Structurally designed shRNAs
AssigneeCold Spring Harbir Laboratory
InventorsGregory J. Hannon, Sihem Cheloufi
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Abstract
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.
Claims20
A pre-miR-451 structural mimic consisting of a short hairpin RNA molecule (shRNA) comprising
- (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 second sequence is fully complementary to the sequence of the first 17 or 18 nucleotides counted from the 5′ end of the first sequence,
- which structural mimic is DICER independent and requires AGO2 catalysis.
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 a pre-miR-451 structural mimic which is DICER independent and requires AGO2 catalysis and which consists of a short hairpin RNA molecule (shRNA) operably linked to an RNA polymerase promoter, the shRNA comprising:
- (i) a first sequence of 21, 22 or 23 nucleotides fully complementary to a sequence in the coding region of the target gene,
- (ii) a second sequence directly following the first sequence, wherein the 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 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.
Dependencies are inferred from the claim text.