Structurally designed shRNAs
Provided is an improved design of shRNA based on structural mimics of miR-451 precursors. These miR-451 shRNA mimics are channeled through a novel small RNA biogenesis pathway, require AGO2 catalysis and are processed by Drosha but are independent of DICER processing. This miRNA pathway feeds active elements only into Ago2 because of its unique catalytic activity. These data demonstrate that this newly identified small RNA biogenesis pathway can be exploited in vivo to produce active molecules.
1. A short hairpin RNA molecule (shRNA) comprising
a) (i) a first sequence of 19, 20 or 21 nucleotides fully complementary to a sequence in a target gene, having a sequence other than the mature sequence of miR-451,
(ii) a second sequence directly following the first sequence, wherein the entire second sequence is fully complementary to the sequence of the first 15 or 16 nucleotides counted from the 5′ end of the first sequence,
wherein the first sequence and the second sequence form the shRNA; or
b) (i) a first sequence of 21, 22 or 23 nucleotides fully complementary to a sequence in the coding region of a target gene,
(ii) a second sequence directly following the first sequence, wherein the entire second sequence is fully complementary to the sequence of the first 17 or 18 nucleotides counted from the 5′ end of the first sequence,
wherein the first sequence and the second sequence form the shRNA.
2. The shRNA of claim 1 , wherein the first sequence of step (b) has a sequence other than the mature sequence of miR-451.
3. The shRNA of claim 1 , wherein the first sequence of step (a) is fully complementary to a coding region of the target gene.
4. The shRNA of claim 3 , wherein the first sequence is fully complementary to a sequence in an mRNA molecule encoded by the gene, wherein the sequence in the mRNA molecule is present in the sequence of the target gene.
5. The shRNA of claim 4 , wherein the first sequence is fully complementary to a 3′ untranslated region (UTR) sequence in an mRNA molecule encoded by the gene, wherein the 3° UTR sequence in the mRNA molecule is present in the sequence of the target gene.
6. The shRNA of claim 1 , wherein the second sequence of step (b) directly following the first sequence is fully complementary to the sequence of the first 18 nucleotides counted from the 5′ end of the first sequence.
7. The shRNA of claim 1 , wherein the last 3 nucleotides of the first sequence form a loop region in the short hairpin molecule.
8. The shRNA of claim 1 , wherein the last 4 nucleotides of the first sequence form a loop region in the short hairpin molecule.
9. The shRNA of claim 1 , wherein the shRNA has a 1, 2, or 3 nucleotide overhang at its 3′ end.
10. The shRNA of claim 1 , wherein the shRNA has more than a 3 nucleotide overhang at its 3′ end.
11. The shRNA of claim 1 , wherein the shRNA has a 1, 2, or 3 nucleotide overhang at its 5′ end.
12. The shRNA of claim 1 , wherein the shRNA has more than a 3 nucleotide overhang at its 5′ end.
13. The shRNA of claim 1 , wherein the shRNA has no 3′ or 5′ overhang.
14. The shRNA of claim 1 , wherein the shRNA consists of from 38 to 50 nucleotides.
15. The shRNA of claim 1 having the structure
wherein X 2 to X 22 are nucleotides complementary to a sequence in a target gene, and are in a sequence other than the mature sequence of miR-451;
Y 4 to Y 20 are nucleotides complementary to X 2 to X 18 ; and
X 1 , Y 1 , Y 2 , and Y 3 , are nucleotides that may be present or absent, wherein, X 1 and Y 3 , when present, may be complementary or not complementary.
16. An expression vector comprising a sequence encoding the shRNA according to claim 1 operably linked to an RNA polymerase promoter.
17. A library of expression vectors, each expression vector encoding the shRNA according to claim 1 operably linked to an RNA polymerase promoter.
18. An isolated mammalian cell comprising the shRNA according to claim 1 .
19. A method of attenuating expression of a target gene in a mammalian cell, the method comprising introducing into the mammalian cell an expression vector comprising a sequence encoding the short hairpin RNA molecule (shRNA) of claim 1 ,
wherein the shRNA molecule is expressed in the mammalian cell in an amount sufficient to attenuate expression of the target gene in a sequence specific manner, whereby expression of the target gene is inhibited.