Methods and compositions for RNA interference
AssigneeCold Spring Harbor Laboratory
InventorsGregory Hannon, Patrick Paddison, Despina Siolas
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Abstract
The present invention provides methods for attenuating gene expression in a cell, especially in a mammalian cell, using gene-targeted double stranded RNA (dsRNA), such as a hairpin RNA. The dsRNA contains a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the gene to be inhibited (the “target” gene).
Claims35
A method for attenuating expression of a target gene in mammalian cells, comprising introducing into the mammalian cells a single-stranded hairpin ribonucleic acid (shRNA) comprising self complementary sequences of 19 to 100 nucleotides that form a duplex region, which self complementary sequences hybridize under intracellular conditions to a target gene, wherein said hairpin RNA:
- (i) is a substrate for cleavage by a RNaseIII enzyme to produce a double-stranded RNA product,
- (ii) does not produce a general sequence-independent killing of the mammalian cells, and
- (iii) reduces expression of said target gene in a manner dependent on the sequence of said complementary regions, and,
- wherein said shRNA comprises a 3′ overhang of about 1-4 nucleotides.
A method for attenuating expression of a target gene in mammalian cells, comprising introducing into the mammalian cells a single-stranded hairpin ribonucleic acid (shRNA) comprising self complementary sequences of 19 to 100 nucleotides that form a duplex region, which self complementary sequences hybridize under intracellular conditions to a target gene, wherein said hairpin RNA:
- (i) is cleaved in the mammalian cells to produce an RNA guide sequence that enters an Argonaut-containing complex,
- (ii) does not produce a general sequence-independent killing of the mammalian cells, and
- (iii) reduces expression of said target gene in a manner dependent on the sequence of said complementary regions, and,
- wherein said shRNA comprises a 3′ overhang of about 1-4 nucleotides.
A method for attenuating expression of one or more target genes in mammalian cells, comprising introducing into the mammalian cells a variegated library of single- stranded hairpin ribonucleic acid (shRNA) species, each shRNA species comprising self complementary sequences of 19 to 100 nucleotides that form duplex regions and which hybridize under intracellular conditions to a target gene, wherein each of said hairpin RNA species:
- (i) is a substrate for cleavage by a RNaseIII enzyme to produce a double-stranded RNA product,
- (ii) does not produce a general sequence-independent killing of the mammalian cells, and
- (iii) if complementary to a target sequence, reduces expression of said target gene in a manner dependent on the sequence of said complementary regions, and,
- wherein said shRNA comprises a 3′ overhang of about 1-4 nucleotides.
A method of designing a short hairpin RNA (shRNA) construct for RNAi, said shRNA comprising a 3′ overhang of about 1-4 nucleotides, the method comprising selecting the nucleotide about 21 bases 5′ to the most 3′-end nucleotide as the first paired nucleotide in a cognate doubled-stranded siRNA with the same 3′ overhang.
Dependencies are inferred from the claim text.