Extended dicer substrate agents and methods for the specific inhibition of gene expression
The invention provides compositions and methods for reducing expression of a target gene in a cell, involving contacting a cell with an isolated double stranded nucleic acid (dsNA) in an amount effective to reduce expression of a target gene in a cell. The dsNAs of the invention possess a pattern of deoxyribonucleotides (in most embodiments, the pattern comprises at least one deoxyribonucleotide-deoxyribonucleotide base pair) designed to direct the site of Dicer enzyme cleavage within the dsNA molecule. Deoxyribonucleotides of the dsNA molecules of the invention are located within a region of the dsNA that can be excised via Dicer cleavage to generate an active siRNA agent that no longer contains the deoxyribonucleotide pattern (e.g., deoxyribonucleotide-deoxyribonucleotide base pairs). Such DNA-extended Dicer-substrate siRNAs (DsiRNAs) were demonstrated to be more effective RNA inhibitory agents than corresponding double stranded RNA-extended DsiRNAs. DsiRNA agents were also found to tolerate guide strand mismatches.
1. A double stranded nucleic acid molecule comprising: a sense strand of 21-50 nucleotides in length; and an antisense strand of 15-22 nucleotides in length, where said sense and antisense strands form a duplex; wherein the sense and antisense strands are separate strands; wherein the sense strand comprises a tetraloop adjacent to said duplex; wherein said tetraloop has a nucleic acid sequence selected from the group consisting of UNCG, GNRA, CUUG, d(GNNA), d(CNNG), and d(TNCG); and wherein said antisense strand is sufficiently complementary to a target mRNA along at least 15 nucleotides of said antisense strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammal or a mammalian cell.
2. The double stranded nucleic acid molecule of claim 1 , wherein the tetraloop has a nucleic acid sequence selected from the group consisting of UUCG, GAAA, d(GTTA), and d(TTCG).
3. The double stranded nucleic acid molecule of claim 1 , wherein the tetraloop is flanked at the 5′ end by a nucleic acid sequence selected from the group consisting of C, CC, G, and GG.
4. The double stranded nucleic acid molecule of claim 1 , wherein the tetraloop is flanked at the 5′ end by a nucleic acid sequence that duplexes with a nucleic sequence selected from the group consisting of C, CC, G, and GG.
5. The double stranded nucleic acid molecule of claim 1 , wherein the tetraloop is flanked at the 3′ end by a nucleic acid sequence selected from the group consisting of C, CC, G, and GG.
6. The double stranded nucleic acid molecule of claim 1 , wherein the tetraloop is flanked at the 3′ end by a nucleic acid sequence that duplexes with a nucleic acid sequence selected from the group consisting of C, CC, G, and GG.
7. The double stranded nucleic acid molecule of claim 1 , wherein the tetraloop has a nucleic acid sequence of GAAA.
8. The double stranded nucleic acid molecule of claim 1 , wherein the double stranded nucleic acid molecule is formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is suitable for human administration.