ShRNA molecules and methods of use thereof
The present invention relates to certain novel shRNA molecules and methods of use thereof. According to certain embodiments of the present invention, methods for reducing the expression level of a target gene are provided. Such methods generally comprise providing a cell with one or more precursor nucleic acid sequences that encode two or more RNA molecules. A first RNA molecule comprises a double stranded sequence, which includes a guide strand sequence that is complementary to a portion of an mRNA transcript encoded by the target gene. In addition, a second RNA molecule comprises a second double stranded sequence, which includes a second guide strand sequence that is partially complementary to a portion of the mRNA transcript encoded by the target gene. Preferably, the second guide strand sequence comprises one or more bases that are mismatched with a nucleic acid sequence of the mRNA transcript encoded by the target gene.
1. A method for reducing the expression level of a target gene, comprising:
providing a cell with a single contiguous bifunctional RNA molecule that comprises:
a first double stranded sequence comprising a guide strand sequence that is fully complementary to a passenger strand and an mRNA transcript encoded by the target gene; and
a second double stranded sequence that is partially complementary to a mismatched passenger strand and complementary to the mRNA transcript encoded by the target gene, wherein the bifunctional RNA molecule activates cleavage-dependent and cleavage-independent RNA-induced silencing complex for reducing the expression level of the target gene.
2. The method of claim 1 , wherein the first and second double stranded sequences reside within a stem portion of separate stem loop structures.
3. The method of claim 1 , wherein the first guide strand sequence is presented to a cleavage dependent RNA-induced silencing complex and binds to an mRNA transcript encoded by the target gene.
4. The method of claim 3 , wherein the binding of the first guide strand sequence to the mRNA transcript causes degradation of the mRNA transcript.
5. The method of claim 3 , wherein the second double stranded sequence is presented to a cleavage independent RNA-induced silencing complex and sequesters the mRNA transcript encoded by the target gene.
6. The method of claim 5 , wherein sequestering the mRNA transcript encoded by the target gene represses the translation of the mRNA transcript.
7. The method of claim 1 , wherein the bifunctional RNA molecule is operably connected to a promoter.
8. The method of claim 7 , wherein the promoter is selected from the group consisting of a tumor cell specific promoter, a regulatable promoter, and an inducible promoter.
9. The method of claim 1 , wherein the first double stranded sequence and the second double stranded sequence initially reside within a single primary transcript.
10. The method of claim 9 , wherein the primary transcript further comprises a third RNA molecule and a fourth RNA molecule which both comprise double stranded sequences within a stem portion of separate stem loop structures.
11. The method of claim 10 , wherein the first and second double stranded sequences comprise a guide strand sequence that is complementary to an mRNA transcript encoded by a first target gene, whereas the third and fourth RNA molecules comprise a guide strand sequence that is complementary to an mRNA transcript encoded by a second target gene.
12. The method of claim 2 , wherein each stem loop structure comprises about 40 to 100 nucleotides, wherein the stem portion thereof comprises about 19-45 nucleotides and a loop portion thereof comprises about 4-25 nucleotides.
13. The method of claim 12 , wherein each stem loop structure comprises about 50 to 75 nucleotides, the stem portion thereof comprises about 20-30 nucleotides, and the loop portion thereof comprises about 6-15 nucleotides.
14. A method for reducing the expression level of a target gene comprising:
providing a cell with a bifunctional. RNA molecule comprising a first and a second portion, wherein:
the first portion comprises a first double stranded sequence comprising a guide strand sequence that is fully complementary to a passenger strand and a portion of an mRNA transcript encoded by the target gene, wherein the guide strand sequence is presented to a cleavage-dependent RNA-induced silencing complex; and
the second portion comprises a second double stranded sequence that is partially complementary to a mismatched passenger strand and to the mRNA transcript encoded by the target gene, wherein the second double stranded sequence is presented to a cleavage-independent RNA-induced silencing complex,
wherein the bifunctional RNA molecule activates both cleavage-dependent and cleavage-independent RNA-induced silencing complex for reducing the expression level of the target gene.
15. The method of claim 14 , wherein the second double stranded sequence comprises a second mismatched guide strand sequence that is partially complementary to a passenger strand and the mRNA transcript encoded by the target gene, wherein the second double stranded sequence is capable of sequestering translation of the mRNA transcript encoded by the target gene.