oligonucleotide with a reverse complement region having 3′ and 5′ ends, and having a reverse complement sequence that is from about 17 to about 37 nucleotides and having at least about 80% complementarity with a target RNA sequence, wherein the target RNA sequence is capable of silencing a target gene; and
a second oligonucleotide annealed to and having at least about 80% complementarity with the first oligonucleotide, said second oligonucleotide having from about 17 to about 37 nucleotides; and
maintaining the cell under conditions in which the silencing of the target gene by the target miRNA is inhibited.
34 . A method as in claim 32 , wherein at least one of the 3′ flanking region or 5′ flanking region includes a duplex region.
35 . A method as in claim 34 , wherein the 3′ flanking region is coupled to the 3′ end of the central region and the 5′ flanking region is coupled to the 5′ end of the central region.
36 . A method as in claim 34 , wherein said at least one of the 3′ flanking region or 5′ flanking region includes a hairpin structure having the duplex region.
37 . A method as in claim 35 , wherein said 3′ flanking region and 5′ flanking region both have a hairpin structure having the duplex region.
38 . A method as in claim 32 , wherein at least one nucleotide of at least one of the oligonucleotides has a 2′ modification.
39 . A method as in claim 38 , wherein the 2′ modification is a 2′-O-alkyl, 2′ orthoester, or 2′ ACE.
40 . A method as in claim 39 , wherein the central region includes more nucleotides with the 2′ modification compared to the 3′ flanking region or 5′ flanking region.
41 . A method as in claim 32 , wherein the target RNA sequence is selected from the group consisting of a region of a RISC-entering strand of a miRNA, a region of pre-miRNA, a mature region and regions bordering the mature region of pri-miRNA, a RISC-entering strand of siRNA, a RISC-entering strand of a short hairpin siRNA, and a RISC-entering strand of a piRNA.
42 . A method as in claim 32 , further comprising a conjugate coupled to at least one oligonucleotide of the RNAi inhibitor.
43 . A method as in claim 42 , wherein the conjugate is coupled to the at least one oligonucleotide via a linker.
44 . An RNAi inhibitor as in claim 23 , further comprises at least one phosphothioate internucleotide linkage in the first oligonucleotide.
45 . An RNAi inhibitor as in claim 31 , wherein the conjugate is cholesterol.
46 . An RNAi inhibitor as in claim 23 , wherein the target RNA sequence is a mature miRNA sequence.
47 . An RNAi inhibitor as in claim 23 , wherein the RNAi inhibitor is characterized by the following:
the first oligonucleotide having about 21 nucleotides in the reverse compliment region each having a 2′-O-methyl modification, at least one phosphothioate internucleotide linkage, and a cholesterol coupled to the 3′ end of the first oligonucleotide through a linker having from 4-8 carbon atoms; and
the second oligonucleotide being unmodified.